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遠山　育夫

  • 理事　理事
Last Updated :2023/02/11

研究者情報

所属

  • 理事　理事

職名

  • 理事

学位

  • 医学博士（1988年05月 京都大学）

ホームページURL

J-Global ID

研究キーワード

  • 神経解剖学・神経病理学   神経科学   神経内科学   

研究分野

  • ライフサイエンス / 神経形態学
  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経科学一般
  • ライフサイエンス / 神経内科学

経歴

  • 2020年04月 - 現在  滋賀医科大学理事・副学長
  • 2010年04月 - 2020年03月  - 滋賀医科大学分子神経科学研究センターセンター長・教授
  • 1999年08月 - 2020年03月  滋賀医科大学神経難病研究センター教授
  • 1995年 - 1999年  滋賀医科大学 助教授
  • 1988年 - 1995年  滋賀医科大学 助手
  • 1988年 - 1995年  滋賀医科大学

学歴

  •         - 1988年   京都大学   医学研究科   内科学
  •         - 1988年   京都大学   Graduate School, Division of Medicine
  •         - 1981年   京都大学   医学部   医学部
  •         - 1981年   京都大学   Faculty of Medicine

所属学協会

  • 日本脳科学会   日本神経科学学会   ニューヨーク科学アカデミー   日本神経学会   日本解剖学会   米国神経科学会   日本認知症学会   日本神経化学会   日本組織細胞化学会   国際神経科学会(IBRO)   

研究活動情報

論文

  • Ayaka Chikugo, Yumi Irie, Chihiro Tsukano, Ayumi Uchino, Takahito Maki, Toshiaki Kume, Taiji Kawase, Kenji Hirose, Yusuke Kageyama, Ikuo Tooyama, Kazuhiro Irie
    ACS Chemical Neuroscience 2022年09月 研究論文（学術雑誌）
  • Asuka Matsuzaki Tada, Hamizah Shahirah Hamezah, Aslina Pahrudin Arrozi, Zulzikry Hafiz Abu Bakar, Daijiro Yanagisawa, Ikuo Tooyama
    Journal of Alzheimer's disease : JAD 2022年08月 研究論文（学術雑誌） 
    BACKGROUND: Tripeptide Met-Lys-Pro (MKP), a component of casein hydrolysates, has effective angiotensin-converting enzyme (ACE) inhibitory activity. Brain angiotensin II enzyme activates the NADPH oxidase complex via angiotensin II receptor type 1 (AT1) and enhances oxidative stress injury. ACE inhibitors improved cognitive function in Alzheimer's disease (AD) mouse models and previous clinical trials. Thus, although undetermined, MKP may be effective against pathological amyloid-β (Aβ) accumulation-induced cognitive impairment. OBJECTIVE: The current study aimed to investigate the potential of MKP as a pharmaceutical against AD by examining MKP's effect on cognitive function and molecular changes in the brain using double transgenic (APP/PS1) mice. METHODS: Experimental procedures were conducted in APP/PS1 mice (n = 38) with a C57BL/6 background. A novel object recognition test was used to evaluate recognition memory. ELISA was used to measure insoluble Aβ 40, Aβ 42, and TNF-α levels in brain tissue. Immunohistochemical analysis allowed the assessment of glial cell activation in MKP-treated APP/PS1 mice. RESULTS: The novel object recognition test revealed that MKP-treated APP/PS1 mice showed significant improvement in recognition memory. ELISA of brain tissue showed that MKP significantly reduced insoluble Aβ 40, Aβ 42, and TNF-α levels. Immunohistochemical analysis indicated the suppression of the marker for microglia and reactive astrocytes in MKP-treated APP/PS1 mice. CONCLUSION: Based on these results, we consider that MKP could ameliorate pathological Aβ accumulation-induced cognitive impairment in APP/PS1 mice. Furthermore, our findings suggest that MKP potentially contributes to preventing cognitive decline in AD.
  • Ayumi Uchino, Yumi Irie, Chihiro Tsukano, Taiji Kawase, Kenji Hirose, Yusuke Kageyama, Ikuo Tooyama, Ryo C. Yanagita, Kazuhiro Irie
    ACS Chemical Neuroscience 2022年08月 研究論文（学術雑誌）
  • Shino Shimizu, Ichiro Tojima, Keigo Nakamura, Hideaki Kouzaki, Takeshi Kanesaka, Norihiro Ogawa, Yoshio Hashizume, Hiroyasu Akatsu, Akira Hori, Ikuo Tooyama, Takeshi Shimizu
    Acta histochemica et cytochemica 55 3 93 - 98 2022年06月 研究論文（学術雑誌） 
    The pathological changes of Alzheimer's disease (AD) begin 10-20 years before clinical onset, and it is therefore desirable to identify effective methods for early diagnosis. The nasal mucosa is a target tissue for measuring AD-related biomarkers because the olfactory nerve is the only cranial nerve that is exposed to the external environment. We describe an autopsy case of rapidly advanced juvenile AD (JAD), focusing on the olfactory system. The formation of senile plaques, neurofibrillary tangles (NFTs), and neuropil threads was examined in the temporal cortex, hippocampus, olfactory bulb, and olfactory and respiratory epithelia in the bilateral olfactory clefts. Neurodegenerative changes in the olfactory and respiratory epithelia and the pathological deposition of amyloid β42 (Aβ42) and phosphorylated tau were also examined. As a result, senile plaques, NFTs, and neuropil threads were found in the temporal cortex, hippocampus, and olfactory bulb. NFTs were also found in the olfactory epithelium. Degenerated olfactory cells and their axons stained positive for phosphorylated tau. Supporting cells in the degenerated olfactory epithelium stained positive for Aβ42. In conclusion, pathological biomarkers of AD were expressed in the degenerated olfactory epithelium of this JAD patient. This observation suggests that nasal samples may be useful for the diagnosis of AD.
  • Kazuyuki Takata, Hiroyuki Kimura, Daijiro Yanagisawa, Koki Harada, Kaneyasu Nishimura, Yoshihisa Kitamura, Shun Shimohama, Ikuo Tooyama
    Molecules (Basel, Switzerland) 27 9 2022年04月 研究論文（学術雑誌） 
    Amyloid-β (Aβ) accumulation and tauopathy are considered the pathological hallmarks of Alzheimer's disease (AD), but attenuation in choline signaling, including decreased nicotinic acetylcholine receptors (nAChRs), is evident in the early phase of AD. Currently, there are no drugs that can suppress the progression of AD due to a limited understanding of AD pathophysiology. For this, diagnostic methods that can assess disease progression non-invasively before the onset of AD symptoms are essential, and it would be valuable to incorporate the concept of neurotheranostics, which simultaneously enables diagnosis and treatment. The neuroprotective pathways activated by nAChRs are attractive targets as these receptors may regulate microglial-mediated neuroinflammation. Microglia exhibit both pro- and anti-inflammatory functions that could be modulated to mitigate AD pathogenesis. Currently, single-cell analysis is identifying microglial subpopulations that may have specific functions in different stages of AD pathologies. Thus, the ability to image nAChRs and microglia in AD according to the stage of the disease in the living brain may lead to the development of new diagnostic and therapeutic methods. In this review, we summarize and discuss the recent findings on the nAChRs and microglia, as well as their methods for live imaging in the context of diagnosis, prophylaxis, and therapy for AD.
  • Yuka Matsushima, Yumi Irie, Yusuke Kageyama, Jean-Pierre Bellier, Ikuo Tooyama, Takahito Maki, Toshiaki Kume, Ryo C Yanagita, Kazuhiro Irie
    Chembiochem : a European journal of chemical biology 23 8 e202200029  2022年02月 研究論文（学術雑誌） 
    Amyloid β (Aβ) oligomers play a critical role in the pathology of Alzheimer's disease. Recently, we reported that a conformation-restricted Aβ42 with an intramolecular disulfide bond through cysteine residues at positions 17/28 formed stable oligomers with potent cytotoxicity. To further optimize this compound as a toxic conformer model, we synthesized three analogues with a combination of cysteine and homocysteine at positions 17/28. The analogues with Cys-Cys, Cys-homoCys, or homoCys-Cys, but not the homoCys-homoCys analogue, exhibited potent cytotoxicity against SH-SY5Y and THP-1 cells even at 10 nM. In contrast, the cytotoxicity of conformation-restricted analogues at positions 16/29 or 18/27 was significantly weaker than that of wild-type Aβ42. Furthermore, thioflavin-T assay, non-denaturing gel electrophoresis, and morphological studies suggested that the majority of these conformation-restricted analogues exists in an oligomeric state in cell culture medium, indicating that the toxic conformation of Aβ42, rather than the oligomeric state, is essential to induce cytotoxicity.
  • Zulzikry Hafiz Abu Bakar, Jean-Pierre Bellier, Daijiro Yanagisawa, Tomoko Kato, Ken-Ichi Mukaisho, Ikuo Tooyama
    International journal of molecular sciences 23 1 2022年01月 研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is a mitochondrial iron storage protein associated with neurodegenerative diseases. In patients with progressive supranuclear palsy (PSP), FtMt was shown to accumulate in nigral neurons. Here, we investigated FtMt and LC3 in the post-mortem midbrain of PSP patients to reveal novel aspects of the pathology. Immunohistochemistry was used to assess the distribution and abnormal changes in FtMt and LC3 immunoreactivities. Colocalization analysis using double immunofluorescence was performed, and subcellular patterns were examined using 3D imaging and modeling. In the substantia nigra pars compacta (SNc), strong FtMt-IR and LC3-IR were observed in the neurons of PSP patients. In other midbrain regions, such as the superior colliculus, the FtMt-IR and LC3-IR remained unchanged. In the SNc, nigral neurons were categorized into four patterns based on subcellular LC3/FtMt immunofluorescence intensities, degree of colocalization, and subcellular overlapping. This categorization suggested that concomitant accumulation of LC3/FtMt is related to mitophagy processes. Using the LC3-IR to stage neuronal damage, we retraced LC3/FtMt patterns and revealed the progression of FtMt accumulation in nigral neurons. Informed by these findings, we proposed a hypothesis to explain the function of FtMt during PSP progression.
  • Nor Faeizah Ibrahim, Hamizah Shahirah Hamezah, Daijiro Yanagisawa, Mayumi Tsuji, Yuji Kiuchi, Kenjiro Ono, Ikuo Tooyama
    Biochemistry and biophysics reports 28 101131 - 101131 2021年12月 研究論文（学術雑誌） 
    One of the neuropathological hallmarks of Alzheimer's disease (AD)-causing neurodegeneration and consequent memory deterioration, and eventually, cognitive decline-is amyloid-β (Aβ) aggregation forming amyloid plaques. Our previous study showed the potential of a tocotrienol-rich fraction-a mixture of naturally occurring of vitamin E analogs-to inhibit Aβ aggregation and restore cognitive function in an AD mouse model. The current study examined the effect of three vitamin E analogs-α-tocopherol (α-TOC), α-tocotrienol (α-T3), and γ-tocotrienol (γ-T3)-on Aβ aggregation, disaggregation, and oligomerization in vitro. Thioflavin T (ThT) assay showed α-T3 reduced Aβ aggregation at 10 μM concentration. Furthermore, both α-T3 and γ-T3 demonstrated Aβ disaggregation, as shown by the reduction of ThT fluorescence. However, α-TOC showed no significant effect. We confirmed the results for ThT assays with scanning electron microscopy imaging. Further investigation in photo-induced cross-linking of unmodified protein assay indicated a reduction in Aβ oligomerization by γ-T3. The present study thus revealed the individual effect of each tocotrienol analog in reducing Aβ aggregation and oligomerization as well as disaggregating preformed fibrils.
  • Natsumi Yamaguchi, Toshinori Sawano, Kae Fukumoto, Jin Nakatani, Shota Inoue, Nobutaka Doe, Daijiro Yanagisawa, Ikuo Tooyama, Takayuki Nakagomi, Tomohiro Matsuyama, Hidekazu Tanaka
    Brain research 1767 147542 - 147542 2021年09月 研究論文（学術雑誌） 
    Cerebral infarction causes motor, sensory, and cognitive impairments. Although rehabilitation enhances recovery of activities of daily living after cerebral infarction, its mechanism remains elusive due to the lack of reproducibility and low survival rate of brain ischemic model animals. Here, to investigate the relationship between rehabilitative intervention, motor function, and pathophysiological remodeling of the tissue in the ipsilateral hemisphere after cerebral infarction, we took advantage of a highly reproducible model of cerebral infarction using C.B-17/Icr-+/+Jcl mice. In this model, we confirmed that voluntary running exercise improved functional recovery after ischemia. Exercise did not alter the volume of infarction or survived cortex, or the number of NeuN-labeled cells in the peri-infarct cortex. In mice who did not exercise, the number of basal dendritic spines of layer 5 pyramidal cells decreased in the peri-infarct motor cortex, whereas in mice who exercised it remained at the normal level. The voluntary exercise intervention maintained basal dendritic spine density within the peri-infarct area, which may reflect an adaptive remodeling of the surviving neural circuitry that might contribute to promoting the recovery of activities of daily living.
  • Yusuke Kageyama, Yumi Irie, Yuka Matsushima, Tatsuya Segawa, Jean-Pierre Bellier, Kumi Hidaka, Hiroshi Sugiyama, Daita Kaneda, Yoshio Hashizume, Hiroyasu Akatsu, Kunio Miki, Akiko Kita, Douglas G Walker, Kazuhiro Irie, Ikuo Tooyama
    ACS chemical neuroscience 12 18 3418 - 3432 2021年09月 研究論文（学術雑誌） 
    Characterization of amyloid β (Aβ) oligomers, the transition species present prior to the formation of Aβ fibrils and that have cytotoxicity, has become one of the major topics in the investigations of Alzheimer's disease (AD) pathogenesis. However, studying pathophysiological properties of Aβ oligomers is challenging due to the instability of these protein complexes in vitro. Here, we report that conformation-restricted Aβ42 with an intramolecular disulfide bond at positions 17 and 28 (SS-Aβ42) formed stable Aβ oligomers in vitro. Thioflavin T binding assays, nondenaturing gel electrophoresis, and morphological analyses revealed that SS-Aβ42 maintained oligomeric structure, whereas wild-type Aβ42 and the highly aggregative Aβ42 mutant with E22P substitution (E22P-Aβ42) formed Aβ fibrils. In agreement with these observations, SS-Aβ42 was more cytotoxic compared to the wild-type and E22P-Aβ42 in cell cultures. Furthermore, we developed a monoclonal antibody, designated TxCo-1, using the toxic conformation of SS-Aβ42 as immunogen. X-ray crystallography of the TxCo-1/SS-Aβ42 complex, enzyme immunoassay, and immunohistochemical studies confirmed the recognition site and specificity of TxCo-1 to SS-Aβ42. Immunohistochemistry with TxCo-1 antibody identified structures resembling senile plaques and vascular Aβ in brain samples of AD subjects. However, TxCo-1 immunoreactivity did not colocalize extensively with Aβ plaques identified with conventional Aβ antibodies. Together, these findings indicate that Aβ with a turn at positions 22 and 23, which is prone to form Aβ oligomers, could show strong cytotoxicity and accumulated in brains of AD subjects. The SS-Aβ42 and TxCo-1 antibody should facilitate understanding of the pathological role of Aβ with toxic conformation in AD.
  • Anarmaa Mendsaikhan, Ikuo Tooyama, Geidy E Serrano, Thomas G Beach, Douglas G Walker
    Journal of neuropathology and experimental neurology 80 8 741 - 753 2021年09月 研究論文（学術雑誌） 
    Alzheimer disease (AD) is a progressive neurodegenerative disease causing cognitive decline in the aging population. To develop disease-modifying treatments, understanding the mechanisms behind the pathology is important, which should include observations using human brain samples. We reported previously on the association of lysosomal proteins progranulin (PGRN) and prosaposin (PSAP) with amyloid plaques in non-demented aged control and AD brains. In this study, we investigated the possible involvement of PGRN and PSAP in tangle formation using human brain tissue sections of non-demented aged control subjects and AD cases and compared with cases of frontotemporal dementia with granulin (GRN) mutations. The study revealed that decreased amounts of PGRN and PSAP proteins were detected even in immature neurofibrillary tangles, while colocalization was still evident in adjacent neurons in all cases. Results suggest that neuronal loss of PGRN preceded loss of PSAP as tangles developed and matured. The GRN mutation cases exhibited almost complete absence of PGRN in most neurons, while PSAP signal was preserved. Although based on correlative data, we suggest that reduced levels of PGRN and PSAP and their interaction in neurons might predispose to accumulation of p-Tau protein.
  • Aslina Pahrudin Arrozi, Zulzikry Hafiz Abu Bakar, Hiroyasu Taguchi, Daijiro Yanagisawa, Ikuo Tooyama
    Molecules (Basel, Switzerland) 26 17 2021年09月 研究論文（学術雑誌） 
    Thioredoxin-interacting protein (TXNIP) is involved in multiple disease-associated functions related to oxidative stress, especially by inhibiting the anti-oxidant- and thiol-reducing activity of thioredoxin (TXN). Shiga-Y5 (SY5), a fluorine-19 magnetic resonance probe for detecting amyloid-β deposition in the brain, previously showed therapeutic effects in a mouse model of Alzheimer's disease; however, the mechanism of action of SY5 remains unclear. SY5 passes the blood-brain barrier and then undergoes hydrolysis to produce a derivative, Shiga-Y6 (SY6), which is a TXNIP-negative regulator. Therefore, this study investigates the therapeutic role of SY5 as the prodrug of SY6 in the thioredoxin system in the brain of a mouse model of Alzheimer's disease. The intraperitoneal injection of SY5 significantly inhibited TXNIP mRNA (p = 0.0072) and protein expression (p = 0.0143) induced in the brain of APP/PS1 mice. In contrast, the levels of TXN mRNA (p = 0.0285) and protein (p = 0.0039) in the brain of APP/PS1 mice were increased after the injection of SY5. The ratio of TXN to TXNIP, which was decreased (p = 0.0131) in the brain of APP/PS1 mice, was significantly increased (p = 0.0072) after the injection of SY5. These results suggest that SY5 acts as a prodrug of SY6 in targeting the thioredoxin system and could be a potential therapeutic compound in oxidative stress-related diseases in the brain.
  • Shogo Higaki, Tomomi Nishie, Takaaki Todo, Reiko Teshima, Kenichiro Kusumi, Risa Mitsumori, Ikuo Tooyama, Yasuhiro Fujioka, Toshihiro Kawasaki, Noriyoshi Sakai, Tatsuyuki Takada
    Journal of fish biology 2021年06月 研究論文（学術雑誌） 
    Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.
  • Zulzikry Hafiz Abu Bakar, Tomoko Kato, Daijiro Yanagisawa, Jean-Pierre Bellier, Ken-Ichi Mukaisho, Ikuo Tooyama
    Acta histochemica et cytochemica 54 3 97 - 104 2021年06月 研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is a novel ferritin that is localized in the mitochondria. FtMt expression is low in the liver and spleen, and high in the heart, testis, and brain. We previously detected FtMt in dopaminergic neurons in the substantia nigra pars compacta (SNc) in human and monkey midbrains. We investigated the localization and expression of FtMt in the midbrain of patients with progressive supranuclear palsy (PSP) and controls using a monoclonal antibody (C65-2) against human FtMt. FtMt immunoreactivity was weakly detected in neuromelanin-containing neurons in the SNc and ventral tegmental area (VTA) of control cases compared with PSP, which exhibited a remarkable increase in FtMt immunoreactivity. Preincubation of C65-2 with the immunizing FtMt peptide significantly reduced the staining, indicating the specificity of C65-2. Several puncta were observed outside the neurons of PSP, in contrast with the control cases. Double immunofluorescence histochemistry for FtMt and tyrosine hydroxylase (TH), glial fibrillary acidic protein, and Iba1 showed localization of FtMt in dopaminergic neurons, microglia, and astrocytes in PSP. Furthermore, FtMt immunoreactivity was detected in a few TH-negative neurons. In the SNc and VTA, FtMt immunoreactivity colocalized with phosphorylated tau immunoreactivity. Our results indicate that FtMt is involved in the pathology of PSP. Clarifying the involvement of FtMt in PSP is of great interest.
  • Daijiro Yanagisawa, Hamizah Shahirah Hamezah, Aslina Pahrudin Arrozi, Ikuo Tooyama
    Scientific reports 11 1 9623 - 9623 2021年05月 研究論文（学術雑誌） 
    Tau, a family of microtubule-associated proteins, forms abnormal intracellular inclusions, so-called tau pathology, in a range of neurodegenerative diseases collectively known as tauopathies. The rTg4510 mouse model is a well-characterized bitransgenic F1 hybrid mouse model of tauopathy, which was obtained by crossing a Camk2α-tTA mouse line (on a C57BL/6 J background) with a tetO-MAPT*P301L mouse line (on a FVB/NJ background). The aim of this study was to investigate the effects of the genetic background and sex on the accumulation of tau pathology in reciprocal F1 hybrids of rTg4510 mice, i.e., rTg4510 on the (C57BL/6 J × FVB/NJ)F1 background (rTg4510_CxF) and on the (FVB/NJ × C57BL/6 J)F1 background (rTg4510_FxC). As compared with rTg4510_CxF mice, the rTg4510_FxC mice showed marked levels of tau pathology in the forebrain. Biochemical analyses indicated that the accumulation of abnormal tau species was accelerated in rTg4510_FxC mice. There were strong effects of the genetic background on the differential accumulation of tau pathology in rTg4510 mice, while sex had no apparent effect. Interestingly, midline-1 (Mid1) was identified as a candidate gene associated with this difference and exhibited significant up/downregulation according to the genetic background. Mid1 silencing with siRNA induced pathological phosphorylation of tau in HEK293T cells that stably expressed human tau with the P301L mutation, suggesting the role of Mid1 in pathological alterations of tau. Elucidation of the underlying mechanisms will provide novel insights into the accumulation of tau pathology and is expected to be especially informative to researchers for the continued development of therapeutic interventions for tauopathies.
  • Aslina Pahrudin Arrozi, Daijiro Yanagisawa, Tomoko Kato, Hiroyasu Akatsu, Yoshio Hashizume, Daita Kaneda, Ikuo Tooyama
    Journal of Alzheimer's disease reports 5 1 263 - 274 2021年04月 研究論文（学術雑誌） 
    Background: Emerging evidence indicates that the misfolded tau protein can propagate aggregates between cells in a prion-like manner. This prion activity has been typically studied in brain extracts of patients with Alzheimer's disease (AD), but not in the olfactory region that can be a potential biomarker in AD. Objective: To investigate the prion seeding activity of tau in nasal mucosa tissues using a cell culture model of tau propagation. Methods: Brain and nasal mucosa homogenates were added to HEK293T cells expressing three repeat or four-repeat domains of tau with the L266V, V337M (3RD*VM) and P301L and V377M mutations (4RD*LM) fused to the enhanced green fluorescence protein (EGFP) respectively. We also measured the level of phosphorylated tau (p-tau), total tau (t-tau), and p-tau/t-tau ratio and performed correlation analysis between tau prion activity and the level of tau. Results: We found that brain and nasal tissue homogenates from patients with AD significantly induced tau aggregation in HEK293T cells either expressing tau 3RD*VM-EGFP or 4RD*LM-EGFP compared with control brain and nasal tissue homogenates. The levels of p-tau and p-tau/t-tau ratio were significantly increased in the brain of patients with AD; however, no significant difference was found in nasal tissue compared with their respective control tissue homogenates. Conclusion: These results suggest that the nasal tissues contain tau seeds, similar to the brain, albeit without changes in the levels of p-tau and t-tau. Therefore, a cellular bioassay using nasal tissues would have great potential as an AD biomarker because of the usefulness of nasal tissue biopsy.
  • Shigeyoshi Shimaoka, Hitomi Hamaoka, Junji Inoue, Masato Asanuma, Ikuo Tooyama, Yoichi Kondo
    Acta medica Okayama 75 2 153 - 167 2021年04月 研究論文（学術雑誌） 
    Lactoferrin (Lf) is an iron-binding glycoprotein mainly found in exocrine secretions and the secondary granules of neutrophils. In the central nervous system (CNS), expression of the Lf protein has been reported in the lesions of some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as in the aged brain. Lf is primarily considered an iron chelator, protecting cells from potentially toxic iron or iron-requiring microorganisms. Other biological functions of Lf include immunomodulation and transcriptional regulation. However, the roles of Lf in the CNS have yet to be fully clarified. In this study, we raised an antiserum against mouse Lf and investigated the immunohistochemical localization of Lf-like immunoreactivity (Lf-LI) throughout the CNS of adult mice. Lf-LI was found in some neuronal populations throughout the CNS. Intense labeling was found in neurons in the olfactory systems, hypothalamic nuclei, entorhinal cortex, and a variety of brainstem nuclei. This study provides detailed information on the Lf-LI distribution in the CNS, and the findings should promote further understanding of both the physiological and pathological significance of Lf in the CNS.
  • Daijiro Yanagisawa, Nor Faeizah Ibrahim, Hiroyasu Taguchi, Shigehiro Morikawa, Takami Tomiyama, Ikuo Tooyama
    Molecules (Basel, Switzerland) 26 5 2021年03月 研究論文（学術雑誌） 
    Recent evidence suggests that the formation of soluble amyloid β (Aβ) aggregates with high toxicity, such as oligomers and protofibrils, is a key event that causes Alzheimer's disease (AD). However, understanding the pathophysiological role of such soluble Aβ aggregates in the brain in vivo could be difficult due to the lack of a clinically available method to detect, visualize, and quantify soluble Aβ aggregates in the brain. We had synthesized a novel fluorinated curcumin derivative with a fixed keto form, named as Shiga-Y51, which exhibited high selectivity to Aβ oligomers in vitro. In this study, we investigated the in vivo detection of Aβ oligomers by fluorine-19 (19F) magnetic resonance imaging (MRI) using Shiga-Y51 in an APP/PS1 double transgenic mouse model of AD. Significantly high levels of 19F signals were detected in the upper forebrain region of APP/PS1 mice compared with wild-type mice. Moreover, the highest levels of Aβ oligomers were detected in the upper forebrain region of APP/PS1 mice in enzyme-linked immunosorbent assay. These findings suggested that 19F-MRI using Shiga-Y51 detected Aβ oligomers in the in vivo brain. Therefore, 19F-MRI using Shiga-Y51 with a 7 T MR scanner could be a powerful tool for imaging Aβ oligomers in the brain.
  • Daijiro Yanagisawa, Tomoko Kato, Hiroyasu Taguchi, Nobuaki Shirai, Koichi Hirao, Takayuki Sogabe, Takami Tomiyama, Keizo Gamo, Yukie Hirahara, Masaaki Kitada, Ikuo Tooyama
    Biomaterials 270 120686 - 120686 2021年03月 研究論文（学術雑誌） 
    The accumulation of β-amyloid (Aβ) aggregates in the brain occurs early in the progression of Alzheimer's disease (AD), and non-fibrillar soluble Aβ oligomers are particularly neurotoxic. During binding to Aβ fibrils, curcumin, which can exist in an equilibrium state between its keto and enol tautomers, exists predominantly in the enol form, and binding activity of the keto form to Aβ fibrils is much weaker. Here we described the strong binding activity the keto form of curcumin derivative Shiga-Y51 shows for Aβ oligomers and its scant affinity for Aβ fibrils. Furthermore, with imaging mass spectrometry we revealed the blood-brain barrier permeability of Shiga-Y51 and its accumulation in the cerebral cortex and the hippocampus, where Aβ oligomers were mainly localized, in a mouse model of AD. The keto form of curcumin derivatives like Shiga-Y51 could be promising seed compounds to develop imaging probes and therapeutic agents targeting Aβ oligomers in the brain.
  • Ali Haidar Syaifullah, Akihiko Shiino, Akira Fujiyoshi, Aya Kadota, Keiko Kondo, Takahiro Ito, Hiroyoshi Segawa, Mohammad Moniruzzaman, Takashi Waki, Naoko Miyagawa, Ikuo Tooyama, Hirotsugu Ueshima, Katsuyuki Miura, Hirotsugu Ueshima, Katsuyuki Miura
    Alcohol (Fayetteville, N.Y.) 90 57 - 65 2021年02月 研究論文（学術雑誌） 
    The clinical implications of alcohol consumption have been extensively examined; however, its effects on brain structures in apparently healthy community-dwellers remain unclear. Therefore, we investigated the relationship between alcohol consumption and brain gray matter volume (GMV) in community-dwelling Japanese men using voxel-based morphometry (VBM). We recruited cognitively intact Japanese men, aged 40-79 years, from a population-based cohort in Shiga, Japan. Brain magnetic resonance imaging was performed, on average, 2 years after demographic and medical information was obtained in 2010-2014. A multivariable linear regression analysis of 639 men was conducted to elucidate the relationship between the amount of alcohol consumed and GMV. VBM statistics were analyzed by threshold-free cluster enhancement with a family-wise error rate of <0.05. The results obtained demonstrated that the amount of alcohol consumed was associated with lower GMV. The VBM analysis showed lower GMV within the parahippocampal, entorhinal, cingulate, insular, temporal, and frontal cortices and cerebellum in very heavy drinkers (≥42 ethanol g/day) than in non-drinkers. Furthermore, alcohol consumption was associated with a higher white matter lesion volume. These results suggest subclinical structural changes similar to alcohol-related neurological diseases.
  • Mohammad Moniruzzaman, Aya Kadota, Akihiko Shiino, Akira Fujiyoshi, Takahiro Ito, Ali Haidar Syaifullah, Naoko Miyagawa, Keiko Kondo, Takashi Hisamatsu, Hiroyoshi Segawa, Ikuo Tooyama, Hirotsugu Ueshima, Katsuyuki Miura
    Journal of physical activity & health 18 2 157 - 164 2021年01月 研究論文（学術雑誌） 
    BACKGROUND: To investigate the association between step counts and brain volumes (BVs)-global and 6 a priori selected cognition-related regions of interest-in Japanese men aged 40-79 years. METHODS: The authors analyzed data from 680 cognitively intact participants of the Shiga Epidemiological Study of Subclinical Atherosclerosis-a population-based observational study. Using multivariable linear regression, the authors assessed cross-sectional associations between 7-day step counts at baseline (2006-2008) and BVs at follow-up (2012-2015) for age-stratified groups (<60 y and ≥60 y). RESULTS: In the older adults ≥60 years, step counts at baseline (per 1000 steps) were associated with total BV at follow-up (β = 1.42, P = .022) while adjusted for potential covariates. Regions of interest-based analyses yielded an association of step counts with both prefrontal cortexes (P < .05) in older adults, while the left entorhinal cortex showed marginally significant association (P = .05). No association was observed with hippocampus, parahippocampal, cingulum, and cerebellum. No association was observed in younger adults (<60 y). CONCLUSIONS: The authors found a positive association between 7-day step counts and BVs, including prefrontal cortexes, and left entorhinal cortex in apparently healthy Japanese men.
  • 山口 菜摘, 福本 佳永, 澤野 俊憲, 中谷 仁, 柳沢 大治郎, 遠山 育夫, 田中 秀和
    日本薬理学会年会要旨集 94 1 - O-C2-2 公益社団法人 日本薬理学会 2021年 

    Many studies report that the rehabilitation after cerebral ischemia influences neural plasticity and improves the functional impairment, but its mechanism is not fully uncovered. The aim of this study is to reveal the detailed mechanisms of functional recovery after the rehabilitation, focusing on the peri-infarct cortex. We induced highly reproducible focal cerebral ischemia in C.B-17/Icr-+/+Jcl mice. These mice were subjected to voluntary running as a rehabilitative exercise. We divided the mice into 4 groups: ① Sham, ② Sham + exercise, ③ Middle cerebral artery occlusion (MCAO), and ④ MCAO + exercise. Voluntary exercise improved functional recovery in grid walking test and wire hang test. The percentage of ipsilateral hemisphere size to the contralateral and the number of NeuN-positive cells in the peri-infarct cortex was not altered by exercise. However, the neuronal morphology in peri-infarct motor cortex layer 5 was changed by exercise. Voluntary exercise suppressed the decrease in the number of dendritic spines after MCAO. Furthermore, voluntary exercise tended to increase the number of c-Fos-labeled cells in the peri-infarct motor cortex layer 5. Our data suggest that voluntary exercise after ischemia alters the morphology of peri-infarct layer 5 neurons, which in turn modifies neuronal activity.

  • Takao Morimune, Ayami Tano, Yuya Tanaka, Haruka Yukiue, Takefumi Yamamoto, Ikuo Tooyama, Yoshihiro Maruo, Masaki Nishimura, Masaki Mori
    PloS one 16 4 e0248517  2021年 研究論文（学術雑誌） 
    It is not fully understood how enzymes are regulated in the tiny reaction field of a cell. Several enzymatic proteins form cytoophidia, a cellular macrostructure to titrate enzymatic activities. Here, we show that the epileptic encephalopathy-associated protein Tbc1d24 forms cytoophidia in neuronal cells both in vitro and in vivo. The Tbc1d24 cytoophidia are distinct from previously reported cytoophidia consisting of inosine monophosphate dehydrogenase (Impdh) or cytidine-5'-triphosphate synthase (Ctps). Tbc1d24 cytoophidia is induced by loss of cellular juvenescence caused by depletion of Gm14230, a juvenility-associated lncRNA (JALNC) and zeocin treatment. Cytoophidia formation is associated with impaired enzymatic activity of Tbc1d24. Thus, our findings reveal the property of Tbc1d24 to form cytoophidia to maintain neuronal cellular juvenescence.
  • Haruka Tsubaki, Ikuo Tooyama, Douglas Gordon Walker
    International journal of molecular sciences 21 24 2020年12月 研究論文（学術雑誌） 
    The development of new therapeutic approaches to diseases relies on the identification of key molecular targets involved in amplifying disease processes. One such molecule is thioredoxin-interacting protein (TXNIP), also designated thioredoxin-binding protein-2 (TBP-2), a member of the α-arrestin family of proteins and a central regulator of glucose and lipid metabolism, involved in diabetes-associated vascular endothelial dysfunction and inflammation. TXNIP sequesters reduced thioredoxin (TRX), inhibiting its function, resulting in increased oxidative stress. Many different cellular stress factors regulate TXNIP expression, including high glucose, endoplasmic reticulum stress, free radicals, hypoxia, nitric oxide, insulin, and adenosine-containing molecules. TXNIP is also directly involved in inflammatory activation through its interaction with the nucleotide-binding domain, leucine-rich-containing family, and pyrin domain-containing-3 (NLRP3) inflammasome complex. Neurodegenerative diseases such as Alzheimer's disease have significant pathologies associated with increased oxidative stress, inflammation, and vascular dysfunctions. In addition, as dysfunctions in glucose and cellular metabolism have been associated with such brain diseases, a role for TXNIP in neurodegeneration has actively been investigated. In this review, we will focus on the current state of the understanding of possible normal and pathological functions of TXNIP in the central nervous system from studies of in vitro neural cells and the brains of humans and experimental animals with reference to other studies. As TXNIP can be expressed by neurons, microglia, astrocytes, and endothelial cells, a complex pattern of regulation and function in the brain is suggested. We will examine data suggesting TXNIP as a therapeutic target for neurodegenerative diseases where further research is needed.
  • 若年性アルツハイマー型認知症患者の嗅上皮における特異的蛋白の発現について
    清水 志乃, 戸嶋 一郎, 中村 圭吾, 神前 英明, 赤津 裕康, 遠山 育夫, 清水 猛史
    日本鼻科学会会誌 59 Suppl. S45 - S45 (一社)日本鼻科学会 2020年12月
  • Undral Buyandelger, Douglas Gordon Walker, Hiroyasu Taguchi, Daijiro Yanagisawa, Ikuo Tooyama
    Biochemical and biophysical research communications 532 4 668 - 674 2020年11月 研究論文（学術雑誌） 
    Thioredoxin-interacting protein (TXNIP) has multiple disease-associated functions including inducing oxidative stress by inhibiting the anti-oxidant and thiol reducing activity of thioredoxin (TRX), reducing cellular glucose transport, and is a component of the activated inflammasome complex. Increased expression of TXNIP is encountered in diabetic conditions of high glucose. Curcumin and chemical derivatives have multiple therapeutic properties as anti-inflammatories, anti-oxidants, amyloid aggregation inhibitors and modulate a number of cellular signaling pathways. Using a fluorinated-derivative of curcumin (designated Shiga-Y6), we showed significant inhibition of TXNIP mRNA and protein expression, and induction of TRX mRNA and protein in ARPE-19 retinal pigment epithelial cells and THP-1-derived macrophages, while the non-fluorinated structural equivalent (Shiga-Y52) and native curcumin did not show these same effects. Shiga-Y6 was effective in reducing high glucose, endoplasmic reticulum stress-induced TXNIP in ARPE-19 cells, and reducing lipopolysaccharide and endoplasmic stress-induced proinflammatory gene expression in THP-1 macrophages. Moreover, TXNIP-knockdown experiments showed that the anti-inflammatory effect of Shiga-Y6 in LPS-stimulated THP-1 macrophages was TXNIP-independent.
  • Faidruz Azura Jam, Takao Morimune, Atsushi Tsukamura, Ayami Tano, Yuya Tanaka, Yasuhiro Mori, Takefumi Yamamoto, Masaki Nishimura, Ikuo Tooyama, Masaki Mori
    Scientific reports 10 1 18044 - 18044 2020年10月 研究論文（学術雑誌） 
    Cell competition is a cell-cell interaction mechanism which maintains tissue homeostasis through selective elimination of unfit cells. During early brain development, cells are eliminated through apoptosis. How cells are selected to undergo elimination remains unclear. Here we aimed to identify a role for cell competition in the elimination of suboptimal cells using an in vitro neuroepithelial model. Cell competition was observed when neural progenitor HypoE-N1 cells expressing RASV12 were surrounded by normal cells in the co-culture. The elimination through apoptosis was observed by cellular changes of RASV12 cells with rounding/fragmented morphology, by SYTOX blue-positivity, and by expression of apoptotic markers active caspase-3 and cleaved PARP. In this model, expression of juvenility-associated genes Srsf7 and Ezh2 were suppressed under cell-competitive conditions. Srsf7 depletion led to loss of cellular juvenescence characterized by suppression of Ezh2, cell growth impairment and enhancement of senescence-associated proteins. The cell bodies of eliminated cells were engulfed by the surrounding cells through phagocytosis. Our data indicates that neuroepithelial cell competition may have an important role for maintaining homeostasis in the neuroepithelium by eliminating suboptimal cells through loss of cellular juvenescence.
  • Nur Zuliani Ramli, Mohamad Fairuz Yahaya, Ikuo Tooyama, Hanafi Ahmad Damanhuri
    Antioxidants (Basel, Switzerland) 9 10 2020年10月 研究論文（学術雑誌） 
    Nutraceuticals have been extensively studied worldwide due to its neuroprotective effects in in vivo and in vitro studies, attributed by the antioxidative properties. Alzheimer (AD) and Parkinson disease (PD) are the two main neurodegenerative disorders that are discussed in this review. Both AD and PD share the similar involvement of oxidative stress in their pathophysiology. Nutraceuticals exert their antioxidative effects via direct scavenging of free radicals, prevent damage to biomolecules, indirectly stimulate the endogenous antioxidative enzymes and gene expressions, inhibit activation of pro-oxidant enzymes, and chelate metals. In addition, nutraceuticals can act as modulators of pro-survival, pro-apoptotic, and inflammatory signaling pathways. They have been shown to be effective particularly in preclinical stages, due to their multiple mechanisms of action in attenuating oxidative stress underlying AD and PD. Natural antioxidants from food sources and natural products such as resveratrol, curcumin, green tea polyphenols, and vitamin E are promising therapeutic agents in oxidative stress-mediated neurodegenerative disease as they have fewer adverse effects, more tolerable, cheaper, and sustainable for long term consumption.
  • 認知症の分子イメージング MRIを用いた認知症の分子イメージング
    遠山 育夫, 柳沢 大治郎, 田口 弘康
    Dementia Japan 34 4 440 - 440 (一社)日本認知症学会 2020年10月
  • 神経炎と血管新生におけるミトコンドリア性フェリチン(FtMt)の役割(Roles of mitochondrial ferritin(FtMt) in neuroinflammation and angiogenesis)
    Buyandelger Undral, ウォカー・ダグラス, 柳沢 大治郎, 守村 敏史, 遠山 育夫
    Dementia Japan 34 4 527 - 527 2020年10月
  • タウ遺伝子改変マウスの遺伝的背景によるタウ蓄積量の変化
    柳沢 大治郎, Hamezah Hamizah Shahirah, Arrozi Aslina Pahrudin, 遠山 育夫
    Dementia Japan 34 4 528 - 528 (一社)日本認知症学会 2020年10月
  • Yosuke Kadota, Faidruz Azura Jam, Haruka Yukiue, Ichiro Terakado, Takao Morimune, Ayami Tano, Yuya Tanaka, Sayumi Akahane, Mayu Fukumura, Ikuo Tooyama, Masaki Mori
    iScience 23 6 101242 - 101242 2020年06月
  • Ali Tanweer Siddiquee, Aya Kadota, Akira Fujiyoshi, Naoko Miyagawa, Yoshino Saito, Harumitsu Suzuki, Keiko Kondo, Hiromi Yamauchi, Takahiro Ito, Hiroyoshi Segawa, Ikuo Tooyama, Katsuyuki Miura, Hirotsugu Ueshima
    Alcohol (Fayetteville, N.Y.) 85 145 - 152 2020年06月 [有り][無し]
     研究論文（学術雑誌） 
    Although heavy alcohol consumption has been identified as a risk factor for adverse cognitive functioning, it currently remains unclear whether moderate alcohol consumption exerts similar effects. Observational studies previously reported the potential benefits of moderate alcohol consumption on cognition, particularly in the elderly; however, these effects have not yet been demonstrated in Asian populations. The aim of the present study was to investigate the relationship between alcohol consumption levels and global and domain-specific cognitive functions in cognitively intact elderly Japanese men. Cross-sectional data from the Shiga Epidemiological Study of Subclinical Atherosclerosis (SESSA), an ongoing prospective, population-based study in Shiga, Japan, were used to examine the relationship between alcohol consumption and cognitive function. Men (n = 585) aged ≥65 years provided information on their weekly consumption of alcohol, and the data obtained were used to construct categories of never, ex- (quit before interview), very light (<14 g/day), light (14-23 g/day), moderate (>23-46 g/day), and heavy (>46 g/day) drinkers. Cognitive function was measured using the Cognitive Abilities Screening Instrument (CASI). A fractional logistic regression model adjusted for age, education, body mass index, smoking, exercise, hypertension, diabetes, and dyslipidemia showed that the CASI scores for global and domain-specific cognitive functions were not significantly different between all subgroups of current drinkers and never-drinkers. However, the CASI score of ex-drinkers (multivariable adjusted mean CASI score [SD]) was significantly lower than that of never-drinkers in the global [never vs. ex: 90.16 (2.21) vs. 88.26 (2.58)] and abstraction and judgment domain [never vs. ex: 9.48 (0.46) vs. 8.61 (0.57)]). The present results do not suggest any beneficial or adverse relationship between current alcohol consumption levels and cognitive functioning (both global and domain specific) in elderly Japanese men; however, low cognitive function among ex-drinkers warrants future investigations to identify the factors causing drinkers to quit.
  • Undral Buyandelger, Douglas G. Walker, Daijiro Yanagisawa, Toshifumi Morimura, Ikuo Tooyama
    International Journal of Molecular Sciences 21 10 3635 - 3635 2020年05月 [有り][無し]
     研究論文（学術雑誌） 
    Aberrant angiogenesis is a pathological feature of a number of diseases and arises from the uncoordinated expression of angiogenic factors as response to different cellular stresses. Age-related macular degeneration (AMD), a leading cause of vision loss, can result from pathological angiogenesis. As a mutation in the mitochondrial ferritin (FTMT) gene has been associated with AMD, its possible role in modulating angiogenic factors and angiogenesis was investigated. FTMT is an iron-sequestering protein primarily expressed in metabolically active cells and tissues with high oxygen demand, including retina. In this study, we utilized the human retinal pigment epithelial cell line ARPE-19, both as undifferentiated and differentiated cells. The effects of proinflammatory cytokines, FTMT knockdown, and transient and stable overexpression of FTMT were investigated on expression of pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial-derived factor (PEDF). Proinflammatory cytokines induced FTMT and VEGF expression, while NF-κB inhibition significantly reduced FTMT expression. VEGF protein and mRNA expression were significantly increased in FTMT-silenced ARPE-19 cells. Using an in vitro angiogenesis assay with endothelial cells, we showed that conditioned media from FTMT-overexpressing cells had significant antiangiogenic effects. Collectively, our findings indicate that increased levels of FTMT inhibit angiogenesis, possibly by reducing levels of VEGF and increasing PEDF expression. The cellular models developed can be used to investigate if increased FTMT may be protective in angiogenic diseases, such as AMD.
  • Liu Ziyi, Zaid Maryam, Hisamatsu Takashi, Tanaka Sachiko, Fujiyoshi Akira, Miyagawa Naoko, Ito Takahiro, Kadota Aya, Tooyama Ikuo, Miura Katsuyuki, Ueshima Hirotsugu, the SESSA Research Group
    Journal of Epidemiology 30 3-4 121 - 127 2020年04月 
    ●日本人男性集団において認知機能と空腹時血糖の関連を検討した。●認知機能はCASIで評価した。●糖尿病のない男性では血糖値が3.97-6.20mmol/Lの範囲で認知機能が最も高かった。●糖尿病のある男性では、血糖値が高いほど認知機能が低かった。●血糖値と認知機能との関連は糖尿病の有無により異なっていた。(著者抄録)
  • Ziyi Liu, Maryam Zaid, Takashi Hisamatsu, Sachiko Tanaka, Akira Fujiyoshi, Naoko Miyagawa, Takahiro Ito, Aya Kadota, Ikuo Tooyama, Katsuyuki Miura, Hirotsugu Ueshima
    Journal of epidemiology 30 3 121 - 127 2020年03月 [有り][無し]
     研究論文（学術雑誌） 
    BACKGROUND: Cognitive dysfunction has been recognized as a diabetes-related complication. Whether hyperglycemia or elevated fasting glucose are associated with cognitive decline remains controversial. We aimed to investigate the relationship between fasting glucose levels and cognitive function in diabetic and non-diabetic individuals. METHODS: Participants were Japanese diabetic (n = 191) and non-diabetic (n = 616) men, aged 46-81 years, from 2010-2014. Blood samples were taken after a 12 h fast. The Cognitive Ability Screening Instrument (CASI), with a maximum score of 100, was used for cognitive assessment. Cognitive domains of CASI were also investigated. Fractional logit regression with covariate adjustment for potential confounders was used to model cross-sectional relationships between fasting blood glucose and CASI score. RESULTS: For diabetic individuals, CASI score was 0.38 (95% confidence interval: 0.66-0.12) lower per 1 mmol/L higher fasting glucose level. Short-term memory domain also exhibited an inverse association. For non-diabetic individuals, a reverse U-shaped relationship was observed between fasting glucose and cognitive function, identifying a threshold for highest cognitive performance of 91.8 CASI score at 3.97-6.20 mmol/L (71.5-111.6 mg/dL) fasting glucose. Language ability domain displayed a similar relationship with fasting glucose. CONCLUSIONS: Elevated fasting glucose levels in diabetic men were associated with lower cognitive function, in which short-term memory was the main associated domain. Interestingly, in non-diabetic men, we identified a threshold for the inverse relationship of elevated fasting glucose with cognitive function. Contrastingly to diabetic men, language ability was the main associated cognitive domain among non-diabetic men.
  • Yosuke Kadota, Faidruz Azura Jam, Haruka Yukiue, Ichiro Terakado, Takao Morimune, Ayami Tano, Yuya Tanaka, Sayumi Akahane, Mayu Fukumura, Ikuo Tooyama, Masaki Mori
    iScience 23 3 100929 - 100929 2020年02月 [有り][無し]
     研究論文（学術雑誌） 
    The juvenile phase is characterized by continuously progressing physiological processes such as growth and maturation, which are accompanied by transitions in gene expression. The contribution of transcriptome dynamics to the establishment of juvenile properties remains unclear. Here, we investigated alternative splicing (AS) events in postnatal growth and elucidated the landscape of age-dependent alternative splicing (ADAS) in C57BL/6 mice. Our analysis of ADAS in the cerebral cortex, cardiomyocytes, and hepatocytes revealed numerous juvenile-specific splicing isoforms that shape the juvenile transcriptome, which in turn functions as a basis for the highly anabolic status of juvenile cells. Mechanistically, the juvenile-expressed splicing factor Srsf7 mediates ADAS, as exemplified by switching from juvenile to adult forms of anabolism-associated genes Eif4a2 and Rbm7. Suppression of Srsf7 results in "fast-forwarding" of this transcriptome transition, causing impaired anabolism and growth in mice. Thus, juvenile-specific AS is indispensable for the anabolic state of juveniles and differentiates juveniles from adults.
  • Douglas G Walker, Tiffany M Tang, Anarmaa Mendsaikhan, Ikuo Tooyama, Geidy E Serrano, Lucia I Sue, Thomas G Beach, Lih-Fen Lue
    International journal of molecular sciences 21 2 2020年01月 [有り][無し]
     研究論文（学術雑誌） 
    Neuroinflammation is considered a key pathological process in neurodegenerative diseases of aging, including Alzheimer's disease (AD). Many studies have defined phenotypes of reactive microglia, the brain-resident macrophages, with different antigenic markers to identify those potentially causing inflammatory damage. We took an alternative approach with the goal of characterizing the distribution of purinergic receptor P2RY12-positive microglia, a marker previously defined as identifying homeostatic or non-activated microglia. We examined the expression of P2RY12 by dual-color light and fluorescence immunohistochemistry using sections of middle temporal gyrus from AD, high plaque and low plaque non-demented cases in relation to amyloid beta (Aβ) plaques and phosphorylated tau, markers of pathology, and HLA-DR, IBA-1, CD68, and progranulin, microglial phenotype markers. In low plaque cases, P2RY12-positive microglia mostly had non-activated morphologies, while the morphologies of P2RY12-positive microglia in AD brains were highly variable, suggesting its expression could encompass a wider range of phenotypes than originally hypothesized. P2RY12 expression by microglia differed depending on the types of plaques or tangles they were associated with. Areas of inflammation characterized by lack of P2RY12-positive microglia around mature plaques could be observed, but many diffuse plaques showed colocalization with P2RY12-positive microglia. Based on these results, P2RY12 expression by microglia should not be considered solely a marker of resting microglia as P2RY12 immunoreactivity was identifying microglia positive for CD68, progranulin and to a limited extent HLA-DR, markers of activation.
  • Asuka Matsuzaki Tada, Hamizah Shahirah Hamezah, Daijiro Yanagisawa, Shigehiro Morikawa, Ikuo Tooyama
    Frontiers in neuroscience 14 845 - 845 2020年 研究論文（学術雑誌） 
    We have previously reported that casein hydrolysate, CH-3, from bovine milk and casein-derived tripeptide Met-Lys-Pro (MKP) has ACE inhibitory activity and reduces blood pressure. In this study, we investigated the therapeutic effects of MKP in a hypertensive rat model (7-week-old male SHRSP/Izm rats). For long term evaluation, rats were fed either a diet containing CH-3 or normal diet. The survival rate of SHRSP rats was significantly improved by intake of CH-3 for 181 days. For short term evaluation, rats were orally administered synthetic tripeptide MKP or distilled water for 4 weeks. MRI study demonstrated that hemorrhagic lesions were observed in two of five rats in the control group, while no hemorrhagic lesions were observed in the MKP group. Volumetric analysis using MRI revealed that MKP administration inhibited atrophy of diencephalic regions. Histological examinations revealed that hemorrhage areas and astrogliosis in the hippocampus and cerebral cortex were lower in the MKP group than in the control group. Gene expression analysis indicated that MKP administration reduced expression of genes related to cerebral circulation insufficiency such as immune responses (Cd74 and Prkcd), response to hypoxia (Ddit4, Apold1, and Prkcd), reactive oxygen species metabolic process (Ddit4 and Pdk4), and apoptotic process (Ddit4, Prkcd, and Sgk1), suggesting that MKP administration prevented cerebral ischemia associated with hypertension. In addition, some genes encoding responses to hormone stimulus (Fos, Dusp1, and Sik1) were also downregulated. Serum aldosterone and corticosterone levels were also significantly decreased following MKP administration. The present study indicates that MKP shows neuroprotective effects in SHRSP rats by regulating cerebral circulation insufficiency and corticoid levels. MKP administration may therefore be a potential therapeutic strategy for hypertensive brain diseases such as cerebrovascular disease.
  • Ryo Kakiuchi, Tasuku Hirayama, Daijiro Yanagisawa, Ikuo Tooyama, Hideko Nagasawa
    Organic & Biomolecular Chemistry 18 30 5843 - 5849 2020年 [有り][無し]
     研究論文（学術雑誌） 

    An activity-based 19F-MRI probe that showed a chemical shift change in response to Fe(ii) was developed.

  • Yasunari Seita, Toshifumi Morimura, Naoki Watanabe, Chizuru Iwatani, Hideaki Tsuchiya, Shinichiro Nakamura, Toshiharu Suzuki, Daijiro Yanagisawa, Tomoyuki Tsukiyama, Masataka Nakaya, Eiichi Okamura, Masanaga Muto, Masatsugu Ema, Masaki Nishimura, Ikuo Tooyama
    Journal of Alzheimer's disease : JAD 75 1 45 - 60 2020年 [有り][無し]
     研究論文（学術雑誌） 
    Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-β precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models. Here we report the generation of two transgenic cynomolgus monkeys overexpressing the human gene for APP with Swedish, Artic, and Iberian mutations, and demonstrated expression of gene tagged green fluorescent protein marker in the placenta, amnion, hair follicles, and peripheral blood. We believe that these nonhuman primate models will be very useful to study the pathogenesis of dementia and AD. However, generated Tg monkeys still have some limitations. We employed the CAG promoter, which will promote gene expression in a non-tissue specific manner. Moreover, we used transgenic models but not knock-in models. Thus, the inserted transgene destroys endogenous gene(s) and may affect the phenotype(s). Nevertheless, it will be of great interest to determine whether these Tg monkeys will develop tauopathy and neurodegeneration similar to human AD.
  • Anarmaa Mendsaikhan, Ikuo Tooyama, Jean-Pierre Bellier, Geidy E Serrano, Lucia I Sue, Lih-Fen Lue, Thomas G Beach, Douglas G Walker
    Acta neuropathologica communications 7 1 215 - 215 2019年12月 [有り][無し]
     研究論文（学術雑誌） 
    Progranulin (PGRN) is a protein encoded by the GRN gene with multiple identified functions including as a neurotrophic factor, tumorigenic growth factor, anti-inflammatory cytokine and regulator of lysosomal function. A single mutation in the human GRN gene resulting in reduced PGRN expression causes types of frontotemporal lobar degeneration resulting in frontotemporal dementia. Prosaposin (PSAP) is also a multifunctional neuroprotective secreted protein and regulator of lysosomal function. Interactions of PGRN and PSAP affect their functional properties. Their roles in Alzheimer's disease (AD), the leading cause of dementia, have not been defined. In this report, we examined in detail the cellular expression of PGRN in middle temporal gyrus samples of a series of human brain cases (n = 45) staged for increasing plaque pathology. Immunohistochemistry showed PGRN expression in cortical neurons, microglia, cerebral vessels and amyloid beta (Aβ) plaques, while PSAP expression was mainly detected in neurons and Aβ plaques, and to a limited extent in astrocytes. We showed that there were increased levels of PGRN protein in AD cases and corresponding increased levels of PSAP. Levels of PGRN and PSAP protein positively correlated with amyloid beta (Aβ), with PGRN levels correlating with phosphorylated tau (serine 205) levels in these samples. Although PGRN colocalized with lysosomal-associated membrane protein-1 in neurons, most PGRN associated with Aβ plaques did not. Aβ plaques with PGRN and PSAP deposits were identified in the low plaque non-demented cases suggesting this was an early event in plaque formation. We did not observe PGRN-positive neurofibrillary tangles. Co-immunoprecipitation studies of PGRN from brain samples identified only PSAP associated with PGRN, not sortilin or other known PGRN-binding proteins, under conditions used. Most PGRN associated with Aβ plaques were immunoreactive for PSAP showing a high degree of colocalization of these proteins that did not change between disease groups. As PGRN supplementation has been considered as a therapeutic approach for AD, the possible involvement of PGRN and PSAP interactions in AD pathology needs to be further considered.
  • Yuyan Liu, Akira Fujiyoshi, Hisatomi Arima, Aya Kadota, Sayaka Kadowaki, Takashi Hisamatsu, Itsuko Miyazawa, Keiko Kondo, Ikuo Tooyama, Katsuyuki Miura, Hirotsugu Ueshima
    Journal of atherosclerosis and thrombosis 26 12 1102 - 1114 2019年12月 [有り][無し]
     研究論文（学術雑誌） 
    AIM: Computed tomography (CT) can directly provide information on body compositions and distributions, compared to anthropometric indices. It has been shown that various obesity indices are associated with carotid intima-media thickness (IMT). However, whether CT-based obesity indices are stronger than anthropometric indices in association with atherosclerosis remains to be determined in a general population. METHODS: We cross-sectionally assessed carotid IMT using ultrasound in 944 community-dwelling Japanese men free of stroke and myocardial infarction. CT image at the L4-L5 level was obtained to compute areas of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT). Anthropometric measures assessed included body mass index (BMI), waist circumference, and waist-to-hip ratio. Using multivariable linear regression, slopes of IMT per 20th to 80th percentile of each index were compared. We also compared the slope of index with simultaneous adjustment for BMI in the same model. RESULTS: Areas of VAT and SAT were positively associated with IMT, but not stronger than those of anthropometric indices in point estimates. Among all obesity indices, BMI was strongest in association with IMT after adjusting for age and lifestyle factors or further adjusting for metabolic factors. In simultaneous adjustment models, BMI, but not CT-based indices, remained significant and showed the strongest association. CONCLUSIONS: In community-dwelling Japanese men, anthropometric obesity indices, BMI in particular, were more strongly associated with carotid atherosclerosis than CT-based obesity indices. The association of general obesity with carotid atherosclerosis was strong and adding CT-based obesity measure did not considerably influence in the association.
  • トコトリエノールリッチフラクションはAPP/PS1マウス脳のプロテオームプロファイルを制御する(Tocotrienol-rich fraction modulates proteome profile in APP/PS1 mouse brain)
    Hamezah Hamizah Shahirah, Durani Lina Wati, 柳沢 大治郎, Ibrahim Nor Faeizah, Damanhuri Hanafi Ahmad, Ngah Wan Zurinah Wan, 遠山 育夫
    Dementia Japan 33 4 520 - 520 2019年10月
  • rTg4510マウスのタウ蓄積量を変動させる因子の探索
    柳沢 大治郎, Hamezah Hamizah Shahirah, Arrozi Aslina Pahrudin, 遠山 育夫
    Dementia Japan 33 4 544 - 544 (一社)日本認知症学会 2019年10月
  • ヒト組織切片を用いたアルツハイマー病の神経原線維変化に結合する新規化合物の探索
    加藤 智子, 柳沢 大治郎, 田口 弘康, 遠山 育夫
    Dementia Japan 33 4 546 - 546 (一社)日本認知症学会 2019年10月
  • Tomoaki Tanaka, Yuriko Ono, Naoki Hikihara, Ayana Yoshida, Hasumi Yamada, Shogo Higaki, Tomomi Nishie, Ikuo Tooyama, Keiko Iida, Akira Hirasawa, Tatsuyuki Takada
    Reproductive toxicology (Elmsford, N.Y.) 88 39 - 47 2019年09月 [有り][無し]
     研究論文（学術雑誌） 
    Effects of endocrine disrupting chemicals (EDCs) on reproduction have not been fully explained comprehensively. In this study, we tried to validate the common effect of Bisphenol A (BPA) and Nonylphenol (NP) on the differentiation of embryonic stem (ES) cells and found that they modify the expression of germ cell specific genes. To elucidate functional significance on biological process, we performed Gene Ontology (GO)-based microarray analysis comparing with published GeneChip data of primordial germ cell development in vivo. Cluster analysis of gene expression profile revealed that EDC treatment and primordial germ cell (PGC) development shared characteristic cluster consists of GO terms related to "germ cell development" and "reproduction". In the GO term "reproduction", meiosis related genes showed high expression level by EDC exposure. These results suggest that BPA and NP affect not only some of the germ cell specific genes, but functionally interferes germ cell development and reproduction.
  • Douglas G Walker, Lih-Fen Lue, Thomas G Beach, Ikuo Tooyama
    Cells 8 7 2019年07月 [有り][無し]
     研究論文（学術雑誌） 
    Inflammation is considered a key pathological process in neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), but there are still mechanisms not understood. In the brain, most microglia are performing essential homeostatic functions, but can also respond to pathogenic stimuli by producing harmful pro-inflammatory cytokines or free radicals. Distinguishing between damaging and homeostatic microglia in human diseased brain tissues is a challenge. This report describes findings using a monoclonal antibody to CD105/Endoglin (R&D Systems MAB1097) that identifies subtypes of activated microglia. CD105/Endoglin is a co-receptor for transforming growth factor beta (TGFβ) receptor that antagonizes TGFβ signaling. CD105/Endoglin is a marker for vascular endothelial cells, but was originally identified as a marker for activated macrophages. This antibody did not identify endothelial cells in brain sections, only microglia-like cells. In this study, we examined with this antibody tissue section from middle temporal gyrus derived from human brains from normal control subjects with low-plaque pathology, high-plaque pathology, and AD cases, and also substantia nigra samples from control and PD cases, in conjunction with antibodies to markers of pathology and microglia. In low-plaque pathology cases, CD105-positive microglia were mostly absent, but noticeably increased with increasing pathology. CD105-positive cells strongly colocalized with amyloid-beta plaques, but not phosphorylated tau positive tangles. In substantia nigra, strong microglial CD105 staining was observed in microglia associated with degenerating dopaminergic neurons and neuromelanin. In PD cases with few surviving dopaminergic neurons, this staining had decreased. By Western blot, this antibody identified polypeptide bands of 70 kDa in brain samples, and samples from microglia, macrophages, and brain endothelial cells. In comparison with other tested CD105 antibodies, this antibody did not recognize the glycosylated forms of CD105 on Western blots. Overall, the data indicate that this antibody and this marker could have utility for subtyping of microglia in pathologically-involved tissue.
  • Akira Fujiyoshi, Katsuyuki Miura, Takayoshi Ohkubo, Naoko Miyagawa, Yoshino Saito, Itsuko Miyazawa, Akihiko Shiino, Aya Kadota, Sayaka Kadowaki, Takashi Hisamatsu, Sayuki Torii, Naoyuki Takashima, Ikuo Tooyama, Hirotsugu Ueshima
    Journal of epidemiology 30 6 244 - 252 2019年05月 [有り][無し]
     研究論文（学術雑誌） 
    BACKGROUND: The association of proteinuria and reduced estimated glomerular filtration rate (eGFR) with cognition needs more clarification. We cross-sectionally examined whether proteinuria and reduced eGFR, even in moderate stages, were independently associated with lower cognition in a community-based sample of elderly men. METHODS: Our cohort initially comprised 1,094 men aged 40-79 years from a random sample from Shiga, Japan in 2006-2008. Of 853 men who returned for the follow-up examination (2009-2014), we analyzed 561 who were ≥65 years, free of stroke, and completed the Cognitive Abilities Screening Instrument (CASI) at follow-up. Higher CASI score (range 0 to 100) indicates better cognition. Proteinuria was assessed by dipstick. eGFR was calculated according to the Chronic Kidney Disease Epidemiology Collaboration Equation. Participants were divided into three groups either by eGFR (≥60, 59-40, and <40 mL/min/1.73 m2) or by proteinuria (no, trace, and positive), considered normal, moderate, and advanced, respectively. Using linear regression, we computed mean CASI score with simultaneous adjustment for proteinuria and eGFR in addition to other potential confounders. RESULTS: Significant trends of lower cognition were observed across the groups of worse proteinuria and lower eGFR independently: multivariable-adjusted mean CASI scores were 90.1, 89.3, and 88.4 for proteinuria (Ptrend=0.029), and 90.0, 88.5, and 88.5 for eGFR (Ptrend=0.015) in mutual adjustment model. CONCLUSIONS: Proteinuria and reduced eGFR, even in their moderate stages, were independently associated with lower cognition in a community-based sample of elderly men. The results suggest the importance of proteinuria and low eGFR for early detection and prevention of cognitive decline.
  • Ayami Tano, Yosuke Kadota, Takao Morimune, Faidruz Azura Jam, Haruka Yukiue, Jean-Pierre Bellier, Tatsuyuki Sokoda, Yoshihiro Maruo, Ikuo Tooyama, Masaki Mori
    Journal of cell science 132 8 2019年04月 [有り][無し]
     研究論文（学術雑誌） 
    Juvenile animals possess distinct properties that are missing in adults. These properties include capabilities for higher growth, faster wound healing, plasticity and regeneration. However, the molecular mechanisms underlying these juvenile physiological properties are not fully understood. To obtain insight into the distinctiveness of juveniles from adults at the molecular level, we assessed long noncoding RNAs (lncRNAs) that are highly expressed selectively in juvenile cells. The noncoding elements of the transcriptome were investigated in hepatocytes and cardiomyocytes isolated from juvenile and adult mice. Here, we identified 62 juvenility-associated lncRNAs (JAlncs), which are selectively expressed in both hepatocytes and cardiomyocytes from juvenile mice. Among these common (shared) JAlncs, Gm14230 is evolutionarily conserved and is essential for cellular juvenescence. Loss of Gm14230 impairs cell growth and causes cellular senescence. Gm14230 safeguards cellular juvenescence through recruiting the histone methyltransferase Ezh2 to Tgif2, thereby repressing the functional role of Tgif2 in cellular senescence. Thus, we identify Gm14230 as a juvenility-selective lncRNA required to maintain cellular juvenescence.
  • Anarmaa Mendsaikhan, Ikuo Tooyama, Douglas G Walker
    Cells 8 3 2019年03月 [有り][無し]
     研究論文（学術雑誌） 
    Neurodegenerative diseases such as Alzheimer's disease have proven resistant to new treatments. The complexity of neurodegenerative disease mechanisms can be highlighted by accumulating evidence for a role for a growth factor, progranulin (PGRN). PGRN is a glycoprotein encoded by the GRN/Grn gene with multiple cellular functions, including neurotrophic, anti-inflammatory and lysosome regulatory properties. Mutations in the GRN gene can lead to frontotemporal lobar degeneration (FTLD), a cause of dementia, and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Both diseases are associated with loss of PGRN function resulting, amongst other features, in enhanced microglial neuroinflammation and lysosomal dysfunction. PGRN has also been implicated in Alzheimer's disease (AD). Unlike FTLD, increased expression of PGRN occurs in brains of human AD cases and AD model mice, particularly in activated microglia. How microglial PGRN might be involved in AD and other neurodegenerative diseases will be discussed. A unifying feature of PGRN in diseases might be its modulation of lysosomal function in neurons and microglia. Many experimental models have focused on consequences of PGRN gene deletion: however, possible outcomes of increasing PGRN on microglial inflammation and neurodegeneration will be discussed. We will also suggest directions for future studies on PGRN and microglia in relation to neurodegenerative diseases.
  • Jean-Pierre Bellier, Pu-Qing Yuan, Kenichi Mukaisho, Ikuo Tooyama, Yvette Taché, Hiroshi Kimura
    Frontiers in neuroanatomy 13 37 - 37 2019年 [有り][無し]
     研究論文（学術雑誌） 
    Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), has an exon-skipping splice variant which is expressed preferentially in the peripheral nervous system (PNS) and thus termed peripheral ChAT (pChAT). A rabbit antiserum previously produced against rat pChAT (rpChAT) has been used for immunohistochemistry (IHC) to study peripheral cholinergic structures in various animals. The present study was undertaken to develop a specific antiserum against a predicted human pChAT (hpChAT) protein. A novel mouse antiserum has been successfully raised against a unique 14-amino acid sequence of hpChAT protein. Our Western blot using this antiserum (termed here anti-hpChAT serum) on human colon extracts revealed only a single band of 47 kDa, matching the deduced size of hpChAT protein. By IHC, the antiserum gave intense staining in many neuronal cells and fibers of human colon but not brain, and such a pattern of staining seemed identical with that reported in colon of various animals using anti-rpChAT serum. In the antibody-absorption test, hpChAT-immunoreactive staining in human colon was completely blocked by using the antiserum pre-absorbed with the antigen peptide. Double immunofluorescence in human colon moreover indicated that structures stained with anti-hpChAT were also stained with anti-rpChAT, and vice versa. hpChAT antiserum allowed the identification of cell types, as Dogiel type cells in intramural plexuses, and fiber innervation of colon muscles and mucosae. The present results demonstrate the specificity and reliability of the hpChAT antiserum as a novel tool for immunohistochemical studies in human colon, opening venues to map cholinergic innervation in other human PNS tissues.
  • Hamizah Shahirah Hamezah, Lina Wati Durani, Daijiro Yanagisawa, Nor Faeizah Ibrahim, Wan Mohd Aizat, Suzana Makpol, Wan Zurinah Wan Ngah, Hanafi Ahmad Damanhuri, Ikuo Tooyama
    Journal of Alzheimer's disease : JAD 72 1 229 - 246 2019年 [有り][無し]
     研究論文（学術雑誌） 
    Tocotrienol-rich fraction (TRF) is a mixture of vitamin E analogs derived from palm oil. We previously demonstrated that supplementation with TRF improved cognitive function and modulated amyloid pathology in AβPP/PS1 mice brains. The current study was designed to examine proteomic profiles underlying the therapeutic effect of TRF in the brain. Proteomic analyses were performed on samples of hippocampus, medial prefrontal cortex (mPFC), and striatum using liquid chromatography coupled to Q Exactive HF Orbitrap mass spectrometry. From these analyses, we profiled a total of 5,847 proteins of which 155 proteins were differentially expressed between AβPP/PS1 and wild-type mice. TRF supplementation of these mice altered the expression of 255 proteins in the hippocampus, mPFC, and striatum. TRF also negatively modulated the expression of amyloid beta A4 protein and receptor-type tyrosine-protein phosphatase alpha protein in the hippocampus. The expression of proteins in metabolic pathways, oxidative phosphorylation, and those involved in Alzheimer's disease were altered in the brains of AβPP/PS1 mice that received TRF supplementation.
  • Wan Nasri WN, Makpol S, Mazlan M, Tooyama I, Wan Zurinah, Wan Ngah WZ, Damanhuri HA
    Journal of Alzheimer's disease : JAD 70 s1 S239-S254  2019年 [有り][無し]
     研究論文（学術雑誌） 
    Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and other cognitive abilities. AD is associated with aggregation of amyloid-β (Aβ) deposited in the hippocampal brain region. Our previous work has shown that tocotrienol rich fraction (TRF) supplementation was able to attenuate the blood oxidative status, improve behavior, and reduce fibrillary-type Aβ deposition in the hippocampus of an AD mouse model. In the present study, we investigate the effect of 6 months of TRF supplementation on transcriptome profile in the hippocampus of APPswe/PS1dE9 double transgenic mice. TRF supplementation can alleviate AD conditions by modulating several important genes in AD. Moreover, TRF supplementation attenuated the affected biological process and pathways that were upregulated in the AD mouse model. Our findings indicate that TRF supplementation can modulate hippocampal gene expression as well as biological processes that can potentially delay the progression of AD.
  • Faidruz Azura Jam, Yosuke Kadota, Anarmaa Mendsaikhan, Ikuo Tooyama, Masaki Mori
    Scientific Reports 8 1 3132 - 3132 2018年12月 [有り][無し]
     研究論文（学術雑誌） 
    Young individuals possess distinct properties that adults do not. The juvenile animals show higher activities for growth, healing, learning and plasticity than adults. The machinery for establishing these juvenile properties is not fully understood. To better understand the molecular constituents for the above properties, we performed a comprehensive transcriptome analysis of differently aged cells of mice by high-throughput sequencing and identified the genes selectively highly expressed in the young cells. These genes, collectively called as juvenility-associated genes (JAGs), show significant enrichments in the functions such as alternative splicing, phosphorylation and extracellular matrix (ECM). This implies the juvenescence might be achieved by these functions at the cell level. The JAG mutations are associated with progeria syndromes and growth disorders. Thus, the JAGs might organize the juvenile property of young animals and analysis of JAGs may provide scientific and therapeutic approaches toward treating the genetic diseases.
  • Yoshitaka Tamaki, Akemi Shodai, Toshifumi Morimura, Ryota Hikiami, Sumio Minamiyama, Takashi Ayaki, Ikuo Tooyama, Yoshiaki Furukawa, Ryosuke Takahashi, Makoto Urushitani
    Scientific Reports 8 1 6030 - 6030 2018年12月 [有り][無し]
     研究論文（学術雑誌） 
    Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following in utero electroporation while causing no overt postnatal brain pathology or developmental anomalies. These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.
  • MRIによるタウ病変の検出法の開発
    柳沢 大治郎, 田口 浩康, 遠山 育夫
    日本脳科学会プログラム・抄録集 45回 33 - 33 日本脳科学会 2018年11月
  • F-methyl-curcumin-1(Shiga-Y5)はアルツハイマー病の画像診断や治療の候補化合物である
    遠山 育夫, 柳沢 大治郎, 田口 弘康
    日本脳科学会プログラム・抄録集 45回 34 - 34 日本脳科学会 2018年11月
  • Hamezah HS, Durani LW, Yanagisawa D, Ibrahim NF, Aizat WM, Bellier JP, Makpol S, Ngah WZW, Damanhuri HA, Tooyama I
    Experimental gerontology 111 53 - 64 2018年10月 [有り][無し]
     研究論文（学術雑誌） 
    Decrease in multiple functions occurs in the brain with aging, all of which can contribute to age-related cognitive and locomotor impairments. Brain atrophy specifically in hippocampus, medial prefrontal cortex (mPFC), and striatum, can contribute to this age-associated decline in function. Our recent metabolomics analysis showed age-related changes in these brain regions. To further understand the aging processes, analysis using a proteomics approach was carried out. This study was conducted to identify proteome profiles in the hippocampus, mPFC, and striatum of 14-, 18-, 23-, and 27-month-old rats. Proteomics analysis using ultrahigh performance liquid chromatography coupled with Q Exactive HF Orbitrap mass spectrometry identified 1074 proteins in the hippocampus, 871 proteins in the mPFC, and 241 proteins in the striatum. Of these proteins, 97 in the hippocampus, 25 in mPFC, and 5 in striatum were differentially expressed with age. The altered proteins were classified into three ontologies (cellular component, molecular function, and biological process) containing 44, 38, and 35 functional groups in the hippocampus, mPFC, and striatum, respectively. Most of these altered proteins participate in oxidative phosphorylation (e.g. cytochrome c oxidase and ATP synthase), glutathione metabolism (e.g. peroxiredoxins), or calcium signaling pathway (e.g. protein S100B and calmodulin). The most prominent changes were observed in the oldest animals. These results suggest that alterations in oxidative phosphorylation, glutathione metabolism, and calcium signaling pathway are involved in cognitive and locomotor impairments in aging.
  • フッ素MRIによるタウイメージングプローブShiga-X34の組織化学的性質
    Buyandelger Undral, 柳沢 大治郎, 田口 弘康, 遠山 育夫
    日本組織細胞化学会総会・学術集会講演プログラム・予稿集 59回 82 - 82 日本組織細胞化学会 2018年09月
  • クルクミン誘導体Shiga-Y5はAβの蓄積を抑制するが、タウの蓄積には作用しない
    柳沢 大治郎, Hamizah Shahirah Hamezah, 田口 弘康, 遠山 育夫
    Dementia Japan 32 3 441 - 441 (一社)日本認知症学会 2018年09月
  • Kaneko K, Kubota Y, Nomura K, Hayashimoto H, Chida T, Yoshino N, Wayama M, Ogasawara K, Nakamura Y, Tooyama I, Furuyama K
    Experimental hematology 65 57 - 68.e2 2018年09月 [有り][無し]
     研究論文（学術雑誌） 
    ALAS2 gene mutations cause X-linked sideroblastic anemia. The presence of ring sideroblasts in a patient's bone marrow is the hallmark of sideroblastic anemia, but the precise mechanisms underlying sideroblast formation are largely unknown. Using a genome-editing system, a mutation was introduced in the erythroid-specific enhancer of the ALAS2 gene in HUDEP2 cells, which were derived from human umbilical stem cells and can produce erythrocytes. The established cell line, termed HA2low, expressed less ALAS2 mRNA than did wild-type cells, even after erythroid differentiation. Although the mRNA expression of α-globin, β-globin, and the mitochondrial iron importer mitoferrin-1 was induced similarly in wild-type and HA2low cells, hemoglobinization of differentiated cells was limited in HA2low cells compared with wild-type cells. Importantly, Prussian blue staining revealed that approximately one-third of differentiated HA2low cells exhibited intracellular iron deposition and these cells looked like ring sideroblasts. Electron microscopy confirmed that the mitochondria in HA2low cells contained high-density deposits that might contain iron. Ring sideroblastic cells appeared among HA2low cells only after differentiation, whereas the induced expression of mitochondrial ferritin was observed in both cell types during differentiation. These results suggest that the induction of mitochondrial ferritin expression might be essential for, but not the primary cause of, ring sideroblast formation. Our results also suggest that the insufficient supply of protoporphyrin IX due to ALAS2 deficiency in combination with increased iron import into mitochondria during erythroid differentiation results in the formation of ring sideroblasts. Furthermore, HA2low cells are a useful tool for characterizing ring sideroblasts in vitro.
  • Takeuchi S, Tooyama I
    Acta histochemica et cytochemica 51 3 111 - 118 2018年06月 [有り][無し]
     研究論文（学術雑誌） 
    The TRK-fused gene (TFG) is reported to be involved in the regulation of cell size, apoptosis, cell growth, ER-Golgi protein secretion, NF-κβ pathway signaling, the ubiquitin-proteasome system, and pancreatic β-cell mass and function. TFG mutations were reported in some neurodegenerative diseases affecting sensory and motor functions. However, the function of TFG in the nervous system and how TFG mutations lead to neurodegeneration remain unclear. In this study, we employed double immunohistochemistry to investigate the details of TFG localization patterns in monoaminergic and cholinergic neurons in the brainstem. Intense TFG immunoreactivity was observed in the dorsal raphe nucleus, the locus coeruleus, and the ventral horn of the spinal cord. TFG immunoreactivity was observed in some serotonergic neurons in all B1-B9 cell groups, and some noradrenergic neurons in all A1-A7 cell groups in the rat brainstem, while no immunoreactivity was observed in the dopaminergic neurons in A8-A10 cell groups. TFG immunoreactivity was observed in all ChAT-positive motor nuclei in the lower corticospinal tract of the rat brainstem.
  • Daijiro Yanagisawa, Nor Faeizah Ibrahim, Hiroyasu Taguchi, Shigehiro Morikawa, Tomoko Kato, Koichi Hirao, Nobuaki Shirai, Takayuki Sogabe, Ikuo Tooyama
    Journal of Neuroscience Research 96 5 841 - 851 2018年05月 [有り][無し]
     研究論文（学術雑誌） 
    Aggregation of tau into neurofibrillary tangles (NFTs) is characteristic of tauopathies, including Alzheimer's disease. Recent advances in tau imaging have attracted much attention because of its potential contributions to early diagnosis and monitoring of disease progress. Fluorine-19 magnetic resonance imaging (19F-MRI) may be extremely useful for tau imaging once a high-quality probe has been formulated. In this investigation, a novel fluorine-19–labeling compound has been developed as a probe for tau imaging using 19F-MRI. This compound is a buta-1,3-diene derivative with a polyethylene glycol side chain bearing a CF3 group and is known as Shiga-X35. Female rTg4510 mice (a mouse model of tauopathy) and wild-type mice were intravenously injected with Shiga-X35, and magnetic resonance imaging of each mouse's head was conducted in a 7.0-T horizontal-bore magnetic resonance scanner. The 19F-MRI in rTg4510 mice showed an intense signal in the forebrain region. Analysis of the signal intensity in the forebrain region revealed a significant accumulation of fluorine-19 magnetic resonance signal in the rTg4510 mice compared with the wild-type mice. Histological analysis showed fluorescent signals of Shiga-X35 binding to the NFTs in the brain sections of rTg4510 mice. Data collected as part of this investigation indicate that 19F-MRI using Shiga-X35 could be a promising tool to evaluate tau pathology in the brain.
  • Shogo Higaki, Takaaki Todo, Reiko Teshima, Ikuo Tooyama, Yasuhiro Fujioka, Noriyoshi Sakai, Tatsuyuki Takada
    Fish Physiology and Biochemistry 44 2 503 - 513 2018年04月 [有り][無し]
     研究論文（学術雑誌） 
    We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.
  • 生体内の鉄(II)イオン検出を指向した19F-MRIプローブ分子の開発
    鈴木 萌恵, 平山 祐, 柳沢 大治郎, 遠山 育夫, 永澤 秀子
    日本薬学会年会要旨集 138年会 2 225 - 225 (公社)日本薬学会 2018年03月
  • Anarmaa Mendsaikhan, Shigeko Takeuchi, Douglas G Walker, Ikuo Tooyama
    Frontiers in molecular neuroscience 11 470 - 470 2018年 [有り][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is an iron-transport protein with ferroxidase properties localized to mitochondria. Levels are generally low in all tissues, while increasing the expression of FtMt in neuronal-like cells has been shown to be protective. To determine whether FtMt has potential as a therapeutic approach, there remains the question of how much FtMt is protective. To address this issue, we transfected SH-SY5Y neuroblastoma cells with a FtMt expression plasmid and isolated cell lines with stable expression of FtMt at high, medium and low levels. Using these cell lines, we examined effects of FtMt on neuronal phenotype, neuroprotective activity and gene expression profiles. The phenotypic properties of high, medium and low FtMt expressors were compared with native untransfected SH-SY5Y cells after differentiation with retinoic acid to a neuronal phenotype. Overexpression of FtMt, even in low expressing cells, showed significant protection from oxidative stress induced by hydrogen peroxide or cobalt chloride. Higher levels of FtMt expression did not appear to offer greater protection, and did not have toxic consequences to cells, even though there were significantly more aggregated mitochondria in the highest expressing clone. The phenotypes differed between cell clones when assessed by cell growth, neurite outgrowth, and expression of neuronal proteins including those associated with neurodegenerative diseases. Microarray analysis of high, medium and negative FtMt-expressing cells identified different patterns of expression of certain genes associated with oxidative stress and neuronal development, amongst others. Validation of microarray analyses was carried out by real time polymerase chain reaction. The results showed significant differences in expression of thioredoxin-interacting protein (TXNIP) and microsomal glutathione transfer-1 (MGST-1), which can have critical roles in the regulation of oxidative stress. Differences in expression of calcitonin-related polypeptide alpha (CALCA), growth differentiation factor-15 (GDF-15) and secretogranin II (SCG2) were also observed. Our findings indicate that even low levels of increased FtMt expression can be protective possibly by alterations of some oxidative stress-related and growth factor genes, while high levels of expression did not appear to offer greater protection from oxidative stress or induce significant toxicity in cells. These experiments provide supporting data that increasing FtMt might be a feasible strategy for therapeutics in certain neurodegenerative and neurological diseases.
  • Liu Z, Kameshima N, Nanjo T, Shiino A, Kato T, Shimizu S, Shimizu T, Tanaka S, Miura K, Tooyama I
    Journal of Alzheimer's disease : JAD 62 2 737 - 744 2018年 [有り][無し]
     研究論文（学術雑誌） 
    Cost-effective and feasible methods for early diagnosis of Alzheimer's disease (AD) are needed. We present two methods to measure AD-related biomarkers simultaneously from one nasal smear for the purpose of diagnosing AD. Japanese men and women aged 63-85 years old were recruited in 2015-2016 for this case-control study. A total of 25 AD cases and 25 controls (22 men and 28 women) participated in this research. Nasal smears were collected from the common nasal meatus, inferior concha, middle nasal meatus, and olfactory cleft, and the proteins in the samples were analyzed by two methods, which we named PGD (Pre-treatment with guanidine- n-Dodecyl-beta-D-maltoside solution) method 1 (PGD-I) and 2 (PGD-II). The PGD-I method measured total tau and amyloid-β (Aβ)42, but no differences in median levels of total tau and Aβ42 between AD cases and controls were found in any of the nasal locations. The PGD-II method measured Aβ42, total tau, and phosphorylated tau, but levels of Aβ40 in all nasal locations of both groups were near zero. Median levels of phosphorylated tau to total tau (p-tau/t-tau) ratios in the middle nasal meatus and in the olfactory cleft were significantly higher in AD cases than in controls, and could significantly predict AD. To assess diagnostic reliability, areas under the ROC curve were 0.74 (95% CL = 0.52-0.95, p = 0.030) for the middle nasal meatus and 0.72 (95% CL = 0.52-0.92, p = 0.029) for the olfactory cleft. Thus, PGD-I and PGD-II can detect AD-related biomarkers in nasal smears and PGD-II may be a useful tool for diagnosing AD.
  • Durani LW, Hamezah HS, Ibrahim NF, Yanagisawa D, Nasaruddin ML, Mori M, Azizan KA, Damanhuri HA, Makpol S, Wan Ngah WZ, Tooyama I
    Journal of Alzheimer's disease : JAD 64 1 249 - 267 2018年 [有り][無し]
     研究論文（学術雑誌） 
    We have recently shown that the tocotrienol-rich fraction (TRF) of palm oil, a mixture of vitamin E analogs, improves amyloid pathology in vitro and in vivo. However, precise mechanisms remain unknown. In this study, we examined the effects of long-term (10 months) TRF treatment on behavioral impairments and brain metabolites in (15 months old) AβPP/PS1 double transgenic (Tg) Alzheimer's disease (AD) mice. The open field test, Morris water maze, and novel object recognition tasks revealed improved exploratory activity, spatial learning, and recognition memory, respectively, in TRF-treated Tg mice. Brain metabolite profiling of wild-type and Tg mice treated with and without TRF was performed using ultrahigh performance liquid chromatography (UHPLC) coupled to high-resolution accurate mass (HRAM)-orbitrap tandem mass spectrometry (MS/MS). Metabolic pathway analysis found perturbed metabolic pathways that linked to AD. TRF treatment partly ameliorated metabolic perturbations in Tg mouse hippocampus. The mechanism of this pre-emptive activity may occur via modulation of metabolic pathways dependent on Aβ interaction or independent of Aβ interaction.
  • Yanagisawa D, Hamezah HS, Durani LW, Taguchi H, Tooyama I
    PloS one 13 12 e0208440  2018年 [有り][無し]
     研究論文（学術雑誌） 
    Intracellular inclusions of aggregated tau appear in neurons and glial cells in a range of neurodegenerative diseases known as tauopathies. Inhibition of pathological changes in tau is a therapeutic target for tauopathy. We recently synthesized a novel curcumin derivative, named Shiga-Y5, and showed that Shiga-Y5 inhibited cognitive impairment and amyloid deposition in a mouse model of Alzheimer's disease. Here we investigated whether Shiga-Y5 inhibited cognitive impairment and tau accumulation in a mouse model of tauopathy, rTg4510. The rTg4510 mouse is a bitransgenic mouse model that uses a system of responder and activator transgenes to express human four-repeat tau with the P301L mutation. This strain is obtained by crossing tetO-MAPT*P301L mouse line (on a FVB/NJ background) with CaMKII-tTA mouse line (on a C57BL/6J background). Male rTg4510 mice and wild-type mice were fed with a standard chow diet with or without Shiga-Y5 (500 ppm) for 4 months. Behavioral tests were conducted from 5.5 months of age, and the mice were sacrificed at 6 months of age. There were no significant changes in behavioral performance in rTg4510 mice fed with SY5-containing chow diet compared with rTg4510 mice fed with control chow diet. Histological and biochemical analyses also showed no significant alterations in tau accumulation by the treatment with SY5. One of noticeable finding in this study was that rTg4510 mice on a F1 female FVB/NJ x male C57BL/6J background showed more severe tau accumulation than rTg4510 mice on a F1 female C57BL/6J x male FVB/NJ background. Further studies to clarify the mechanisms underlying tau aggregation may help to develop therapeutic approaches aimed at preventing this pathological feature.
  • フッ素MRIによるタウオパチーマウスモデルにおけるタウ病変の検出
    柳沢 大治郎, 田口 弘康, 森川 茂廣, 遠山 育夫
    JSMI Report 11 1 29 - 33 日本分子イメージング学会 2017年12月 
    タウオパチーと称される神経変性疾患では、微小管結合タンパク質であるタウの細胞内凝集体の蓄積が認められる。我々はタウ病変を検出するためのフッ素magnetic resonance(MR)imaging(MRI)プローブとしてbuta-1,3-diene誘導体Shiga-X35を合成した。タウオパチーモデルマウスrTg4510にShiga-X35を投与してフッ素MRを測定し、前脳領域における信号強度を野生型マウスと比較したところ、rTg4510マウスでは有意に強いフッ素MR信号が検出された。MR測定後に脳切片を組織学的に検索したところ、大脳皮質や海馬など前脳領域においてタウ病変のマーカーであるAT8と共存してShiga-X35の蛍光が観察された。以上より、Shiga-X35は脳に移行してタウ病変に結合し、フッ素MRを測定することでタウ病変のイメージングが可能であることが示された。フッ素MRIを利用した分子イメージングの将来の実用化が待たれる。(著者抄録)
  • Hamizah Shahirah Hamezah, Lina Wati Durani, Nor Faeizah Ibrahim, Daijiro Yanagisawa, Tomoko Kato, Akihiko Shiino, Sachiko Tanaka, Hanafi Ahmad Damanhuri, Wan Zurinah Wan Ngah, Ikuo Tooyama
    EXPERIMENTAL GERONTOLOGY 99 69 - 79 2017年12月 [有り][無し]
     研究論文（学術雑誌） 
    Impairments in cognitive and locomotor functions usually occur with advanced age, as do changes in brain volume. This study was conducted to assess changes in brain volume, cognitive and locomotor functions, and oxidative stress levels in middle-to late-aged rats. Forty-four male Sprague-Dawley rats were divided into four groups: 14, 18, 23, and 27 months of age. H-1 magnetic resonance imaging (MRI) was performed using a 7.0-Tesla MR scanner system. The volumes of the lateral ventricles, medial prefrontal cortex (mPFC), hippocampus, striatum, cerebellum, and whole brain were measured. Open field, object recognition, and Morris water maze tests were conducted to assess cognitive and locomotor functions. Blood was taken for measurements of malondialdehyde (MDA), protein carbonyl content, and antioxidant enzyme activity. The lateral ventricle volumes were larger, whereas the mPFC, hippocampus, and striatum volumes were smaller in 27-month-old rats than in 14-month-old rats. In behavioral tasks, the 27-month-old rats showed less exploratory activity and poorer spatial learning and memory than did the 14-month-old rats. Biochemical measurements likewise showed increased MDA and lower glutathione peroxidase (GPx) activity in the 27-month-old rats. In conclusion, age-related increases in oxidative stress, impairment in cognitive and locomotor functions, and changes in brain volume were observed, with the most marked impairments observed in later age.
  • Lina Wati Durani, Hamizah Shahirah Hamezah, Nor Faeizah Ibrahim, Daijiro Yanagisawa, Suzana Makpol, Hanafi Ahmad Damanhuri, Ikuo Tooyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 493 3 1356 - 1363 2017年11月 [有り][無し]
     研究論文（学術雑誌） 
    We have recently shown that age-dependent regional brain atrophy and lateral ventricle expansion may be linked with impaired cognitive and locomotor functions. However, metabolic profile transformation in different brain regions during aging is unknown. This study examined metabolic changes in the hippocampus, medial prefrontal cortex (mPFC) and striatum of middle- and late-aged Sprague-Dawley rats using ultrahigh performance liquid chromatography coupled with high-resolution accurate mass-orbitrap tandem mass spectrometry. Thirty-eight potential metabolites were altered in hippocampus, 29 in mPFC, and 14 in striatum. These alterations indicated that regional metabolic mechanisms in lated-aged rats are related to multiple pathways including glutathione, sphingolipid, tyrosine, and purine metabolism. Thus, our findings might be useful for understanding the complexity of metabolic mechanisms in aging and provide insight for aging and health span. (C) 2017 The Authors. Published by Elsevier Inc.
  • フッ素MRIによるタウ病変のイメージング
    柳沢 大治郎, 田口 弘康, 森川 茂廣, 遠山 育夫
    Dementia Japan 31 4 554 - 554 (一社)日本認知症学会 2017年10月
  • 中脳黒質ドパミン細胞に存在するミトコンドリアフェリチンの神経細胞保護作用
    高畑 翔吾, 関 宏鵬, 柳沢 大治郎, ベリエ・ジャンピエール, 野中 隆, 遠山 育夫
    Dementia Japan 31 4 570 - 570 2017年10月
  • Shogo Higaki, Natsue Kuwata, Kotaro Tanaka, Ikuo Tooyama, Yasuhiro Fujioka, Noriyoshi Sakai, Tatsuyuki Takada
    ZYGOTE 25 5 652 - 661 2017年10月 [有り][無し]
     研究論文（学術雑誌） 
    Sperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis via in vitro spermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed by in vitro fertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.
  • Jean-Pierre Bellier, Yu Xie, Sameh Mohamed Farouk, Yuko Sakaue, Ikuo Tooyama, Hiroshi Kimura
    BRAIN STRUCTURE & FUNCTION 222 7 3043 - 3061 2017年09月 [有り][無し]
     研究論文（学術雑誌） 
    The octopus arm contains a tridimensional array of muscles with a massive sensory-motor system. We herein provide the first evidence for the existence of serotonin (5-HT) in the octopus arm nervous system and investigated its distribution using immunohistochemistry. 5-HT-like immunoreactive (5-HT-lir) nerve cell bodies were exclusively localized in the cellular layer of the axial nerve cord. Those cell bodies emitted 5-HT-lir nerve fibers in the direction of the sucker, the intramuscular nerves cords, the ganglion of the sucker, and the intrinsic musculature. Others 5-HT-lir nerve fibers were observed in various tissues, including the cerebrobrachial tract, the skin, and the blood vessels. 5-HT was detected by high-performance liquid chromatography in various regions of the octopus arm at levels matching the density of 5-HT-lir staining. The absence of 5-HT-lir interconnections between the cerebrobrachial tract and the other components of the axial nerve cord suggests that two types of 5-HT-lir innervation exist in the arm. One type, which originates from the brain, may innervate the periphery through the cerebrobrachial tract. Another type, which originates in the cellular layer of the axial nerve cord, may form an intrinsic network in the arm. In addition, 5-HT-lir fibers likely emitted from the neuropil of the axial nerve cord were found to project into cells showing staining for peripheral choline acetyltransferase, a marker of sensory cells of the sucker. Taken together, these observations suggest that intrinsic 5-HT-lir innervation may participate in the sensory transmission in the octopus arm.
  • Daijiro Yanagisawa, Keisuke Oda, Masatoshi Inden, Shigehiro Morikawa, Toshiro Inubushi, Takashi Taniguchi, Masanori Hijioka, Yoshihisa Kitamura, Ikuo Tooyama
    JOURNAL OF NEUROSCIENCE RESEARCH 95 7 1485 - 1494 2017年07月 [有り][無し]
     研究論文（学術雑誌） 
    Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra projecting to the striatum. It has been estimated that approximately 80% of the striatal dopamine and 50% of nigral dopaminergic neurons are lost before the onset of typical motor symptoms, indicating that early diagnosis of PD using noninvasive imaging is feasible. Fluorine-19 (F-19) magnetic resonance imaging (MRI) represents a highly sensitive, easily available, low-background, and cost-effective approach to evaluate dopaminergic function using non-radioactive fluorine-containing dopaminergic agents. The aim of this study was to find a potent F-19 MRI probe to evaluate dopaminergic presynaptic function in the striatum. To select candidates for F-19 MRI probes, we investigated the following eight non-radioactive fluorine-containing dopaminergic agents: fluorodopa (F-DOPA), F-tyrosine, haloperidol, GBR13069 duhydrochloride, GBR12909 duhydrochloride, 3-bis-(4-fluorophenyl) methoxytropane hydrochloride, flupenthixol, and fenfluramine. In F-19 nuclear magnetic resonance measurements, F-tyrosine and FDOPA displayed a relatively higher signal-to-noise ratio value in brain homogenates than in others. F-DOPA, but not F-tyrosine, induced the rotational behavior in a 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonian rat model. In addition, a significantly high amount of FDOPA accumulated in the ipsilateral striatum of hemiparkinsonian rats after the injection. We performed F-19 MRI in PC12 cells and isolated rat brain using a 7T MR scanner. Our findings suggest that F-DOPA is a promising F-19 MRI probe for evaluating dopaminergic presynaptic function in the striatum of hemiparkinsonian rats. (C) 2016 Wiley Periodicals, Inc.
  • Guan H, Yang H, Yang M, Yanagisawa D, Bellier JP, Mori M, Takahata S, Nonaka T, Zhao S, Tooyama I
    Experimental neurology 291 51 - 61 2017年05月 [有り][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is a type of ferritin that sequesters iron. Previous studies have shown that FtMt is expressed by dopaminergic neurons in the substantia nigra and that it may be involved in the pathology of Parkinson's disease. However, the functional roles of FtMt in dopaminergic neurons remain unclear. In this study, we investigated the function of FtMt in a-synuclein regulation and its antioxidant roles in dopaminergic cells using human dopaminergic neuroblastoma cells, SH-SY5Y. In physiological conditions, FtMt knockdown increased a-synuclein expression at the protein level but not at the mRNA level. By contrast, FtMt overexpression reduced alpha-synuclein expression at the protein level but not at the mRNA level. FtMt enhanced the iron levels in mitochondria but decreased the iron levels in the intracellular labile iron pool. We found that FeCl2 could abolish the effects of FtMt overexpression on a-synuclein expression. Under oxidative stress conditions induced by H2O2, we found that H2O2 treatment induced FtMt and a-synuclein expression at both the mRNA and protein levels in a dose-dependent manner. FtMt overexpression protected cells against oxidative stress and alleviated the enhanced a-synuclein expression induced by H2O2 at the posttranscriptional level. Our results indicate that FtMt modulates alpha-synudein expression at the posttranscriptional level via iron regulation in physiological conditions. FtMt expression is enhanced under oxidative stress conditions, where FtMt protects cells against the oxidative stress as well as plays an important role in maintaining a-synuclein levels. (C) 2017 Published by Elsevier Inc.
  • Takao Ogawa, Naoya Irikawa, Daijiro Yanagisawa, Akihiko Shiino, Ikuo Tooyama, Takeshi Shimizu
    Auris Nasus Larynx 44 2 168 - 173 2017年04月 [有り][無し]
     研究論文（学術雑誌） 
    Objective Alzheimer-type dementia (AD) is pathologically characterized by massive neuronal loss in the brain, and the taste cortex is thought to be affected. However, there are only a few reports regarding the gustatory function of AD patients, and the conclusions of this research are inconsistent. Methods This prospective study enrolled 22 consecutive patients with mild to moderately severe Alzheimer-type dementia (AD) with mean age of 84.0 years, and 49 elderly volunteers without dementia with mean age of 71.0 years as control subjects. The control subjects were divided into two groups according to age: a younger group (N = 28, mean age: 68.5) and an older group (N = 21, mean age: 83.0). The gustatory function was investigated using the filter paper disc method (FPD) and electrogustometry (EGM). Results The gustatory function as measured by the FPD was significantly impaired in patients with AD as compared with age-matched control subjects no such difference was found between the younger and the older control groups. On the other hand, as for the EGM thresholds, there were no differences between the AD patient group and the age-matched controls. Conclusion The FPD method demonstrated decreased gustatory function in AD patients beyond that of aging. On the other hand, EGM thresholds did not differ between the AD patient group and the age-matched controls. These results suggest that failure of taste processing in the brain, but not taste transmission in the peripheral taste system, occurs in patients with AD.
  • Shogo Higaki, Manami Shimada, Kazuaki Kawamoto, Takaaki Todo, Toshihiro Kawasaki, Ikuo Tooyama, Yasuhiro Fujioka, Noriyoshi Sakai, Tatsuyuki Takada
    SCIENTIFIC REPORTS 7 42852 - 42852 2017年02月 [有り][無し]
     研究論文（学術雑誌） 
    Many endemic fish species are threatened with extinction. Conservation strategies and the restoration of endemic fish after extinction must therefore be investigated. Although sperm cryopreservation is indispensable for the conservation of endangered fishes, the limited number of mature fish and limited availability (volume and period) of sperm from small endemic fish hinders the optimization and practical use of this material. In this report, we demonstrate the in vitro differentiation of fertile sperm from cryopreserved spermatogonia of juveniles of the endangered small cyprinid honmoroko (Gnathopogon caerulescens), which is endemic to Lake Biwa in Japan. The entire process of spermatogenesis was recapitulated in vitro using cryopreserved spermatogonia of non-spawning adult and juvenile fish. The differentiation of sperm from spermatogonia was captured as a time-lapse video and confirmed by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into sperm. Fertility was demonstrated by artificial insemination. These results suggest that the combination of cryopreservation of spermatogonia and in vitro sperm differentiation will provide a new and promising strategy for the preservation of paternal genetic materials.
  • Akihiko Shiino, Yen-wei Chen, Kenji Tanigaki, Atsushi Yamada, Piers Vigers, Toshiyuki Watanabe, Ikuo Tooyama, Ichiro Akiguchi
    SCIENTIFIC REPORTS 7 39818 - 39818 2017年01月 [有り][無し]
     研究論文（学術雑誌） 
    It has been contended that any observed difference of the corpus callosum (CC) size between men and women is not sex-related but brain-size-related. A recent report, however, showed that the midsagittal CC area was significantly larger in women in 37 brain-size-matched pairs of normal young adults. Since this constituted strong evidence of sexual dimorphism and was obtained from publicly available data in OASIS, we examined volume differences within the CC and in other white matter using voxel-based morphometry (VBM). We created a three-dimensional region of interest of the CC and measured its volume. The VBM statistics were analyzed by permutation test and threshold-free cluster enhancement (TFCE) with the significance levels at FWER < 0.05. The CC volume was significantly larger in women in the same 37 brain-size-matched pairs. We found that the CC genu was the subregion showing the most significant sex-related difference. We also found that white matter in the bilateral anterior frontal regions and the left lateral white matter near to Broca's area were larger in women, whereas there were no significant larger regions in men. Since we used brain-size-matched subjects, our results gave strong volumetric evidence of localized sexual dimorphism of white matter.
  • Takahiro Ito, Sachiko Tanaka-Mizuno, Narihito Iwashita, Ikuo Tooyama, Akihiko Shiino, Katsuyuki Miura, Sei Fukui
    JOURNAL OF PAIN RESEARCH 10 287 - 293 2017年 [有り][無し]
     研究論文（学術雑誌） 
    Background: Chronic pain is a common cause of reduced quality of life. Recent studies suggest that chronic pain patients have a different brain neurometabolic status to healthy people. Proton magnetic resonance spectroscopy (H-1-MRS) can determine the concentrations of metabolites in a specific region of the brain without being invasive. Patients and methods: We recruited 56 chronic pain patients and 60 healthy controls to compare brain metabolic characteristics. The concentrations of glutamic acid (Glu), myo-inositol (Ins), N-acetylaspartate (NAA), Glu + glutamine (Glx), and creatine + phosphocreatine (total creatine [tCr]) in the anterior cingulate cortex of participants were measured using (II)-I-1-MRS. We used age-and gender-adjusted general linear models and receiver-operating characteristic analyses for this investigation. Patients were also assessed using the Hospital Anxiety and Depression Scale (HADS) to reveal the existence of any mental health issues. Results: Our analysis indicates that pain patients have statistically significantly higher levels of Glu/tCr (p=0.039) and Glx/tCr (p<0.001) and lower levels of NAA/tCr than controls, although this did not reach statistical significance (p=0.052). Receiver-operating characteristic analysis performed on the combination of Glx/tCr, Ins/tCr, and NAA/tCr effectively discriminated chronic pain patients from healthy controls. Patients with higher HADS-Depression scores had increased Glx/rCr levels (p=0.015), and those with higher HADS-Anxiety scores had increased NAA/tCr levels (p=0.018). Conclusion: Chronic pain patients have a different metabolite status in the anterior cingulate cortex to controls. Within the pain patient group, HADS scores had a positive relationship with NAA/tCr and Glx/tCr levels. 1H-MRS successfully detected metabolic changes in patients' brains in a noninvasive manner, revealing its potential as a superior diagnostic tool for pain patients.
  • Mingchun Yang, Hongkuan Yang, Hongpeng Guan, Tomoko Kato, Kenichi Mukaisho, Hiroyuki Sugihara, Kazumasa Ogasawara, Tomohiro Terada, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 50 1 49 - 55 2017年 [有り][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is a novel iron storage protein with high homology to H-ferritin. Unlike the ubiquitously expressed H-and L-ferritin, FtMt is expressed in specific tissues such as the testis, heart, and brain. The function of FtMt is not fully understood; however, evidence suggests that it has a neuroprotective role in neurodegenerative diseases. We have previously reported that FtMt is expressed in catecholaminergic neurons of the monkey brainstem. To explore FtMt expression in human dopaminergic neurons, we designed a novel monoclonal antibody, C65-2, directed against human FtMt. Here, we report the properties of our C65-2 antibody. Western blots analysis and immunoabsorption tests demonstrated that the C65-2 antibody specifically recognized FtMt with no cross-reactivity to H-ferritin. Immunohistochemistry showed that the C65-2 antibody detected FtMt in neurons of the substantia nigra pars compacta (SNc) in humans and monkeys. We confirmed that FtMt is expressed in dopaminergic neurons of the human SNc. Our results suggest that FtMt is involved in various physiological and pathological mechanisms in human dopaminergic neurons, and the C65-2 monoclonal antibody promises to be a useful tool for determining the localization and biological functions of FtMt in the brain.
  • Yoshihisa Kitamura, Masatoshi Inden, Yasuto Kimoto, Kazuyuki Takata, Daijiro Yanagisawa, Masanori Hijioka, Eishi Ashihara, Ikuo Tooyama, Shun Shimohama, Hiroyoshi Ariga
    JOURNAL OF ALZHEIMERS DISEASE 55 1 67 - 72 2017年 [有り][無し]
     研究論文（学術雑誌） 
    Previously, DJ-1 modulator UCP0054278/comp-B was identified by virtual screening, where comp-B interacts with DJ-1 to produce antioxidant and neuroprotective responses in Parkinson's disease models. However, the effect of comp-B in an in vivo Alzheimer's disease (AD) model is yet undetermined. Thus, we examined the effect of comp-B on spatial learning, memory, and amyloid-beta (A beta) clearance in a transgenic mouse model of AD. We found that comp-B resolved the cognitive deficits, reduced insoluble A beta(42) levels, and prevented the degeneration of synaptic functions, thereby suggesting that comp-B may become a major compound for AD treatment.
  • Ibrahim NF, Yanagisawa D, Durani LW, Hamezah HS, Damanhuri HA, Wan Ngah WZ, Tsuji M, Kiuchi Y, Ono K, Tooyama I
    Journal of Alzheimer's disease : JAD 55 2 597 - 612 2017年 [有り][無し]
     研究論文（学術雑誌） 
    Alzheimer's disease (AD) is the most common cause of dementia. The cardinal neuropathological characteristic of AD is the accumulation of amyloid-beta (A beta) into extracellular plaques that ultimately disrupt neuronal function and lead to neurodegeneration. One possible therapeutic strategy therefore is to prevent A beta aggregation. Previous studies have suggested that vitamin E analogs slow AD progression in humans. In the present study, we investigated the effects of the tocotrienol-rich fraction (TRF), a mixture of vitamin E analogs from palm oil, on amyloid pathology in vitro and in vivo. TRF treatment dose-dependently inhibited the formation of A beta fibrils and A beta oligomers in vitro. Moreover, daily TRF supplementation to A beta PPswe/PS1dE9 double transgenic mice for 10 months attenuated A beta immunoreactive depositions and thioflavin S- positive fibrillar type plaques in the brain, and eventually improved cognitive function in the novel object recognition test compared with control A beta PPswe/PS1dE9 mice. The present result indicates that TRF reduced amyloid pathology and improved cognitive functions, and suggests that TRF is a potential therapeutic agent for AD.
  • Essam M. Abdelalim, Jean-Pierre Bellier, Ikuo Tooyama
    FRONTIERS IN NEUROANATOMY 10 116 - 116 2016年12月 [有り][無し]
     研究論文（学術雑誌） 
    Brain natriuretic peptide (BNP) exerts its functions through NP receptors. Recently, BNP has been shown to be involved in a wide range of functions. Previous studies reported BNP expression in the sensory afferent fibers in the dorsal horn (DH) of the spinal cord. However, BNP expression and function in the neurons of the central nervous system are still controversial. Therefore, in this study, we investigated BNP expression in the rat spinal cord in detail using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RT-PCR analysis showed that BNP mRNA was present in the spinal cord and dorsal root ganglion (DRG). BNP immunoreactivity was observed in different structures of the spinal cord, including the neuronal cell bodies and neuronal processes. BNP immunoreactivity was observed in the DH of the spinal cord and in the neurons of the intermediate column (IC) and ventral horn (VH). Double-immunolabeling showed a high level of BNP expression in the afferent fibers (laminae I-II) labeled with calcitonin gene-related peptide (CGRP), suggesting BNP involvement in sensory function. In addition, BNP was co-localized with CGRP and choline acetyltransferase (ChAT) in the motor neurons of the VH. Together, these results indicate that BNP is expressed in sensory and motor systems of the spinal cord, suggesting its involvement in several biological actions on sensory and motor neurons via its binding to NP receptor-A (NPR-A) and/or NP receptor-B (NPR-B) at the spinal cord level.
  • Wang X, Yang H, Yanagisawa D, Bellier JP, Morino K, Zhao S, Liu P, Vigers P, Tooyama I
    Neurobiology of aging 47 168 - 179 2016年11月 [有り][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is believed to play an antioxidant role via iron regulation, and FtMt gene mutation has been reported in age-related macular degeneration (AMD). However, little is known about FtMt's functions in the retina and any links to AMD. In this study, we observed age-related increase in FtMt and hypoxia-inducible factor-1 alpha (HIF-1 alpha) in murine retinal pigment epithelium (RPE). FtMt overexpression in ARPE-19 cells stabilized HIF-1 alpha, and increased the secretion of vascular endothelial growth factor. Conversely, HIF-1 alpha stabilization reduced the protein level of the mature, functional form of FtMt. FtMt-overexpressing ARPE-19 cells exhibited less oxidative phosphorylation but unchanged production of adenosine triphosphate, enhanced mitochondrial fission, and triggered mitophagy in a HIF-1 alpha-dependent manner. These findings suggest that increased FtMt in RPE may be protective via triggering mitophagy but cause wet AMD by inducing neovascularization due to increased vascular endothelial growth factor secretion. However, reduced level of functional FtMt in RPE under hypoxia may allow dry AMD through susceptibility to age-related stress. (C) 2016 The Author(s). Published by Elsevier Inc.
  • ミトコンドリアフェリチンは細胞を保護してα-Synuclein発現を調節する(Mitochondrial Ferritin Protects Cells and Modulates α-Synuclein Expression)
    関 宏鵬, 楊 宏寛, 楊 明春, 柳沢 大治郎, Bellier Jean-Pierre, 野中 隆, 趙 世光, 遠山 育夫
    Dementia Japan 30 4 563 - 563 2016年10月
  • Ikuo Tooyama, Daijiro Yanagisawa, Hiroyasu Taguchi, Tomoko Kato, Koichi Hirao, Nobuaki Shirai, Takayuki Sogabe, Nor Faeizah Ibrahim, Toshiro Inubushi, Shigehiro Morikawa
    AGEING RESEARCH REVIEWS 30 85 - 94 2016年09月 [有り][無し]
     
    The formation of senile plaques followed by the deposition of amyloid-beta is the earliest pathologicalchange in Alzheimer's disease. Thus, the detection of senile plaques remains the most important early diagnostic indicator of Alzheimer's disease. Amyloid imaging is a noninvasive technique for visualizing senile plaques in the brains of Alzheimer's patients using positron emission tomography (PET) or magnetic resonance imaging (MRI). Because fluorine-19 (F-19) displays an intense nuclear magnetic resonance signal and is almost non-existent in the body, targets are detected with a higher signal-to-noise ratio using appropriate fluorinated contrast agents. The recent introduction of high-field MRI allows us to detect amyloid depositions in the brain of living mouse using F-19-MRI. So far, at least three probes have been reported to detect amyloid deposition in the brain of transgenic mouse models of Alzheimer's disease; (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), 1,7-bis(4'-hydroxy-3'-trifluoromethoxyphenyl)-4-methoxycarbonylethyl-1,6-heptadiene3,5-dione (FMeCl, Shiga-Y5) and 6-(3',6',9',15',18',21'-heptaoxa-23',23',23'-trifluorotricosanyloxy)-2-(4'dimethylaminostyryl)benzoxazole (XP7, Shiga-X22). This review presents the recent advances in amyloid imaging using F-19-MRI, including our own studies. (C) 2016 Elsevier B.V. All rights reserved.
  • Yuma Sonoda, Ikuo Tooyama, Hideyuki Mukai, Kiyoshi Maeda, Haruhiko Akiyama, Toshio Kawamata
    NEUROPATHOLOGY 36 4 325 - 332 2016年08月 [有り][無し]
     研究論文（学術雑誌） 
    The 70-kDa ribosomal protein S6 kinase (S6K), a serine/threonine kinase that modulates the phosphorylation of the 40S ribosomal protein S6, regulates cell cycle progression and is known as a tau kinase in Alzheimer's disease (AD). In AD brains, neurofibrillary tangles (NFTs) have been shown to be positively stained with antibodies against S6K proteins phosphorylated at T389 (pT389-S6K) or T421/S424 (pT421/S424-S6K) by the mammalian target of rapamycin and mitogen-activated protein kinase pathways, respectively. However, there is little information available about S6K proteins directly phosphorylated at T229 (pT229-S6K) by the PI3K-PDK1 pathway. In the present study, we investigated the distribution of pT229-S6K in post mortem human brain tissues from elderly (control) and patients with AD using immnunoblotting and immunohistochemistry. pT229-S6K immunoreactivity was localized to small granular structures in neurons and endothelial cells in control and AD brains. In AD brains, intense pT229-S6K immunoreactivity was detected in 16.3% of AT8-positive NFTs, neuropil threads, and dystrophic neurites in the hippocampus and other vulnerable brain areas. In addition, Hirano bodies were also positive for pT229-S6K but were negative for pT389-S6K or pT421/S424-S6K. The present results indicate that S6K phosphorylation via the PI3K-PD1 pathway is involved in tau pathology in NFTs and abnormal neurites as well as actin pathology in Hirano bodies.
  • 免疫組織化学の基礎と抗体の上手な使い方
    遠山 育夫, 加藤 智子, 柳沢 大治郎, 相見 良成, 安原 治
    組織細胞化学 2016 1 - 17 日本組織細胞化学会 2016年07月
  • Mingchun Yang, Hongkuan Yang, Hongpeng Guan, Jean-Pierre Bellier, Shiguang Zhao, Ikuo Tooyama
    NEUROSCIENCE 328 92 - 106 2016年07月 [有り][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt), a recently-studied iron storage protein, which we suspect is an important defense against oxidative stress in neurons and elsewhere. The 242-amino acid FtMt precursor protein is cleaved to mature protein (of molecular weight about 22-kDa) in the mitochondrial matrix. Compared with the ubiquitously expressed traditional ferritin (H-ferritin and L-ferritin), FtMt has been found in fewer locations including the testis, heart and brain. Previous studies have reported that the expression of FtMt in mouse and human brain is predominantly localized to neurons and partly to glial cells, and FtMt exerts protective effects on neurons by maintaining normal function and regulates apoptosis in Alzheimer's disease and Parkinson's disease. To find out the function of FtMt in neurodegenerative disease, we had a novel antibody made against human FtMt and characterized it via Western blot analysis, immunoabsorption testing, and double immunofluorescence histochemistry. Then we used this new FtMt antibody to map the distribution of FtMt in the monkey brainstem. We demonstrated widespread distribution of FtMt immunoreactivity throughout the monkey brainstem, with variable staining intensity. FtMt immunoreactivity was observed in the extrapyramidal system, sensory trigeminal nerve nuclei, some motor nuclei including ambiguous nucleus, dorsal motor nucleus of the vagus and hypoglossal nucleus, and some dorsal column nuclei such as the gracile nucleus and cuneate nucleus. In addition, double immunohistochemical stainings confirmed that FtMt immunoreactivity was co-localized with catecholaminergic neurons in the locus coeruleus (63.64%), substantia nigra pars compacta (69.18%), and ventral tegmental area (56.89%). The distribution involvement of FtMt in several physiological mechanisms, especially in the catecholaminergic neurons, and the possibility of significant involvement in neurodegenerative disease. (C) 2016 The Author(s). Published by Elsevier Ltd on behalf of IBRO.
  • Ikuo Tooyama, Nor Faeizah Ibrahim, Lina Wati Durani, Hamizah Shahirah Hamezah, Mohd Hanafi Ahmad Damanhuri, Wan Zurinah Wan Ngah, Hiroyasu Taguchi, Daijiro Yanagisawa
    Fruits, Vegetables, and Herbs: Bioactive Foods in Health Promotion 487 - 505 2016年05月 [有り][無し]
     論文集(書籍)内論文 
    Recent evidence suggests that amyloid pathology occurs 10-20 years before the clinical onset of Alzheimer's disease (AD). Accordingly, modulating abnormal amyloid-β (Aβ) aggregation is now considered as a potential therapeutic target in AD. Curcumin, a low molecular weight polyphenol derived from the well-known spice turmeric, has various pharmacological properties, including antitumor, antioxidative, antiinflammatory, and antiamyloid effects. Curcumin binds both Aβ fibrils and oligomers and inhibits Aβ aggregation as well as Aβ-related oxidative stress and inflammation. The keto-enol tautomerism of curcumin is involved in its binding to Aβ aggregates. The enol form, but not the keto form, of curcumin can bind to Aβ aggregates. When curcumin was administered to transgenic mouse models of AD, it effectively suppressed amyloid pathology in the mouse brain. However, the results of early clinical trials of curcumin for AD have been negative, probably due to its low bioavailability. Several approaches have been proposed to improve the bioavailability of curcumin.
  • アルツハイマー病の病態解明のためのフッ素MRIによるダブルプローブイメージング法の開発
    柳沢 大治郎, Ibrahim Nor Faeizah, 田口 弘康, 森川 茂廣, 遠山 育夫
    JSMI Report 9 2 110 - 110 日本分子イメージング学会 2016年04月
  • Yasunari Seita, Tomoyuki Tsukiyama, Chizuru Iwatani, Hideaki Tsuchiya, Jun Matsushita, Takuya Azami, Junko Okahara, Shinichiro Nakamura, Yoshitaka Hayashi, Seiji Hitoshi, Yasushi Itoh, Takeshi Imamura, Masaki Nishimura, Ikuo Tooyama, Hiroyuki Miyoshi, Mitinori Saitou, Kazumasa Ogasawara, Erika Sasaki, Masatsugu Ema
    SCIENTIFIC REPORTS 6 24868  2016年04月 [有り][無し]
     研究論文（学術雑誌） 
    Nonhuman primates are valuable for human disease modelling, because rodents poorly recapitulate some human diseases such as Parkinson's disease and Alzheimer's disease amongst others. Here, we report for the first time, the generation of green fluorescent protein (GFP) transgenic cynomolgus monkeys by lentivirus infection. Our data show that the use of a human cytomegalovirus immediateearly enhancer and chicken beta actin promoter (CAG) directed the ubiquitous expression of the transgene in cynomolgus monkeys. We also found that injection into mature oocytes before fertilization achieved homogenous expression of GFP in each tissue, including the amnion, and fibroblasts, whereas injection into fertilized oocytes generated a transgenic cynomolgus monkey with mosaic GFP expression. Thus, the injection timing was important to create transgenic cynomolgus monkeys that expressed GFP homogenously in each of the various tissues. The strategy established in this work will be useful for the generation of transgenic cynomolgus monkeys for transplantation studies as well as biomedical research.
  • Inden M, Takata K, Yanagisawa D, Ashihara E, Tooyama I, Shimohama S, Kitamura Y
    Neurochemistry international 94 74 - 81 2016年03月 [有り][無し]
     研究論文（学術雑誌） 
    Galantamine, an acetylcholine esterase (AChE) inhibitor used to treat dementia symptoms, also acts as an allosteric potentiating ligand (APL) at nicotinic acetylcholine receptors (nAChRs). This study was designed to evaluate the allosteric effect of galantamine on nAChR regulation of nigrostrial dopaminergic neuronal function in the hemiparkinsonian rat model established by unilateral nigral 6-hydroxydopamine (6-OHDA) injection. Methamphetamine, a dopamine releaser, induced ipsilateral rotation, whereas dopamine agonists apomorphine (a non-selective dopamine receptor agonist), SKF38393 (a selective dopamine D1 receptor agonist), and quinpirole (a selective dopamine D2 receptor agonist) induced contralateral rotation. When 6-OHDA-injected rats were co-treated with nomifensine, a dopamine transporter inhibitor, a more pronounced and a remarkable effect of nicotine and galantamine was observed. Under these conditions, the combination of nomifensine with nicotine or galantamine induced the ipsilateral rotation similar to the methamphetamine-induced rotational behavior, indicating that nicotine and galantamine also induce dopamine release from striatal terminals. Both nicotine- and galantamine-induced rotations were significantly blocked by flupenthixol (an antagonist of both D1 and D2 dopamine receptors) and mecamylamine (an antagonist of nAChRs), suggesting that galantamine modulation of nAChRs on striatal dopaminergic terminals regulates dopamine receptor-mediated movement. Immunohistochemical staining showed that alpha 4 nAChRs were highly expressed on striatal dopaminergic terminals, while no alpha 7 nAChRs were detected. Pretreatment with the alpha 4 nAChR antagonist dihydroxy-beta-erythroidine significantly inhibited nicotine- and galantamine-induced rotational behaviors, whereas pretreatment with the alpha 7 nAChR antagonist methyllycaconitine was ineffective. Moreover, the alpha 4 nAChR agonist ABT-418 induced ipsilateral rotation, while the alpha 7 nAChR agonist PNU282987 had no significant effect on rotational behavior. These results suggest that galantamine can enhance striatal dopamine release through allosteric modulation of alpha 4 nAChRs on nigrostriatal dopaminergic terminals. (C) 2016 Elsevier Ltd. All rights reserved.
  • Li Zhao, Hongkuan Yang, Tsukuru Amano, Hongmei Qin, Luyi Zheng, Akimasa Takahashi, Shiguang Zhao, Ikuo Tooyama, Takashi Murakami, Naoki Komatsu
    Journal of Materials Chemistry B 4 47 7741 - 7748 2016年 [有り][無し]
     研究論文（学術雑誌） 
    This journal is © The Royal Society of Chemistry 2016. In cancer treatment, efficient delivery of active anticancer drugs into cancer cells is highly desirable for maximizing therapeutic effects and alleviating side effects. In this work, a nanocarrier consisting of an Fe3O4 core, a polyglycerol coating, and an octalysine functionality (SPION-PG-Lys8) has been designed, synthesized and used to deliver a photosensitizer, chlorin e6 (Ce6), into cancer cells for photodynamic therapy (PDT) of cancer cells. SPION-PG-Lys8 is colloidally stable in various aqueous solutions, showing a high positive zeta potential of 47.2 ± 6.9 mV in pure water. In vitro characterization reveals that SPION-PG-Lys8 is efficiently taken up by SKOV3 ovarian cancer cells, exhibiting low cytotoxicity, and suppressed autophagy compared to bare SPIONs. Negatively charged Ce6 is thus loaded on the SPION-PG-Lys8 through electrostatic attraction to yield a SPION-PG-Lys8/Ce6 nanocomplex with a positive zeta potential of 22.4 ± 4.3 mV. SPION-PG-Lys8/Ce6 is more easily taken up by the cells than free Ce6, and surprisingly, the internalized SPION-PG-Lys8/Ce6 is found to be enriched in the mitochondria. SPION-PG-Lys8/Ce6 exhibits almost no cytotoxicity under dark conditions, but strong photocytotoxicity due to the light-triggered production of reactive oxygen species (ROS) destroying the mitochondria. Taken together, our results highlight the great potential of SPION-PG-Lys8 as an efficient carrier of Ce6 for photodynamic cancer therapy.
  • Yanagisawa D, Taguchi H, Morikawa S, Kato T, Hirao K, Shirai N, Tooyama I
    Biochemistry and biophysics reports 4 357 - 368 2015年12月 [有り][無し]
     研究論文（学術雑誌） 
    Modulation of abnormal amyloid β (Aβ) aggregation is considered to be a potential therapeutic target for Alzheimer's disease (AD). Recent in vitro and in vivo experiments suggest that inhibition of Aβ aggregation by curcumin would exert favorable effects for preventing or treating AD. We have previously synthesized a series of novel curcumin derivatives. In this study, we investigated the effects of our curcumin derivatives on Aβ aggregation and the cell toxicities of Aβ aggregates. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles, 14 of 41 compounds showed a significant increase in the densities of the bands of Aβ (1-42) by incubation during the aggregation process relative to those of Aβ (1-42) prepared in the presence of the vehicle control. Of the 14 compounds, four compounds additionally reduced cell toxicity of the Aβ aggregates by incubation during the aggregation process. A significant positive correlation was observed between the cell viability and densities of the bands at ranges of 15-20, 20-37, 37-75, and 75-200. kDa in SDS-PAGE. On the basis of these results, we propose four curcumin derivatives with potential for preventing AD. These curcumin derivatives exhibited high inhibitory effects on Aβ aggregation and induced the formation of lower molecular size Aβ species that have weaker cell toxicity. These compounds may exert therapeutic effects on AD in future in vivo studies.
  • フッ素MRIを利用したAβオリゴマーの画像化法の開発
    柳沢 大治郎, Ibrahim Nor Faeizah, 田口 弘康, 森川 茂廣, 遠山 育夫
    JSMI Report 8 2 137 - 137 日本分子イメージング学会 2015年04月
  • Kato T, Konishi Y, Shimohama S, Beach TG, Akatsu H, Tooyama I
    Neuroscience Letters 591 19 - 24 2015年03月 [有り][無し]
     研究論文（学術雑誌） 
    Alpha1-chimaerin is a GTPase-activating protein (GAP) for Rac1, a member of the Rho small GTPase family, whose action leads to the inactivation of Rac1. Rac1 activity is upregulated in Alzheimer's disease, but little is known about the role of alpha 1-chimaerin. In this study, we investigated the expression and localization of alpha 1-chimaerin mRNA in postmortem human brains from patients with Alzheimer's disease and control subjects. In situ hybridization studies demonstrated that alpha 1 -chimaerin was expressed by neurons in the neo-cortex of the temporal lobe and the hippocampus of both controls and Alzheimer's disease cases, with the signal intensity dramatically decreased in patients with Alzheimer's disease. Realtime PCR analysis confirmed a significant reduction of alpha 1-chimaerin mRNA expression in the temporal cortex of Alzheimer's disease cases. In contrast, alpha 2-chimaerin mRNA levels showed no significant difference between the groups. The present study showed reduced alpha 1-chimaerin expression in the brain of Alzheimer's disease cases, suggesting a role in the upregulation of Rac1 activity during the disease process. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
  • Yoshiko Wada, Yuki Iwasaki, Takashi Abe, Kennosuke Wada, Ikuo Tooyama, Toshimichi Ikemura
    GENES & GENETIC SYSTEMS 90 1 43 - 53 2015年02月 [有り][無し]
     研究論文（学術雑誌） 
    Unsupervised data mining capable of extracting a wide range of information from big sequence data without prior knowledge or particular models is highly desirable in an era of big data accumulation for research on genes, genomes and genetic systems. By handling oligonucleotide compositions in genomic sequences as high-dimensional data, we have previously modified the conventional SOM (self-organizing map) for genome informatics and established BLSOM for oligonucleotide composition, which can analyze more than ten million sequences simultaneously and is thus suitable for big data analyses. Oligonucleotides often represent motif sequences responsible for sequence-specific binding of proteins such as transcription factors. The distribution of such functionally important oligonucleotides is probably biased in genomic sequences, and may differ among genomic regions. When constructing BLSOMs to analyze pentanucleotide composition in 50-kb sequences derived from the human genome in this study, we found that BLSOMs did not classify human sequences according to chromosome but revealed several specific zones, which are enriched for a class of CG-containing pentanucleotides; these zones are composed primarily of sequences derived from pericentric regions. The biological significance of enrichment of these pentanucletides in pericentric regions is discussed in connection with cell type- and stage-dependent formation of the condensed heterochromatin in the chromocenter, which is formed through association of pericentric regions of multiple chromosomes.
  • Gender Difference of the Corpus Callosum-subregional Inspection, International Society for Cerebral Blood Flow & Metabolism
    Shiino, A, K. Tanigaki, Y. Chen, A. Yamada, I. Tooyama
    ISBFM Brain 2015 2015年 [有り][無し]
     研究論文（国際会議プロシーディングス）
  • Daijiro Yanagisawa, Nor Faeizah Ibrahim, Hiroyasu Taguchi, Shigehiro Morikawa, Koichi Hirao, Nobuaki Shirai, Takayuki Sogabe, Ikuo Tooyama
    NEUROBIOLOGY OF AGING 36 1 201 - 210 2015年01月 [有り][無し]
     研究論文（学術雑誌） 
    Recent evidence supports the amyloid cascade hypothesis that a pathological change of amyloid beta (A beta) in the brain is an initiating event in Alzheimer's disease (AD). Accordingly, modulating the abnormal A beta aggregation is considered a potential therapeutic target in AD. Curcumin, a low-molecular-weight polyphenol derived from the well-known curry spice turmeric, has shown favorable effects on preventing or treating AD pathology. The present study investigated the effects of curcumin and 2 novel curcumin derivatives, FMeC1 and FMeC2, on AD pathology in APPswe/PS1dE9 double transgenic mice. Mice fed a chow diet that contained FMeC1 for 6 months showed a reduction in insoluble A beta deposits and glial cell activity together with reduced cognitive deficits, compared to animals receiving a control diet or with curcumin or FMeC2 in their diet. Both curcumin and FMeC1 modulated the formation of A beta aggregates; however, only FMeC1 significantly attenuated the cell toxicity of A beta. These results indicate that FMeC1 may have potential for preventing AD. (C) 2015 Elsevier Inc. All rights reserved.
  • Richard McClure, Daijiro Yanagisawa, Donald Stec, Dave Abdollahian, Dmitry Koktysh, Dritan Xhillari, Rudolph Jaeger, Gregg Stanwood, Eduard Chekmenev, Ikuo Tooyama, John C. Gore, Wellington Pham
    JOURNAL OF ALZHEIMERS DISEASE 44 1 283 - 295 2015年 [有り][無し]
     研究論文（学術雑誌） 
    Curcumin is a promising compound that can be used as a theranostic agent to aid research in Alzheimer's disease. Beyond its ability to bind to amyloid plaques, the compound can also cross the blood-brain barrier. Presently, curcumin can be applied only to animal models, as the formulation needed for iv injection renders it unfit for human use. Here, we describe a novel technique to aerosolize a curcumin derivative, FMeC1, and facilitate its safe delivery to the brain. Aside from the translational applicability of this approach, a study in the 5XFAD mouse model suggested that inhalation exposure to an aerosolized FMeC1 modestly improved the distribution of the compound in the brain. Additionally, immunohistochemistry data confirms that following aerosol delivery, FMeC1 binds amyloid plaques expressed in the hippocampal areas and cortex.
  • Hongkuan Yang, Hongpeng Guan, Mingchun Yang, Ziyi Liu, Shigeko Takeuchi, Daijiro Yanagisawa, Steven R. Vincent, Shiguang Zhao, Ikuo Tooyama
    JOURNAL OF ALZHEIMERS DISEASE 45 3 797 - 811 2015年 [有り][無し]
     研究論文（学術雑誌） 
    Studies have shown an increased expression of mitochondrial ferritin (FtMt) and an antioxidant role for the protein in the brains of Alzheimer's disease (AD) patients. However, little information is available concerning the role of FtMt in other AD pathologies, including inflammation and amyloidogenesis. Therefore, we investigated the regulation and function of FtMt in inflammation and amyloidogenesis. FtMt protein expression was increased by proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin 6 (IL-6), whereas FtMt mRNA levels were increased by TNF-alpha but not by IL-1 beta or IL-6 in IMR-32 cells. The transcription factor nuclear factor-kappa B (NF-kappa B) inhibitor, Bay 11-7082, suppressed this TNF-alpha-induced FtMt expression. FtMt overexpression increased NF-kappa B activity and translocation of p65 into the nucleus in HEK293 cells. Conversely, knockdown of FtMt attenuated TNF-alpha-induced NF-kappa B activity. Overexpression of FtMt inhibited TNF-alpha-induced apoptosis in the cell culture. FtMt overexpression reduced iron-mediated expression of amyloid-beta protein precursor and decreased NF-kappa B-dependent increases in beta- and gamma-secretase, leading to decreased amyloid-beta production. Our data provide new insights into the mechanism underlying the regulation of FtMt expression by proinflammatory cytokines and indicate further roles for FtMt in AD.
  • Hiroyasu Taguchi, Daijiro Yanagisawa, Shigehiro Morikawa, Koichi Hirao, Nobuaki Shirai, Ikuo Tooyama
    AUSTRALIAN JOURNAL OF CHEMISTRY 68 2 224 - 229 2015年 [無し][無し]
     研究論文（学術雑誌） 
    1,7-Bis(4-hydroxy-3-trifluoromethoxyphenyl)-1,6-heptadiene-3,5-dione (2a), related to curcumin, and thirteen 4-substituted derivatives were prepared and their keto/enol ratio in DMSO[D6] was determined by F-19 NMR because the enolic form of these related curcumins had been shown to bind to amyloid plaques in the Alzheimer brain. The parent compound and the 4-ethoxycarbonyl derivative were almost 100% in the enolic form that contains a conjugated hepta-1,4,6-trien-3-on-5-ol backbone. Enolisation decreased to varying amounts in the derivatives that had 4-substituted alkyl groups. Attempts to prepare the 4-hydroxypropyl derivative by hydrolysis of O-methoxymethyl 2m or O-tetrahydropyranyloxy 2n protected derivatives led to cyclised products. A related pyrimidine compound 6b that mimicked a fixed enol form was also prepared.
  • Hiroyasu Taguchi, Daijiro Yanagisawa, Shigehiro Morikawa, Koichi Hirao, Nobuaki Shirai, Ikuo Tooyama
    AUSTRALIAN JOURNAL OF CHEMISTRY 68 2 224 - 229 2015年 [無し][無し]
     研究論文（学術雑誌） 
    1,7-Bis(4-hydroxy-3-trifluoromethoxyphenyl)-1,6-heptadiene-3,5-dione (2a), related to curcumin, and thirteen 4-substituted derivatives were prepared and their keto/enol ratio in DMSO[D6] was determined by F-19 NMR because the enolic form of these related curcumins had been shown to bind to amyloid plaques in the Alzheimer brain. The parent compound and the 4-ethoxycarbonyl derivative were almost 100% in the enolic form that contains a conjugated hepta-1,4,6-trien-3-on-5-ol backbone. Enolisation decreased to varying amounts in the derivatives that had 4-substituted alkyl groups. Attempts to prepare the 4-hydroxypropyl derivative by hydrolysis of O-methoxymethyl 2m or O-tetrahydropyranyloxy 2n protected derivatives led to cyclised products. A related pyrimidine compound 6b that mimicked a fixed enol form was also prepared.
  • 【脳内環境-維持機構と破綻がもたらす疾患研究】(第3章)脳内環境をモニターするイメージング フッ素MR画像法によるアミロイドオリゴマーのin vivo病態解析
    遠山 育夫, 柳沢 大治郎, Ibrahim Nor Faeizah, 田口 弘康
    遺伝子医学MOOK 26 190 - 194 (株)メディカルドゥ 2014年11月 
    7テスラ高磁場MR画像装置を用い,フッ素MR画像法によるAβオリゴマーの画像化を試みた。まず,Aβ凝集体のみならずAβオリゴマーにも結合して画像化ができるShiga-Y5とAβ凝集体のみに強く結合するShiga-X22を開発した。ついでShiga-Y5とShiga-X22の混合液をAPP/PS1マウスに投与した。それぞれの化合物に固有のフッ素ケミカルシフト値を用いることで,2つの化合物それぞれのMR画像を得た。また,両者の画像の差分を算出することにより,Aβオリゴマーの候補画像を作成することに成功した。(著者抄録)
  • ミトコンドリアフェリチンはパーキンソン病において保護的な役割を果たす(Mitochondrial Ferritin plays a protective role in Parkinson Disease)
    関 宏鵬, 陽 宏寛, 陽 銘春, 劉 子儀, 趙 世光, 柳沢 大治郎, 遠山 育夫
    Dementia Japan 28 4 482 - 482 2014年10月
  • アルツハイマー病遺伝子改変マウスにおける新規クルクミン誘導体の治療効果の解析
    柳沢 大治郎, Faeizah Ibrahim Nor, Wati Durani Lina, 田口 弘康, 遠山 育夫
    Dementia Japan 28 4 483 - 483 (一社)日本認知症学会 2014年10月
  • Gen Tamiya, Satoshi Makino, Makiko Hayashi, Akiko Abe, Chikahiko Numakura, Masao Ueki, Atsushi Tanaka, Chizuru Ito, Kiyotaka Toshimori, Nobuhiro Ogawa, Tomoya Terashima, Hiroshi Maegawa, Daijiro Yanagisawa, Ikuo Tooyama, Masayoshi Tada, Osamu Onodera, Kiyoshi Hayasaka
    AMERICAN JOURNAL OF HUMAN GENETICS 95 3 294 - 300 2014年09月 [有り][無し]
     研究論文（学術雑誌） 
    Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy characterized by clinical and genetic heterogeneity. Although more than 30 loci harboring CMT-causing mutations have been identified, many other genes still remain to be discovered for many affected individuals. For two consanguineous families with CMT (axonal and mixed phenotypes), a parametric linkage analysis using genome-wide SNP chip identified a 4.3 Mb region on 12q24 showing a maximum multipoint LOD score of 4.23. Subsequent whole-genome sequencing study in one of the probands, followed by mutation screening in the two families, revealed a disease-specific 5 bp deletion (c.247-10_247-6de1CACTC) in a splicing element (pyrimidine tract) of intron 2 adjacent to the third exon of cytochrome c oxidase subunit VIa polypeptide 1 (COX6A1), which is a component of mitochondrial respiratory complex IV (cytochrome c oxidase [COX]), within the autozygous linkage region. Functional analysis showed that expression of COX6A1 in peripheral white blood cells from the affected individuals and COX activity in their EB-virus-transformed lymphoblastoid cell lines were significantly reduced. In addition, Cox6a1-null mice showed significantly reduced COX activity and neurogenic muscular atrophy leading to a difficulty in walking. Those data indicated that COX6A1 mutation causes the autosomal-recessive axonal or mixed CMT.
  • Sameh Magdeldin, Tadashi Yamamoto, Ikuo Tooyama, Essam M. Abdelalim
    STEM CELL REVIEWS AND REPORTS 10 4 561 - 572 2014年08月 [有り][無し]
     研究論文（学術雑誌） 
    Embryonic stem cells (ESCs) have the ability to self-renew indefinitely and they can give unlimited source of cells and tissues for cellular therapies. Recently, the natriuretic peptide receptor A (NPR-A) has been recognized as an important regulator for the self-renewal of ESCs. To gain insights into possible novel mechanisms involved in NPR-A pathway that presumably regulates self-renewal and survival of ESCs, we utilized a comprehensive label-free proteomics technology in our study. Targeting of NPR-A gene with small interfering RNA (siRNA) resulted in the inhibition of ESCs self-renewal. Coherently, quantitative label-free shotgun proteomic analysis identified differentially expressed proteins involved in several biological processes, including cell cycle regulation, cell proliferation, cell fate specification, and apoptosis. Interestingly, in addition to Oct4 Nanog, and Sox2, other proteins involved in ESCs self-renewal were down-regulated after NPR-A knockdown, such as heterogeneous nuclear ribonucleoprotein A2/B1 (ROA2), non-POU domain-containing octamer-binding protein (Nono), nucleoplasmin (Npm1), histone H2A type 1-B/E (histone H2A.2), SW1/SNF complex (Brg1), polycomb protein Suz12 (Suz12), and cyclin-dependent kinase 4 (Cdk4). Furthermore, several protein candidates involved in early differentiation and cell death were up-regulated or down-regulated as a result of NPR-A knockdown, including importin subunit alpha-4 (Imp alpha 4), importin-5 (Ipo5), H3 histones, core histone macro-H2A.1 (H2A.y), apurine/apyrimidine endonuclease 1 (Apex1), 78-kDa glucose-regulated protein (Grp78), and programmed cell death 5 (Pdcd5). Overall, these findings depict a comprehensive view to our understanding of the pathways involved in the role of NPR-A in maintaining ESC functions.
  • Masanao Miwa, Gyokukou You, Hideaki Tanaka, Seigo Taniguchi, Takahiro Fujii, Kazuo Kamemura, Masafumi Suzaki, Takahiro Isono, Ikuo Tooyama, Masakazu Tanaka, Petcharin Srivatanakul, Chutiwan Viwatthanasittiphong, Suleeporn Sangrajrang, Thiravud Khuhaprema
    JOURNAL OF HEPATO-BILIARY-PANCREATIC SCIENCES 21 6 397 - 398 2014年06月 [無し][無し]
     研究論文（学術雑誌） 
    Cholangiocarcinoma is one of the most serious diseases in northeast Thailand, where its incidence is reported to be the highest in the world. We tried to develop a new method to detect cholangiocarcinoma in the early stages using serum proteins. We found that after fluorescent labeling of the sugar moiety of serum proteins, a new peak was identified, which might be a promising marker for cholangiocarcinoma.
  • Hasegawa H, Liu L, Tooyama I, Murayama S, Nishimura M
    Nat Commun 5 3917  2014年06月 [有り][無し]
     研究論文（学術雑誌） 
    Accumulation of amyloid-beta peptide (A beta) in the brain underlies the pathogenesis of Alzheimer's disease (AD). A beta is produced by beta- and gamma-secretase-mediated sequential proteolysis of amyloid-beta precursor protein (APP). Here we identify a secretory protein named interleukin-like epithelial-mesenchymal transition inducer (ILEI, also known as FAM3 superfamily member C) as a negative regulator of A beta production. ILEI destabilizes the beta-secretase-cleaved APP carboxy-terminal fragment, the penultimate precursor of A beta, by binding to the gamma-secretase complex and interfering with its chaperone properties. Notch signalling and gamma-secretase activity are not affected by ILEI. We also show neuronal expression of ILEI and its induction by transforming growth factor-beta signalling. The level of secreted ILEI is markedly decreased in the brains of AD patients. Transgenic (Tg) overexpression of ILEI significantly reduces the brain A beta burden and ameliorates the memory deficit in AD model mice. ILEI may be a plausible target for the development of disease-modifying therapies.
  • Masanao Miwa, Gyokukou You, Hideaki Tanaka, Seigo Taniguchi, Takahiro Fujii, Kazuo Kamemura, Masafumi Suzaki, Takahiro Isono, Ikuo Tooyama, Masakazu Tanaka, Petcharin Srivatanakul, Chutiwan Viwatthanasittiphong, Suleeporn Sangrajrang, Thiravud Khuhaprema
    JOURNAL OF HEPATO-BILIARY-PANCREATIC SCIENCES 21 6 397 - 398 2014年06月 [無し][無し]
     研究論文（学術雑誌） 
    Cholangiocarcinoma is one of the most serious diseases in northeast Thailand, where its incidence is reported to be the highest in the world. We tried to develop a new method to detect cholangiocarcinoma in the early stages using serum proteins. We found that after fluorescent labeling of the sugar moiety of serum proteins, a new peak was identified, which might be a promising marker for cholangiocarcinoma.
  • 高磁場MR装置を利用したフッ素MRIアミロイドイメージングのためのstyrylbenzoxazole誘導体の開発
    柳沢 大治郎, 田口 弘康, 森川 茂廣, 椎野 顯彦, 犬伏 俊郎, 遠山 育夫
    JSMI Report 7 2 81 - 81 日本分子イメージング学会 2014年05月
  • 椎野 顯彦, 渡辺 俊之, 白樫 義知, 山田 篤史, 谷 徹, 遠山 育夫
    28 3 339 - 347 2014年 [有り][無し]
     研究論文（学術雑誌）
  • Daijiro Yanagisawa, Hiroyasu Taguchi, Nor Faeizah Ibrahim, Shigehiro Morikawa, Akihiko Shiino, Toshiro Inubushi, Koichi Hirao, Nobuaki Shirai, Takayuki Sogabe, Ikuo Tooyama
    JOURNAL OF ALZHEIMERS DISEASE 39 3 617 - 631 2014年 [無し][無し]
     研究論文（学術雑誌） 
    Fluorine-19 magnetic resonance imaging (F-19 MRI) could be a promising approach for imaging amyloid deposition in the brain. However, the required features of a F-19 MRI probe for amyloid detection remain unclear. In the present study, we investigated a series of compounds as potent F-19 probes that could prevent the reduction in MR signal when bound to amyloid plaques in the brain. Each compound consists of styrylbenzoxazole as a core structure linked by a different length of polyethylene glycol (PEG) chain to one of three types of fluorine-labeled group: a trifluoroethoxy group, a hexafluoroisopropoxy group, or a 3',5'-bis(trifluoromethyl) benzylamino group. Among these compounds, 6-(3',6',9',15',18',21'-heptaoxa-23',23',23'-trifluorotricosanyloxy)-2-(4'-dimethylaminostyryl)benzoxazole [compound 3b (m = 6)], which has a trifluoroethoxy group with seven ethylene glycol groups in the PEG chain, showed significant F-19 MR signals in the brains of A beta PPswe/PS1dE9 double-transgenic mice, but not wild-type mice. This suggested that compound 3b (m = 6) could be a useful F-19 MRI probe for amyloid detection. Furthermore, this study identified the most effective length of PEG chain between the fluorine-labeled group and the core structure to ensure a strong MR signal when the probe is bound to amyloid plaques.
  • Essam Mohamed Abdelalim, Ikuo Tooyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 443 2 652 - 657 2014年01月 [無し][無し]
     研究論文（学術雑誌） 
    Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.
  • Daijiro Yanagisawa, Hiroyasu Taguchi, Nor Faeizah Ibrahim, Shigehiro Morikawa, Akihiko Shiino, Toshiro Inubushi, Koichi Hirao, Nobuaki Shirai, Takayuki Sogabe, Ikuo Tooyama
    JOURNAL OF ALZHEIMERS DISEASE 39 3 617 - 631 2014年 [無し][無し]
     研究論文（学術雑誌） 
    Fluorine-19 magnetic resonance imaging (F-19 MRI) could be a promising approach for imaging amyloid deposition in the brain. However, the required features of a F-19 MRI probe for amyloid detection remain unclear. In the present study, we investigated a series of compounds as potent F-19 probes that could prevent the reduction in MR signal when bound to amyloid plaques in the brain. Each compound consists of styrylbenzoxazole as a core structure linked by a different length of polyethylene glycol (PEG) chain to one of three types of fluorine-labeled group: a trifluoroethoxy group, a hexafluoroisopropoxy group, or a 3',5'-bis(trifluoromethyl) benzylamino group. Among these compounds, 6-(3',6',9',15',18',21'-heptaoxa-23',23',23'-trifluorotricosanyloxy)-2-(4'-dimethylaminostyryl)benzoxazole [compound 3b (m = 6)], which has a trifluoroethoxy group with seven ethylene glycol groups in the PEG chain, showed significant F-19 MR signals in the brains of A beta PPswe/PS1dE9 double-transgenic mice, but not wild-type mice. This suggested that compound 3b (m = 6) could be a useful F-19 MRI probe for amyloid detection. Furthermore, this study identified the most effective length of PEG chain between the fluorine-labeled group and the core structure to ensure a strong MR signal when the probe is bound to amyloid plaques.
  • Essam Mohamed Abdelalim, Ikuo Tooyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 443 2 652 - 657 2014年01月 [無し][無し]
     研究論文（学術雑誌） 
    Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression. (C) 2013 The Authors. Published by Elsevier Inc. All rights reserved.
  • Chiaki Masuda, Shigeko Takeuchi, Naomi J. Bisem, Steven R. Vincent, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 47 2 75 - 83 2014年 [有り][無し]
     研究論文（学術雑誌） 
    The TRK-fused gene (TFG) was originally identified in chromosome translocation events, creating a pair of oncogenes in some cancers, and was recently demonstrated as the causal gene of hereditary motor and sensory neuropathy with proximal dominant involvement. Recently, we cloned an alternative splicing variant of Tfg from a cDNA library of the rat retina, tentatively naming it retinal Tfg (rTfg). Although the common form of Tfg is ubiquitously expressed in most rat tissues, rTfg expression is localized to the central nervous system. In this study, we produced an antibody against an rTFG-specific amino acid sequence and used it to examine the localization of rTFG-like protein in the rat retina by imnnunohistochennistry and Western blots. Western blot analysis showed that the antibody detected a single band of 24 kDa in the rat retina. When we examined rTFG recombinant protein, the antibody detected two bands of about 42 kDa and 24 kDa. The results suggest that the 24 kDa rTFG-like protein is a fragment of rTFG. In our immunohistochemical studies of the rat retina, rTFG-like immunoreactivity was observed in all calbindin D-28K-positive horizontal cells and in some syntaxin 1-positive amacrine cells (ACs). In addition, the rTFGlike innmunopositive ACs were actually glycine transporter 1-positive glycinergic or glutamate decarboxylase-positive GABAergic ACs. Our findings indicate that this novel 24 kDa rTFGlike protein may play a specific role in retinal inhibitory interneurons.
  • アルツハイマー病遺伝子改変マウスの鼻粘膜Aβ42量は脳内Aβ42の蓄積量に比例する
    遠山 育夫, 亀島 直子, 南條 俊文, 福原 崇臣, 柳沢 大治郎
    臨床神経学 53 12 1464 - 1464 (一社)日本神経学会 2013年12月
  • モデル動物を用いた脳アミロイド蓄積と鼻粘膜Aβとの関連性の評価
    亀島 直子, 南條 俊文, 福原 崇臣, 柳沢 大治郎, 遠山 育夫
    Dementia Japan 27 4 478 - 478 (一社)日本認知症学会 2013年10月
  • Shogo Higaki, Yoshie Koyama, Manami Shimada, Yuriko Ono, Ikuo Tooyama, Yasuhiro Fujioka, Noriyoshi Sakai, Toshitaka Ikeuchi, Tatsuyuki Takada
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 191 65 - 73 2013年09月 [無し][無し]
     研究論文（学術雑誌） 
    Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17 alpha,20 beta-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish. (C) 2013 Elsevier Inc. All rights reserved.
  • Hongkuan Yang, Mingchun Yang, Hongpeng Guan, Ziyi Liu, Shiguang Zhao, Shigeko Takeuchi, Daijiro Yanagisawa, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 77 1-2 1 - 7 2013年09月 [無し][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is a novel protein encoded by an intronless gene mapped to chromosome 5q23.1. Ferritin is ubiquitously expressed; however, FtMt expression is restricted to specific tissues such as the testis and the brain. The distribution pattern of FtMt suggests a functional role for this protein in the brain; however, data concerning the roles of FtMt in neurodegenerative diseases remain scarce. In the human cerebral cortex, FtMt expression was increased in Alzheimer's disease patients compared to control cases. Cultured neuroblastoma cells showed low-level expression of FtMt, which was increased by H2O2 treatment. FtMt overexpression showed a neuroprotective effect against H2O2-induced oxidative stress and A beta-induced neurotoxicity in neuroblastoma cells. FtMt expression was also detected in dopaminergic neurons in the substantia nigra and was increased in patients with restless legs syndrome, while FtMt had a protective effect against cell death in a neuroblastoma cell line model of Parkinson's disease. FtMt is involved in other neurodegenerative diseases such as age-related macular degeneration (AMD), with an FtMt gene mutation identified in AMD patients, and Friedreich's ataxia, which is caused by a deficiency in frataxin. FtMt overexpression in frataxin-deficient cells increased cell resistance to H2O2 damage. These results implicate a neuroprotective role of FtMt in neurodegenerative diseases. (C) 2013 The Authors. Published by Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Shogo Higaki, Yoshie Koyama, Manami Shimada, Yuriko Ono, Ikuo Tooyama, Yasuhiro Fujioka, Noriyoshi Sakai, Toshitaka Ikeuchi, Tatsuyuki Takada
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 191 65 - 73 2013年09月 [無し][無し]
     研究論文（学術雑誌） 
    Fish Sertoli cells play a critical role in spermatogenesis by mediating androgen and progestogen signaling. Their hormonal response, however, considerably differ among species. Therefore it would be ideal to use Sertoli cells originated from the fish of interest to investigate the effects of hormones as well as endocrine disrupting chemicals (EDCs). The aim of this study was to investigate the responses to reproductive hormones and EDCs of a Sertoli cell line that we established from an endemic cyprinid Gnathopogon caerulescens. As the Sertoli cell line expressed endogenous androgen and progestogen receptors, we were able to detect hormone responses by transfecting only a reporter vector (pGL4.36) expressing luciferase under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter into the cell line. Unlike previous reporter gene assays using fish steroid hormone receptors expressed in mammalian cell lines, luciferase activities were induced by the fish specific androgen (11-ketotestosterone) and progestogen (17 alpha,20 beta-dihydroxy-4-pregnen-3-one), but not by testosterone and progesterone, at physiologically relevant concentrations. Furthermore, we found 4-nonylphenol (NP) but not bisphenol A showed strong anti-androgenic effects, implying that NP may have direct anti-androgenic effects on fish Sertoli cells in vivo. This is the first evidence, to the best of our knowledge, of anti-androgenic effects of NP in a fish Sertoli cell line. In addition, neither NP nor BPA showed anti-progestogenic effects. These results suggest that the Sertoli cell line established from the fish of interest can be a useful in vitro tool for investigating the mechanisms of reproductive hormones and EDCs in the specific fish. (C) 2013 Elsevier Inc. All rights reserved.
  • Hongkuan Yang, Mingchun Yang, Hongpeng Guan, Ziyi Liu, Shiguang Zhao, Shigeko Takeuchi, Daijiro Yanagisawa, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 77 1-2 1 - 7 2013年09月 [無し][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (FtMt) is a novel protein encoded by an intronless gene mapped to chromosome 5q23.1. Ferritin is ubiquitously expressed; however, FtMt expression is restricted to specific tissues such as the testis and the brain. The distribution pattern of FtMt suggests a functional role for this protein in the brain; however, data concerning the roles of FtMt in neurodegenerative diseases remain scarce. In the human cerebral cortex, FtMt expression was increased in Alzheimer's disease patients compared to control cases. Cultured neuroblastoma cells showed low-level expression of FtMt, which was increased by H2O2 treatment. FtMt overexpression showed a neuroprotective effect against H2O2-induced oxidative stress and A beta-induced neurotoxicity in neuroblastoma cells. FtMt expression was also detected in dopaminergic neurons in the substantia nigra and was increased in patients with restless legs syndrome, while FtMt had a protective effect against cell death in a neuroblastoma cell line model of Parkinson's disease. FtMt is involved in other neurodegenerative diseases such as age-related macular degeneration (AMD), with an FtMt gene mutation identified in AMD patients, and Friedreich's ataxia, which is caused by a deficiency in frataxin. FtMt overexpression in frataxin-deficient cells increased cell resistance to H2O2 damage. These results implicate a neuroprotective role of FtMt in neurodegenerative diseases. (C) 2013 The Authors. Published by Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Naoko Kameshima, Daijiro Yanagisawa, Ikuo Tooyama
    Neuroscience Research 76 3 169 - 172 2013年07月 [無し][無し]
     研究論文（学術雑誌） 
    We examined the distribution patterns of human β-amyloid (1-40) peptide labeled with iodine 125 (125I-Aβ40) after injections into the cerebral ventricle or tail vein of rats. In rats receiving an intravenous injection, the radioactive concentration of 125I-Aβ40 in the nasal area was similar to other extracranial organs. In contrast, the caudal part of the nasal area in rats receiving an intracerebroventricular injection displayed a high level of 125I-Aβ40 radioactivity. These results suggest that Aβ reaches the nasal cavity from the brain via a non-blood pathway. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Eric M. Yezdimer, Tomohiro Umemoto, Hiroshi Yamada, Satoshi Makino, Ikuo Tooyama
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 170 5 1138 - 1150 2013年07月 [無し][無し]
     研究論文（学術雑誌） 
    The potential utility of an imaging agent for the detection of hepatic copper was investigated in a Wilson's disease animal model. Solid-phase peptide synthesis was used to construct an imaging agent which consisted of a copper-binding moiety, taken from the prion protein, and a gamma ray-emitting indium radiolabel. Long-Evans Cinnamon (LEC) rats were used for the Wilson's disease animal model. Our evaluation methodology consisted of administering the indium-labeled agent to both LEC and genetically healthy Long-Evans (LE) cohorts via a tail vein injection and following the pharmacokinetics with single-photon emission computed tomography (SPECT) over the course of an hour. The animals were then sacrificed and their livers necropsied. An additional control agent, lacking the copper-binding moiety, was used to gauge whether any change in the hepatic uptake might be caused by other physiological differences between the two animal models. LEC rats injected with the indium-labeled agent had roughly double the amount of hepatic radioactivity as compared to the healthy control animals. The control agent, without the copper-binding moiety, displayed a hepatic signal similar to that of the control LE animals. Additional intraperitoneal spiking with CuSO4 in C57BL/6 mice also found that the pharmacokinetics of the indium-labeled, prion-based imaging agent is profoundly altered by exposure to in vivo pools of extracellular copper. The described SPECT application with this compound represented a significant improvement over a previous MRI application using the same base peptide sequence.
  • Kameshima N, Yanagisawa D, Tooyama I
    Neurosci Res 76 3 169 - 172 2013年07月 [無し][無し]
     研究論文（学術雑誌） 
    We examined the distribution patterns of human β-amyloid (1-40) peptide labeled with iodine 125 (125I-Aβ40) after injections into the cerebral ventricle or tail vein of rats. In rats receiving an intravenous injection, the radioactive concentration of 125I-Aβ40 in the nasal area was similar to other extracranial organs. In contrast, the caudal part of the nasal area in rats receiving an intracerebroventricular injection displayed a high level of 125I-Aβ40 radioactivity. These results suggest that Aβ reaches the nasal cavity from the brain via a non-blood pathway. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society.
  • Eric M. Yezdimer, Tomohiro Umemoto, Hiroshi Yamada, Satoshi Makino, Ikuo Tooyama
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 170 5 1138 - 1150 2013年07月 [無し][無し]
     研究論文（学術雑誌） 
    The potential utility of an imaging agent for the detection of hepatic copper was investigated in a Wilson's disease animal model. Solid-phase peptide synthesis was used to construct an imaging agent which consisted of a copper-binding moiety, taken from the prion protein, and a gamma ray-emitting indium radiolabel. Long-Evans Cinnamon (LEC) rats were used for the Wilson's disease animal model. Our evaluation methodology consisted of administering the indium-labeled agent to both LEC and genetically healthy Long-Evans (LE) cohorts via a tail vein injection and following the pharmacokinetics with single-photon emission computed tomography (SPECT) over the course of an hour. The animals were then sacrificed and their livers necropsied. An additional control agent, lacking the copper-binding moiety, was used to gauge whether any change in the hepatic uptake might be caused by other physiological differences between the two animal models. LEC rats injected with the indium-labeled agent had roughly double the amount of hepatic radioactivity as compared to the healthy control animals. The control agent, without the copper-binding moiety, displayed a hepatic signal similar to that of the control LE animals. Additional intraperitoneal spiking with CuSO4 in C57BL/6 mice also found that the pharmacokinetics of the indium-labeled, prion-based imaging agent is profoundly altered by exposure to in vivo pools of extracellular copper. The described SPECT application with this compound represented a significant improvement over a previous MRI application using the same base peptide sequence.
  • Essam M. Abdelalim, Jean-Pierre Bellier, Ikuo Tooyama
    Peptides 43 56 - 61 2013年05月 [無し][無し]
     研究論文（学術雑誌） 
    C-type natriuretic peptide (CNP) is an abundant neuropeptide in the central nervous system, which exerts its physiological effects through natriuretic peptide receptor B (NPR-B). Recently, the CNP/NPR-B system has been recognized as an important regulator for the development of sensory axons. The dorsal root ganglion (DRG) contains neurons transmitting several kinds of spinal sensory stimuli to the central nervous system. In this study, we characterized NPR-B receptor expression in the rat DRG, using reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. Immunostaining revealed that NPR-B was expressed in neuronal cell bodies and processes of the DRG, with NPR-B immunoreactivity mainly prominent in small and medium-sized DRG neurons. Double-immunolabeling showed that NPR-B was expressed in calcitonin gene-related peptide- and isolectin B4-positive neurons. Furthermore, NPR-B expression was co-localized with calcitonin gene-related peptide in the dorsal horn of the spinal cord. Together, our data suggest that the natriuretic peptides may perform several biological actions on sensory neurons via their binding to NPR-B in the DRG. © 2013 Elsevier Inc.
  • Essam M. Abdelalim, Jean-Pierre Bellier, Ikuo Tooyama
    Peptides 43 56 - 61 2013年05月 [無し][無し]
     研究論文（学術雑誌） 
    C-type natriuretic peptide (CNP) is an abundant neuropeptide in the central nervous system, which exerts its physiological effects through natriuretic peptide receptor B (NPR-B). Recently, the CNP/NPR-B system has been recognized as an important regulator for the development of sensory axons. The dorsal root ganglion (DRG) contains neurons transmitting several kinds of spinal sensory stimuli to the central nervous system. In this study, we characterized NPR-B receptor expression in the rat DRG, using reverse transcription-polymerase chain reaction, Western blotting and immunohistochemistry. Immunostaining revealed that NPR-B was expressed in neuronal cell bodies and processes of the DRG, with NPR-B immunoreactivity mainly prominent in small and medium-sized DRG neurons. Double-immunolabeling showed that NPR-B was expressed in calcitonin gene-related peptide- and isolectin B4-positive neurons. Furthermore, NPR-B expression was co-localized with calcitonin gene-related peptide in the dorsal horn of the spinal cord. Together, our data suggest that the natriuretic peptides may perform several biological actions on sensory neurons via their binding to NPR-B in the DRG. © 2013 Elsevier Inc.
  • Generation of a monoclonal antibody specifically recognizing with neuron-specific TATA-box binding protein-associated factor 1 (TAF-1).
    Makino S, Masuda C, Ando S, Tamiya G, Tooyama I
    Antibodies 2 1 - 8 2013年 [無し][無し]
     研究論文（学術雑誌）
  • Tsuneyuki Koga, Jean-Pierre Bellier, Hiroshi Kimura, Ikuo Tooyama
    Acta Histochemica et Cytochemica 46 2 59 - 64 2013年 [無し][無し]
     研究論文（学術雑誌） 
    Transcripts of the choline of acetyltransferase (ChAT) gene reveal a number of different splice variants including ChAT of a peripheral type (pChAT). Immunohistochemical staining of the brain using an antibody against pChAT clearly revealed peripheral cholinergic neurons, but failed to detect cholinergic neurons in the central nervous system. In rodents, pChATimmunoreactivity has been detected in cholinergic parasympathetic postganglionic and enteric ganglion neurons. In addition, pChAT has been observed in non-cholinergic neurons such as peripheral sensory neurons in the trigeminal and dorsal root ganglia. The common type of ChAT (cChAT) has been investigated in many parts of the brain and the spinal cord of non-human primates, but little information is available about the localization of pChAT in primate species. Here, we report the detection of pChAT immunoreactivity in trigeminal ganglion (TG) neurons and its co-localization with Substance P (SP) and/or calcitonin generelated peptide (CGRP) in the cynomolgus monkey, Macaca fascicularis. Neurons positive for pChAT were observed in a rather uniform pattern in approximately half of the trigeminal neurons throughout the TG. Most pChAT-positive neurons had small or medium-sized cell bodies. Double-immunofluorescence staining showed that 85.1% of SP-positive cells and 74.0% of CGRP-positive cells exhibited pChAT immunoreactivity. Most pChAT-positive cells were part of a larger population of neurons that co-expressed SP and/or CGRP. © 2013 The Japan Society of Histochemistry and Cytochemistry.
  • Shigeko Takeuchi, Wakoto Matsuda, Ikuo Tooyama, Osamu Yasuhara
    Folia Histochemica et Cytobiologica 51 1 25 - 30 2013年 [無し][無し]
     研究論文（学術雑誌） 
    Caveolin-1, a major constituent of caveolae, has been implicated in endocytosis, signal transduction and cholesterol transport in a wide variety of cells. In the present study, the expression of caveolin-1 was examined by immunohistochemistry in rat brain with or without systemic injection of kainic acid (KA). Caveolin-1 immunoreactivity was observed in capillary walls in brains of control rats. From one to seven days after KA injection, caveolin-1 immunoreactivity appeared in activated microglia in the cerebral cortex, hippocampus and other brain regions. The strongest immunoreactivity of microglia was seen after 3 days after KA administration. The expression of caveolin-1 was confirmed by RT-PCR and Western blot analysis, respectively. The induction of caveolin-1 expression in microglia activated in response to kainic acid administration suggests its possible role in a modulation of inflammation. © Polish Society for Histochemistry and Cytochemistry Folia Histochem Cytobiol. 2013.
  • Tsuyoshi Kitanishi, Yoshinari Aimi, Hiroya Kitano, Mikio Suzuki, Hiroshi Kimura, Atsushi Saito, Takeshi Shimizu, Ikuo Tooyama
    Acta Histochemica et Cytochemica 46 5 145 - 152 2013年 [無し][無し]
     研究論文（学術雑誌） 
    We previously discovered a splice variant of choline acetyltransferase (ChAT) mRNA, and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, we examined the immunohistochemical localization of pChAT in rat cochlea and compared the distribution pattern to those of common ChAT (cChAT) and acetylcholinesterase. Some neuronal cell bodies and fibers in the spiral ganglia showed immunoreactivity for pChAT, predominantly the small spiral ganglion cells, indicating outer hair cell type II neurons. In contrast, cChAT- and acetylcholinesterase-positive structures were localized to fibers and not apparent in ganglion cells. After ablation of the cochlear nuclei, many pChAT-positive cochlear nerve fibers became clearly visible, whereas fibers immunopositive for cChAT and acetylcholine esterase disappeared. These results suggested that pChAT and cChAT are localized in different systems of the rat cochlea pChAT in the afferent and cChAT in the efferent structures. © 2013 The Japan Society of Histochemistry and Cytochemistry.
  • Generation of a monoclonal antibody specifically recognizing with neuron-specific TATA-box binding protein-associated factor 1 (TAF-1).
    Makino S, Masuda C, Ando S, Tamiya G, Tooyama I
    Antibodies 2 1 - 8 2013年 [無し][無し]
     研究論文（学術雑誌）
  • Tsuneyuki Koga, Jean-Pierre Bellier, Hiroshi Kimura, Ikuo Tooyama
    Acta Histochemica et Cytochemica 46 2 59 - 64 2013年 [無し][無し]
     研究論文（学術雑誌） 
    Transcripts of the choline of acetyltransferase (ChAT) gene reveal a number of different splice variants including ChAT of a peripheral type (pChAT). Immunohistochemical staining of the brain using an antibody against pChAT clearly revealed peripheral cholinergic neurons, but failed to detect cholinergic neurons in the central nervous system. In rodents, pChATimmunoreactivity has been detected in cholinergic parasympathetic postganglionic and enteric ganglion neurons. In addition, pChAT has been observed in non-cholinergic neurons such as peripheral sensory neurons in the trigeminal and dorsal root ganglia. The common type of ChAT (cChAT) has been investigated in many parts of the brain and the spinal cord of non-human primates, but little information is available about the localization of pChAT in primate species. Here, we report the detection of pChAT immunoreactivity in trigeminal ganglion (TG) neurons and its co-localization with Substance P (SP) and/or calcitonin generelated peptide (CGRP) in the cynomolgus monkey, Macaca fascicularis. Neurons positive for pChAT were observed in a rather uniform pattern in approximately half of the trigeminal neurons throughout the TG. Most pChAT-positive neurons had small or medium-sized cell bodies. Double-immunofluorescence staining showed that 85.1% of SP-positive cells and 74.0% of CGRP-positive cells exhibited pChAT immunoreactivity. Most pChAT-positive cells were part of a larger population of neurons that co-expressed SP and/or CGRP. © 2013 The Japan Society of Histochemistry and Cytochemistry.
  • Shigeko Takeuchi, Wakoto Matsuda, Ikuo Tooyama, Osamu Yasuhara
    FOLIA HISTOCHEMICA ET CYTOBIOLOGICA 51 1 25 - 30 2013年 [無し][無し]
     研究論文（学術雑誌） 
    Caveolin-1, a major constituent of caveolae, has been implicated in endocytosis, signal transduction and cholesterol transport in a wide variety of cells. In the present study, the expression of caveolin-1 was examined by immunohistochemistry in rat brain with or without systemic injection of kainic acid (KA). Caveolin-1 immunoreactivity was observed in capillary walls in brains of control rats. From one to seven days after KA injection, caveolin-1 immunoreactivity appeared in activated microglia in the cerebral cortex, hippocampus and other brain regions. The strongest immunoreactivity of microglia was seen after 3 days after KA administration. The expression of caveolin-1 was confirmed by RT-PCR and Western blot analysis, respectively. The induction of caveolin-1 expression in microglia activated in response to kainic acid administration suggests its possible role in a modulation of inflammation.
  • Kitanishi T, Aimi Y, Kitano H, Suzuki M, Kimura H, Saito A, Shimizu T, Tooyama I
    Acta Histochem Cytochem 46 5 142 - 145 JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 2013年 [無し][無し]
     研究論文（学術雑誌） 
    We previously discovered a splice variant of choline acetyltransferase (ChAT) mRNA, and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, we examined the immunohistochemical localization of pChAT in rat cochlea and compared the distribution pattern to those of common ChAT (cChAT) and acetylcholinesterase. Some neuronal cell bodies and fibers in the spiral ganglia showed immunoreactivity for pChAT, predominantly the small spiral ganglion cells, indicating outer hair cell type II neurons. In contrast, cChAT- and acetylcholinesterase-positive structures were localized to fibers and not apparent in ganglion cells. After ablation of the cochlear nuclei, many pChAT-positive cochlear nerve fibers became clearly visible, whereas fibers immunopositive for cChAT and acetylcholine esterase disappeared. These results suggested that pChAT and cChAT are localized in different systems of the rat cochlea; pChAT in the afferent and cChAT in the efferent structures.
  • クルクミン骨格をもつ高磁場MR用アミロイドイメージング試薬
    遠山 育夫, 柳沢 大治郎, 田口 弘康, 森川 茂廣, 椎野 顕彦
    臨床神経学 52 12 1480 - 1480 (一社)日本神経学会 2012年12月
  • フッ素MRIを用いたアルツハイマー病モデルマウスにおける脳内アミロイド斑の検出
    柳沢 大治郎, 椎野 顯彦, 田口 弘康, 森川 茂廣, 犬伏 俊郎, 遠山 育夫
    脳循環代謝 24 1 131 - 131 (一社)日本脳循環代謝学会 2012年11月
  • Masaki Nishimura, Shin-ichiro Nakamura, Nobuyuki Kimura, Lei Liu, Toshiharu Suzuki, Ikuo Tooyama
    JOURNAL OF NEUROCHEMISTRY 123 1 21 - 28 2012年10月 [無し][無し]
     研究論文（学術雑誌） 
    Age-dependent accumulation of the amyloid-beta peptide (A beta) in the brain is a pre-condition for development of Alzheimers disease. A relative increase in the generation of longer A beta species such as A beta 42 and A beta 43 is critical for A beta deposition, but the underlying mechanism remains unresolved. Here, we performed a cell-free assay using microsome fractions of temporal cortex tissues from 42 cynomolgus monkeys and found that A beta 40-generating ?-secretase activity (?40) decreased with age, whereas A beta 42-generating ?-secretase activity (?42) was unaltered. In ELISAs, more than 80% of monkeys over 20-years old showed evidence of A beta accumulation in the temporal cortex. The ratio of ?42 to ?40 increased with age and correlated with the level of accumulated A beta. These results suggest that ?-secretase activity undergoes age-related, non-genetic modulation and that this modulation may cause A beta accumulation in aging brains. Similar modulation may predispose aged human brains to Alzheimers disease.
  • Satoshi Makino, Tomohiro Umemoto, Hiroshi Yamada, Eric M. Yezdimer, Ikuo Tooyama
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 168 3 504 - 518 2012年10月 [無し][無し]
     研究論文（学術雑誌） 
    Abnormal distributions of transition metals inside the body are potential diagnostic markers for several diseases, including Alzheimer's disease, Parkinson's disease, Wilson's disease, and cancer. In this article, we demonstrate that P57/Gd, a novel prion-based contrast agent, can selectively image tissues with excessive copper accumulation using magnetic resonance imaging (MRI). P57/Gd selectivity binds copper(II) over other physiologically relevant cations such as zinc, iron, manganese, and calcium. To simulate a metabolic copper disorder, we treated mice with an intraperitoneal injection of a CuSO4 solution to induce a renal copper overload. The MRI signal intensities from the renal cortex and medulla of copper spiked animals that were administered P57/Gd were found to correlate with the ex vivo copper concentrations determined by inductively coupled plasma mass spectrometry.
  • Nishimura M, Nakamura S, Kimura N, Liu L, Suzuki T, Tooyama I
    J Neurochem 123 1 21 - 28 2012年10月 [無し][無し]
     研究論文（学術雑誌） 
    Age-dependent accumulation of the amyloid-beta peptide (A beta) in the brain is a pre-condition for development of Alzheimers disease. A relative increase in the generation of longer A beta species such as A beta 42 and A beta 43 is critical for A beta deposition, but the underlying mechanism remains unresolved. Here, we performed a cell-free assay using microsome fractions of temporal cortex tissues from 42 cynomolgus monkeys and found that A beta 40-generating ?-secretase activity (?40) decreased with age, whereas A beta 42-generating ?-secretase activity (?42) was unaltered. In ELISAs, more than 80% of monkeys over 20-years old showed evidence of A beta accumulation in the temporal cortex. The ratio of ?42 to ?40 increased with age and correlated with the level of accumulated A beta. These results suggest that ?-secretase activity undergoes age-related, non-genetic modulation and that this modulation may cause A beta accumulation in aging brains. Similar modulation may predispose aged human brains to Alzheimers disease.
  • Satoshi Makino, Tomohiro Umemoto, Hiroshi Yamada, Eric M. Yezdimer, Ikuo Tooyama
    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 168 3 504 - 518 2012年10月 [無し][無し]
     研究論文（学術雑誌） 
    Abnormal distributions of transition metals inside the body are potential diagnostic markers for several diseases, including Alzheimer's disease, Parkinson's disease, Wilson's disease, and cancer. In this article, we demonstrate that P57/Gd, a novel prion-based contrast agent, can selectively image tissues with excessive copper accumulation using magnetic resonance imaging (MRI). P57/Gd selectivity binds copper(II) over other physiologically relevant cations such as zinc, iron, manganese, and calcium. To simulate a metabolic copper disorder, we treated mice with an intraperitoneal injection of a CuSO4 solution to induce a renal copper overload. The MRI signal intensities from the renal cortex and medulla of copper spiked animals that were administered P57/Gd were found to correlate with the ex vivo copper concentrations determined by inductively coupled plasma mass spectrometry.
  • Akihiko Shiino, Ichiro Akiguchi, Toshiyuki Watanabe, Yoshitomo Shirakashi, Kazuhiko Nozaki, Ikuo Tooyama, Toshiro Inubushi
    EUROPEAN JOURNAL OF RADIOLOGY 81 9 2375 - 2379 2012年09月 [無し][無し]
     研究論文（学術雑誌） 
    Background and purpose: Dementia due to hypertensive vascular disease is a potential target to treat prophylactively before it progresses insidiously. Binswanger's disease (BD) is a type of subcortical vascular dementia, but its clinical features and pathophysiology are still obscure. We therefore tried to find a topographic distribution of brain atrophy in BD by morphometric analysis. Methods: Twenty patients with BD, 50 patients with AD, and 80 elderly controls were recruited. We contrasted the gray matter atrophy of BD to that of AD to identify a pathognomic pattern using magnetic resonance imaging. We used DARTEL (diffeomorphic anatomical registration through exponential Lie algebra) for voxel-based morphometry, expecting that its sophisticated algorithm would work well to deal with the subjects with brain atrophy. Results: Atrophy of cortices was predominant in the posterior cortices in AD but was in the anterior cortices in BD. Atrophy of amygdala and hippocampus was similar in each disease. In contrast, thalamus, caudate nucleus, insula, anterior cingulate cortex, and frontal cortices were significantly more atrophied in BD than in AD (z-score > 3). Conclusions: We demonstrated topographic patterns of brain atrophy in BD. Since affected regions of BD match with the anatomical connections of frontal-subcortical circuits, it seems reasonable to suppose that BD pathology is the result of hypertensive vascular disease and subsequent regression from the white matter injuries. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • 脳 高磁場MRIを用いた脳内アミロイドベータ検出法の開発
    柳沢 大治郎, 森川 茂廣, 田口 弘康, 犬伏 俊郎, 遠山 育夫
    JSMI Report 5 2 36 - 36 日本分子イメージング学会 2012年05月
  • 19F MRIによるアルツハイマー病モデルマウスの脳内アミロイド蓄積の検出
    柳沢 大治郎, 森川 茂廣, 田口 弘康, 犬伏 俊郎, 遠山 育夫
    JSMI Report 5 2 123 - 123 日本分子イメージング学会 2012年05月
  • Masatoshi Inden, Mari Abe, Hideaki Minamino, Kazuyuki Takata, Kanji Yoshimoto, Ikuo Tooyama, Yoshihisa Kitamura
    JOURNAL OF PHARMACOLOGICAL SCIENCES 119 1 10 - 19 2012年05月 [無し][無し]
     研究論文（学術雑誌） 
    L-Dihydroxyphenylalanine (L-DOPA) is considered the gold standard for the treatment of Parkinson's disease (PD). However, long-term administration of L-DOPA can induce abnormal side effects. On the other hand, selective serotonin reuptake inhibitors (SSRIs) including fluoxetine have gained tremendous popularity in the treatment of depression in PD. SSRIs are thought to influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic networks, which are complex and not yet fully understand. In this study, intranigral injection of 6-hydroxydopamine (6-OHDA) in rats caused a significant loss of tyrosine hydroxylase immunoreactivity in the striatum and substantia nigra. However, tryptophan hydroxylase immunoreactivity of the striatum and raphe nucleus was unaffected by 6-OHDA. Immunohistochemical analysis reveal that the serotonergic system was unaffected by the injection of 6-OHDA. We demonstrated also that pre-treatment with fluoxetine significantly suppressed L-DOPA-induced rotational behavior. Additionally, fluoxetine suppressed L-DOPA-induced ERK1/2 and histone H3 phosphorylation. These effects of fluoxetine were abolished by pre-treatment with WAY 100135, a 5-HT1A antagonist. These results suggest that fluoxetine may influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic neuronal networks.
  • Essam M. Abdelalim, Ikuo Tooyama
    STEM CELLS AND DEVELOPMENT 21 8 1264 - 1271 2012年05月 [無し][無し]
     研究論文（学術雑誌） 
    The identification of intrinsic factors required for propagation of self-renewing embryonic stem (ES) cells is important to improve the efficiency of expansion of ES cells for therapeutic purposes. Here, we report a novel role for natriuretic peptide receptor-C (NPR-C) in the survival of murine ES cells. We found that NPR-C was highly expressed in ES cells and was downregulated during ES cell differentiation. Knockdown of NPR-C in ES cells by using a small-interfering RNA resulted in apoptotic cell death, and the induction of p53 protein expression. Conversely, chemical inhibition of p53 by alpha-pifithrin significantly reduced apoptosis in NPR-C-deficient cells. cANF((4-23)), a selective NPR-C agonist, protected ES cells against oxidative stress-induced apoptosis, and blocked activation of p53 and Nanog suppression in the presence of DNA-damaging agents. Thus, NPR-C is required to control DNA damage-induced p53 levels to maintain ES cell self-renewal.
  • Masatoshi Inden, Mari Abe, Hideaki Minamino, Kazuyuki Takata, Kanji Yoshimoto, Ikuo Tooyama, Yoshihisa Kitamura
    JOURNAL OF PHARMACOLOGICAL SCIENCES 119 1 10 - 19 2012年05月 [無し][無し]
     研究論文（学術雑誌） 
    L-Dihydroxyphenylalanine (L-DOPA) is considered the gold standard for the treatment of Parkinson's disease (PD). However, long-term administration of L-DOPA can induce abnormal side effects. On the other hand, selective serotonin reuptake inhibitors (SSRIs) including fluoxetine have gained tremendous popularity in the treatment of depression in PD. SSRIs are thought to influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic networks, which are complex and not yet fully understand. In this study, intranigral injection of 6-hydroxydopamine (6-OHDA) in rats caused a significant loss of tyrosine hydroxylase immunoreactivity in the striatum and substantia nigra. However, tryptophan hydroxylase immunoreactivity of the striatum and raphe nucleus was unaffected by 6-OHDA. Immunohistochemical analysis reveal that the serotonergic system was unaffected by the injection of 6-OHDA. We demonstrated also that pre-treatment with fluoxetine significantly suppressed L-DOPA-induced rotational behavior. Additionally, fluoxetine suppressed L-DOPA-induced ERK1/2 and histone H3 phosphorylation. These effects of fluoxetine were abolished by pre-treatment with WAY 100135, a 5-HT1A antagonist. These results suggest that fluoxetine may influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic neuronal networks.
  • Essam M. Abdelalim, Ikuo Tooyama
    STEM CELLS AND DEVELOPMENT 21 8 1264 - 1271 2012年05月 [無し][無し]
     研究論文（学術雑誌） 
    The identification of intrinsic factors required for propagation of self-renewing embryonic stem (ES) cells is important to improve the efficiency of expansion of ES cells for therapeutic purposes. Here, we report a novel role for natriuretic peptide receptor-C (NPR-C) in the survival of murine ES cells. We found that NPR-C was highly expressed in ES cells and was downregulated during ES cell differentiation. Knockdown of NPR-C in ES cells by using a small-interfering RNA resulted in apoptotic cell death, and the induction of p53 protein expression. Conversely, chemical inhibition of p53 by alpha-pifithrin significantly reduced apoptosis in NPR-C-deficient cells. cANF((4-23)), a selective NPR-C agonist, protected ES cells against oxidative stress-induced apoptosis, and blocked activation of p53 and Nanog suppression in the presence of DNA-damaging agents. Thus, NPR-C is required to control DNA damage-induced p53 levels to maintain ES cell self-renewal.
  • Dongsun Park, Hong Jun Lee, Seong Soo Joo, Dae-Kwon Bae, Goeun Yang, Yun-Hui Yang, Inja Lim, Akinori Matsuo, Ikuo Tooyama, Yun-Bae Kim, Seung U. Kim
    EXPERIMENTAL NEUROLOGY 234 2 521 - 526 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model. (C) 2012 Elsevier Inc. All rights reserved.
  • Naoko Kameshima, Toshifumi Nanjou, Takaomi Fukuhara, Daijiro Yanagisawa, Ikuo Tooyama
    NEUROSCIENCE LETTERS 513 2 166 - 169 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    The deposition of beta-amyloid peptides (A beta) is commonly reported in the nasal cavity of Alzheimer's disease (AD) patients, although the pathological significance of this finding is unknown. This study compared A beta concentrations in the nasal area with those in the brain, blood, and cerebrospinal fluid, respectively. Immunohistochemical analysis identified A beta deposits in the nasal epithelium of Tg2576 mice. Enzyme-linked immunosorbent assay measurements revealed a correlation between the content of A beta 42 in the nasal area and that in the brain, but not with that in the blood. These results suggest that the highly accessible nasal cavity could be a useful site for diagnostic analysis of AD based on A beta content. (c) 2012 Elsevier Ireland Ltd. All rights reserved.
  • Hiroyasu Akatsu, Akira Hori, Takayuki Yamamoto, Mari Yoshida, Maya Mimuro, Yoshio Hashizume, Ikuo Tooyama, Eric M. Yezdimer
    BIOMETALS 25 2 337 - 350 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    Abnormal distributions of transition metals inside the brain are potential diagnostic markers for several central nervous system diseases, including Alzheimer's disease (AD), Parkinson's disease, dementia with Lewy bodies (DLB), bipolar disorders and depression. To further explore this possibility, the total concentrations of iron, zinc, copper, manganese, aluminum, chromium and cadmium were measured in post-mortem hippocampus and amygdala tissues taken from AD, DLB and Control patients. A statistically significant near fifty percent reduction in the total copper levels of AD patients was observed in both the hippocampus and amygdala. The statistical power of the hippocampus and amygdala copper analysis was found to be 86 and 74% respectively. No statistically significant deviations in the total metal concentrations were found for zinc, manganese, chromium or aluminum. Iron was found to be increased by 38% in AD amygdala tissues, but was unchanged in AD hippocampus tissues. Accounting for differences in tissue water content, as a function of both tissue type and disease state, revealed more consistencies with previous literature. To aid in the design of future experiments, the effect sizes for all tissue types and metals studied are also presented.
  • Essam Mohamed Abdelalim, Ikuo Tooyama
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 420 3 605 - 610 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-alpha, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-alpha resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-alpha caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions. (C) 2012 Elsevier Inc. All rights reserved.
  • Dongsun Park, Hong Jun Lee, Seong Soo Joo, Dae-Kwon Bae, Goeun Yang, Yun-Hui Yang, Inja Lim, Akinori Matsuo, Ikuo Tooyama, Yun-Bae Kim, Seung U. Kim
    EXPERIMENTAL NEUROLOGY 234 2 521 - 526 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    A human neural stem cell (NSC) line over-expressing human choline acetyltransferase (ChAT) gene was generated and these F3.ChAT NSCs were transplanted into the brain of rat Alzheimer disease (AD) model which was induced by application of ethylcholine mustard aziridinium ion (AF64A) that specifically denatures cholinergic nerves and thereby leads to memory deficit as a salient feature of AD. Transplantation of F3.ChAT human NSCs fully recovered the learning and memory function of AF64A animals, and induced elevated levels of acetylcholine (ACh) in cerebrospinal fluid (CSF). Transplanted F3.ChAT human NSCs were found to migrate to various brain regions including cerebral cortex, hippocampus, striatum and septum, and differentiated into neurons and astrocytes. The present study demonstrates that brain transplantation of human NSCs over-expressing ChAT ameliorates complex learning and memory deficits in AF64A-cholinotoxin-induced AD rat model. (C) 2012 Elsevier Inc. All rights reserved.
  • Kameshima N, Nanjou T, Fukuhara T, Yanagisawa D, Tooyama I
    Neurosci Lett 513 2 166 - 169 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    The deposition of beta-amyloid peptides (A beta) is commonly reported in the nasal cavity of Alzheimer's disease (AD) patients, although the pathological significance of this finding is unknown. This study compared A beta concentrations in the nasal area with those in the brain, blood, and cerebrospinal fluid, respectively. Immunohistochemical analysis identified A beta deposits in the nasal epithelium of Tg2576 mice. Enzyme-linked immunosorbent assay measurements revealed a correlation between the content of A beta 42 in the nasal area and that in the brain, but not with that in the blood. These results suggest that the highly accessible nasal cavity could be a useful site for diagnostic analysis of AD based on A beta content. (c) 2012 Elsevier Ireland Ltd. All rights reserved.
  • Hiroyasu Akatsu, Akira Hori, Takayuki Yamamoto, Mari Yoshida, Maya Mimuro, Yoshio Hashizume, Ikuo Tooyama, Eric M. Yezdimer
    BIOMETALS 25 2 337 - 350 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    Abnormal distributions of transition metals inside the brain are potential diagnostic markers for several central nervous system diseases, including Alzheimer's disease (AD), Parkinson's disease, dementia with Lewy bodies (DLB), bipolar disorders and depression. To further explore this possibility, the total concentrations of iron, zinc, copper, manganese, aluminum, chromium and cadmium were measured in post-mortem hippocampus and amygdala tissues taken from AD, DLB and Control patients. A statistically significant near fifty percent reduction in the total copper levels of AD patients was observed in both the hippocampus and amygdala. The statistical power of the hippocampus and amygdala copper analysis was found to be 86 and 74% respectively. No statistically significant deviations in the total metal concentrations were found for zinc, manganese, chromium or aluminum. Iron was found to be increased by 38% in AD amygdala tissues, but was unchanged in AD hippocampus tissues. Accounting for differences in tissue water content, as a function of both tissue type and disease state, revealed more consistencies with previous literature. To aid in the design of future experiments, the effect sizes for all tissue types and metals studied are also presented.
  • Abdelalim EM, Tooyama I
    Biochem Biophys Res Commun 420 3 605 - 610 2012年04月 [無し][無し]
     研究論文（学術雑誌） 
    Recent studies have reported the role of p53 in suppressing the pluripotency of embryonic stem (ES) cells after DNA damage and blocking the reprogramming of somatic cells into induced pluripotent stem (iPS) cells. However, to date no evidence has been presented to support the function of p53 in unstressed ES cells. In this study, we investigated the effect of pifithrin (PFT)-alpha, an inhibitor of p53-dependent transcriptional activation, on self-renewal of ES cells. Our results revealed that treatment of ES cells with PFT-alpha resulted in the inhibition of ES cell propagation in a dose-dependent manner, as indicated by a marked reduction in the cell number and colony size. Also, PFT-alpha caused a cell cycle arrest and significant reduction in DNA synthesis. In addition, inhibition of p53 activity reduced the expression levels of cyclin D1 and Nanog. These findings indicate that p53 pathway in ES cells rather than acting as an inactive gene, is required for ES cell proliferation and self-renewal under unstressful conditions. (C) 2012 Elsevier Inc. All rights reserved.
  • Shigeko Takeuchi, Chiaki Masuda, Hisae Maebayashi, Ikuo Tooyama
    Acta Histochemica et Cytochemica 45 4 240  2012年 [有り][無し]
     研究論文（学術雑誌）
  • Kato T, Tamiya G, Koyama S, Nakamura T, Makino S, Arawaka S, Kawanami T, Tooyama I
    ISRN neurology 2012 508308 - 4 2012年 [有り][無し]
     研究論文（学術雑誌） 
    The causal gene(s) for familial adult myoclonic epilepsy (FAME) remains undetermined. To identify it, an exome analysis was performed for the proband in a Japanese FAME family. Of the 383 missense/nonsense variants examined, only c.5720G>A mutation (p.Arg1907His) in the UBR5 gene was found in all of the affected individuals in the family, but not in the nonaffected members. Such mutation was not found in any of the 85 healthy individuals in the same community nor in any of the 24 individuals of various ethnicities. The present study demonstrated an FAME-associated mutation in the UBR5 gene, which is located close to the reported locus linked to Japanese FAME families.
  • Hisae Maebayashi, Shigako Takeuchi, Chiaki Masuda, Satoshi Makino, Kenji Fukui, Hiroshi Kimura, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 45 1 15 - 23 2012年 [無し][無し]
     研究論文（学術雑誌） 
    The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It has been reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. However, no information regarding the localization of Tfg in rat tissues is available. In this study, we investigated the expression of Tfg mRNA in normal rat tissues using reverse transcription-polymerase chain reaction (RT-PCR). We also produced an antibody against Tfg gene products and examined the localization of TFG in the rat brain and retina. The RTPCR experiments demonstrated that two types of Tfg mRNA were expressed in rat tissues: the conventional form of Tfg (cTfg) and a novel variant form, retinal Tfg (rTfg). RT-PCR analyses demonstrated that cTfg was ubiquitously expressed in rat tissues, while rTfg was predominantly expressed in the brain and retina. Western blot analysis demonstrated two bands with molecular weights of about 30 kDa and 50 kDa in the rat brain. Immunohistochemistry indicated that TFG proteins were predominantly expressed by neurons in the brain. In the rat retina, intense TFG-immunoreactivity was detected in the layer of rods and cones and the outer plexiform layer.
  • Shigeko Takeuchi, Chiaki Masuda, Hisae Maebayashi, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 45 1 57 - 64 2012年 [無し][無し]
     研究論文（学術雑誌） 
    The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It was since reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. As shown in the accompanying paper, we produced an antibody to rat TFG and used it to localize TFG to selected neurons in specific regions. In the present study, we mapped the TFG-positive neurons in the brainstem, cerebellum, and spinal cord of rats. In the brainstem, neurons intensely positive for TFG were distributed in the raphe nuclei, the gigantocellular reticular nucleus, the reticulotegmental nucleus of the pons, and some cranial nerve nuclei such as the trigeminal nuclei, the vestibulocochlear nuclei, and the dorsal motor nucleus of the vagus. Purkinje cells in the cerebellum and motor neurons in the spinal anterior horn were also positive for TFG. These results provide fundamental data for studying the functions of TFG in the brain.
  • Dongsun Park, Seong Soo Joo, Tae Kyun Kim, Sun Hee Lee, Hyomin Kang, Hong Jun Lee, Inja Lim, Akinori Matsuo, Ikuo Tooyama, Yun-Bae Kim, Seung U. Kim
    CELL TRANSPLANTATION 21 1 365 - 371 2012年 [無し][無し]
     研究論文（学術雑誌） 
    Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.
  • Naomi J. Bisem, Shigeko Takeuchi, Toru Imamura, Essam M. Abdelalim, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 45 6 325 - 334 2012年 [無し][無し]
     研究論文（学術雑誌） 
    FGF1 is highly expressed in neurons and it has been proposed to play a role in the neuroprotection and in regeneration. Low FGF1 expression in neurons has been linked to increased vulnerability in cholinergic neurons. Previous reports have shown that the expression of FGF1 in rat brain is localized to the cholinergic nuclei of the medulla oblongata, with low ratio of neurons positive for FGF1 in the dorsal motor nucleus of the vagus (DMNV). The role of FGF1 in the primate brain has yet to be clarified. In this study, we mapped FGF1 immunoreactivity in the medulla oblongata of cynomolgus monkey brainstems. Our results demonstrated that FGF1 immunoreactivity follows the pattern of distribution of cholinergic nuclei in the medulla oblongata; with strong localization of FGF1 to cholinergic neurons of the hypoglossal nucleus, the facial nucleus and the nucleus ambiguus. In contrast, the DMNV shows markedly lower FGF1 immunoreactivity. Localization of FGF1 to cholinergic neurons was only observed in the lateral region of the DMNV, with higher immunoreactivity in the rostral ventral-lateral region of the DMNV. These findings are consistent with the distribution of FGF1 immunoreactivity in previous studies of the rat brain.
  • Hisae Maebayashi, Shigako Takeuchi, Chiaki Masuda, Satoshi Makino, Kenji Fukui, Hiroshi Kimura, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 45 1 15 - 23 2012年 [無し][無し]
     研究論文（学術雑誌） 
    The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It has been reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. However, no information regarding the localization of Tfg in rat tissues is available. In this study, we investigated the expression of Tfg mRNA in normal rat tissues using reverse transcription-polymerase chain reaction (RT-PCR). We also produced an antibody against Tfg gene products and examined the localization of TFG in the rat brain and retina. The RTPCR experiments demonstrated that two types of Tfg mRNA were expressed in rat tissues: the conventional form of Tfg (cTfg) and a novel variant form, retinal Tfg (rTfg). RT-PCR analyses demonstrated that cTfg was ubiquitously expressed in rat tissues, while rTfg was predominantly expressed in the brain and retina. Western blot analysis demonstrated two bands with molecular weights of about 30 kDa and 50 kDa in the rat brain. Immunohistochemistry indicated that TFG proteins were predominantly expressed by neurons in the brain. In the rat retina, intense TFG-immunoreactivity was detected in the layer of rods and cones and the outer plexiform layer.
  • Shigeko Takeuchi, Chiaki Masuda, Hisae Maebayashi, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 45 1 57 - 64 2012年 [無し][無し]
     研究論文（学術雑誌） 
    The TRK-fused gene (TFG in human, Tfg in rat) was originally identified in human papillary thyroid cancer as a chimeric form of the NTRK1 gene. It was since reported that the gene product (TFG) plays a role in regulating phosphotyrosine-specific phosphatase-1 activity. As shown in the accompanying paper, we produced an antibody to rat TFG and used it to localize TFG to selected neurons in specific regions. In the present study, we mapped the TFG-positive neurons in the brainstem, cerebellum, and spinal cord of rats. In the brainstem, neurons intensely positive for TFG were distributed in the raphe nuclei, the gigantocellular reticular nucleus, the reticulotegmental nucleus of the pons, and some cranial nerve nuclei such as the trigeminal nuclei, the vestibulocochlear nuclei, and the dorsal motor nucleus of the vagus. Purkinje cells in the cerebellum and motor neurons in the spinal anterior horn were also positive for TFG. These results provide fundamental data for studying the functions of TFG in the brain.
  • Dongsun Park, Seong Soo Joo, Tae Kyun Kim, Sun Hee Lee, Hyomin Kang, Hong Jun Lee, Inja Lim, Akinori Matsuo, Ikuo Tooyama, Yun-Bae Kim, Seung U. Kim
    CELL TRANSPLANTATION 21 1 365 - 371 2012年 [無し][無し]
     研究論文（学術雑誌） 
    Alzheimer disease (AD) is a progressive neurodegenerative disease, which is characterized by loss of memory and cognitive function. In AD patients dysfunction of the cholinergic system is the main cause of cognitive disorders, and decreased activity of choline acetyltransferase (ChAT), an enzyme responsible for acetylcholine (ACh) synthesis, is observed. In the present study we investigated if brain transplantation of human neural stem cells (NSCs) genetically modified to encode ChAT gene improves cognitive function of kainic acid (KA)-induced learning deficit rats. Intrahippocampal injection of KA to hippocampal CA3 region caused severe neuronal loss, resulting in profound learning and memory deficit. F3.ChAT human NSCs transplanted intracerebroventricularly improved fully the learning and memory function of KA-induced learning deficit animals, in parallel with the elevation of ACh levels in cerebrospinal fluid. F3.ChAT human NSCs migrated to the KA-induced injury site (CA3) and differentiated into neurons and astrocytes. The present study demonstrates that human NSCs expressing ChAT have lesion-tropic property and improve cognitive function of learning deficit model rats with hippocampal injury by increasing ACh level.
  • Naomi J. Bisem, Shigeko Takeuchi, Toru Imamura, Essam M. Abdelalim, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 45 6 325 - 334 2012年 [無し][無し]
     研究論文（学術雑誌） 
    FGF1 is highly expressed in neurons and it has been proposed to play a role in the neuroprotection and in regeneration. Low FGF1 expression in neurons has been linked to increased vulnerability in cholinergic neurons. Previous reports have shown that the expression of FGF1 in rat brain is localized to the cholinergic nuclei of the medulla oblongata, with low ratio of neurons positive for FGF1 in the dorsal motor nucleus of the vagus (DMNV). The role of FGF1 in the primate brain has yet to be clarified. In this study, we mapped FGF1 immunoreactivity in the medulla oblongata of cynomolgus monkey brainstems. Our results demonstrated that FGF1 immunoreactivity follows the pattern of distribution of cholinergic nuclei in the medulla oblongata; with strong localization of FGF1 to cholinergic neurons of the hypoglossal nucleus, the facial nucleus and the nucleus ambiguus. In contrast, the DMNV shows markedly lower FGF1 immunoreactivity. Localization of FGF1 to cholinergic neurons was only observed in the lateral region of the DMNV, with higher immunoreactivity in the rostral ventral-lateral region of the DMNV. These findings are consistent with the distribution of FGF1 immunoreactivity in previous studies of the rat brain.
  • クルクミンのケト・エノール互変異性とアミロイド親和性を利用したアルツハイマー病診断薬の検討
    遠山 育夫, 柳沢 大治郎, 田口 弘康
    臨床神経学 51 12 1397 - 1397 (一社)日本神経学会 2011年12月
  • Essam M. Abdelalim, Ikuo Tooyama
    BRAIN STRUCTURE & FUNCTION 216 4 387 - 402 2011年11月 [無し][無し]
     研究論文（学術雑誌） 
    C-type natriuretic peptide (CNP), the most abundant natriuretic peptide hormone in the brain, plays an important role in neuroendocrine function. The physiological effects of CNP are mediated by the natriuretic peptide receptor-B (NPR-B). Although CNP and NPR-B have been detected in several brain regions, little is known about the neuroanatomical localization of NPR-B protein in the brainstem. In the present study, we investigated the topographical distribution of NPR-B immunoreactivity in the monkey brainstem. The data demonstrate widespread NPR-B immunoreactivity throughout the brainstem. NPR-B immunoreactivity was located in the superior colliculus, inferior colliculus, periaqueductal gray, oculomotor nucleus, red nucleus, ventral tegmental area, substantia nigra, and cerebral peduncle of the midbrain, as well as in the abducens nucleus, medial vestibular nucleus, lateral vestibular nucleus, parabrachial nucleus, locus coeruleus, trigeminal motor nucleus, pontine reticular nucleus, facial nucleus, oral part of the spinal trigeminal nucleus, cochlear nucleus, raphe magnus nucleus, raphe pallidus nucleus, pontine nucleus of the pons, the dorsal motor nucleus of the vagus, hypoglossal nucleus, nucleus tractus solitarius, gracile nucleus, cuneate nucleus, medial vestibular nucleus, spinal trigeminal nucleus, nucleus ambiguus, lateral paragigantocellular nucleus, lateral reticular nucleus, and the inferior olivary nucleus of the medulla oblongata. The widespread distribution of NPR-B-immunoreactive structures throughout the monkey brainstem indicates that CNP may be involved in several physiological mechanisms, acting as a neurotransmitter and/or neuromodulator.
  • Essam M. Abdelalim, Ikuo Tooyama
    BRAIN STRUCTURE & FUNCTION 216 4 387 - 402 2011年11月 [無し][無し]
     研究論文（学術雑誌） 
    C-type natriuretic peptide (CNP), the most abundant natriuretic peptide hormone in the brain, plays an important role in neuroendocrine function. The physiological effects of CNP are mediated by the natriuretic peptide receptor-B (NPR-B). Although CNP and NPR-B have been detected in several brain regions, little is known about the neuroanatomical localization of NPR-B protein in the brainstem. In the present study, we investigated the topographical distribution of NPR-B immunoreactivity in the monkey brainstem. The data demonstrate widespread NPR-B immunoreactivity throughout the brainstem. NPR-B immunoreactivity was located in the superior colliculus, inferior colliculus, periaqueductal gray, oculomotor nucleus, red nucleus, ventral tegmental area, substantia nigra, and cerebral peduncle of the midbrain, as well as in the abducens nucleus, medial vestibular nucleus, lateral vestibular nucleus, parabrachial nucleus, locus coeruleus, trigeminal motor nucleus, pontine reticular nucleus, facial nucleus, oral part of the spinal trigeminal nucleus, cochlear nucleus, raphe magnus nucleus, raphe pallidus nucleus, pontine nucleus of the pons, the dorsal motor nucleus of the vagus, hypoglossal nucleus, nucleus tractus solitarius, gracile nucleus, cuneate nucleus, medial vestibular nucleus, spinal trigeminal nucleus, nucleus ambiguus, lateral paragigantocellular nucleus, lateral reticular nucleus, and the inferior olivary nucleus of the medulla oblongata. The widespread distribution of NPR-B-immunoreactive structures throughout the monkey brainstem indicates that CNP may be involved in several physiological mechanisms, acting as a neurotransmitter and/or neuromodulator.
  • クルクミンとアミロイドβオリゴマーの結合性の解析
    柳沢 大治郎, 田口 弘康, 山本 章嗣, 白井 伸明, 平尾 浩一, 遠山 育夫
    Dementia Japan 25 3 422 - 422 (一社)日本認知症学会 2011年10月
  • Ikuo Tooyama, Daijiro Yanagisawa
    Nihon rinsho. Japanese journal of clinical medicine 69 Suppl 8 214 - 218 2011年10月 研究論文（学術雑誌）
  • W. Sako, R. Morigaki, R. Kaji, I. Tooyama, S. Okita, K. Kitazato, S. Nagahiro, A. M. Graybiel, S. Goto
    NEUROSCIENCE 189 100 - 107 2011年08月 [無し][無し]
     研究論文（学術雑誌） 
    The neuron-specific isoform of the TAF1 gene (N-TAF1) is thought to be involved in the pathogenesis of DYT3 dystonia, which leads to progressive neurodegeneration in the striatum. To determine the expression pattern of N-TAF1 transcripts, we developed a specific monoclonal antibody against the N-TAF1 protein. Here we show that in the rat brain, N-TAF1 protein appears as a nuclear protein within subsets of neurons in multiple brain regions. Of particular interest is that in the striatum, the nuclei possessing N-TAF1 protein are largely within medium spiny neurons, and they are distributed preferentially, though not exclusively, in the striosome compartment. The compartmental preference and cell type-selective distribution of N-TAF1 protein in the striatum are strikingly similar to the patterns of neuronal loss in the striatum of DYT3 patients. Our findings suggest that the distribution of N-TAF1 protein could represent a key molecular characteristic contributing to the pattern of striatal degeneration in DYT3 dystonia. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
  • W. Sako, R. Morigaki, R. Kaji, I. Tooyama, S. Okita, K. Kitazato, S. Nagahiro, A. M. Graybiel, S. Goto
    NEUROSCIENCE 189 100 - 107 2011年08月 [無し][無し]
     研究論文（学術雑誌） 
    The neuron-specific isoform of the TAF1 gene (N-TAF1) is thought to be involved in the pathogenesis of DYT3 dystonia, which leads to progressive neurodegeneration in the striatum. To determine the expression pattern of N-TAF1 transcripts, we developed a specific monoclonal antibody against the N-TAF1 protein. Here we show that in the rat brain, N-TAF1 protein appears as a nuclear protein within subsets of neurons in multiple brain regions. Of particular interest is that in the striatum, the nuclei possessing N-TAF1 protein are largely within medium spiny neurons, and they are distributed preferentially, though not exclusively, in the striosome compartment. The compartmental preference and cell type-selective distribution of N-TAF1 protein in the striatum are strikingly similar to the patterns of neuronal loss in the striatum of DYT3 patients. Our findings suggest that the distribution of N-TAF1 protein could represent a key molecular characteristic contributing to the pattern of striatal degeneration in DYT3 dystonia. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Ligang Wang, Hongkuan Yang, Shiguang Zhao, Haruhisa Sato, Yoshihiro Konishi, Thomas G. Beach, Essam Mohamed Abdelalim, Naomi J. Bisem, Ikuo Tooyama
    PLOS ONE 6 7 e22325  2011年07月 [無し][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (MtF) has been identified as a novel ferritin encoded by an intron-lacking gene with specific mitochondrial localization located on chromosome 5q23.1. MtF has been associated with neurodegenerative disorders such as Friedreich ataxia and restless leg syndrome. However, little information is available about MtF in Alzheimer's disease (AD). In this study, therefore, we investigated the expression and localization of MtF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) as well as in situ hybridization histochemistry. We also examined protein expression using western-blot assay. In addition, we used in vitro methods to further explore the effect of oxidative stress and beta-amyloid peptide (A beta) on MtF expression. To do this we examined MtF mRNA and protein expression changes in the human neuroblastoma cell line, IMR-32, after treatment with A beta, H2O2, or both. The neuroprotective effect of MtF on oxidative stress induced by H2O2 was measured by MTT assay. The in situ hybridization studies revealed that MtF mRNA was detected mainly in neurons to a lesser degree in glial cells in the cerebral cortex. The staining intensity and the number of positive cells were increased in the cerebral cortex of AD patients. Real-time PCR and western-blot confirmed that MtF expression levels in the cerebral cortex were significantly higher in AD cases than that in control cases at both the mRNA and the protein level. Cell culture experiments demonstrated that the expression of both MtF mRNA and protein were increased by treatment with H2O2 or a combination of A beta and H2O2, but not with A beta alone. Finally, MtF expression showed a significant neuroprotective effect against H2O2-induced oxidative stress (p<0.05). The present study suggests that MtF is involved in the pathology of AD and may play a neuroprotective role against oxidative stress.
  • Wang L, Yang H, Zhao S, Sato H, Konishi Y, Beach TG, Abdelalim EM, Bisem NJ, Tooyama I
    PLOS ONE 6 7 e22325  2011年07月 [無し][無し]
     研究論文（学術雑誌） 
    Mitochondrial ferritin (MtF) has been identified as a novel ferritin encoded by an intron-lacking gene with specific mitochondrial localization located on chromosome 5q23.1. MtF has been associated with neurodegenerative disorders such as Friedreich ataxia and restless leg syndrome. However, little information is available about MtF in Alzheimer's disease (AD). In this study, therefore, we investigated the expression and localization of MtF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) as well as in situ hybridization histochemistry. We also examined protein expression using western-blot assay. In addition, we used in vitro methods to further explore the effect of oxidative stress and beta-amyloid peptide (A beta) on MtF expression. To do this we examined MtF mRNA and protein expression changes in the human neuroblastoma cell line, IMR-32, after treatment with A beta, H2O2, or both. The neuroprotective effect of MtF on oxidative stress induced by H2O2 was measured by MTT assay. The in situ hybridization studies revealed that MtF mRNA was detected mainly in neurons to a lesser degree in glial cells in the cerebral cortex. The staining intensity and the number of positive cells were increased in the cerebral cortex of AD patients. Real-time PCR and western-blot confirmed that MtF expression levels in the cerebral cortex were significantly higher in AD cases than that in control cases at both the mRNA and the protein level. Cell culture experiments demonstrated that the expression of both MtF mRNA and protein were increased by treatment with H2O2 or a combination of A beta and H2O2, but not with A beta alone. Finally, MtF expression showed a significant neuroprotective effect against H2O2-induced oxidative stress (p<0.05). The present study suggests that MtF is involved in the pathology of AD and may play a neuroprotective role against oxidative stress.
  • Yanagisawa D, Amatsubo T, Morikawa S, Taguchi H, Urushitani M, Shirai N, Hirao K, Shiino A, Inubushi T, Tooyama I
    Neuroscience. 2011 Jun 16;184:120-7. Epub 2011 Apr 12. 184 120 - 127 2011年06月 [無し][無し]
     研究論文（学術雑誌） 
    Amyloid β (Aβ) deposition in the brain is considered the initiating event in the progression of Alzheimer's disease (AD). Amyloid imaging is widely studied in diagnosing AD and evaluating the disease stage, with considerable advances achieved in recent years. We have developed a novel 19F-containing curcumin derivative (named FMeC1) as a potential imaging agent. This compound can exist in equilibrium between keto and enol tautomers, with the enol form able to bind Aβ aggregates while the keto form cannot. This study investigated whether FMeC1 is suitable as a 19F magnetic resonance imaging (MRI) probe to detect Aβ deposition in the Tg2576 mouse, a model of AD. In 19F nuclear magnetic resonance (NMR) spectra obtained from the whole head, a delayed decreased rate of 19F signal was observed in Tg2576 mice that were peripherally injected with FMeC1 in comparison to wild-type mice. Furthermore, 19F MRI displayed remarkable levels of 19F signal in the brain of Tg2576 mice after the injection of FMeC1. Histological analysis of FMeC1-injected mouse brain showed penetration of the compound across the blood-brain barrier and binding to Aβ plaques in peripherally injected Tg2576 mice. Moreover, the distribution of Aβ deposits in Tg2576 mice was in accordance with the region of the brain in which the 19F signal was imaged. FMeC1 also exhibited an affinity for senile plaques in human brain sections. These findings suggest the usefulness of FMeC1 as a 19F MRI probe for the detection of amyloid deposition in the brain. Furthermore, the properties of FMeC1 could form the basis for further novel amyloid imaging probes. © 2011 IBRO.
  • E. M. Abdelalim, I. Tooyama
    CELL DEATH & DISEASE 2 2 e127  2011年03月 [無し][無し]
     研究論文（学術雑誌） 
    Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells. Cell Death and Disease (2011) 2, e127; doi: 10.1038/cddis.2011.10; published online 10 March 2011
  • E. M. Abdelalim, I. Tooyama
    CELL DEATH & DISEASE 2 2 e127  2011年03月 [無し][無し]
     研究論文（学術雑誌） 
    Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells. Cell Death and Disease (2011) 2, e127; doi: 10.1038/cddis.2011.10; published online 10 March 2011
  • Daijiro Yanagisawa, Shigehiro Morikawa, Hiroyasu Taguchi, Akihiko Shiino, Toshiro Inubushi, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 71 E85 - E85 2011年 [有り][無し]
  • Daijiro Yanagisawa, Hiroyasu Taguchi, Akitsugu Yamamoto, Nobuaki Shirai, Koichi Hirao, Ikuo Tooyama
    JOURNAL OF ALZHEIMERS DISEASE 24 33 - 42 2011年 [無し][無し]
     研究論文（学術雑誌） 
    Studies of Alzheimer's disease (AD) strongly support the hypothesis that amyloid-beta (A beta) deposition in the brain is the initiating event in the progression of AD. A beta peptides easily form long insoluble amyloid fibrils, which accumulate in deposits known as senile plaques. On the other hand, recent work indicated that soluble A beta oligomers, rather than monomers or insoluble A beta fibrils, might be responsible for neuronal and synaptic dysfunction in AD. Curcumin, a low molecular weight yellow-orange pigment derived from the turmeric plant, has shown therapeutic effects in transgenic mouse models of AD. However, it remains unclear whether curcumin interacts directly with the A beta oligomers. This study investigated any interaction between curcumin and A beta oligomers such as globulomer and A beta-derived diffusible ligand (ADDL). Globulomer was observed as a cluster of spherical structures by electron microscopic analysis, and ADDL was also detected as small spherical structures. Fluorescence analysis revealed a significant increase in the fluorescence of curcumin when reacted with both oligomers. Furthermore quartz crystal microbalance analysis showed significant frequency decreases in oligomer-immobilized electrodes following the addition of curcumin. These results strongly suggested that curcumin binds to A beta oligomers and to A beta fibrils. The association of curcumin with A beta oligomers may contribute to the therapeutic effect on AD. Based on these findings, curcumin could provide the basis of a novel concept in AD therapies targeting A beta oligomers.
  • In vivo detection of amyloid β deposition using (19)F magnetic resonance imaging with a (19)F-containing curcumin derivative in a mouse model of Alzheimer's disease.
    Yanagisawa D, Amatsubo T, Morikawa S, Taguchi H, Urushitani M, Shirai N, Hirao K, Shiino A, Inubushi T, Tooyama I
    Neuroscience. 2011 Jun 16;184:120-7. Epub 2011 Apr 12. 184 120 - 1277 2011年 [無し][無し]
     研究論文（学術雑誌）
  • Kaori Morimoto, Juri Horio, Haruhisa Satoh, Lucia Sue, Thomas Beach, Seizaburo Arita, Ikuo Tooyama, Yoshihiro Konishi
    JOURNAL OF ALZHEIMERS DISEASE 25 1 59 - 76 2011年 [無し][無し]
     研究論文（学術雑誌） 
    Neuroinflammation is involved in the pathology of Alzheimer's disease (AD). Our major focus was to clarify whether neuroinflammation plays an important role in AD pathogenesis, particularly prior to the manifestation of overt dementia. We analyzed cytokine expression profiles of the brain, with focus on non-demented patients with increasing AD pathology, referred to as high pathology control (HPC) patients, who provide an intermediate subset between AD and normal control subjects, referred to as low pathology control (LPC) patients. With real-time PCR techniques, we found significant differences in interleukin (IL)-1 beta, 10, 13, 18, and 33, tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE), and transforming growth factor beta 1 (TGF beta 1) mRNA expression ratios between HPC and AD patients, while no significant differences in the expression ratios of any cytokine tested here were observed between LPC and HPC patients. The cytokine mRNA expression ratios were determined as follows: first, cytokine mRNA levels were normalized to mRNA levels of a housekeeping gene, peptidyl-prolyl isomerase A (PPIA), which showed the most stable expression among ten housekeeping genes tested here; then, the normalized data of cytokine levels in the temporal cortex were divided by those in the cerebellum, which is resistant to AD pathology. Subsequently, the expression ratios of the temporal cortex to cerebellum were compared among LPC, HPC, and AD patient groups. Our results indicate that cytokines are more mobilized and implicated in the later AD stage when a significant cognitive decline occurs and develops than in the developmental course of AD pathology prior to the manifestation of overt dementia.
  • Yanagisawa D, Taguchi H, Yamamoto A, Shirai N, Hirao K, Tooyama I
    J Alzheimer 24 33 - 42 2011年 [無し][無し]
     研究論文（学術雑誌） 
    Studies of Alzheimer's disease (AD) strongly support the hypothesis that amyloid-beta (A beta) deposition in the brain is the initiating event in the progression of AD. A beta peptides easily form long insoluble amyloid fibrils, which accumulate in deposits known as senile plaques. On the other hand, recent work indicated that soluble A beta oligomers, rather than monomers or insoluble A beta fibrils, might be responsible for neuronal and synaptic dysfunction in AD. Curcumin, a low molecular weight yellow-orange pigment derived from the turmeric plant, has shown therapeutic effects in transgenic mouse models of AD. However, it remains unclear whether curcumin interacts directly with the A beta oligomers. This study investigated any interaction between curcumin and A beta oligomers such as globulomer and A beta-derived diffusible ligand (ADDL). Globulomer was observed as a cluster of spherical structures by electron microscopic analysis, and ADDL was also detected as small spherical structures. Fluorescence analysis revealed a significant increase in the fluorescence of curcumin when reacted with both oligomers. Furthermore quartz crystal microbalance analysis showed significant frequency decreases in oligomer-immobilized electrodes following the addition of curcumin. These results strongly suggested that curcumin binds to A beta oligomers and to A beta fibrils. The association of curcumin with A beta oligomers may contribute to the therapeutic effect on AD. Based on these findings, curcumin could provide the basis of a novel concept in AD therapies targeting A beta oligomers.
  • Morimoto K, Horio J, Sato H, Sue L, Beach T, Arita S, Tooyama I, Konishi Y
    J Alzheimer 25 1 59 - 76 2011年 [無し][無し]
     研究論文（学術雑誌） 
    Neuroinflammation is involved in the pathology of Alzheimer's disease (AD). Our major focus was to clarify whether neuroinflammation plays an important role in AD pathogenesis, particularly prior to the manifestation of overt dementia. We analyzed cytokine expression profiles of the brain, with focus on non-demented patients with increasing AD pathology, referred to as high pathology control (HPC) patients, who provide an intermediate subset between AD and normal control subjects, referred to as low pathology control (LPC) patients. With real-time PCR techniques, we found significant differences in interleukin (IL)-1 beta, 10, 13, 18, and 33, tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE), and transforming growth factor beta 1 (TGF beta 1) mRNA expression ratios between HPC and AD patients, while no significant differences in the expression ratios of any cytokine tested here were observed between LPC and HPC patients. The cytokine mRNA expression ratios were determined as follows: first, cytokine mRNA levels were normalized to mRNA levels of a housekeeping gene, peptidyl-prolyl isomerase A (PPIA), which showed the most stable expression among ten housekeeping genes tested here; then, the normalized data of cytokine levels in the temporal cortex were divided by those in the cerebellum, which is resistant to AD pathology. Subsequently, the expression ratios of the temporal cortex to cerebellum were compared among LPC, HPC, and AD patient groups. Our results indicate that cytokines are more mobilized and implicated in the later AD stage when a significant cognitive decline occurs and develops than in the developmental course of AD pathology prior to the manifestation of overt dementia.
  • Shigeko Takeuchi, Noriko Fujiwara, Akemi Ido, Miki Oono, Yuki Takeuchi, Minako Tateno, Keiichiro Suzuki, Ryosuke Takahashi, Ikuo Tooyama, Naoyuki Taniguchi, Jean-Pierre Julien, Makoto Urushitani
    JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY 69 10 1044 - 1056 2010年10月 [無し][無し]
     研究論文（学術雑誌） 
    Vaccinations targeting extracellular superoxide dismutase 1 (SOD1) mutants are beneficial in mouse models of amyotrophic lateral sclerosis (ALS). Because of its misfolded nature, wild-type nonmetallated SOD1 protein (WT-apo) may have therapeutic application for vaccination of various SOD1 mutants. We compared the effects of WT-apo to those of a G93A SOD1 vaccine in low-copy G93A SOD1 transgenic mice. Both SOD1 vaccines induced antibody against G93A SOD1 and significantly delayed disease onset compared with saline/adjuvant controls. WT-apo SOD1 significantly extended the life span of vaccinated mice. The vaccines potentiated T(H)2 deviation in the spinal cord as determined by the ratio of interleukin-4 to interferon-gamma (IFN gamma) or tumor necrosis factor and induced C1q deposition around motor neurons. Transgenic mice had abundant microglial expression of signal transducers and activators of transcription 4, an activator of transcription of IFN gamma, in the spinal cord implicating IFN gamma in the pathogenesis. On the other hand, the sera from G93A SOD1-vaccinated mice showed higher IFN gamma or tumor necrosis factor and yielded a lower IgG1/IgG2c ratio than the sera from WT-apo-vaccinated mice. These results indicate that the T(H)1/T(H)2 milieu is affected by specific vaccinations and that antigenicity might counteract beneficial effects by enhancing T(H)1 immunity. Thus, because of its lower T(H)1 induction, WT-apo may be a therapeutic option and have broader application in ALS associated with diverse SOD1 mutations.
  • Shigeko Takeuchi, Noriko Fujiwara, Akemi Ido, Miki Oono, Yuki Takeuchi, Minako Tateno, Keiichiro Suzuki, Ryosuke Takahashi, Ikuo Tooyama, Naoyuki Taniguchi, Jean-Pierre Julien, Makoto Urushitani
    JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY 69 10 1044 - 1056 2010年10月 [無し][無し]
     研究論文（学術雑誌） 
    Vaccinations targeting extracellular superoxide dismutase 1 (SOD1) mutants are beneficial in mouse models of amyotrophic lateral sclerosis (ALS). Because of its misfolded nature, wild-type nonmetallated SOD1 protein (WT-apo) may have therapeutic application for vaccination of various SOD1 mutants. We compared the effects of WT-apo to those of a G93A SOD1 vaccine in low-copy G93A SOD1 transgenic mice. Both SOD1 vaccines induced antibody against G93A SOD1 and significantly delayed disease onset compared with saline/adjuvant controls. WT-apo SOD1 significantly extended the life span of vaccinated mice. The vaccines potentiated T(H)2 deviation in the spinal cord as determined by the ratio of interleukin-4 to interferon-gamma (IFN gamma) or tumor necrosis factor and induced C1q deposition around motor neurons. Transgenic mice had abundant microglial expression of signal transducers and activators of transcription 4, an activator of transcription of IFN gamma, in the spinal cord implicating IFN gamma in the pathogenesis. On the other hand, the sera from G93A SOD1-vaccinated mice showed higher IFN gamma or tumor necrosis factor and yielded a lower IgG1/IgG2c ratio than the sera from WT-apo-vaccinated mice. These results indicate that the T(H)1/T(H)2 milieu is affected by specific vaccinations and that antigenicity might counteract beneficial effects by enhancing T(H)1 immunity. Thus, because of its lower T(H)1 induction, WT-apo may be a therapeutic option and have broader application in ALS associated with diverse SOD1 mutations.
  • クルクミン系化合物のケト・エノール互変異性とアミロイド親和性
    柳沢 大治郎, 雨坪 知音, 田口 弘康, 漆谷 真, 森川 茂廣, 犬伏 俊郎, 椎野 顯彦, 遠山 育夫
    Dementia Japan 24 3 396 - 396 (一社)日本認知症学会 2010年09月
  • Ligang Wang, Haruhisa Sato, Shiguang Zhao, Ikuo Tooyama
    NEUROSCIENCE LETTERS 481 3 164 - 167 2010年09月 [無し][無し]
     研究論文（学術雑誌） 
    We and others have previously reported that lactoferrin (LF), which acts as both an iron-binding protein and an inflammatory modulator, is strongly up-regulated in the brains of patients with Alzheimer's disease (AD). We have also studied the expression and localization of LF mRNA in the brain cortices of patients with AD. In this study, we investigated immunohistochemically the localization of LF in the brains of APP-transgenic mice, representing a model of AD. No LF immunoreactivity was detected in the brains of the wild-type mice. In the transgenic AD mice, LF deposition was detected in the brains. Double-immunofluorescence staining with antibodies directed against the amyloid-beta peptide (A beta) and LF localized the LF depositions to amyloid deposits (senile plaques) and regions of amyloid angiopathy. Senile plaque formation precedes LF deposition in AD. In the transgenic mice aged <18 months, most of senile plaques were negative for LF. LF deposits appeared weakly at about 18 months of age in these mice. Both the intensity and number of LF-positive depositions in the transgenic mice increased with age. Double-staining for LF and thioflavin-S revealed that LF accumulated in thioflavin-S-positive, fibrillar-type senile plaques. The up-regulation of LF in the brains of both AD patients and the transgenic mouse model of AD provides evidence of an important role for LF in AD-affected brain tissues. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
  • Wang L, Sato H, Zhao S, Tooyama I
    NEUROSCIENCE LETTERS 481 3 164 - 167 2010年09月 [無し][無し]
     研究論文（学術雑誌） 
    We and others have previously reported that lactoferrin (LF), which acts as both an iron-binding protein and an inflammatory modulator, is strongly up-regulated in the brains of patients with Alzheimer's disease (AD). We have also studied the expression and localization of LF mRNA in the brain cortices of patients with AD. In this study, we investigated immunohistochemically the localization of LF in the brains of APP-transgenic mice, representing a model of AD. No LF immunoreactivity was detected in the brains of the wild-type mice. In the transgenic AD mice, LF deposition was detected in the brains. Double-immunofluorescence staining with antibodies directed against the amyloid-beta peptide (A beta) and LF localized the LF depositions to amyloid deposits (senile plaques) and regions of amyloid angiopathy. Senile plaque formation precedes LF deposition in AD. In the transgenic mice aged <18 months, most of senile plaques were negative for LF. LF deposits appeared weakly at about 18 months of age in these mice. Both the intensity and number of LF-positive depositions in the transgenic mice increased with age. Double-staining for LF and thioflavin-S revealed that LF accumulated in thioflavin-S-positive, fibrillar-type senile plaques. The up-regulation of LF in the brains of both AD patients and the transgenic mouse model of AD provides evidence of an important role for LF in AD-affected brain tissues. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
  • パーキンソン病モデルラットにおけるfluoro-dopaのMR画像
    遠山 育夫, 小田 恵輔, 柳沢 大治郎, 高田 和幸, 北村 佳久, 谷口 隆之, 森川 茂廣, 犬伏 俊郎
    JSMI Report 3 2 54 - 54 日本分子イメージング学会 2010年05月
  • クルクミン誘導体のアミロイド結合活性におけるケト・エノール互変異性の関与
    柳沢 大治郎, 雨坪 知音, 田口 弘康, 漆谷 真, 森川 茂廣, 犬伏 俊郎, 遠山 育夫
    JSMI Report 3 2 82 - 82 日本分子イメージング学会 2010年05月
  • パーキンソン病モデルラットにおけるfluoro-dopaのMR画像
    遠山 育夫, 小田 恵輔, 柳沢 大治郎, 高田 和幸, 北村 佳久, 谷口 隆之, 森川 茂廣, 犬伏 俊郎
    JSMI Report 3 2 98 - 98 日本分子イメージング学会 2010年05月
  • Daijiro Yanagisawa, Nobuaki Shirai, Tomone Amatsubo, Hiroyasu Taguchi, Koichi Hirao, Makoto Urushitani, Shigehiro Morikawa, Toshiro Inubushi, Masanari Kato, Fuminori Kato, Kyuya Morino, Hirohiko Kimura, Ichiro Nakano, Chikako Yoshida, Takashi Okada, Mitsuo Sano, Yoshiko Wada, Ken-nosuke Wada, Akitsugu Yamamoto, Ikuo Tooyama
    BIOMATERIALS 31 14 4179 - 4185 2010年05月 [無し][無し]
     研究論文（学術雑誌） 
    Curcumin, which can exist in an equilibrium between keto and enol tautomers, binds to beta-amyloid (A beta) fibrils/aggregates. The aim of this study was to assess the relationship between the tautomeric structures of curcumin derivatives and their A beta-binding activities. Curcumin derivatives with keto-enol tautomerism showed high levels of binding to A beta aggregates but not to All monomers. The binding activity of the keto form analogue of curcumin to All aggregates was found to be much weaker than that of curcumin derivatives with keto-enol tautomerism. The color of a curcumin derivative with keto-enol tautomerism, which was substituted at the C-4 position, changed from yellow to orange within 30 min of being combined with All aggregates in physiological buffer. This resulted from a remarkable increase in the enol form with extended conjugation of double bonds upon binding. These findings suggest that curcumin derivatives exist predominantly in the enol form during binding to A beta aggregates, and that the enolization of curcumin derivatives is crucial for binding to A beta aggregates. The keto-enol tautomerism of curcumin derivatives may be a novel target for the design of amyloid-binding agents that can be used both for therapy and for amyloid detection in Alzheimer's disease. (C) 2010 Elsevier Ltd. All rights reserved.
  • Yanagisawa D, Shirai N, Amatsubo T, Taguchi H, Hirao K, Urushitani M, Morikawa S, Inubushi T, Kato M, Kato F, Morino K, Kimura H, Nakano I, Yoshida C, Okada T, Sano M, Wada Y, Wada KN, Yamamoto A, Tooyama I
    BIOMATERIALS 31 14 4179 - 4185 2010年05月 [無し][無し]
     研究論文（学術雑誌） 
    Curcumin, which can exist in an equilibrium between keto and enol tautomers, binds to beta-amyloid (A beta) fibrils/aggregates. The aim of this study was to assess the relationship between the tautomeric structures of curcumin derivatives and their A beta-binding activities. Curcumin derivatives with keto-enol tautomerism showed high levels of binding to A beta aggregates but not to All monomers. The binding activity of the keto form analogue of curcumin to All aggregates was found to be much weaker than that of curcumin derivatives with keto-enol tautomerism. The color of a curcumin derivative with keto-enol tautomerism, which was substituted at the C-4 position, changed from yellow to orange within 30 min of being combined with All aggregates in physiological buffer. This resulted from a remarkable increase in the enol form with extended conjugation of double bonds upon binding. These findings suggest that curcumin derivatives exist predominantly in the enol form during binding to A beta aggregates, and that the enolization of curcumin derivatives is crucial for binding to A beta aggregates. The keto-enol tautomerism of curcumin derivatives may be a novel target for the design of amyloid-binding agents that can be used both for therapy and for amyloid detection in Alzheimer's disease. (C) 2010 Elsevier Ltd. All rights reserved.
  • Makoto Urushitani, Takashi Sato, Hitoshi Bamba, Yasuo Hisa, Ikuo Tooyama
    JOURNAL OF NEUROSCIENCE RESEARCH 88 4 784 - 797 2010年03月 [無し][無し]
     研究論文（学術雑誌） 
    Cytoplasmic aggregates of ubiquitinated TAR DNA-binding protein 43 (TDP-43) are a pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism of TDP-43 polyubiquitination remains elusive. We investigated the effect of nuclear exclusion of TDP-43 on aggregate formation and fragmentation, using TDP-43 expression constructs for WT or mutant TDP-43 with a modified nuclear localizing signal (LQ-NLS). Overexpression of the LQ-NLS mutant alone induced no detectable cytoplasmic aggregates during a 72-hr period. Polyubiquitination of both WT TDP-43 and the LQ-NLS mutant was similar in total cell lysates exposed to the proteasome inhibitor lactacystin. However, analysis of subcellular fractions demonstrated a higher concentration of polyubiquitinated TDP-43 in the nuclear fraction than in the cytosol for WT, and vice versa for the LQ-NLS mutant. Polyubiquitin-charged WT and mutant TDP-43 were highly concentrated in the membrane/microsome fraction, which was also positive for the autophagosome marker LC3. In addition, the autophagy inhibitor 3-methyladenine (3MA) blocked degradation of both TDP-43 types, whereas lactacystin was minimally restorative. Furthermore, lactacystin plus 3MA induced prominent cytoplasmic aggregates. We also demonstrated mediation of TDP-43 polyubiquitination by lysine 48 of ubiquitin, indicating a degradation signal in both TDP-43 types. This is the first report delineating the distribution of polyubiquitinated TDP-43 and the degradation pathway of TDP-43 and clarifying the crucial role of autophagosomes in TDP-43 clearance. We also demonstrate that nuclear exclusion itself is not an immediate trigger for ALS pathology. Further clarification of the mechanism of polyubiquitination of TDP-43 and the role of autophagosomes may help in understanding and treating ALS. (C) 2009 Wiley-Liss, Inc.
  • Essam Mohamed Abdelalim, Ikuo Tooyama
    PEPTIDES 31 1 180 - 183 2010年01月 [無し][無し]
     研究論文（学術雑誌） 
    Natriuretic peptide receptor C (NPR-C) is known to bind all natriuretic peptides with similar affinity. Given their biological role it is interesting that natriuretic peptides and their activated guanylate cyclases (NPR-A and NPR-B) are expressed in retinal amacrine cells. The purpose of this study is to examine the presence of NPR-C in the rat retina and its relationship to cholinergic and dopaminergic amacrine cells using immunofluorescence techniques. NPR-C immunoreactivity was found in several layers of the retina including the ganglion cell layer (GCL), inner nuclear layer (INL), outer plexiform layer (OPL), and inner segments of photoreceptors (IS). Immunofluorescence double-labeling showed the co-localization of NPR-C with tyrosine hydroxylase, a marker of dopaminergic cells, and with choline acetyltransferase (ChAT), a marker of cholinergic cells. These data suggest that natriuretic peptides may play a role in maintaining the retinal functions via interaction with NPR-C. (C) 2009 Elsevier Inc. All rights reserved
  • Hiroaki Nakahara, Yoshihiro Konishi, Thomas G. Beach, Naoto Yamada, Satoshi Makino, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 43 6 157 - 162 2010年 [無し][無し]
     研究論文（学術雑誌） 
    We and others have previously shown that reactive microglia express the major histocompatibility complex (MHC) class I and class II antigens in the hippocampus of patients suffering from epilepsy. Although the MHC glycoproteins serve as restriction elements for T lymphocytes, there is little information available regarding T lymphocytes in hippocampal sclerosis. In the present study, we investigated T lymphocyte infiltration in human hippocampi in four cases of epilepsy with hippocampal sclerosis, as well as in four control cases without neurological disease. No CD8- or CD4-positive T lymphocytes were seen in hippocampi from the control cases. In contrast, CD8- and CD4-positive T lymphocytes had infiltrated into the hippocampi of patients with hippocampal sclerosis. In addition, expression of intercellular adhesion molecule-1 was diffusely upregulated in the hippocampi with hippocampal sclerosis. These results indicate that T lymphocyte infiltration is involved in the pathology of hippocampal sclerosis.
  • Essam Mohamed Abdelalim, Ikuo Tooyama
    PEPTIDES 31 1 180 - 183 2010年01月 [無し][無し]
     研究論文（学術雑誌） 
    Natriuretic peptide receptor C (NPR-C) is known to bind all natriuretic peptides with similar affinity. Given their biological role it is interesting that natriuretic peptides and their activated guanylate cyclases (NPR-A and NPR-B) are expressed in retinal amacrine cells. The purpose of this study is to examine the presence of NPR-C in the rat retina and its relationship to cholinergic and dopaminergic amacrine cells using immunofluorescence techniques. NPR-C immunoreactivity was found in several layers of the retina including the ganglion cell layer (GCL), inner nuclear layer (INL), outer plexiform layer (OPL), and inner segments of photoreceptors (IS). Immunofluorescence double-labeling showed the co-localization of NPR-C with tyrosine hydroxylase, a marker of dopaminergic cells, and with choline acetyltransferase (ChAT), a marker of cholinergic cells. These data suggest that natriuretic peptides may play a role in maintaining the retinal functions via interaction with NPR-C. (C) 2009 Elsevier Inc. All rights reserved
  • Hiroaki Nakahara, Yoshihiro Konishi, Thomas G. Beach, Naoto Yamada, Satoshi Makino, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 43 6 157 - 162 2010年 [無し][無し]
     研究論文（学術雑誌） 
    We and others have previously shown that reactive microglia express the major histocompatibility complex (MHC) class I and class II antigens in the hippocampus of patients suffering from epilepsy. Although the MHC glycoproteins serve as restriction elements for T lymphocytes, there is little information available regarding T lymphocytes in hippocampal sclerosis. In the present study, we investigated T lymphocyte infiltration in human hippocampi in four cases of epilepsy with hippocampal sclerosis, as well as in four control cases without neurological disease. No CD8- or CD4-positive T lymphocytes were seen in hippocampi from the control cases. In contrast, CD8- and CD4-positive T lymphocytes had infiltrated into the hippocampi of patients with hippocampal sclerosis. In addition, expression of intercellular adhesion molecule-1 was diffusely upregulated in the hippocampi with hippocampal sclerosis. These results indicate that T lymphocyte infiltration is involved in the pathology of hippocampal sclerosis.
  • T. Sato, S. Takeuchi, A. Saito, W. Ding, H. Bamba, H. Matsuura, Y. Hisa, I. Tooyama, M. Urushitani
    NEUROSCIENCE 164 4 1565 - 1578 2009年12月 [無し][無し]
     研究論文（学術雑誌） 
    Nuclear exclusion of TAR DNA binding protein 43 (TDP-43) and formation of cytosolic aggregates are a pathological characteristic of amyotrophic lateral sclerosis (ALS). However, the molecular basis of the aberrant distribution of TDP-43 remains elusive. Here, we show evidence that axonal ligation induced transient nuclear exclusion and peripheral accumulation of TDP-43, without apparent cytosolic aggregates in hypoglossal neurons in mice. Immunohistochemistry showed marked loss of nuclear TDP-43 7-14 days after ligation, which was accompanied by reduction of choline acetyltransferase (ChAT). TDP-43 staining was restored in the nucleus on day 28 exclusively in the neurons with normalized ChAT expression. We also showed that importin beta, which was shown to mediate nuclear transport of TDP-43 was downregulated transiently by nerve ligation. The analysis of the peripheral nerves proximal to the ligation revealed that TDP-43 markedly accumulated with a concomitant decrease in active autophagosome. Moreover, we showed that TDP-43 was present in the microsome fraction containing endoplasmic reticulum (ER) or autophagosomes in the brainstem section, indicating that TDP-43 is axonally transported with vesicles. These results indicate that axonal damage is associated with redistribution of TDP-43 through the combination of defective axonal autophagy periphery and the impaired nuclear transport system in the soma. Moreover, it was also shown that transient redistribution of TDP-43 does not prevent motor neurons from axonal regeneration. Therefore, our data suggest that the subcellular distribution of TDP-43 correlates to the innervation status of motor neurons, which may be governed by unidentified cause of ALS. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Keisuke Oda, Satoshi Makino, Chiaki Masuda, Tatsuhiro Yoshiki, Yoshihisa Kitamura, Kazuyuki Takata, Daijiro Yanagisawa, Takashi Taniguchi, Ikuo Tooyama
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 57 12 1121 - 1126 2009年12月 [無し][無し]
     研究論文（学術雑誌） 
    The putative protein C7orf24 is encoded by Homo sapiens chromosome 7 open reading frame 24. C7orF24 was first identified as a 21-kDa cytochrome c-releasing factor detected in the cytosolic fraction of human leukemia U937 cells after treatment with geranylgeraniol. C7orf24 protein was recently identified as a gamma-glutamyl cyclotransferase, an enzyme in the gamma-glutamyl cycle. However, the exact localization of C7orf24 mRNA in normal tissues remains unknown. The present study examined the distribution pattern of C7orf24 mRNA in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. The RT-PCR experiments demonstrated that C7orf24 and a variant C7orf24 mRNA were expressed in various tissues. Quantitative RT-PCR analysis revealed significantly high levels of both C7orf24 mRNAs in the liver and kidney, compared with other tissues examined. In situ hybridization histochemistry localized C7orf24 mRNA to hepatocytes in the liver and renal tubules in the kidney. The present results thus implicated an important role for C7orf24 in liver and kidney. This manuscript contains online supplemental material at httpj/www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 57:1121-1126, 2009)
  • T. Sato, S. Takeuchi, A. Saito, W. Ding, H. Bamba, H. Matsuura, Y. Hisa, I. Tooyama, M. Urushitani
    NEUROSCIENCE 164 4 1565 - 1578 2009年12月 [無し][無し]
     研究論文（学術雑誌） 
    Nuclear exclusion of TAR DNA binding protein 43 (TDP-43) and formation of cytosolic aggregates are a pathological characteristic of amyotrophic lateral sclerosis (ALS). However, the molecular basis of the aberrant distribution of TDP-43 remains elusive. Here, we show evidence that axonal ligation induced transient nuclear exclusion and peripheral accumulation of TDP-43, without apparent cytosolic aggregates in hypoglossal neurons in mice. Immunohistochemistry showed marked loss of nuclear TDP-43 7-14 days after ligation, which was accompanied by reduction of choline acetyltransferase (ChAT). TDP-43 staining was restored in the nucleus on day 28 exclusively in the neurons with normalized ChAT expression. We also showed that importin beta, which was shown to mediate nuclear transport of TDP-43 was downregulated transiently by nerve ligation. The analysis of the peripheral nerves proximal to the ligation revealed that TDP-43 markedly accumulated with a concomitant decrease in active autophagosome. Moreover, we showed that TDP-43 was present in the microsome fraction containing endoplasmic reticulum (ER) or autophagosomes in the brainstem section, indicating that TDP-43 is axonally transported with vesicles. These results indicate that axonal damage is associated with redistribution of TDP-43 through the combination of defective axonal autophagy periphery and the impaired nuclear transport system in the soma. Moreover, it was also shown that transient redistribution of TDP-43 does not prevent motor neurons from axonal regeneration. Therefore, our data suggest that the subcellular distribution of TDP-43 correlates to the innervation status of motor neurons, which may be governed by unidentified cause of ALS. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Keisuke Oda, Satoshi Makino, Chiaki Masuda, Tatsuhiro Yoshiki, Yoshihisa Kitamura, Kazuyuki Takata, Daijiro Yanagisawa, Takashi Taniguchi, Ikuo Tooyama
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 57 12 1121 - 1126 2009年12月 [無し][無し]
     研究論文（学術雑誌） 
    The putative protein C7orf24 is encoded by Homo sapiens chromosome 7 open reading frame 24. C7orF24 was first identified as a 21-kDa cytochrome c-releasing factor detected in the cytosolic fraction of human leukemia U937 cells after treatment with geranylgeraniol. C7orf24 protein was recently identified as a gamma-glutamyl cyclotransferase, an enzyme in the gamma-glutamyl cycle. However, the exact localization of C7orf24 mRNA in normal tissues remains unknown. The present study examined the distribution pattern of C7orf24 mRNA in rat tissues using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. The RT-PCR experiments demonstrated that C7orf24 and a variant C7orf24 mRNA were expressed in various tissues. Quantitative RT-PCR analysis revealed significantly high levels of both C7orf24 mRNAs in the liver and kidney, compared with other tissues examined. In situ hybridization histochemistry localized C7orf24 mRNA to hepatocytes in the liver and renal tubules in the kidney. The present results thus implicated an important role for C7orf24 in liver and kidney. This manuscript contains online supplemental material at httpj/www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 57:1121-1126, 2009)
  • 新規アルツハイマー病画像診断薬の合成と19F-MRIによる脳内19Fシグナル検出のための検討
    雨坪 知音, 森川 茂廣, 柳沢 大治郎, 田口 弘康, 白井 伸明, 平尾 浩一, 加藤 文法, 木村 博彦, 森野 久弥, 吉田 稚加子, 遠山 育夫
    Dementia Japan 23 2 229 - 229 (一社)日本認知症学会 2009年08月
  • Essam Mohamed Abdelalim, Ikuo Tooyama
    PLOS ONE 4 4 e5341  2009年04月 [無し][無し]
     研究論文（学術雑誌） 
    Background: Embryonic stem (ES) cells have unlimited proliferation potential, and can differentiate into several cell types, which represent ideal sources for cell-based therapy. This high-level proliferative ability is attributed to an unusual type of cell cycle. The Signaling pathways that regulate the proliferation of ES cells are of great interest. Methodology/Principal Findings: In this study, we show that murine ES cells specifically express brain natriuretic peptide (BNP), and its signaling is essential for ES cell proliferation. We found that BNP and its receptor (NPR-A, natriuretic peptide receptor-A) were highly expressed in self-renewing murine ES cells, whereas the levels were markedly reduced after ES cell differentiation by the withdrawal of LIF. Targeting of BNP with short interfering RNA ( siRNA) resulted in the inhibition of ES cell proliferation, as indicated by a marked reduction in the cell number and colony size, a significant reduction in DNA synthesis, and decreased numbers of cells in S phase. BNP knockdown in ES cells led to the up-regulation of gamma-aminobutyric acid receptor A (GABAAR) genes, and activation of phosphorylated histone (gamma-H2AX), which negatively affects ES cell proliferation. In addition, knockdown of BNP increased the rate of apoptosis and reduced the expression of the transcription factor Ets-1. Conclusions/Significance: Appropriate BNP expression is essential for the maintenance of ES cell propagation. These findings establish BNP as a novel endogenous regulator of ES cell proliferation.
  • Essam Mohamed Abdelalim, Ikuo Tooyama
    PLOS ONE 4 4 e5341  2009年04月 [無し][無し]
     研究論文（学術雑誌） 
    Background: Embryonic stem (ES) cells have unlimited proliferation potential, and can differentiate into several cell types, which represent ideal sources for cell-based therapy. This high-level proliferative ability is attributed to an unusual type of cell cycle. The Signaling pathways that regulate the proliferation of ES cells are of great interest. Methodology/Principal Findings: In this study, we show that murine ES cells specifically express brain natriuretic peptide (BNP), and its signaling is essential for ES cell proliferation. We found that BNP and its receptor (NPR-A, natriuretic peptide receptor-A) were highly expressed in self-renewing murine ES cells, whereas the levels were markedly reduced after ES cell differentiation by the withdrawal of LIF. Targeting of BNP with short interfering RNA ( siRNA) resulted in the inhibition of ES cell proliferation, as indicated by a marked reduction in the cell number and colony size, a significant reduction in DNA synthesis, and decreased numbers of cells in S phase. BNP knockdown in ES cells led to the up-regulation of gamma-aminobutyric acid receptor A (GABAAR) genes, and activation of phosphorylated histone (gamma-H2AX), which negatively affects ES cell proliferation. In addition, knockdown of BNP increased the rate of apoptosis and reduced the expression of the transcription factor Ets-1. Conclusions/Significance: Appropriate BNP expression is essential for the maintenance of ES cell propagation. These findings establish BNP as a novel endogenous regulator of ES cell proliferation.
  • Atsushi Saito, Takashi Sato, Hiroyuki Okano, Ken-Ichiro Toyoda, Hitoshi Bamba, Shin Kimura, Jean-Pierre Bellier, Akinori Matsuo, Hiroshi Kimura, Yasuo Hisa, Ikuo Tooyama
    JOURNAL OF COMPARATIVE NEUROLOGY 513 2 237 - 248 2009年03月 [無し][無し]
     研究論文（学術雑誌） 
    Choline acetyltransferase of the peripheral type (pChAT) is a splice variant that lacks exons 6-9 of the common-type ChAT (cChAT); the role of pChAT remains unknown. We investigated the expression of pChAT and cChAT after axotomy to try to elucidate its function. In the dorsal motor nucleus of the vagus nerve (DMNV), nucleus ambiguus (NA), and hypoglossal nucleus (HN) of control rats, we observed neural expression of cChAT but no pChAT-positive neurons. Following nerve transection, we clearly detected pChAT-labeled neurons in the DMNV and weakly labeled neurons in the NA, but pChAT was not seen in the HN. In the DMNV, the mean number of cChAT-positive neurons decreased rapidly to 40.5% of control at 3 days post transection, and to 5.0% of control after 7 days. The number of cChAT-positive neurons then gradually increased and reached a plateau of about 25% of control value at 28 days post transection. pChAT-positive neurons did not appear until 7 days after transection. On the same day, pChAT mRNA was detected in the DMNV neurons by reverse transcription-polymerase chain reaction (RT-PCR) by using laser capture microdissection. The number of pChAT-positive neurons gradually decreased, and only 10% of the cholinergic neurons retained pChAT expression 56 days post transection. Double-immunofluorescence analysis showed that some of the DMNV neurons expressed both cChAT and pChAT upon recovery from axotomy. These results suggest that the expression of pChAT is associated with the regenerative or degenerative processes of motoneurons especially for general visceral efferents. J. Comp. Neurol. 513:237-248, 2009. (c) 2009 Wiley-Liss, Inc.
  • Li An, Haruhisha Sato, Yoshihiro Konishi, Douglas G. Walker, Thomas G. Beach, Joseph Rogers, Ikuo Tooyama
    NEUROSCIENCE LETTERS 452 3 277 - 280 2009年03月 [無し][無し]
     研究論文（学術雑誌） 
    We and others have previously reported that lactotransferrin (LF), acting both as an iron-binding protein and inflammatory modulator, is greatly up-regulated in the brain of patients with Alzheimer's disease (AD). However, it remains unknown which type of cells express LF in the brain of AD. In this study, therefore, we investigated the expression and localization of LF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) and in situ hybridization histochemistry. Real-time PCR demonstrated that LF mRNA expression in the cortex of AD cases was significantly greater than that in control cases. LF mRNA-positive granules were observed in the cortex by in situ hybridization histochemistry, and the number of positive granules was increased in AD cases compared to controls. The double staining technique of LF mRNA in situ hybridisation and D-related human leukocyte antigen (HLA-DR) immunohistochemistry revealed that positive granules were localized in a subpopulation of HLA-DR-positive reactive microglia. In addition, LF mRNA-positive granules were observed in some cells that were negative for HLA-DR. These cells were also negative for CD4 and CD8 but positive for leukocyte common antigen (CD45RB), suggesting they were monocytes/macrophages. These results indicate that reactive microglia, in the cerebral cortex and monocytes/macrophages infiltrating from the circulation might be responsible for synthesizing LF in AD brain. (c) 2009 Elsevier Ireland Ltd. All rights reserved.
  • Atsushi Saito, Takashi Sato, Hiroyuki Okano, Ken-Ichiro Toyoda, Hitoshi Bamba, Shin Kimura, Jean-Pierre Bellier, Akinori Matsuo, Hiroshi Kimura, Yasuo Hisa, Ikuo Tooyama
    JOURNAL OF COMPARATIVE NEUROLOGY 513 2 237 - 248 2009年03月 [無し][無し]
     研究論文（学術雑誌） 
    Choline acetyltransferase of the peripheral type (pChAT) is a splice variant that lacks exons 6-9 of the common-type ChAT (cChAT); the role of pChAT remains unknown. We investigated the expression of pChAT and cChAT after axotomy to try to elucidate its function. In the dorsal motor nucleus of the vagus nerve (DMNV), nucleus ambiguus (NA), and hypoglossal nucleus (HN) of control rats, we observed neural expression of cChAT but no pChAT-positive neurons. Following nerve transection, we clearly detected pChAT-labeled neurons in the DMNV and weakly labeled neurons in the NA, but pChAT was not seen in the HN. In the DMNV, the mean number of cChAT-positive neurons decreased rapidly to 40.5% of control at 3 days post transection, and to 5.0% of control after 7 days. The number of cChAT-positive neurons then gradually increased and reached a plateau of about 25% of control value at 28 days post transection. pChAT-positive neurons did not appear until 7 days after transection. On the same day, pChAT mRNA was detected in the DMNV neurons by reverse transcription-polymerase chain reaction (RT-PCR) by using laser capture microdissection. The number of pChAT-positive neurons gradually decreased, and only 10% of the cholinergic neurons retained pChAT expression 56 days post transection. Double-immunofluorescence analysis showed that some of the DMNV neurons expressed both cChAT and pChAT upon recovery from axotomy. These results suggest that the expression of pChAT is associated with the regenerative or degenerative processes of motoneurons especially for general visceral efferents. J. Comp. Neurol. 513:237-248, 2009. (c) 2009 Wiley-Liss, Inc.
  • Li An, Haruhisha Sato, Yoshihiro Konishi, Douglas G. Walker, Thomas G. Beach, Joseph Rogers, Ikuo Tooyama
    NEUROSCIENCE LETTERS 452 3 277 - 280 2009年03月 [無し][無し]
     研究論文（学術雑誌） 
    We and others have previously reported that lactotransferrin (LF), acting both as an iron-binding protein and inflammatory modulator, is greatly up-regulated in the brain of patients with Alzheimer's disease (AD). However, it remains unknown which type of cells express LF in the brain of AD. In this study, therefore, we investigated the expression and localization of LF messenger RNA (mRNA) in the cerebral cortex of AD and control cases using real-time polymerase chain reaction (PCR) and in situ hybridization histochemistry. Real-time PCR demonstrated that LF mRNA expression in the cortex of AD cases was significantly greater than that in control cases. LF mRNA-positive granules were observed in the cortex by in situ hybridization histochemistry, and the number of positive granules was increased in AD cases compared to controls. The double staining technique of LF mRNA in situ hybridisation and D-related human leukocyte antigen (HLA-DR) immunohistochemistry revealed that positive granules were localized in a subpopulation of HLA-DR-positive reactive microglia. In addition, LF mRNA-positive granules were observed in some cells that were negative for HLA-DR. These cells were also negative for CD4 and CD8 but positive for leukocyte common antigen (CD45RB), suggesting they were monocytes/macrophages. These results indicate that reactive microglia, in the cerebral cortex and monocytes/macrophages infiltrating from the circulation might be responsible for synthesizing LF in AD brain. (c) 2009 Elsevier Ireland Ltd. All rights reserved.
  • Tomone Amatsubo, Shigehiro Morikawa, Toshio Inubushi, Makoto Urushitani, Hiroyasu Taguchi, Nobuaki Shirai, Koichi Hirao, Masanari Kato, Kyuya Morino, Hirohiko Kimura, Ichiro Nakano, Chikako Yoshida, Takashi Okada, Mitsuo Sano, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 63 1 76 - 81 2009年01月 [無し][無し]
     研究論文（学術雑誌） 
    The chemical properties of probes that improve amyloid detection by non-invasive F-19 magnetic resonance imaging (MRI)are of interest. We synthesized benzoxazole compounds with trifluoromethoxy groups, and found that these compounds displayed sharp F-19 nuclear magnetic resonance (NMR) signals in an assay buffer. However, the intensities of the F-19 NMR signals were dramatically reduced in mouse brain lysates. Our results indicate that the inhibitory effect of brain tissue on the F-19 NMR signals from these probes can be attributed to the hydrophobicity of the tissue. These results highlight the importance of using hydrophilic F-19-MRI agents to avoid the inhibitory effects of brain tissues on F-19 NMR signals. (C) 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Oxidative stress induction of DJ-1 protein in reactive astrocytes scavengers free radicals and reduces cell injury
    Yanagida T, Tsushima J, Kitamura Y, Yanagisawa D, Takata K, Shibaike T, Yamamoto A, Tniguchi T, Ysui H, Tairs T, Morikawa S, Inubushi T, Tooyama I, Ariga H
    Oxid Med Cell Longev 2 1 36 - 42 2009年 [無し][無し]
     研究論文（学術雑誌）
  • Tomone Amatsubo, Shigehiro Morikawa, Toshio Inubushi, Makoto Urushitani, Hiroyasu Taguchi, Nobuaki Shirai, Koichi Hirao, Masanari Kato, Kyuya Morino, Hirohiko Kimura, Ichiro Nakano, Chikako Yoshida, Takashi Okada, Mitsuo Sano, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 63 1 76 - 81 2009年01月 [無し][無し]
     研究論文（学術雑誌） 
    The chemical properties of probes that improve amyloid detection by non-invasive F-19 magnetic resonance imaging (MRI)are of interest. We synthesized benzoxazole compounds with trifluoromethoxy groups, and found that these compounds displayed sharp F-19 nuclear magnetic resonance (NMR) signals in an assay buffer. However, the intensities of the F-19 NMR signals were dramatically reduced in mouse brain lysates. Our results indicate that the inhibitory effect of brain tissue on the F-19 NMR signals from these probes can be attributed to the hydrophobicity of the tissue. These results highlight the importance of using hydrophilic F-19-MRI agents to avoid the inhibitory effects of brain tissues on F-19 NMR signals. (C) 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Takashi Yanagida, Jun Tsushima, Yoshihisa Kitamura, Daijiro Yanagisawa, Kazuyuki Takata, Tomonori Shibaike, Atsuko Yamamoto, Takashi Taniguchi, Hiroyuki Yasui, Takahiro Taira, Shigehiro Morikawa, Toshihiro Inubushi, Ikuo Tooyama, Hiroyoshi Ariga
    OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2 1 36 - 42 2009年01月 [無し][無し]
     研究論文（学術雑誌） 
    Astrocytes, one of the predominant types of glial cells, function as both supportive and metabolic cells for the brain. Under cerebral ischemia/reperfusion-induced oxidative conditions, astrocytes accumulate and activate in the ischemic region. DJ-1 has recently been shown to be a sensor of oxidative stress in living cells. However, the function of astrocytic DJ-1 is still unknown. In the present study, to clarify the effect of astrocytic DJ-1 protein under massive oxidative insult, we used a focal ischemic rat model that had been subjected to middle cerebral artery occlusion (MCAO) and reperfusion. We then investigated changes in the distribution of DJ-1 in astrocytes, DJ-1 release from cultured astrocytes, and the effects of recombinant DJ-1 protein on hydrogen peroxide (H(2)O(2))- induced death in normal and DJ-1-knockdown SH-SY5Y cells and on in vitro scavenging of hydroxyl radicals ((center dot)OH) by electron spin resonance spectrometry. At 24 h after 2-h MCAO and reperfusion, an infarct lesion was markedly observed using magnetic resonance imaging and 2,3,5-triphenyltetrazolium chloride staining. In addition, reactive astrocytes enhanced DJ-1 expression in the penumbral zone of the ischemic core and that DJ-1 protein was extracellularly released from astrocytes by H(2)O(2) in in vitro primary cultures. Although DJ-1-knockdown SH-SY5Y cells were markedly vulnerable to oxidative stress, treatment with glutathione S-transferase-tagged recombinant human DJ-1 protein (GST-DJ-1) significantly inhibited H(2)O(2)-induced cell death. In addition, GST-DJ-1 protein directly scavenged (center dot)OH. These results suggest that oxidative stress induces the release of astrocytic DJ-1 protein, which may contribute to astrocyte-mediated neuroprotection.
  • Makoto Urushitani, Samer Abou Ezzi, Akinori Matsuo, Ikuo Tooyama, Jean-Pierre Julien
    FASEB JOURNAL 22 7 2476 - 2487 2008年07月 [無し][無し]
     研究論文（学術雑誌） 
    Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% cases of familial amyotrophic lateral sclerosis (ALS). However, the mechanism of motor neuron degeneration caused by ALS-linked SOD1 mutants is not fully understood. Here, we used novel live cell imaging techniques to demonstrate the subcellular localization of EGFP-fused SOD1 of both wild-type (WT) and ALS-linked mutant forms in the endoplasmic reticulum (ER) and Golgi. The presence of WT and mutant SOD1 species in luminal structures was further confirmed by immunoblotting analysis of microsomal fractions from spinal cord lysates of SOD1 transgenic mice prepared by sucrose density-gradient ultracentrifugation. Chemical cross-linking studies also revealed an age-dependent aggregation of mutant SOD1, but not of WT SOD1, prominently in the microsomal fraction. Cell-free translocation assays provided evidence that monomeric SOD1 is a molecular form that can be translocated into luminal structures in the presence of ATP. Our finding that the ER-Golgi pathway is a predominant cellular site of aggregation of mutant SOD1 suggests that secretion could play a key role in pathogenesis, which is in line with the view that the disease is non-cell autonomous.
  • E. M. Abdelalim, C. Masuda, J. P. Bellier, A. Saito, S. Yamamoto, N. Mori, I. Tooyama
    NEUROSCIENCE 155 1 192 - 202 2008年07月 [無し][無し]
     研究論文（学術雑誌） 
    The natriuretic peptide receptor type C (NPR-C) binds all natriuretic peptides. It is thought to be involved in the clearance of natriuretic peptides and more recently has been defined as essential for the neuromodulatory effects of natriuretic peptides. Although the distribution of NPR-C mRNA has been reported in the rat forebrain, there are no data on the distribution of NPR-C in the brainstem. We report an immunofluorescence study on the distribution of NPR-C immunoreactivity in the rat brainstem, and its presence in cholinergic and catecholaminergic neurons. NPR-C immunoreactivity was detected in several regions, including the periaqueductal gray, oculomotor nucleus, red nucleus and trochlear nucleus of the midbrain; the Pontine nucleus, dorsal tegmental nucleus, vestibular nucleus, locus coeruleus, trigeminal motor nucleus, nucleus of the trapezoid body, abducens nucleus and facial nucleus of the pons; and the dorsal motor nucleus of the vagus, hypoglossal nucleus, lateral reticular nucleus, nucleus ambiguus and inferior olivary nucleus of the medulla oblongata. Interestingly, NPR-C immunoreactivity was detected in the cholinergic neurons of the oculomotor nucleus, trochlear nucleus, dorsal tegmental nucleus, motor trigeminal nucleus, facial nucleus, dorsal motor nucleus of the vagus, nucleus ambiguus and hypoglossal nucleus. Furthermore, NPR-C immunoreactivity was detected in several catecholaminergic neuronal groups including the A6, A5, A1, C3 and C1 cell groups. These results are consistent with an important role for natriuretic peptides in neuroendocrine regulation and central cardiovascular integration. The extensive distribution of NPR-C in the brainstem supports the hypothesis that NPR-C is involved in the neuromodulatory effect of natriuretic peptides. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Makoto Urushitani, Samer Abou Ezzi, Akinori Matsuo, Ikuo Tooyama, Jean-Pierre Julien
    FASEB JOURNAL 22 7 2476 - 2487 2008年07月 [無し][無し]
     研究論文（学術雑誌） 
    Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% cases of familial amyotrophic lateral sclerosis (ALS). However, the mechanism of motor neuron degeneration caused by ALS-linked SOD1 mutants is not fully understood. Here, we used novel live cell imaging techniques to demonstrate the subcellular localization of EGFP-fused SOD1 of both wild-type (WT) and ALS-linked mutant forms in the endoplasmic reticulum (ER) and Golgi. The presence of WT and mutant SOD1 species in luminal structures was further confirmed by immunoblotting analysis of microsomal fractions from spinal cord lysates of SOD1 transgenic mice prepared by sucrose density-gradient ultracentrifugation. Chemical cross-linking studies also revealed an age-dependent aggregation of mutant SOD1, but not of WT SOD1, prominently in the microsomal fraction. Cell-free translocation assays provided evidence that monomeric SOD1 is a molecular form that can be translocated into luminal structures in the presence of ATP. Our finding that the ER-Golgi pathway is a predominant cellular site of aggregation of mutant SOD1 suggests that secretion could play a key role in pathogenesis, which is in line with the view that the disease is non-cell autonomous.
  • E. M. Abdelalim, C. Masuda, J. P. Bellier, A. Saito, S. Yamamoto, N. Mori, I. Tooyama
    NEUROSCIENCE 155 1 192 - 202 2008年07月 [無し][無し]
     研究論文（学術雑誌） 
    The natriuretic peptide receptor type C (NPR-C) binds all natriuretic peptides. It is thought to be involved in the clearance of natriuretic peptides and more recently has been defined as essential for the neuromodulatory effects of natriuretic peptides. Although the distribution of NPR-C mRNA has been reported in the rat forebrain, there are no data on the distribution of NPR-C in the brainstem. We report an immunofluorescence study on the distribution of NPR-C immunoreactivity in the rat brainstem, and its presence in cholinergic and catecholaminergic neurons. NPR-C immunoreactivity was detected in several regions, including the periaqueductal gray, oculomotor nucleus, red nucleus and trochlear nucleus of the midbrain; the Pontine nucleus, dorsal tegmental nucleus, vestibular nucleus, locus coeruleus, trigeminal motor nucleus, nucleus of the trapezoid body, abducens nucleus and facial nucleus of the pons; and the dorsal motor nucleus of the vagus, hypoglossal nucleus, lateral reticular nucleus, nucleus ambiguus and inferior olivary nucleus of the medulla oblongata. Interestingly, NPR-C immunoreactivity was detected in the cholinergic neurons of the oculomotor nucleus, trochlear nucleus, dorsal tegmental nucleus, motor trigeminal nucleus, facial nucleus, dorsal motor nucleus of the vagus, nucleus ambiguus and hypoglossal nucleus. Furthermore, NPR-C immunoreactivity was detected in several catecholaminergic neuronal groups including the A6, A5, A1, C3 and C1 cell groups. These results are consistent with an important role for natriuretic peptides in neuroendocrine regulation and central cardiovascular integration. The extensive distribution of NPR-C in the brainstem supports the hypothesis that NPR-C is involved in the neuromodulatory effect of natriuretic peptides. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Essam Mohamed Abdelalim, Chiaki Masuda, Ikuo Tooyama
    PEPTIDES 29 4 622 - 628 2008年04月 [無し][無し]
     研究論文（学術雑誌） 
    Recently, the natriuretic peptides were detected in the cholinergic and dopaminergic amacrine cells of the retina. We performed immunofluorescence labeling of rat retinal sections to examine the immunoreactivity of natriuretic peptide-activated guanylate cyclases (NPR-A and NPR-B) in the rat retina, in particular whether they were localized to dopaminergic and cholinergic amacrine cells. NPR-A and NPR-B immunoreactivity was detected in several layers of the retina including amacrine cells. In amacrine cells, both NPRA and NPR-B were co-localized with tyrosine hydroxylase, a marker of dopaminergic cells. NPR-B, but not NPR-A, was localized to amacrine cells expressing choline acetyltransferase (ChAT), a marker of cholinergic cells. These findings suggest that natriuretic peptides have different regulatory systems in dopaminergic and cholinergic amacrine cells in rat retina. (C) 2008 Elsevier Inc. All rights reserved.
  • Essam Mohamed Abdelalim, Chiaki Masuda, Ikuo Tooyama
    PEPTIDES 29 4 622 - 628 2008年04月 [無し][無し]
     研究論文（学術雑誌） 
    Recently, the natriuretic peptides were detected in the cholinergic and dopaminergic amacrine cells of the retina. We performed immunofluorescence labeling of rat retinal sections to examine the immunoreactivity of natriuretic peptide-activated guanylate cyclases (NPR-A and NPR-B) in the rat retina, in particular whether they were localized to dopaminergic and cholinergic amacrine cells. NPR-A and NPR-B immunoreactivity was detected in several layers of the retina including amacrine cells. In amacrine cells, both NPRA and NPR-B were co-localized with tyrosine hydroxylase, a marker of dopaminergic cells. NPR-B, but not NPR-A, was localized to amacrine cells expressing choline acetyltransferase (ChAT), a marker of cholinergic cells. These findings suggest that natriuretic peptides have different regulatory systems in dopaminergic and cholinergic amacrine cells in rat retina. (C) 2008 Elsevier Inc. All rights reserved.
  • Daijiro Yanagisawa, Yoshihisa Kitamura, Masatoshi Inden, Kazuyuki Takata, Takashi Taniguchi, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Ikuo Tooyama, Takahiro Taira, Sanae M. M. Iguchi-Ariga, Akinori Akaike, Hiroyoshi Ariga
    JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 28 3 563 - 578 2008年03月 [無し][無し]
     研究論文（学術雑誌） 
    Reactive oxygen species (ROS) is massively produced in the brain after cerebral ischemia and reperfusion. It reacts strongly with cellular components, which has detrimental effects and leads to neuronal cell death. DJ-1, which was found to be the causative gene of familial Parkinson's disease PARK7, is a multifunction protein, which plays a key role in transcriptional regulation, and a molecular chaperone. In this study, we investigated the neuroprotective effect of DJ-1 against neurodegeneration caused by ischemia/reperfusion injury. Cerebral ischemia was induced in rats by 120 mins of middle cerebral artery occlusion (MCAO) using an intraluminal introduction method. The intrastriatal injection of recombinant glutathione S-transferase-tagged human DJ-1 (GST-DJ-1) markedly reduced infarct size in 2,3,5-triphenyltetrazolium chloride staining at 3 days after MCAO. In addition, we performed a noninvasive evaluation of ischemic size using magnetic resonance imaging and found a significant reduction of infarct size with the administration of GST-DJ-1. In GST-DJ-1-treated rats, behavioral dysfunction and nitrotyrosine formation were significantly inhibited. Furthermore, GST-DJ-1 markedly inhibited H2O2-mediated ROS production in SH-SY5Y cells. These results indicate that GST-DJ-1 exerts a neuroprotective effect by reducing ROS-mediated neuronal injury, suggesting that DJ-1 may be a useful therapeutic target for ischemic neurodegeneration.
  • Daijiro Yanagisawa, Yoshihisa Kitamura, Masatoshi Inden, Kazuyuki Takata, Takashi Taniguchi, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Ikuo Tooyama, Takahiro Taira, Sanae M. M. Iguchi-Ariga, Akinori Akaike, Hiroyoshi Ariga
    JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM 28 3 563 - 578 2008年03月 [無し][無し]
     研究論文（学術雑誌） 
    Reactive oxygen species (ROS) is massively produced in the brain after cerebral ischemia and reperfusion. It reacts strongly with cellular components, which has detrimental effects and leads to neuronal cell death. DJ-1, which was found to be the causative gene of familial Parkinson's disease PARK7, is a multifunction protein, which plays a key role in transcriptional regulation, and a molecular chaperone. In this study, we investigated the neuroprotective effect of DJ-1 against neurodegeneration caused by ischemia/reperfusion injury. Cerebral ischemia was induced in rats by 120 mins of middle cerebral artery occlusion (MCAO) using an intraluminal introduction method. The intrastriatal injection of recombinant glutathione S-transferase-tagged human DJ-1 (GST-DJ-1) markedly reduced infarct size in 2,3,5-triphenyltetrazolium chloride staining at 3 days after MCAO. In addition, we performed a noninvasive evaluation of ischemic size using magnetic resonance imaging and found a significant reduction of infarct size with the administration of GST-DJ-1. In GST-DJ-1-treated rats, behavioral dysfunction and nitrotyrosine formation were significantly inhibited. Furthermore, GST-DJ-1 markedly inhibited H2O2-mediated ROS production in SH-SY5Y cells. These results indicate that GST-DJ-1 exerts a neuroprotective effect by reducing ROS-mediated neuronal injury, suggesting that DJ-1 may be a useful therapeutic target for ischemic neurodegeneration.
  • A. Saito, H. Okano, H. Bamba, Y. Hisa, Y. Oomura, T. Imamura, I. Tooyama
    HISTOLOGY AND HISTOPATHOLOGY 22 12 1327 - 1335 2007年12月 [無し][無し]
     研究論文（学術雑誌） 
    Fibroblast growth factor-1 (FGF1), a member of the FGF family of growth factors, is localized in cholinergic neurons where it has trophic activity. We recently reported that cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) contain little FGF1, raising the possibility that FGF1 is not localized to parasympathetic preganglionic cholinergic neurons. To clarify this issue, we investigated the co-localization of FGF1 with cholinergic neuron markers in the Edinger-Westphal nucleus (EWN), salivatory nucleus, DMNV, and sacral parasympathetic nucleus by double immunofluorescence using antibodies to FGF1 and choline acetyltransferase (ChAT). The neurons in the EWN were devoid of FGF1. In the salivatory nucleus, 13% of ChAT-positive neurons were also positive for FGF1. In the DMNV, only 8% of ChAT-positive neurons contained FGF1, and in the sacral parasympathetic nucleus, 18% of ChAT-positive neurons were FGF1-positive. We also confirmed that a large number of ChAT-positive motor neurons in the oculomotor nucleus, facial nucleus, hypoglossal nucleus, and spinal motor neurons contained FGF1. The results confirmed that parasympathetic preganglionic neurons are largely devoid of FGF1, which is a unique feature among cholinergic neurons.
  • A. Saito, H. Okano, H. Bamba, Y. Hisa, Y. Oomura, T. Imamura, I. Tooyama
    HISTOLOGY AND HISTOPATHOLOGY 22 12 1327 - 1335 2007年12月 [無し][無し]
     研究論文（学術雑誌） 
    Fibroblast growth factor-1 (FGF1), a member of the FGF family of growth factors, is localized in cholinergic neurons where it has trophic activity. We recently reported that cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) contain little FGF1, raising the possibility that FGF1 is not localized to parasympathetic preganglionic cholinergic neurons. To clarify this issue, we investigated the co-localization of FGF1 with cholinergic neuron markers in the Edinger-Westphal nucleus (EWN), salivatory nucleus, DMNV, and sacral parasympathetic nucleus by double immunofluorescence using antibodies to FGF1 and choline acetyltransferase (ChAT). The neurons in the EWN were devoid of FGF1. In the salivatory nucleus, 13% of ChAT-positive neurons were also positive for FGF1. In the DMNV, only 8% of ChAT-positive neurons contained FGF1, and in the sacral parasympathetic nucleus, 18% of ChAT-positive neurons were FGF1-positive. We also confirmed that a large number of ChAT-positive motor neurons in the oculomotor nucleus, facial nucleus, hypoglossal nucleus, and spinal motor neurons contained FGF1. The results confirmed that parasympathetic preganglionic neurons are largely devoid of FGF1, which is a unique feature among cholinergic neurons.
  • E. M. Abdelalim, A. H. K. Osman, T. Takada, R. Torii, I. Tooyama
    NEUROSCIENCE 145 3 1087 - 1096 2007年03月 [有り][無し]
     研究論文（学術雑誌） 
    Natriuretic peptide receptor-A (NPR-A) mediates the biological effects of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and is involved in maintaining cardiovascular homeostasis. In this immunohistochemical study we examined the distribution of NPR-A in the brainstem of the cynomoigus monkey. NPR-A immunoreactivity was localized to neurons in specific brainstem regions. NPR-A-immunoreactive perikarya were found in the red nucleus and the oculomotor nucleus in the midbrain, the parabrachial nucleus and the locus coeruleus in the pons, and the dorsal motor nucleus of the vagus, the hypoglossal nucleus, the cuneate nucleus, the gracile nucleus, the nucleus ambiguus, the lateral reticular nucleus, the reticular formation, and the inferior olivary nucleus in the medulla oblongata. Extensive networks of immunoreactive fibers were apparent in the red nucleus, the oculomotor nucleus, the principal sensory trigeminal nucleus, and the parabrachial nucleus. Double immunostaining revealed NPR-A immunoreactivity in cholinergic neurons of the parabrachial nucleus, the dorsal motor nucleus of vagus, the hypoglossal nucleus, and the nucleus ambiguus. However, there was no colocalization of NPR-A and tyrosine hydroxylase in the locus coeruleus. The wide anatomical distribution of NPR-A-immunoreactive structures suggests that natriuretic peptides, besides having a role in the central regulation of endocrine and cardiovascular homeostasis, may also mediate diverse physiological functions. (c) 2007 IBRO. Published by Elsevier Ltd. All rights reserved.
  • Satoshi Makino, Ryuji Kaji, Satoshi Ando, Maiko Tomizawa, Katsuhito Yasuno, Satoshi Goto, Shinnichi Matsumoto, Ma. Daisy Tabuena, Elma Maranon, Marita Dantes, Lillian V. Lee, Kazumasa Ogasawara, Ikuo Tooyama, Hiroyasu Akatsu, Masataka Nishimura, Gen Tamiya
    AMERICAN JOURNAL OF HUMAN GENETICS 80 3 393 - 406 2007年03月 [無し][無し]
     研究論文（学術雑誌） 
    X-linked dystonia- parkinsonism (XDP) is a movement disorder endemic to the Philippines. The disease locus, DYT3, has been mapped to Xq13.1. In a search for the causative gene, we performed genomic sequencing analysis, followed by expression analysis of XDP brain tissues. We found a disease- specific SVA ((s) under bar hort interspersed nuclear element, (v) under bar ariable number of tandem repeats, and (A) under bar lu composite) retrotransposon insertion in an intron of the TATA- binding protein-associated factor 1 gene (TAF1), which encodes the largest component of the TFIID complex, and significantly decreased expression levels of TAF1 and the dopamine receptor D2 gene (DRD2) in the caudate nucleus. We also identified an abnormal pattern of DNA methylation in the retrotransposon in the genome from the patient's caudate, which could account for decreased expression of TAF1. Our findings suggest that the reduced neuron-specific expression of the TAF1 gene is associated with XDP.
  • Satoshi Makino, Ryuji Kaji, Satoshi Ando, Maiko Tomizawa, Katsuhito Yasuno, Satoshi Goto, Shinnichi Matsumoto, Ma. Daisy Tabuena, Elma Maranon, Marita Dantes, Lillian V. Lee, Kazumasa Ogasawara, Ikuo Tooyama, Hiroyasu Akatsu, Masataka Nishimura, Gen Tamiya
    AMERICAN JOURNAL OF HUMAN GENETICS 80 3 393 - 406 2007年03月 [無し][無し]
     研究論文（学術雑誌） 
    X-linked dystonia- parkinsonism (XDP) is a movement disorder endemic to the Philippines. The disease locus, DYT3, has been mapped to Xq13.1. In a search for the causative gene, we performed genomic sequencing analysis, followed by expression analysis of XDP brain tissues. We found a disease- specific SVA ((s) under bar hort interspersed nuclear element, (v) under bar ariable number of tandem repeats, and (A) under bar lu composite) retrotransposon insertion in an intron of the TATA- binding protein-associated factor 1 gene (TAF1), which encodes the largest component of the TFIID complex, and significantly decreased expression levels of TAF1 and the dopamine receptor D2 gene (DRD2) in the caudate nucleus. We also identified an abnormal pattern of DNA methylation in the retrotransposon in the genome from the patient's caudate, which could account for decreased expression of TAF1. Our findings suggest that the reduced neuron-specific expression of the TAF1 gene is associated with XDP.
  • Kazuyuki Takata, Yoshihisa Kitamura, Daijiro Yanagisawa, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Daiju Tsuchiya, Saori Chishiro, Mana Saeki, Takashi Taniguchi, Shun Shimohama, Ikuo Tooyama
    FEBS LETTERS 581 3 475 - 478 2007年02月 [無し][無し]
     研究論文（学術雑誌） 
    Immunization with amyloid-beta (A beta) peptides, a therapeutic approach in Alzheimer's disease (AD), reduces brain A beta, and microglial A beta phagocytosis has been proposed as an AD-lowering mechanism. We transplanted rat microglia into the rat lateral ventricle just after intra-hippocampal A beta injection, and then investigated the contribution of exogenous microglia to A beta clearance. Migration of exogenous microglia from the lateral ventricle to A beta plaque was detected by magnetic resonance imaging and histochemical analysis, and the clearance of A beta was increased by transplantation. These results suggest the possible usefulness of exogenous microglia to the therapeutic approach in AD. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Kazuyuki Takata, Yoshihisa Kitamura, Daijiro Yanagisawa, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Daiju Tsuchiya, Saori Chishiro, Mana Saeki, Takashi Taniguchi, Shun Shimohama, Ikuo Tooyama
    FEBS LETTERS 581 3 475 - 478 2007年02月 [無し][無し]
     研究論文（学術雑誌） 
    Immunization with amyloid-beta (A beta) peptides, a therapeutic approach in Alzheimer's disease (AD), reduces brain A beta, and microglial A beta phagocytosis has been proposed as an AD-lowering mechanism. We transplanted rat microglia into the rat lateral ventricle just after intra-hippocampal A beta injection, and then investigated the contribution of exogenous microglia to A beta clearance. Migration of exogenous microglia from the lateral ventricle to A beta plaque was detected by magnetic resonance imaging and histochemical analysis, and the clearance of A beta was increased by transplantation. These results suggest the possible usefulness of exogenous microglia to the therapeutic approach in AD. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Ikuo Tooyama, Hiroyasu Taguchi, Shigehiro Morikawa, Toshiro Inubushi
    Journal of Brain Science 33 1 12 - 17 2007年 研究論文（国際会議プロシーディングス） 
    The therapeutic use of microglial cells has received some attention for the treatment of Alzheimer's disease. However, few non-invasive techniques exist for monitoring the cells after administration. Magnetic resonance imaging (MRI) may provide a useful tool for non-invasive tracking of transplanted cells. The present study used MRI technologies for track microglia injected intra-arterially or intra-ventricularly in rat models of Alzheimer's disease. The β-amyloid peptide aβ1-42 (Aβ42) was microinjected into the left hippocampus of rats and saline into the right hippocampus. Microglia were labeled with a superparamagnetic iron oxide (Resovist) and then administered into either the carotid artery or the lateral ventricle. MRI revealed clear signal changes attributable to the Resovist-containing microglia that gathered in the Aβ42-injected areas. Histochemistry demonstrated that Resovist-positive microglia had accumulated around the Aβ deposits and had internalized some of the peptide. The resultant amounts of Aβ42 injected into the rat hippocampi were then measured with and without the microglial transplantation into the lateral ventricle. Although the amounts of Aβ42 were gradually reduced, even in the control rats, the reductions were significantly enhanced by the transplantation. Thus, the Resovist-labeled exogenous microglia can migrate from the lateral ventricle to the Aβ42-injected site and phagocytose the Aβ42. This study has demonstrated that MRJ is effective for the non-invasive monitoring of exogenous microglia, and that these microglia/macrophages could provide a promising future as therapeutic tools for Alzheimer's disease.
  • Junichi Maki, Chiaki Masuda, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Yoshitaka Matsusue, Hiroyasu Taguchi, Ikuo Tooyama
    BIOMATERIALS 28 3 434 - 440 2007年01月 [無し][無し]
     研究論文（学術雑誌） 
    Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. However, since SPIOs are negative contrast agents it is difficult to track transplanted cells in bone and cartilage that originally display low signals. In this study, we examined the feasibility of tracking with fluorescein isothiocyanate (FITC)-labeled poly-L-lysine-CF3 (PLK-CF3) using mouse ATDC5 cells, a stein cell line of bone and cartilage cells. FITC-labeled PLK-CF3 was easily internalized by ATDC5 cells by adding it into culture medium. No acute or long-term toxicities were seen at less than 160 mu g/ml. Labeled cells transplanted into the cranial bone of mice were detected for at least 7 days by MR images. FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking in bone and cartilage. (c) 2006 Elsevier Ltd. All rights reserved.
  • Shin Kimura, Jean-Pierre Bellier, Akinori Matsuo, Ikuo Tooyama, Hiroshi Kimura
    NEUROCHEMISTRY INTERNATIONAL 50 1 251 - 255 2007年01月 [無し][無し]
     研究論文（学術雑誌） 
    To produce antibodies that permit the immunohistochemical discrimination of choline acetyltransferase of the common type (cChAT) from its splice variant of a peripheral type (pChAT), we immunized rabbits with a cChAT specific recombinant protein encoded by ChAT exons 7 and 8 of the rat cChAT gene. Successful antibody production was proved by Western blotting on rat brain and on HEK293 cells expressing green fluorescent protein (GFP), cChAT-GFP and pChAT-GFP. By immunohistochemistry our antiserum clearly labeled known cholinergic structures in rat brain, but gave no positive staining in the trigeminal ganglion which contained many neurons positive with pChAT antiserum. (c) 2006 Elsevier Ltd. All rights reserved.
  • Essam M. Abdelalim, Abdel-Hamid K. Osman, Tatsuyuki Takada, Ryuzo Torii, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 58 S219 - S219 2007年 [無し][無し]
     研究論文（学術雑誌）
  • Shigehiro Morikawa, Toshiro Inubushi, Masahito Morita, Koichiro Murakami, Chiaki Masuda, Jun-ichi Maki, Ikuo Tooyama
    Magnetic Resonance in Medical Sciences 6 4 235 - 240 2007年 [無し][無し]
     研究論文（学術雑誌） 
    We investigated the eFfects of fast recovery (FR) to increase the sensitivity of fiuorine-19 (19F) fast spin echo (FSE) in mapping 5-fiuorouracil (5-FU) and its metabolites. We added an additional 90°pulse (which fiips back longitudinal magnetization at the end of the sequence) to the chemical shift selective 19F FSE pulse sequence. In 5-FU solution, FR remarkably improved the signal-to-noise (S/N) ratio of 19F 5-FU images, having higher eFfects with shorter repetition time and smaller echo train numbers. In animal studies, FR produced a conspicuous increase in 19F signals in the urinary bladder. FR eFfects for 19F signals in the liver were smaller than those in other organs but still substantial. Utilization of FR in 19F FSE images promises more sensitive observation of 19F metabolite maps of 5-FU and other 19F-containing compounds that have relatively long relaxation times.
  • Junichi Maki, Chiaki Masuda, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Yoshitaka Matsusue, Hiroyasu Taguchi, Ikuo Tooyama
    BIOMATERIALS 28 3 434 - 440 2007年01月 [無し][無し]
     研究論文（学術雑誌） 
    Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. However, since SPIOs are negative contrast agents it is difficult to track transplanted cells in bone and cartilage that originally display low signals. In this study, we examined the feasibility of tracking with fluorescein isothiocyanate (FITC)-labeled poly-L-lysine-CF3 (PLK-CF3) using mouse ATDC5 cells, a stein cell line of bone and cartilage cells. FITC-labeled PLK-CF3 was easily internalized by ATDC5 cells by adding it into culture medium. No acute or long-term toxicities were seen at less than 160 mu g/ml. Labeled cells transplanted into the cranial bone of mice were detected for at least 7 days by MR images. FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking in bone and cartilage. (c) 2006 Elsevier Ltd. All rights reserved.
  • Shin Kimura, Jean-Pierre Bellier, Akinori Matsuo, Ikuo Tooyama, Hiroshi Kimura
    NEUROCHEMISTRY INTERNATIONAL 50 1 251 - 255 2007年01月 [無し][無し]
     研究論文（学術雑誌） 
    To produce antibodies that permit the immunohistochemical discrimination of choline acetyltransferase of the common type (cChAT) from its splice variant of a peripheral type (pChAT), we immunized rabbits with a cChAT specific recombinant protein encoded by ChAT exons 7 and 8 of the rat cChAT gene. Successful antibody production was proved by Western blotting on rat brain and on HEK293 cells expressing green fluorescent protein (GFP), cChAT-GFP and pChAT-GFP. By immunohistochemistry our antiserum clearly labeled known cholinergic structures in rat brain, but gave no positive staining in the trigeminal ganglion which contained many neurons positive with pChAT antiserum. (c) 2006 Elsevier Ltd. All rights reserved.
  • Essam M. Abdelalim, Abdel-Hamid K. Osman, Tatsuyuki Takada, Ryuzo Torii, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 58 S219 - S219 2007年 [無し][無し]
     研究論文（学術雑誌）
  • Shigehiro Morikawa, Toshiro Inubushi, Masahito Morita, Koichiro Murakami, Chiaki Masuda, Jun-ichi Maki, Ikuo Tooyama
    Magnetic Resonance in Medical Sciences 6 4 235 - 240 2007年 [無し][無し]
     研究論文（学術雑誌） 
    We investigated the eFfects of fast recovery (FR) to increase the sensitivity of fiuorine-19 (19F) fast spin echo (FSE) in mapping 5-fiuorouracil (5-FU) and its metabolites. We added an additional 90°pulse (which fiips back longitudinal magnetization at the end of the sequence) to the chemical shift selective 19F FSE pulse sequence. In 5-FU solution, FR remarkably improved the signal-to-noise (S/N) ratio of 19F 5-FU images, having higher eFfects with shorter repetition time and smaller echo train numbers. In animal studies, FR produced a conspicuous increase in 19F signals in the urinary bladder. FR eFfects for 19F signals in the liver were smaller than those in other organs but still substantial. Utilization of FR in 19F FSE images promises more sensitive observation of 19F metabolite maps of 5-FU and other 19F-containing compounds that have relatively long relaxation times.
  • Chiaki Masuda, Zyunichi Maki, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Yoshitaka Matsusue, Sonoko Yamagata, Hiroyasu Taguchi, Yukio Doi, Nobuaki Shirai, Koichi Hirao, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 56 2 224 - 228 2006年10月 [無し][無し]
     研究論文（学術雑誌） 
    Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. Since, however, SPIOs are negative contrast agents, positive agents have been explored. In this study, we examined the feasibility of FITC-labeled poly-L-lysine-CF3 (PLK-CF3) using glial cells. FITC-labeled PLK-CF3 was easily internalized by neuroblastoma cells and glia as adding it into culture medium. No toxicity was seen at the concentration of less than 80 mu g/ml. MR images positively detected labeled cells transplanted in the brain of living mouse. The results indicate that FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Chiaki Masuda, Zyunichi Maki, Shigehiro Morikawa, Masahito Morita, Toshiro Inubushi, Yoshitaka Matsusue, Sonoko Yamagata, Hiroyasu Taguchi, Yukio Doi, Nobuaki Shirai, Koichi Hirao, Ikuo Tooyama
    NEUROSCIENCE RESEARCH 56 2 224 - 228 2006年10月 [無し][無し]
     研究論文（学術雑誌） 
    Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. Since, however, SPIOs are negative contrast agents, positive agents have been explored. In this study, we examined the feasibility of FITC-labeled poly-L-lysine-CF3 (PLK-CF3) using glial cells. FITC-labeled PLK-CF3 was easily internalized by neuroblastoma cells and glia as adding it into culture medium. No toxicity was seen at the concentration of less than 80 mu g/ml. MR images positively detected labeled cells transplanted in the brain of living mouse. The results indicate that FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
  • Hiroyuki Okano, Ken-ichiro Toyoda, Hitoshi Bamba, Yasuo Hisa, Yutaka Oomura, Toru Imamura, Shoei Furukawa, Hiroshi Kimura, Ikuo Tooyama
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 54 9 1061 - 1071 2006年09月 [無し][無し]
     研究論文（学術雑誌） 
    Cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) are particularly vulnerable to laryngeal nerve damage, possibly because they lack fibroblast growth factor-1 (FGF1). To test this hypothesis, we investigated the localization of FGH in cholinergic neurons innervating the rat larynx by immunohistochemistry using central-type antibodies to choline acetyltransferase (cChAT) and peripheral type (pChAT) antibodies, as well as tracer experiments. In the DMNV, only 9% of cChAT-positive neurons contained FGF1, and 71% of FGF1-positive neurons colocalized with cChAT. In the nucleus ambiguus, 100% of cChAT-positive neurons were FGE1 positive. In the intralaryngeal ganglia, all ganglionic neurons contained both pChAT and FGF1. In the nodose ganglia, 66% of pChAT-positive neurons were also positive for FGF1, and 90% of FGF1-positive ganglionic cells displayed pChAT immunoreactivity. Neuronal tracing using cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV and nucleus ambiguus to the superior laryngeal nerve were FGF1 negative and FGF1 positive, respectively. In the nodose ganglia, some FGF1-positive cells were labeled with CTb. The results indicate that for innervation of the rat larynx, FGF1 is localized to motor neurons, postganglionic parasympathetic neurons, and sensory neurons, but expression is very low in preganglionic parasympathetic cholinergic neurons.
  • Hiroyuki Okano, Ken-ichiro Toyoda, Hitoshi Bamba, Yasuo Hisa, Yutaka Oomura, Toru Imamura, Shoei Furukawa, Hiroshi Kimura, Ikuo Tooyama
    JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY 54 9 1061 - 1071 2006年09月 [無し][無し]
     研究論文（学術雑誌） 
    Cholinergic neurons in the dorsal motor nucleus of the vagus (DMNV) are particularly vulnerable to laryngeal nerve damage, possibly because they lack fibroblast growth factor-1 (FGF1). To test this hypothesis, we investigated the localization of FGH in cholinergic neurons innervating the rat larynx by immunohistochemistry using central-type antibodies to choline acetyltransferase (cChAT) and peripheral type (pChAT) antibodies, as well as tracer experiments. In the DMNV, only 9% of cChAT-positive neurons contained FGF1, and 71% of FGF1-positive neurons colocalized with cChAT. In the nucleus ambiguus, 100% of cChAT-positive neurons were FGE1 positive. In the intralaryngeal ganglia, all ganglionic neurons contained both pChAT and FGF1. In the nodose ganglia, 66% of pChAT-positive neurons were also positive for FGF1, and 90% of FGF1-positive ganglionic cells displayed pChAT immunoreactivity. Neuronal tracing using cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV and nucleus ambiguus to the superior laryngeal nerve were FGF1 negative and FGF1 positive, respectively. In the nodose ganglia, some FGF1-positive cells were labeled with CTb. The results indicate that for innervation of the rat larynx, FGF1 is localized to motor neurons, postganglionic parasympathetic neurons, and sensory neurons, but expression is very low in preganglionic parasympathetic cholinergic neurons.
  • Y Song, S Morikawa, M Morita, T Inubushi, T Takada, R Toril, Y Kitamura, T Taniguchi, Tooyama, I
    HISTOLOGY AND HISTOPATHOLOGY 21 7 705 - 711 2006年07月 [無し][無し]
     研究論文（学術雑誌） 
    The therapeutic use of microglial cells has recently received some attention for the treatment of Alzheimer disease (AD), but few non-invasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technique for tracking microglia injected intra-arterially in vivo. We micro-injected A beta 42 into the left hippocampus and saline into the right hippocampus of rats. We then administered microglia, which were labeled with enhanced green fluorescent protein (EGFP) gene and Resovist, into the carotid artery. After monitoring exogenously administered microglia using MRI, we compared the MR images and the histochemical localization of administered microglia. MRI revealed clear signal changes attributable to Resovist-containing microglia in A beta-injected areas. Histochemistry demonstrated that EGFP-positive microglia accumulated around A beta deposits and internalized the peptide. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for AD.
  • EM Abdelalim, T Takada, R Torii, Tooyama, I
    PEPTIDES 27 7 1886 - 1893 2006年07月 [無し][無し]
     研究論文（学術雑誌） 
    Previous physiological studies have suggested central roles of brain natriuretic peptide (BNP). However, little information is available about the localization of BNP in the brain. In this study, we determined cDNA sequence encoding the entire coding region of prepro-BNP of Japanese and cynomologus monkeys, and then examined the immunohistochemical localization of BNP in the monkey hypothalamus. Japanese and cynomologus monkey prepro-BNP consisted of 132 amino acid residues with biologically active C-terminal 32 amino acids. Comparisons of deduced amino acid sequences among different species revealed high homology between monkey and human (91% in prerpro-BNP and 97% in the mature region). Immunohistochemical examination showed that BNP immunoreactive dots were observed in the paraventricular, periventricular, and supraoptic nuclei of the monkey hypothalamus. The present result suggests the central role of BNP in the neuroendocrine system in the hypothalamus. (c) 2006 Elsevier Inc. All rights reserved.
  • Y Song, S Morikawa, M Morita, T Inubushi, T Takada, R Toril, Y Kitamura, T Taniguchi, Tooyama, I
    HISTOLOGY AND HISTOPATHOLOGY 21 7 705 - 711 2006年07月 [無し][無し]
     研究論文（学術雑誌） 
    The therapeutic use of microglial cells has recently received some attention for the treatment of Alzheimer disease (AD), but few non-invasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technique for tracking microglia injected intra-arterially in vivo. We micro-injected A beta 42 into the left hippocampus and saline into the right hippocampus of rats. We then administered microglia, which were labeled with enhanced green fluorescent protein (EGFP) gene and Resovist, into the carotid artery. After monitoring exogenously administered microglia using MRI, we compared the MR images and the histochemical localization of administered microglia. MRI revealed clear signal changes attributable to Resovist-containing microglia in A beta-injected areas. Histochemistry demonstrated that EGFP-positive microglia accumulated around A beta deposits and internalized the peptide. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for AD.
  • EM Abdelalim, T Takada, R Torii, Tooyama, I
    PEPTIDES 27 7 1886 - 1893 2006年07月 [無し][無し]
     研究論文（学術雑誌） 
    Previous physiological studies have suggested central roles of brain natriuretic peptide (BNP). However, little information is available about the localization of BNP in the brain. In this study, we determined cDNA sequence encoding the entire coding region of prepro-BNP of Japanese and cynomologus monkeys, and then examined the immunohistochemical localization of BNP in the monkey hypothalamus. Japanese and cynomologus monkey prepro-BNP consisted of 132 amino acid residues with biologically active C-terminal 32 amino acids. Comparisons of deduced amino acid sequences among different species revealed high homology between monkey and human (91% in prerpro-BNP and 97% in the mature region). Immunohistochemical examination showed that BNP immunoreactive dots were observed in the paraventricular, periventricular, and supraoptic nuclei of the monkey hypothalamus. The present result suggests the central role of BNP in the neuroendocrine system in the hypothalamus. (c) 2006 Elsevier Inc. All rights reserved.
  • EM Abdelalim, T Takada, F Toyoda, M Omatsu-Kanbe, H Matsuura, Tooyama, I, R Torii
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 340 2 689 - 695 2006年02月 [無し][無し]
     研究論文（学術雑誌） 
    Functional characterization of ES cell-derived cardiomyocytes is important for differentiation control and application to the cell therapy. One of the crucial functions of cardiomyocytes is a production of atrial and brain natriuretic peptides (ANP and BNP, respectively), which have important endocrine, autocrine, and paracrine functions. In this study, we focused on the functional aspect of the cardiomyocytes differentiated from monkey ES cells in vitro and investigated the expression of ANP and BNP. Spontaneously contracting cells showed nodal-like action potentials, and expression of ANP and BNP by RT-PCR and immunocytochemistry. Interestingly, ANP and BNP expressions were detected as immunoreactive granules in the perinuclear area and these signals appeared to co-localize with trans-Golgi network. These findings suggest that monkey ES cells were able to differentiate into cardiomyocytes with functional characteristics in vitro and therefore can be used as a useful model to study mechanisms and functions in early cardiogenesis. (c) 2005 Elsevier Inc. All rights reserved.
  • EM Abdelalim, T Takada, F Toyoda, M Omatsu-Kanbe, H Matsuura, Tooyama, I, R Torii
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 340 2 689 - 695 2006年02月 [無し][無し]
     研究論文（学術雑誌） 
    Functional characterization of ES cell-derived cardiomyocytes is important for differentiation control and application to the cell therapy. One of the crucial functions of cardiomyocytes is a production of atrial and brain natriuretic peptides (ANP and BNP, respectively), which have important endocrine, autocrine, and paracrine functions. In this study, we focused on the functional aspect of the cardiomyocytes differentiated from monkey ES cells in vitro and investigated the expression of ANP and BNP. Spontaneously contracting cells showed nodal-like action potentials, and expression of ANP and BNP by RT-PCR and immunocytochemistry. Interestingly, ANP and BNP expressions were detected as immunoreactive granules in the perinuclear area and these signals appeared to co-localize with trans-Golgi network. These findings suggest that monkey ES cells were able to differentiate into cardiomyocytes with functional characteristics in vitro and therefore can be used as a useful model to study mechanisms and functions in early cardiogenesis. (c) 2005 Elsevier Inc. All rights reserved.
  • Ken-ichiro Toyoda, Hiroyuki Okano, Hitoshi Bamba, Yasuo Hisa, Yutaka Oomura, Toru Imamura, Shoei Furukawa, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 39 1 1 - 7 2006年 [無し][無し]
     研究論文（学術雑誌） 
    Neurons in the dorsal motor nucleus of the vagus (DMNV) are more severely affected by axonal injury than most other nerves, such as those of the hypoglossal nucleus. However, the mechanism underlying such a response remains unclear. In this study, we compared the expression of fibroblast growth factor 1 (FGF1), a neurotrophic factor, between the DMNV and the hypoglossal nucleus by RT-PCR and immunohistochemical analyses. RT-PCR showed that the level of FGF1 mRNA expression in the DMNV was lower than that in the hypoglossal nucleus (P < 0.01). Immunohistochemistry revealed that FGF1 was localized to neurons. FGF1-positive neurons in large numbers were evenly distributed in the hypoglossal nucleus, whereas FGF1-positive neurons were located in the lateral part of the DMNV. Double immunostaining for FGF1 and choline acetyltransferase demonstrated that 22.7% and 78% of cholinergic neurons were positive for FGF1 in the DMNV and hypoglossal nucleus, respectively. A tracing study with cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV to the superior laryngeal nerve were FGF1-negative. The results suggest that the low expression of FGF1 in the DMNV is due to severe damage of neurons in the DMNV.
  • Hiroyasu Akatsu, Hidehisa D. Yamagata, Jun Kawamata, Kouzin Kamino, Masatoshi Takeda, Takayuki Yamamoto, Tetsuro Miki, Ikuo Tooyama, Shun Shimohama, Kenji Kosaka
    DEMENTIA AND GERIATRIC COGNITIVE DISORDERS 22 3 216 - 222 2006年 [無し][無し]
     研究論文（学術雑誌） 
    Background/Aim: Brain-derived neurotrophic factor ( BDNF) is associated with the hippocampus and the nigrostriatal dopaminergic function. Data showing that its level was reduced in Alzheimer's disease ( AD) and Parkinson's disease (PD) suggested that the BDNF function must play an important role in the pathogenetics of these diseases. Indeed, variation in the BDNF gene may confer susceptibility to AD and PD development. Recently, a functional BDNF Val66Met polymorphism has been found to be associated with episodic memory and hippocampal function, with intracellular trafficking, and with activity-dependent secretion of BDNF. To date, there have been several conflicting reports on the correlation between AD or PD and Val66Met or C270T polymorphism in the BDNF promoter region, although no data on this relationship have been published with respect to dementia with Lewy bodies (DLB). In the present study, we investigated a possible association between such BDNF polymorphisms and susceptibility to AD or DLB. Methods: BDNF genotyping was carried out by the polymerase chain reaction-restriction fragment length polymorphism method in autopsy-confirmed human samples. Results and Conclusion: On comparing patients and controls, the distribution of BDNF genotypes and alleles did not differ significantly. Our findings suggest that it is unlikely that these BDNF polymorphisms play a major role in the pathogenesis of AD and DLB in the Japanese population. Copyright (C) 2006 S. Karger AG, Basel.
  • Ken-ichiro Toyoda, Hiroyuki Okano, Hitoshi Bamba, Yasuo Hisa, Yutaka Oomura, Toru Imamura, Shoei Furukawa, Ikuo Tooyama
    ACTA HISTOCHEMICA ET CYTOCHEMICA 39 1 1 - 7 2006年 [無し][無し]
     研究論文（学術雑誌） 
    Neurons in the dorsal motor nucleus of the vagus (DMNV) are more severely affected by axonal injury than most other nerves, such as those of the hypoglossal nucleus. However, the mechanism underlying such a response remains unclear. In this study, we compared the expression of fibroblast growth factor 1 (FGF1), a neurotrophic factor, between the DMNV and the hypoglossal nucleus by RT-PCR and immunohistochemical analyses. RT-PCR showed that the level of FGF1 mRNA expression in the DMNV was lower than that in the hypoglossal nucleus (P < 0.01). Immunohistochemistry revealed that FGF1 was localized to neurons. FGF1-positive neurons in large numbers were evenly distributed in the hypoglossal nucleus, whereas FGF1-positive neurons were located in the lateral part of the DMNV. Double immunostaining for FGF1 and choline acetyltransferase demonstrated that 22.7% and 78% of cholinergic neurons were positive for FGF1 in the DMNV and hypoglossal nucleus, respectively. A tracing study with cholera toxin B subunit (CTb) demonstrated that cholinergic neurons sending their axons from the DMNV to the superior laryngeal nerve were FGF1-negative. The results suggest that the low expression of FGF1 in the DMNV is due to severe damage of neurons in the DMNV.
  • Akatsu H, Yamagata H, Kawamata J, Kamino K, Takeda M, Yamamoto T, Miki T, Tooyama I, Shimohama S, Kosaka K, Okada H
    Dementia Geriatric Cogn Dis 22 3 216 - 222 2006年 [無し][無し]
     研究論文（学術雑誌） 
    Background/Aim: Brain-derived neurotrophic factor ( BDNF) is associated with the hippocampus and the nigrostriatal dopaminergic function. Data showing that its level was reduced in Alzheimer's disease ( AD) and Parkinson's disease (PD) suggested that the BDNF function must play an important role in the pathogenetics of these diseases. Indeed, variation in the BDNF gene may confer susceptibility to AD and PD development. Recently, a functional BDNF Val66Met polymorphism has been found to be associated with episodic memory and hippocampal function, with intracellular trafficking, and with activity-dependent secretion of BDNF. To date, there have been several conflicting reports on the correlation between AD or PD and Val66Met or C270T polymorphism in the BDNF promoter region, although no data on this relationship have been published with respect to dementia with Lewy bodies (DLB). In the present study, we investigated a possible association between such BDNF polymorphisms and susceptibility to AD or DLB. Methods: BDNF genotyping was carried out by the polymerase chain reaction-restriction fragment length polymorphism method in autopsy-confirmed human samples. Results and Conclusion: On comparing patients and controls, the distribution of BDNF genotypes and alleles did not differ significantly. Our findings suggest that it is unlikely that these BDNF polymorphisms play a major role in the pathogenesis of AD and DLB in the Japanese population. Copyright (C) 2006 S. Karger AG, Basel.
  • GI Hazama, O Yasuhara, H Morita, Y Aimi, Tooyama, I, H Kimura
    JOURNAL OF COMPARATIVE NEUROLOGY 492 2 234 - 249 2005年11月 [無し][無し]
     研究論文（学術雑誌） 
    Unlike the brains of most mammals, the mouse brain appears unique in the massive appearance of cells showing IgG-like immunoreactivity, which has repeatedly been shown via immunohistochemistry. In the present study, we first examined possible species differences in IgG-like immunohistochemical staining in the brains of various rodents, including mice. In four of six mouse strains examined (ICR, Balb/c, C57BL/6, and AKR/J), antibodies against mouse IgG revealed positive staining in many brain microglia. However, no such positive staining was detected in brains of the rat, hamster, guinea pig, or two other mouse strains (CBA/N and CBA/J). We purified IgG-like-immunoreactive molecule(s) biochemically from brain of the ICR mouse as a representative mouse strain. Our amino-acid-sequence analysis proved that the purified protein was identical to serum IgG. The possibility of IgG synthesis by brain microglia in the ICR mouse was denied by our RT-PCR experiments and in situ hybridization histochemistry. In addition, Fc gamma-receptor-deficient double-knockout mice of the C57BL/6 genetic background contained no IgG-immunoreactive microglia in the brain. These results clearly indicate that microglial IgG staining is due to the uptake of serum IgG through Fc gamma receptors. However, the strain-specific mechanisms resulting in microglial IgG uptake remain to be elucidated, in that Fc gamma receptors are omnipresent in microglia of all rodents examined here.
  • GI Hazama, O Yasuhara, H Morita, Y Aimi, Tooyama, I, H Kimura
    JOURNAL OF COMPARATIVE NEUROLOGY 492 2 234 - 249 2005年11月 [無し][無し]
     研究論文（学術雑誌） 
    Unlike the brains of most mammals, the mouse brain appears unique in the massive appearance of cells showing IgG-like immunoreactivity, which has repeatedly been shown via immunohistochemistry. In the present study, we first examined possible species differences in IgG-like immunohistochemical staining in the brains of various rodents, including mice. In four of six mouse strains examined (ICR, Balb/c, C57BL/6, and AKR/J), antibodies against mouse IgG revealed positive staining in many brain microglia. However, no such positive staining was detected in brains of the rat, hamster, guinea pig, or two other mouse strains (CBA/N and CBA/J). We purified IgG-like-immunoreactive molecule(s) biochemically from brain of the ICR mouse as a representative mouse strain. Our amino-acid-sequence analysis proved that the purified protein was identical to serum IgG. The possibility of IgG synthesis by brain microglia in the ICR mouse was denied by our RT-PCR experiments and in situ hybridization histochemistry. In addition, Fc gamma-receptor-deficient double-knockout mice of the C57BL/6 genetic background contained no IgG-immunoreactive microglia in the brain. These results clearly indicate that microglial IgG staining is due to the uptake of serum IgG through Fc gamma receptors. However, the strain-specific mechanisms resulting in microglial IgG uptake remain to be elucidated, in that Fc gamma receptors are omnipresent in microglia of all rodents examined here.
  • ジャンピエール・ベリエ, 相見 良成, 松尾 明典, 安原 治, 木村 新, 遠山 育夫, 木村 宏
    日本組織細胞化学会総会・学術集会講演プログラム・予稿集 46回 56 - 56 日本組織細胞化学会 2005年10月
  • H Okano, H Bamba, Y Hisa, S Makino, S Ando, G Tamiya, S Goto, R Kaji, H Kimura, Tooyama, I
    HISTOLOGY AND HISTOPATHOLOGY 20 4 1029 - 1035 2005年10月 [無し][無し]
     研究論文（学術雑誌） 
    The cause of spasmodic dysphonia, a dystonic disorder of the larynx, remains unclear. Recently, TAFII250, TATA-box binding protein associated factor, was suggested to be involved in dystonia parkinsonism. There is a possibility that TA-FII250 is involved in spasmodic dysphonia, but little information is available about the expression of TAFII250 in the laryngeal nervous system. In this study, we investigated the localization of TAFII250 protein in the rat laryngeal nervous system by immunohistochemistry. TAFII250-immunoreactivity was detected in the nodose ganglion and superior cervical ganglion. In these nuclei, TAFII250 was localized in the nucleus of NeuroTrace-positive neurons but not in GFAP-positive glial cells. No positive cells were detected in the motor and parasympathetic nervous system. TAFII250-immunoreactivity was sustained between 3 and 7 days after vagotomy, but at 14 days expression was downregulated in the distal part of the nodose ganglion. These findings suggest that TAFII250 plays an important role in the laryngeal innervation of the sensory and sympathetic nervous systems.
  • H Okano, H Bamba, Y Hisa, S Makino, S Ando, G Tamiya, S Goto, R Kaji, H Kimura, Tooyama, I
    HISTOLOGY AND HISTOPATHOLOGY 20 4 1029 - 1035 2005年10月 [無し][無し]
     研究論文（学術雑誌） 
    The cause of spasmodic dysphonia, a dystonic disorder of the larynx, remains unclear. Recently, TAFII250, TATA-box binding protein associated factor, was suggested to be involved in dystonia parkinsonism. There is a possibility that TA-FII250 is involved in spasmodic dysphonia, but little information is available about the expression of TAFII250 in the laryngeal nervous system. In this study, we investigated the localization of TAFII250 protein in the rat laryngeal nervous system by immunohistochemistry. TAFII250-immunoreactivity was detected in the nodose ganglion and superior cervical ganglion. In these nuclei, TAFII250 was localized in the nucleus of NeuroTrace-positive neurons but not in GFAP-positive glial cells. No positive cells were detected in the motor and parasympathetic nervous system. TAFII250-immunoreactivity was sustained between 3 and 7 days after vagotomy, but at 14 days expression was downregulated in the distal part of the nodose ganglion. These findings suggest that TAFII250 plays an important role in the laryngeal innervation of the sensory and sympathetic nervous systems.
  • S Goto, LV Lee, EL Munoz, Tooyama, I, G Tamiya, S Makino, S Ando, MB Dantes, K Yamada, S Matsumoto, H Shimazu, J Kuratsu, A Hirano, R Kaji
    ANNALS OF NEUROLOGY 58 1 7 - 17 2005年07月 [無し][無し]
     研究論文（学術雑誌） 
    Dystonia is a neurological syndrome characterized by sustained muscle contractions that produce repetitive twisting movements or abnormal postures. X-linked recessive dystonia parkinsonism (XDP; DYT3; Lubag) is an adult-onset disorder that manifests severe and progressive dystonia with a high frequency of generalization. In search for the anatomical basis for dystonia, we performed postmortem analyses of the functional anatomy of the basal ganglia based on the striatal compartments (ie, the striosomes and the matrix compartment) in XDP. Here, we provide anatomopatho-logical evidence that, in the XDP neostriatum, the matrix compartment is relatively spared in a unique fashion, whereas the striosomes are severely depleted. We also document that there is a differential loss of striatal neuron subclasses in XDP. In view of the three-pathway basal ganglia model, we postulate that the disproportionate involvement of neostriatal compartments and their efferent projections may underlie the manifestation of dystonia in patients with XDP. This study is the first to our knowledge to show specific basal ganglia pathology that could explain the genesis of dystonia in human heredodegenerative movement disorders, suggesting that dystonia may result from an imbalance in the activity between the striosomal and matrix-based pathways.
  • S Goto, LV Lee, EL Munoz, Tooyama, I, G Tamiya, S Makino, S Ando, MB Dantes, K Yamada, S Matsumoto, H Shimazu, J Kuratsu, A Hirano, R Kaji
    ANNALS OF NEUROLOGY 58 1 7 - 17 2005年07月 [無し][無し]
     研究論文（学術雑誌） 
    Dystonia is a neurological syndrome characterized by sustained muscle contractions that produce repetitive twisting movements or abnormal postures. X-linked recessive dystonia parkinsonism (XDP; DYT3; Lubag) is an adult-onset disorder that manifests severe and progressive dystonia with a high frequency of generalization. In search for the anatomical basis for dystonia, we performed postmortem analyses of the functional anatomy of the basal ganglia based on the striatal compartments (ie, the striosomes and the matrix compartment) in XDP. Here, we provide anatomopatho-logical evidence that, in the XDP neostriatum, the matrix compartment is relatively spared in a unique fashion, whereas the striosomes are severely depleted. We also document that there is a differential loss of striatal neuron subclasses in XDP. In view of the three-pathway basal ganglia model, we postulate that the disproportionate involvement of neostriatal compartments and their efferent projections may underlie the manifestation of dystonia in patients with XDP. This study is the first to our knowledge to show specific basal ganglia pathology that could explain the genesis of dystonia in human heredodegenerative movement disorders, suggesting that dystonia may result from an imbalance in the activity between the striosomal and matrix-based pathways.
  • A Matsuo, JP Bellier, T Hisano, Y Aimi, O Yasuhara, Tooyama, I, N Saito, H Kimura
    NEUROCHEMISTRY INTERNATIONAL 46 5 423 - 433 2005年04月 [無し][無し]
     研究論文（学術雑誌） 
    Choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of, ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type (pChAT), because of its. preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type (cChAT) using green fluorescent protein (GFP) in living human embryonic kidney cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B. treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT. (c) 2004 Elsevier Ltd. All rights reserved.
  • A Matsuo, JP Bellier, T Hisano, Y Aimi, O Yasuhara, Tooyama, I, N Saito, H Kimura
    NEUROCHEMISTRY INTERNATIONAL 46 5 423 - 433 2005年04月 [無し][無し]
     研究論文（学術雑誌） 
    Choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of, ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type (pChAT), because of its. preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type (cChAT) using green fluorescent protein (GFP) in living human embryonic kidney cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B. treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT. (c) 2004 Elsevier Ltd. All rights reserved.
  • Two types of choline acetyltransferase of rat differ in intracellular translocation.
    MATSUO A, JEAN-PIERRE Bellier J P, HISANO T, AIMI Y, YASUHARA O, TOOYAMA I, SAITO Naoaki, KIMURA H
    Neurochem. Int vol. 46, pp 423-433 2005年 [無し][無し]
     研究論文（学術雑誌）
  • 木村 新, 遠山 育夫, 松尾 明典, ジャンピエール・ベリエ, 相見 良成, 安原 治, 木村 宏
    日本組織細胞化学会総会・学術集会講演プログラム・予稿集 45回 55 - 55 日本組織細胞化学会 2004年10月
  • ラットエックリン汗腺におけるpChAT含有神経線維の局在
    花田 圭司, Bellier J・P, 松尾 明典, 相見 良成, 安原 治, 遠山 育夫, 木村 宏
    解剖学雑誌 79 3 111 - 111 (一社)日本解剖学会 2004年09月
  • ラット眼組織のコリン神経支配 新規アセチルコリン合成酵素pChATによる再評価(Re-evaluation of cholinergic innervation of rat ocular tissues using choline acetyltransferase of a peripheral type as a marker)
    安原 治, 相見 良成, 松尾 明典, Bellier Jean-Pierre, Elnasharty Mohamed, 遠山 育夫, 木村 宏
    神経化学 43 2-3 401 - 401 2004年08月
  • ラットの2つのタイプのコリンアセチル基転移酵素の細胞内動態の差異(Differences in intracellular translocation between two types of choline acetyltransferase of the rat)
    松尾 明典, Bellier Jean Pierre, 久野 正, 相見 良成, 安原 治, 遠山 育夫, 齋藤 尚亮, 木村 宏
    神経化学 43 2-3 401 - 401 2004年08月
  • O Yasuhara, Y Aimi, A Shibano, A Matsuo, JP Bellier, M Park, Tooyama, I, H Kimura
    JOURNAL OF COMPARATIVE NEUROLOGY 472 2 232 - 245 2004年04月 [無し][無し]
     研究論文（学術雑誌） 
    We have recently discovered a splice variant of choline acetyltransferase (ChAT) mRNA and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, the presence of pChAT in rat iris was examined by immunohistochemistry and Western blot using a pChAT antiserum, in combination with RT-PCR analysis and ChAT enzyme assay. For comparison, the conventional ChAT (cChAT) was studied in parallel. By pChAT immunohistochemistry, intense labeling was found to occur in nerve fibers of the iris and in neurons of the ciliary and trigeminal ganglia. Denervation studies, analyzed by semiquantitative morphometry, indicated that these iridial pChAT fibers originated about half from the ciliary ganglion and the other half from the trigeminal ganglion. The presence of pChAT protein in the iris and trigeminal ganglion was confirmed by Western blot. The expression of pChAT mRNA in the ciliary and trigeminal ganglia was proved by RT-PCR. Although cChAT protein and mRNA were detected in the ciliary ganglion, neither was detectable in the trigeminal ganglion. The contributions of the ciliary and trigeminal ganglia to the iridial ChAT enzyme activity were verified by the present ChAT assay. Here, we provide evidence that iridial pChAT nerves are composed of postganglionic parasympathetic efferents from the ciliary ganglion and, more interestingly, somatic sensory afferents of the trigeminal ophthalmic nerve. (C) 2004 Wiley-Liss, Inc.
  • A Casini, A Pinna, Tooyama, I, H Kimura, G Di Chiara, TG Renda
    EUROPEAN JOURNAL OF HISTOCHEMISTRY 48 2 135 - 140 2004年04月 [無し][無し]
     研究論文（学術雑誌） 
    The use of a polyclonal antiserum specific to C-terminal tetrapeptide amide of (D-Ala(2))deltorphin-I, a naturally occurring amphibian skin opioid peptide, has already demonstrated the presence of immunoreactive neurons in rat midbrain. Double immunostaining identified these neurons as a sub-population of the mesencephalic dopaminergic neurons that were also tyrosine hydroxylase-immunopositive and calbindin-D28kD- negative, namely, the neurons predominantly affected in Parkinson disease. We followed the fate of these neurons after a monolateral injection of 6-hydroxy-dopamine into rat brain. Almost all the immunopositive neurons and their nigrostriatal, mesolimbic and mesocortical projections on the side ipsilateral to the lesion disappeared. Only a few scattered immunopositive neurons within the substantia nigra, pars compacta, and those of supramammillary nucleus remained unaffected. The consistent overlap of dopamine and this new molecule provides a further key to identifying the mammalian counterpart of these amphibian skin opioid peptides.
  • O Yasuhara, Y Aimi, A Shibano, A Matsuo, JP Bellier, M Park, Tooyama, I, H Kimura
    JOURNAL OF COMPARATIVE NEUROLOGY 472 2 232 - 245 2004年04月 [無し][無し]
     研究論文（学術雑誌） 
    We have recently discovered a splice variant of choline acetyltransferase (ChAT) mRNA and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, the presence of pChAT in rat iris was examined by immunohistochemistry and Western blot using a pChAT antiserum, in combination with RT-PCR analysis and ChAT enzyme assay. For comparison, the conventional ChAT (cChAT) was studied in parallel. By pChAT immunohistochemistry, intense labeling was found to occur in nerve fibers of the iris and in neurons of the ciliary and trigeminal ganglia. Denervation studies, analyzed by semiquantitative morphometry, indicated that these iridial pChAT fibers originated about half from the ciliary ganglion and the other half from the trigeminal ganglion. The presence of pChAT protein in the iris and trigeminal ganglion was confirmed by Western blot. The expression of pChAT mRNA in the ciliary and trigeminal ganglia was proved by RT-PCR. Although cChAT protein and mRNA were detected in the ciliary ganglion, neither was detectable in the trigeminal ganglion. The contributions of the ciliary and trigeminal ganglia to the iridial ChAT enzyme activity were verified by the present ChAT assay. Here, we provide evidence that iridial pChAT nerves are composed of postganglionic parasympathetic efferents from the ciliary ganglion and, more interestingly, somatic sensory afferents of the trigeminal ophthalmic nerve. (C) 2004 Wiley-Liss, Inc.
  • A Casini, A Pinna, Tooyama, I, H Kimura, G Di Chiara, TG Renda
    EUROPEAN JOURNAL OF HISTOCHEMISTRY 48 2 135 - 140 2004年04月 [無し][無し]
     研究論文（学術雑誌） 
    The use of a polyclonal antiserum specific to C-terminal tetrapeptide amide of (D-Ala(2))deltorphin-I, a naturally occurring amphibian skin opioid peptide, has already demonstrated the presence of immunoreactive neurons in rat midbrain. Double immunostaining identified these neurons as a sub-population of the mesencephalic dopaminergic neurons that were also tyrosine hydroxylase-immunopositive and calbindin-D28kD- negative, namely, the neurons predominantly affected in Parkinson disease. We followed the fate of these neurons after a monolateral injection of 6-hydroxy-dopamine into rat brain. Almost all the immunopositive neurons and their nigrostriatal, mesolimbic and mesocortical projections on the side ipsilateral to the lesion disappeared. Only a few scattered immunopositive neurons within the substantia nigra, pars compacta, and those of supramammillary nucleus remained unaffected. The consistent overlap of dopamine and this new molecule provides a further key to identifying the mammalian counterpart of these amphibian skin opioid peptides.
  • A Brehmer, F Schrodl, W Neuhuber, I Tooyama, H Kimura
    JOURNAL OF CHEMICAL NEUROANATOMY 27 1 33 - 41 2004年03月 [無し][無し]
     研究論文（学術雑誌） 
    Cholinergic enteric neurons were demonstrated immunohistochemically so far by using antibodies staining the common choline acetyl-transferase (cChAT) in neurons of the central nervous system. The results of staining in the enteric nervous system of various species were, however, not satisfactory. We describe here findings obtained with a newly raised antibody against a peripheral variant of choline acetyltransferase (pChAT) in myenteric neurons of the pig small intestine. Triple labelling for pChAT/cChAT/neuronal nitric oxide synthase (nNOS) revealed 19.7% of 1664 neurons (within 40 ganglia) to be immunoreactive exclusively for pChAT whereas 29.6% were positive for cChAT alone and 18.8% were reactive only for nNOS. Colocalization of pChAT and cChAT was found in 22.4%, of pChAT and nNOS in 8.1% and of cChAT and nNOS in 1.4%. All three markers were simultaneously found in only I of 1664 neurons. To investigate the presence and possible colocalization of the above markers within morphologically defined neuron types, triple labelling of cChAT or nNOS with pChAT and a neurofilament (NF) antibody pool was applied and the coexpression patterns of pChAT and cChAT as well as of pChAT and nNOS in 120 neurons of each type were recorded. All type I, II, IV and V neurons displayed immunoreactivity either for one or both cholinergic markers. These neuron types were considered to be cholinergic. All type VI neurons, a descending neuron population, were negative for cChAT but positive for nNOS. However, 95% were immunoreactive for both pChAT and nNOS. The physiological significance of the possible co-existence of acetylcholine and nitric oxide within type VI neurons remains to be clarified. It is concluded that the pChAT and cChAT antibodies used here recognize partly different populations of enteric neurons in the pig. Thus, for total immunohistochemical characterization of cholinergic enteric neurons both forms of choline acetyltransferase have to be considered. (C) 2003 Elsevier B.V. All rights reserved.
  • KI Toyoda, Tooyama, I, M Kato, H Sato, S Morikawa, Y Hisa, T Inubushi
    NEUROREPORT 15 4 589 - 593 2004年03月 [無し][無し]
     研究論文（学術雑誌） 
    Magnetic labeling of transplanted cells permits us to monitor their localization non-invasively using MRI. Since most transfection agents for magnetic labeling have the same cationic charge as Fe3+, the efficiency may be reduced. The hemagglutinating virus-envelope has no charge and utilizes membrane fusion activity to deliver internalized materials. In this study, we investigated the feasibility of using the envelope to incorporate paramagnetic Fe3+ particles into PC12 cells and astrocytes. The envelope effectively labeled both cells with Fe, which showed significant decreases of signal intensity in T2-weighted MRI. Labeled cells transplanted into the rat striatum were clearly visualized by T2*-weighted MRI at a magnetic field of 2T. The results indicate that the hemagglutinating virus-envelope is a powerful tool for magnetic labeling.
  • A Brehmer, F Schrodl, W Neuhuber, I Tooyama, H Kimura
    JOURNAL OF CHEMICAL NEUROANATOMY 27 1 33 - 41 2004年03月 [無し][無し]
     研究論文（学術雑誌） 
    Cholinergic enteric neurons were demonstrated immunohistochemically so far by using antibodies staining the common choline acetyl-transferase (cChAT) in neurons of the central nervous system. The results of staining in the enteric nervous system of various species were, however, not satisfactory. We describe here findings obtained with a newly raised antibody against a peripheral variant of choline acetyltransferase (pChAT) in myenteric neurons of the pig small intestine. Triple labelling for pChAT/cChAT/neuronal nitric oxide synthase (nNOS) revealed 19.7% of 1664 neurons (within 40 ganglia) to be immunoreactive exclusively for pChAT whereas 29.6% were positive for cChAT alone and 18.8% were reactive only for nNOS. Colocalization of pChAT and cChAT was found in 22.4%, of pChAT and nNOS in 8.1% and of cChAT and nNOS in 1.4%. All three markers were simultaneously found in only I of 1664 neurons. To investigate the presence and possible colocalization of the above markers within morphologically defined neuron types, triple labelling of cChAT or nNOS with pChAT and a neurofilament (NF) antibody pool was applied and the coexpression patterns of pChAT and cChAT as well as of pChAT and nNOS in 120 neurons of each type were recorded. All type I, II, IV and V neurons displayed immunoreactivity either for one or both cholinergic markers. These neuron types were considered to be cholinergic. All type VI neurons, a descending neuron population, were negative for cChAT but positive for nNOS. However, 95% were immunoreactive for both pChAT and nNOS. The physiological significance of the possible co-existence of acetylcholine and nitric oxide within type VI neurons remains to be clarified. It is concluded that the pChAT and cChAT antibodies used here recognize partly different populations of enteric neurons in the pig. Thus, for total immunohistochemical characterization of cholinergic enteric neurons both forms of choline acetyltransferase have to be considered. (C) 2003 Elsevier B.V. All rights reserved.
  • KI Toyoda, Tooyama, I, M Kato, H Sato, S Morikawa, Y Hisa, T Inubushi
    NEUROREPORT 15 4 589 - 593 2004年03月 [無し][無し]
     研究論文（学術雑誌） 
    Magnetic labeling of transplanted cells permits us to monitor their localization non-invasively using MRI. Since most transfection agents for magnetic labeling have the same cationic charge as Fe3+, the efficiency may be reduced. The hemagglutinating virus-envelope has no charge and utilizes membrane fusion activity to deliver internalized materials. In this study, we investigated the feasibility of using the envelope to incorporate paramagnetic Fe3+ particles into PC12 cells and astrocytes. The envelope effectively labeled both cells with Fe, which showed significant decreases of signal intensity in T2-weighted MRI. Labeled cells transplanted into the rat striatum were clearly visualized by T2*-weighted MRI at a magnetic field of 2T. The results indicate that the hemagglutinating virus-envelope is a powerful tool for magnetic labeling.
  • Ameliorative effects of acetyl-L-carnitine and acidic fibroblast growth factor fragment analog on brain lipid hydroperoxide level, passive avoidance learning and/or immunoreactivity for choline acetyltransferse in the medial septum in senesence-acceler・・・
    Sasaki K, Ishibashi M, Yasui F, Matsugo S, Tooyama I, Kimura H, Oomura Y
    International Congress Series 1260 123 - 128 2004年 [無し][無し]
     研究論文（学術雑誌） 
    Ameliorative effects of acetyl-L-carnitine and acidic fibroblast growth factor fragment analog on brain lipid hydroperoxide level, passive avoidance learning and/or immunoreactivity for choline acetyltransferse in the medial septum in senesence-accelerated mice.
  • Ameliorative effects of acetyl-L-carnitine and acidic fibroblast growth factor fragment analog on brain lipid hydroperoxide level, passive avoidance learning and/or immunoreactivity for choline acetyltransferse in the medial septum in senesence-acceler・・・
    Sasaki K, Ishibashi M, Yasui F, Matsugo S, Tooyama I, Kimura H, Oomura Y
    International Congress Series 1260 123 - 128 2004年 [無し][無し]
     研究論文（学術雑誌） 
    Ameliorative effects of acetyl-L-carnitine and acidic fibroblast growth factor fragment analog on brain lipid hydroperoxide level, passive avoidance learning and/or immunoreactivity for choline acetyltransferse in the medial septum in senesence-accelerated mice.
  • Y Yazawa, M Suzuki, M Hanamitsu, H Kimura, Tooyama, I
    ORL-JOURNAL FOR OTO-RHINO-LARYNGOLOGY AND ITS RELATED SPECIALTIES 65 3 162 - 168 2003年05月 [無し][無し]
     研究論文（学術雑誌） 
    The main purpose of this study is to search for a viral etiology in Meniere's disease by examining the presence or absence of herpes family virus DNA in the endolymphatic sac (ES) using the in situ hybridization method. This was a prospective study with the ES from 10 patients with Meniere's disease and from 7 control cases without any pre-mortem ear diseases except a case of acoustic tumor. These 10 patients underwent the ES surgery. The presence of herpes family virus DNA, such as herpes simplex virus types 1 and 2 (HSV1&2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and human cytomegalovirus (CMV), was examined using the in situ hybridization method. Serum antibody titers against these viruses just before the ES surgery were studied in these patients. Of the 10 specimens from the patients with Meniere's disease, 7 were positive for VZV, 4 for EBV, 1 for CMV and none for HSV1&2, although the serum antibody titers against these viruses did not show any significant elevation in these patients just before the ES surgery. This result suggests that the viral DNA in the ES is inactive and is present in a latent form. From the statistical analysis, it can be postulated that VZV infection in early childhood may reach the ES and play a role in the pathogenesis of Meniere's disease (p = 0.0235). The double infection with both VZV and EBV tended to be another candidate for the pathogenesis of Meniere's disease (p = 0.0557). Copyright (C) 2003 S. Karger AG, Basel.
  • Y Yazawa, M Suzuki, M Hanamitsu, H Kimura, Tooyama, I
    ORL-JOURNAL FOR OTO-RHINO-LARYNGOLOGY AND ITS RELATED SPECIALTIES 65 3 162 - 168 2003年05月 [無し][無し]
     研究論文（学術雑誌） 
    The main purpose of this study is to search for a viral etiology in Meniere's disease by examining the presence or absence of herpes family virus DNA in the endolymphatic sac (ES) using the in situ hybridization method. This was a prospective study with the ES from 10 patients with Meniere's disease and from 7 control cases without any pre-mortem ear diseases except a case of acoustic tumor. These 10 patients underwent the ES surgery. The presence of herpes family virus DNA, such as herpes simplex virus types 1 and 2 (HSV1&2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and human cytomegalovirus (CMV), was examined using the in situ hybridization method. Serum antibody titers against these viruses just before the ES surgery were studied in these patients. Of the 10 specimens from the patients with Meniere's disease, 7 were positive for VZV, 4 for EBV, 1 for CMV and none for HSV1&2, although the serum antibody titers against these viruses did not show any significant elevation in these patients just before the ES surgery. This result suggests that the viral DNA in the ES is inactive and is present in a latent form. From the statistical analysis, it can be postulated that VZV infection in early childhood may reach the ES and play a role in the pathogenesis of Meniere's disease (p = 0.0235). The double infection with both VZV and EBV tended to be another candidate for the pathogenesis of Meniere's disease (p = 0.0557). Copyright (C) 2003 S. Karger AG, Basel.
  • コリンアセチル基転移酵素のスプライス・バリアントpChATのラット脳内分布
    松尾 明典, 金山 秀彦, 相見 良成, 安原 治, 遠山 育夫, 木村 宏
    解剖学雑誌 78 Suppl. 340 - 340 (一社)日本解剖学会 2003年04月
  • ラットカイニン酸てんかんモデルでは海馬神経細胞によるIgGの取り込みが神経細胞傷害に先行する
    森田 浩之, 安原 治, 相見 良成, 遠山 育夫, 森 則夫, 木村 宏
    てんかん研究 21 1 78 - 78 (一社)日本てんかん学会 2003年02月
  • T Kondo, Tooyama, I
    ACTA HISTOCHEMICA ET CYTOCHEMICA 36 3 215 - 220 2003年 [無し][無し]
     研究論文（学術雑誌） 
    Alpha2-macroglobulin (A2M), a pan-proteinase inhibitor, accumulates in senile plaques of Alzheimer disease. In this study, we investigated the immunohistochemical localization of A2M in the brain of PS/APP mice. In control mice, A2M immunoreactivity was restricted to the vessels of brain parenchyma. In PS/APP mice, A2M deposition occurred in the brain parenchyma. A2M-positive deposits appeared after three months of age when senile plaque formation began to occur. The number of A2M-positive deposits increased with age. Double-staining for A2M and thioflavin S revealed that A2M accumulated in a subpopulation of thioflavin S-positive fibrillar type of amyloid beta deposits. These results indicate that A2M is implicated in the formation of fibrillar type senile plaques in PS/APP mice.
  • Demonstration of cholinergic ganglion cells in rat retina; expression of an alternative splice variant of choline acetyltransfer
    Yasuhara O, Tooyama I, Aimi Y, Bellier JP, Hisano T, Matsuo A, Park M, Kimura H
    J Neurosci 23 2872 - 2881 2003年 [無し][無し]
     研究論文（学術雑誌）
  • Mika Iwami, Ikuo Tooyama, Ayae Kinoshita, Akinori Matsuo, Yutaka Oomura, Kazuo Sasaki, Hiroshi Kimura
    Acta Histochemica et Cytochemica 36 4 353 - 359 2003年 [無し][無し]
     研究論文（学術雑誌） 
    Recent studies have shown that FGF-1 (aFGF) and FGF-2 (bFGF) are expressed in the adrenal glands. In order to understand the roles of FGFs in the adrenal gland, we investigated the expression of the fibroblast growth factor receptor-1 (FGFR1) in rat adrenal gland using reverse transcription-polymerase chain reaction (RT-PCR) method, Western blot and immunohistochemistry. RT-PCR experiment using a primer set to amplify the acidic region of FGFR1 gave two bands of about 426 bp and 160 bp in both the adrenal cortex and medulla. The sizes of the large and small PCR products corresponded well to the three IgG-like domain (long form) and two IgG-like domain (short, or secreted form) FGFR1, respectively. Western blotting analysis using an antibody capable of detecting the acidic region of FGFR1, gave two bands in the adrenal gland with molecular weights of about 130 kDa and 75 kDa, corresponding to the long and the secreted form of FGFR1, respectively. Immunohistochemical study using the anti-body demonstrated that FGFR1-positive cells were distributed mainly in the zona glomerulosa of the adrenal cortex and medulla. A few positive cells were scattered in the zona fasciculata and reticularis. In the adrenal medulla, almost all cells were positive for FGFR1. Double immunostaining revealed that FGFR1 colocalized with tyrosine hydroxylase-immunoreactive catecholamine cells in the adrenal medulla.
  • Shigeru Tsuji, Ikuo Tooyama, Tomoko Kato, Gabriel Peltre, Hiroshi Kimura
    Biomedical Research 24 5 217 - 221 2003年 [無し][無し]
     研究論文（学術雑誌） 
    On nitrocellulose membrane (NCM), a spot of acetylcholine droplet (ACh) was precipitated with silicotungstic acid (STA). NCM was incubated either with rabbit polyclonal antiserum against ACh or with rabbit non-immune serum. When visualized by an immunostaining procedure using goat anti-rabbit IgG and rabbit peroxidase-anti-peroxydase (PAP) complex, both sera gave similar positive reaction products on the spot. The second antibody (goat anti-rabbit IgG) alone, namely omitting the first antibody, also gave positive immunoperoxidase reaction. The application of PAP alone gave faint but similarly positive staining. The binding of IgG molecules appears to be concerned with the ACh moiety, but not with STA moiety of the precipitate of acetylcholine silicotungstate (ACh-STA) complex. We thus consider this phenomenon as avidity of IgG for ACh. Other quaternary ammonium compounds such as decamethonium, eserine, d-tubocurarine and atropine are also targets of IgG avidity. The solution of ACh at 10-5 M facilitated the avidity, while 10-2 to 1 M ACh inhibited the avidity. Purified rabbit IgG and Fab revealed a distinct avidity, while Fc had only a weak avidity. The IgG avidity for ACh was discussed in relation to two biological phenomena: autoimmunity against ACh of the experimental animals and structural similarity of IgG molecule with nicotinic ACh receptor.
  • H Kanayama, O Yasuhara, A Matsuo, Tooyama, I, Y Aimi, JP Bellier, JI Nagy, K Fukui, H Kimura
    NEUROSCIENCE 118 1 243 - 251 2003年 [無し][無し]
     研究論文（学術雑誌） 
    A splice variant of choline acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this, nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of choline acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a cholinergic component. (C) 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
  • T Kondo, Tooyama, I
    ACTA HISTOCHEMICA ET CYTOCHEMICA 36 3 215 - 220 2003年 [無し][無し]
     研究論文（学術雑誌） 
    Alpha2-macroglobulin (A2M), a pan-proteinase inhibitor, accumulates in senile plaques of Alzheimer disease. In this study, we investigated the immunohistochemical localization of A2M in the brain of PS/APP mice. In control mice, A2M immunoreactivity was restricted to the vessels of brain parenchyma. In PS/APP mice, A2M deposition occurred in the brain parenchyma. A2M-positive deposits appeared after three months of age when senile plaque formation began to occur. The number of A2M-positive deposits increased with age. Double-staining for A2M and thioflavin S revealed that A2M accumulated in a subpopulation of thioflavin S-positive fibrillar type of amyloid beta deposits. These results indicate that A2M is implicated in the formation of fibrillar type senile plaques in PS/APP mice.
  • Demonstration of cholinergic ganglion cells in rat retina; expression of an alternative splice variant of choline acetyltransfer
    Yasuhara O, Tooyama I, Aimi Y, Bellier JP, Hisano T, Matsuo A, Park M, Kimura H
    J Neurosci 23 2872 - 2881 2003年 [無し][無し]
     研究論文（学術雑誌）
  • Mika Iwami, Ikuo Tooyama, Ayae Kinoshita, Akinori Matsuo, Yutaka Oomura, Kazuo Sasaki, Hiroshi Kimura
    Acta Histochemica et Cytochemica 36 4 353 - 359 2003年 [無し][無し]
     研究論文（学術雑誌） 
    Recent studies have shown that FGF-1 (aFGF) and FGF-2 (bFGF) are expressed in the adrenal glands. In order to understand the roles of FGFs in the adrenal gland, we investigated the expression of the fibroblast growth factor receptor-1 (FGFR1) in rat adrenal gland using reverse transcription-polymerase chain reaction (RT-PCR) method, Western blot and immunohistochemistry. RT-PCR experiment using a primer set to amplify the acidic region of FGFR1 gave two bands of about 426 bp and 160 bp in both the adrenal cortex and medulla. The sizes of the large and small PCR products corresponded well to the three IgG-like domain (long form) and two IgG-like domain (short, or secreted form) FGFR1, respectively. Western blotting analysis using an antibody capable of detecting the acidic region of FGFR1, gave two bands in the adrenal gland with molecular weights of about 130 kDa and 75 kDa, corresponding to the long and the secreted form of FGFR1, respectively. Immunohistochemical study using the anti-body demonstrated that FGFR1-positive cells were distributed mainly in the zona glomerulosa of the adrenal cortex and medulla. A few positive cells were scattered in the zona fasciculata and reticularis. In the adrenal medulla, almost all cells were positive for FGFR1. Double immunostaining revealed that FGFR1 colocalized with tyrosine hydroxylase-immunoreactive catecholamine cells in the adrenal medulla.
  • Shigeru Tsuji, Ikuo Tooyama, Tomoko Kato, Gabriel Peltre, Hiroshi Kimura
    Biomedical Research 24 5 217 - 221 2003年 [無し][無し]
     研究論文（学術雑誌） 
    On nitrocellulose membrane (NCM), a spot of acetylcholine droplet (ACh) was precipitated with silicotungstic acid (STA). NCM was incubated either with rabbit polyclonal antiserum against ACh or with rabbit non-immune serum. When visualized by an immunostaining procedure using goat anti-rabbit IgG and rabbit peroxidase-anti-peroxydase (PAP) complex, both sera gave similar positive reaction products on the spot. The second antibody (goat anti-rabbit IgG) alone, namely omitting the first antibody, also gave positive immunoperoxidase reaction. The application of PAP alone gave faint but similarly positive staining. The binding of IgG molecules appears to be concerned with the ACh moiety, but not with STA moiety of the precipitate of acetylcholine silicotungstate (ACh-STA) complex. We thus consider this phenomenon as avidity of IgG for ACh. Other quaternary ammonium compounds such as decamethonium, eserine, d-tubocurarine and atropine are also targets of IgG avidity. The solution of ACh at 10-5 M facilitated the avidity, while 10-2 to 1 M ACh inhibited the avidity. Purified rabbit IgG and Fab revealed a distinct avidity, while Fc had only a weak avidity. The IgG avidity for ACh was discussed in relation to two biological phenomena: autoimmunity against ACh of the experimental animals and structural similarity of IgG molecule with nicotinic ACh receptor.
  • H Kanayama, O Yasuhara, A Matsuo, Tooyama, I, Y Aimi, JP Bellier, JI Nagy, K Fukui, H Kimura
    NEUROSCIENCE 118 1 243 - 251 2003年 [無し][無し]
     研究論文（学術雑誌） 
    A splice variant of choline acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this, nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of choline acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a cholinergic component. (C) 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved.
  • L D'Este, A Casini, S Puglisi-Allegra, S Cabib, Tooyama, I, H Kimura, TG Renda
    JOURNAL OF CHEMICAL NEUROANATOMY 24 3 189 - 198 2002年09月 [無し][無し]
     研究論文（学術雑誌） 
    D-Ala(2)-deltorphin I (DADTI) is a heptapeptide amide first extracted from frog skin that displays a high selectivity and affinity for delta opioid receptors. Previous studies using a polyclonal antiserum specific for its C-terminal tetrapeptide-amide (DVVG) have already described in rat and mouse brain the presence of immunoreactive neurons, most of them belonging to the mesencephalic dopaminergic neurons. C57BL/6J (C57) and DBA/2J (DBA) are two inbred strains of mice well known for showing marked genotype-dependent differences for phenotypes related to differential brain dopamine functioning. Brain specimens of both inbred mouse strains were frozen, cut and immunostained using the same antiserum. Some sections were also double immunostained with monoclonal anti-tyrosine hydroxylase (TH). DVVG-immunoreactive neurons were observed among both dopaminergic and nondopaminergic neurons. DVVG- and TH-immunoreactive neurons were observed among the dopaminergic A8, A9 and A10 mesencephalic nuclei. They were on average 21.9% more numerous in DBA than in C57 mice. DVVG-immunoreactive nerve fibres could be seen in limbic, striatal, cortical and thalamic areas. The distribution patterns of DVVG-IR and TH-IR nerve fibres differed most conspicuously within the infralimbic, prelimbic and cingulate cortices, forming a dense network in DBA but rare in C57 mice. Non-dopaminergic DVVG-immunoreactive neurons did not differ significantly in the two strains. Our finding that the number and distribution pattern of this dopaminergic neuronal subpopulation differed in the two mouse strains could provide morphological support for the known behavioural differences between the DBA and C57 strains under normal and experimental conditions. (C) 2002 Elsevier Science B.V. All rights reserved.
  • L D'Este, A Casini, S Puglisi-Allegra, S Cabib, Tooyama, I, H Kimura, TG Renda
    JOURNAL OF CHEMICAL NEUROANATOMY 24 3 189 - 198 2002年09月 [無し][無し]
     研究論文（学術雑誌） 
    D-Ala(2)-deltorphin I (DADTI) is a heptapeptide amide first extracted from frog skin that displays a high selectivity and affinity for delta opioid receptors. Previous studies using a polyclonal antiserum specific for its C-terminal tetrapeptide-amide (DVVG) have already described in rat and mouse brain the presence of immunoreactive neurons, most of them belonging to the mesencephalic dopaminergic neurons. C57BL/6J (C57) and DBA/2J (DBA) are two inbred strains of mice well known for showing marked genotype-dependent differences for phenotypes related to differential brain dopamine functioning. Brain specimens of both inbred mouse strains were frozen, cut and immunostained using the same antiserum. Some sections were also double immunostained with monoclonal anti-tyrosine hydroxylase (TH). DVVG-immunoreactive neurons were observed among both dopaminergic and nondopaminergic neurons. DVVG- and TH-immunoreactive neurons were observed among the dopaminergic A8, A9 and A10 mesencephalic nuclei. They were on average 21.9% more numerous in DBA than in C57 mice. DVVG-immunoreactive nerve fibres could be seen in limbic, striatal, cortical and thalamic areas. The distribution patterns of DVVG-IR and TH-IR nerve fibres differed most conspicuously within the infralimbic, prelimbic and cingulate cortices, forming a dense network in DBA but rare in C57 mice. Non-dopaminergic DVVG-immunoreactive neurons did not differ significantly in the two strains. Our finding that the number and distribution pattern of this dopaminergic neuronal subpopulation differed in the two mouse strains could provide morphological support for the known behavioural differences between the DBA and C57 strains under normal and experimental conditions. (C) 2002 Elsevier Science B.V. All rights reserved.
  • Molecular mechanisms in Alzheimer's disease
    Kimura H, Aimi Y, Minnash P, Yasuhara O, Bellier JP, Tooyama I
    Res Legal Medicine 27 161 - 174 2002年 [無し][無し]
     研究論文（学術雑誌）
  • Ikuo Tooyama, Jean-Pierre Bellier, Masami Park, Petra Minnasch, Shuji Uemura, Tadashi Hisano, Mika Iwami, Yoshinari Aimi, Osamu Yasuhara, Hiroshi Kimura
    Epilepsia 43 9 39 - 43 2002年 [無し][無し]
     研究論文（国際会議プロシーディングス） 
    Purpose: To clarify the relationship of neuronal death to cellular responses, we studied neuronal death as well as reactions of glia and progenitor cells in the hippocampus of two rat models of epilepsy. Methods: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neuronal degeneration was assessed by in situ DNA fragmentation analysis. Reactions of glial cells were studied by immunohistochemistry. Progenitor cell division was evaluated using the bromodeoxyuridine (BrdU) labeling method. Results: DNA fragmentation and reactive microglia were observed in the CA1, CA3, and hilus region for 24 h to 4 weeks after KA injection, but not detected in the kindling model. Reactive astrocytes and enhancement of progenitor cell division were seen in both animal models. The number of BrdU-positive cells began to increase on day 3 after KA injection, peaked on day 5, and returned to baseline on day 10. After kindling, the number of BrdU-positive cells began to increase after five consecutive experience of stage I seizures. Conclusions: These observations show that neuronal degeneration is not necessary for triggering the upregulation. Microglial activation is closely related to the neuronal death process induced by KA.
  • Molecular mechanisms in Alzheimer's disease
    Kimura H, Aimi Y, Minnash P, Yasuhara O, Bellier JP, Tooyama I
    Res Legal Medicine 27 161 - 174 2002年 [無し][無し]
     研究論文（学術雑誌）
  • Tooyama, I, JP Bellier, M Park, P Minnasch, S Uemura, T Hisano, M Iwami, Y Aimi, O Yasuhara, H Kimura
    EPILEPSIA 43 9 39 - 43 2002年 [無し][無し]
     研究論文（学術雑誌） 
    Purpose: To clarify the relationship of neuronal death to cellular responses, we studied neuronal death as well as reactions of glia and progenitor cells in the hippocampus of two rat models of epilepsy. Methods: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neuronal degeneration was assessed by in situ DNA fragmentation analysis. Reactions of glial cells were studied by immunohistochemistry. Progenitor cell division was evaluated using the bromodeoxyuridine (BrdU) labeling method. Results: DNA fragmentation and reactive microglia were observed in the CA1, CA3, and hilus region for 24 h to 4 weeks after KA injection, but not detected in the kindling model. Reactive astrocytes and enhancement of progenitor cell division were seen in both animal models. The number of BrdU-positive cells began to increase on day 3 after KA injection, peaked on day 5, and returned to baseline on day 10. After kindling, the number of BrdU-positive cells began to increase after five consecutive experience of stage I seizures. Conclusions: These observations show that neuronal degeneration is not necessary for triggering the upregulation. Microglial activation is closely related to the neuronal death process induced by KA.
  • Makoto Kage, Qing Yang, Haruhisa Sato, Sadayuki Matsumoto, Ryuji Kaji, Ichiro Akiguchi, Hiroshi Kimura, Ikuo Tooyama
    NeuroReport 12 17 3799 - 3803 2001年12月 [有り][無し]
     研究論文（学術雑誌） 
    The expression and localization of acidic fibroblast growth factor (aFGF FGF-I) were examined in the spinal cord of patients with amyotrophic lateral sclerosis (ALS) and controls by reverse transcription-polymerase chain reaction (RT-PCR) method and immunohistochemistry. The RT-PCR experiments demonstrated that aFGF amplification products were clearly detected in all control cases but could be scarcely seen in ALS patients. aFGF immunoreactivity was detected in the anterior horn cells of the spinal cord. Double immunostaining for aFGF and choline acetyltransferase revealed that the maiority (95.9%) of cholinergic neurons expressed aFGF. In ALS cases, the number and the staining intensity of aFGF-positive neurons were markedly decreased. These results suggest that aFGF is involved in ALS pathology. © 2001 Lippincott Williams & Wilkins.
  • XL Li, S Aou, AJ Li, T Hori, Tooyama, I, Y Oomura
    BRAIN RESEARCH BULLETIN 56 6 531 - 536 2001年12月 [無し][無し]
     研究論文（学術雑誌） 
    Endogenous sugar acid 2-buten-4-olide, a satiety substance, has been shown to increase the blood glucose, norepinephrine, and glucocorticoid concentrations that are known to modulate learning and memory processes. The glucose-induced release of acidic fibroblast growth factor facilitated the hippocampus-dependent memory function. In the present study, we investigated the effect of 2-buten-4-olide on the spatial performance of male DDY mice undergoing the water maze task. The intraperitoneal injection of 2-buten-4-olide (5 mg/kg) facilitated the spatial performance, which was indicated by a reduction in the escape latency in which the mouse finds and climbs the goal platform in comparison to the vehicle-injected control mice. In the probe test after removing the platform, the 2-buten-4-olide-treated mice stayed a longer time in the quadrant where the platform was originally located and crossed more frequently at the platform location than did the control mice. The pretreatment of acidic fibroblast growth factor antibody injected into the lateral ventricle eliminated the effect of 2-buten-4-olide both during the training sessions and during the probe test. Therefore, 2-buten-4-olide was found to improve the spatial performance, and this effect is mediated, at least in part, by acidic fibroblast growth factor. (C) 2002 Elsevier Science Inc.
  • XL Li, S Aou, AJ Li, T Hori, Tooyama, I, Y Oomura
    BRAIN RESEARCH BULLETIN 56 6 531 - 536 2001年12月 [無し][無し]
     研究論文（学術雑誌） 
    Endogenous sugar acid 2-buten-4-olide, a satiety substance, has been shown to increase the blood glucose, norepinephrine, and glucocorticoid concentrations that are known to modulate learning and memory processes. The glucose-induced release of acidic fibroblast growth factor facilitated the hippocampus-dependent memory function. In the present study, we investigated the effect of 2-buten-4-olide on the spatial performance of male DDY mice undergoing the water maze task. The intraperitoneal injection of 2-buten-4-olide (5 mg/kg) facilitated the spatial performance, which was indicated by a reduction in the escape latency in which the mouse finds and climbs the goal platform in comparison to the vehicle-injected control mice. In the probe test after removing the platform, the 2-buten-4-olide-treated mice stayed a longer time in the quadrant where the platform was originally located and crossed more frequently at the platform location than did the control mice. The pretreatment of acidic fibroblast growth factor antibody injected into the lateral ventricle eliminated the effect of 2-buten-4-olide both during the training sessions and during the probe test. Therefore, 2-buten-4-olide was found to improve the spatial performance, and this effect is mediated, at least in part, by acidic fibroblast growth factor. (C) 2002 Elsevier Science Inc.
  • ME Sunday, KJ Haley, RL Emanuel, JS Torday, N Asokananthan, KA Sikorski, Tooyama, I, H Kimura, T Renda, Erspamer, V
    AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY 25 4 447 - 456 2001年10月 [無し][無し]
     研究論文（学術雑誌） 
    Opiate-like peptides can regulate many cellular functions. We now map [D-Ala(2)]deltorphin I (DADTI)-like immunoreactivity (DADTI-LI) in developing mouse lung and analyze potential functional roles. Most DADTI-Li-positive cells were alveolar ce s negative for prosurfactant protein (proSP)-C immunoreactivity. Peak numbers of DADTI-Li-positive cells occurred on embryonic Day 18, decreasing postnatally. To analyze developmental effects of DADTI, e17-18 lung explants were treated with [D-Ala(2)]deltorphin II (DADTII, soluble DADTI analogue, delta -receptor-specific) versus dermorphin (R-receptor-specific). Type II pneumocyte differentiation, assessed by [H-3]choline incorporation into saturated phosphatidylcholine and proSP-C immunostaining, was inhibited by DADTII but stimulated by dermorphin. Cell proliferation, measured as [H-3]-thymidine incorporation and proliferating cell nuclear antigen immunostaining, was stimulated by DADTII and inhibited by dermorpin. All effects were dose-dependent. DADTII-inhibited choline incorporation was reversed by the delta -blocker, naltrindole. Unexpectedly, DADTII-stimulated thymidine incorporation was augmented by naltrindole and reversed by naloxone (mu -blocker). Although dermorphin-stimulated choline incorporation was appropriately blocked by bin alto rphimi ne, dermorphin-inhibited thymidine incorporation was reversed by delta, K-, or mu -blockers. The delta- and mu -receptor messenger RNAs occurred pre- and postnatally, whereas K-receptor transcripts occurred mainly prenatally. All three receptor proteins were present in epithelial and mesenchymal cells in e18 lung. Thus, DADTI-LI from pro SP-C-immunonegative alveolar cells could regulate development via both direct and indirect effects involving multiple opiate receptors.
  • ME Sunday, KJ Haley, RL Emanuel, JS Torday, N Asokananthan, KA Sikorski, Tooyama, I, H Kimura, T Renda, Erspamer, V
    AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY 25 4 447 - 456 2001年10月 [無し][無し]
     研究論文（学術雑誌） 
    Opiate-like peptides can regulate many cellular functions. We now map [D-Ala(2)]deltorphin I (DADTI)-like immunoreactivity (DADTI-LI) in developing mouse lung and analyze potential functional roles. Most DADTI-Li-positive cells were alveolar ce s negative for prosurfactant protein (proSP)-C immunoreactivity. Peak numbers of DADTI-Li-positive cells occurred on embryonic Day 18, decreasing postnatally. To analyze developmental effects of DADTI, e17-18 lung explants were treated with [D-Ala(2)]deltorphin II (DADTII, soluble DADTI analogue, delta -receptor-specific) versus dermorphin (R-receptor-specific). Type II pneumocyte differentiation, assessed by [H-3]choline incorporation into saturated phosphatidylcholine and proSP-C immunostaining, was inhibited by DADTII but stimulated by dermorphin. Cell proliferation, measured as [H-3]-thymidine incorporation and proliferating cell nuclear antigen immunostaining, was stimulated by DADTII and inhibited by dermorpin. All effects were dose-dependent. DADTII-inhibited choline incorporation was reversed by the delta -blocker, naltrindole. Unexpectedly, DADTII-stimulated thymidine incorporation was augmented by naltrindole and reversed by naloxone (mu -blocker). Although dermorphin-stimulated choline incorporation was appropriately blocked by bin alto rphimi ne, dermorphin-inhibited thymidine incorporation was reversed by delta, K-, or mu -blockers. The delta- and mu -receptor messenger RNAs occurred pre- and postnatally, whereas K-receptor transcripts occurred mainly prenatally. All three receptor proteins were present in epithelial and mesenchymal cells in e18 lung. Thus, DADTI-LI from pro SP-C-immunonegative alveolar cells could regulate development via both direct and indirect effects involving multiple opiate receptors.
  • ME Sunday, KJ Haley, RL Emanuel, JS Torday, N Asokananthan, KA Sikorski, Tooyama, I, H Kimura, T Renda, Erspamer, V
    AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY 25 4 447 - 456 2001年10月 [無し][無し]
     研究論文（学術雑誌） 
    Opiate-like peptides can regulate many cellular functions. We now map [D-Ala(2)]deltorphin I (DADTI)-like immunoreactivity (DADTI-LI) in developing mouse lung and analyze potential functional roles. Most DADTI-Li-positive cells were alveolar ce s negative for prosurfactant protein (proSP)-C immunoreactivity. Peak numbers of DADTI-Li-positive cells occurred on embryonic Day 18, decreasing postnatally. To analyze developmental effects of DADTI, e17-18 lung explants were treated with [D-Ala(2)]deltorphin II (DADTII, soluble DADTI analogue, delta -receptor-specific) versus dermorphin (R-receptor-specific). Type II pneumocyte differentiation, assessed by [H-3]choline incorporation into saturated phosphatidylcholine and proSP-C immunostaining, was inhibited by DADTII but stimulated by dermorphin. Cell proliferation, measured as [H-3]-thymidine incorporation and proliferating cell nuclear antigen immunostaining, was stimulated by DADTII and inhibited by dermorpin. All effects were dose-dependent. DADTII-inhibited choline incorporation was reversed by the delta -blocker, naltrindole. Unexpectedly, DADTII-stimulated thymidine incorporation was augmented by naltrindole and reversed by naloxone (mu -blocker). Although dermorphin-stimulated choline incorporation was appropriately blocked by bin alto rphimi ne, dermorphin-inhibited thymidine incorporation was reversed by delta, K-, or mu -blockers. The delta- and mu -receptor messenger RNAs occurred pre- and postnatally, whereas K-receptor transcripts occurred mainly prenatally. All three receptor proteins were present in epithelial and mesenchymal cells in e18 lung. Thus, DADTI-LI from pro SP-C-immunonegative alveolar cells could regulate development via both direct and indirect effects involving multiple opiate receptors.
  • ME Sunday, KJ Haley, RL Emanuel, JS Torday, N Asokananthan, KA Sikorski, Tooyama, I, H Kimura, T Renda, Erspamer, V
    AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY 25 4 447 - 456 2001年10月 [無し][無し]
     研究論文（学術雑誌） 
    Opiate-like peptides can regulate many cellular functions. We now map [D-Ala(2)]deltorphin I (DADTI)-like immunoreactivity (DADTI-LI) in developing mouse lung and analyze potential functional roles. Most DADTI-Li-positive cells were alveolar ce s negative for prosurfactant protein (proSP)-C immunoreactivity. Peak numbers of DADTI-Li-positive cells occurred on embryonic Day 18, decreasing postnatally. To analyze developmental effects of DADTI, e17-18 lung explants were treated with [D-Ala(2)]deltorphin II (DADTII, soluble DADTI analogue, delta -receptor-specific) versus dermorphin (R-receptor-specific). Type II pneumocyte differentiation, assessed by [H-3]choline incorporation into saturated phosphatidylcholine and proSP-C immunostaining, was inhibited by DADTII but stimulated by dermorphin. Cell proliferation, measured as [H-3]-thymidine incorporation and proliferating cell nuclear antigen immunostaining, was stimulated by DADTII and inhibited by dermorpin. All effects were dose-dependent. DADTII-inhibited choline incorporation was reversed by the delta -blocker, naltrindole. Unexpectedly, DADTII-stimulated thymidine incorporation was augmented by naltrindole and reversed by naloxone (mu -blocker). Although dermorphin-stimulated choline incorporation was appropriately blocked by bin alto rphimi ne, dermorphin-inhibited thymidine incorporation was reversed by delta, K-, or mu -blockers. The delta- and mu -receptor messenger RNAs occurred pre- and postnatally, whereas K-receptor transcripts occurred mainly prenatally. All three receptor proteins were present in epithelial and mesenchymal cells in e18 lung. Thus, DADTI-LI from pro SP-C-immunonegative alveolar cells could regulate development via both direct and indirect effects involving multiple opiate receptors.
  • 選択的スプライシングによる脳内アセチルコリン合成酵素の多様性
    安原 治, 遠山 育夫, 相見 良成, 松尾 明典, 金山 秀彦, 木村 宏
    神経化学 40 2-3 248 - 248 日本神経化学会 2001年09月
  • Tooyama, I, H Sato, O Yasuhara, H Kimura, Y Konishi, Y Shen, DG Walker, TG Beach, LI Sue, J Rogers
    DEMENTIA AND GERIATRIC COGNITIVE DISORDERS 12 4 237 - 242 2001年07月 [有り][無し]
     研究論文（学術雑誌） 
    We compared the expression level of Clq mRNA and the number of Clq-positive plaques in adjacent or nearby brain sections from Alzheimer disease (AD) and control cases, Small blocks of temporal cortex were fixed wi th 4% paraformaldehyde for 2 days at 4 degreesC. After cryoprotection with solutions containing 10-20% glycerol and 2% dimethylsulfoxide, 40-mum sections were cut from the tissue blocks. A section from each case was stained by immunohistochemistry using a C1q antibody, while RNA was purified from adjacent or nearby sections using a combination of proteinase K pretreatment followed by extraction using Trizol reagent. The expression of C1q B chain mRNA was analyzed in these samples by the reverse-transcription polymerase chain reaction (RTPCR), The intensities of the PCR products were measured by an image analyzer. The expression of C1q B chain mRNA was significantly more abundant in AD than in control cases (p < 0.05). Immunohistochemical analysis showed that C1q protein was localized in senile plaques in the AD brain. The number of C1q-positive plaques correlated with the expression level of C1q gene (p < 0.05). The present results suggest that C1q protein in senile plaques originates is endogenously produced in the AD brain. Copyright (C) 2001 S. Karger AG. Basel.
  • 芝埜 彰, 北嶋 和智, 相見 良成, 遠山 育夫, 木村 宏
    日本耳鼻咽喉科学会会報 104 4 429 - 429 (一社)日本耳鼻咽喉科頭頸部外科学会 2001年04月
  • 培養後根神経節におけるアセチルコリン合成能の証明(Demonstration of cholinergic properties in cultured rat dorsal root ganglion neurons)
    相見 良成, 二宮 孝文, 遠山 育夫, 安原 治, Bellier Jean-Pierre, 木村 宏
    解剖学雑誌 76 1 147 - 147 2001年02月
  • H Kitano, T Takeda, S Takeda, M Suzuki, T Kitanishi, K Kitajima, H Kimura, Tooyama, I
    ACTA HISTOCHEMICA ET CYTOCHEMICA 34 4 229 - 233 2001年 [無し][無し]
     研究論文（学術雑誌） 
    The anti-diuretic hormone, vasopressin, has been shown to be important in regulating inner ear fluid. We report on the involvement between Meniere's disease and vasopressin. We designed an experiment to determine whether vasopressin directly affects the fluid level. We chronically infused this hormone in rats and examined them for morphological signs of endolymphatic hydrops. Vasopressin infusion significantly increased the scala media area by 5.9 +/- 7.1% (P <0.01), and the length of Reissner's membrane by 5.9 +/- 3.2% (P <0.001). The results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps due to down-regulating the number of vasopressin receptors.
  • Y Matsuoka, Y Aimi, H Kimura, T Taniguchi, Y Oomura, K Sasaki, Tooyama, I
    ACTA HISTOCHEMICA ET CYTOCHEMICA 34 2 129 - 134 2001年 [無し][無し]
     研究論文（学術雑誌） 
    Expression of acidic fibroblast growth factor (aFGF; FGF-1) in rat adrenal gland was investigated using a reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. FGF-1 mRNA was detected in the adrenal medulla but not in the adrenal cortex. Analysis of the PCR product indicated that the sequence was identical to that of FGF-1 in the midbrain. FGF-1 immunoreactivity was detected in the adrenal medulla. Double immunostaining revealed that FGF-1 colocalized with ethanolamine-N-methyltransferase-, an enzyme of adrenaline synthesis, immunoreactive adrenergic cells. These results indicate that FGF-1 is expressed by adrenergic cells in the adrenal medulla.
  • H Kitano, T Takeda, S Takeda, M Suzuki, T Kitanishi, K Kitajima, H Kimura, Tooyama, I
    ACTA HISTOCHEMICA ET CYTOCHEMICA 34 4 229 - 233 2001年 [無し][無し]
     研究論文（学術雑誌） 
    The anti-diuretic hormone, vasopressin, has been shown to be important in regulating inner ear fluid. We report on the involvement between Meniere's disease and vasopressin. We designed an experiment to determine whether vasopressin directly affects the fluid level. We chronically infused this hormone in rats and examined them for morphological signs of endolymphatic hydrops. Vasopressin infusion significantly increased the scala media area by 5.9 +/- 7.1% (P <0.01), and the length of Reissner's membrane by 5.9 +/- 3.2% (P <0.001). The results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps due to down-regulating the number of vasopressin receptors.
  • Y Matsuoka, Y Aimi, H Kimura, T Taniguchi, Y Oomura, K Sasaki, Tooyama, I
    ACTA HISTOCHEMICA ET CYTOCHEMICA 34 2 129 - 134 2001年 [無し][無し]
     研究論文（学術雑誌） 
    Expression of acidic fibroblast growth factor (aFGF; FGF-1) in rat adrenal gland was investigated using a reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. FGF-1 mRNA was detected in the adrenal medulla but not in the adrenal cortex. Analysis of the PCR product indicated that the sequence was identical to that of FGF-1 in the midbrain. FGF-1 immunoreactivity was detected in the adrenal medulla. Double immunostaining revealed that FGF-1 colocalized with ethanolamine-N-methyltransferase-, an enzyme of adrenaline synthesis, immunoreactive adrenergic cells. These results indicate that FGF-1 is expressed by adrenergic cells in the adrenal medulla.
  • S Yu, T Zhao, M Fan, Tooyama, I, H Kimura, TG Renda
    PEPTIDES 21 11 1657 - 1662 2000年11月 [無し][無し]
     研究論文（学術雑誌） 
    A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals. (C) 2000 Published by Elsevier Science Inc.
  • S Yu, T Zhao, M Fan, Tooyama, I, H Kimura, TG Renda
    PEPTIDES 21 11 1657 - 1662 2000年11月 [無し][無し]
     研究論文（学術雑誌） 
    A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals. (C) 2000 Published by Elsevier Science Inc.
  • Tooyama, I, H Abe, TG Renda, H Kimura
    PEPTIDES 21 11 1649 - 1655 2000年11月 [無し][無し]
     研究論文（学術雑誌） 
    Tyr-D-Ala-Phe is a N-terminal sequence commonly found in a peptide family including dermorphin and deltorphin. The tripeptide was synthesized and conjugated with poly L-lysine. Nuclear magnetic resonance (NMR) indicated that approximately 38 molecules of the tripeptide were bound to each molecule of poly L-lysine. The conjugate was used to immunize rabbits, and high titer antisera were obtained. An IgG fraction was purified by protein G affinity chromatography. A specific antibody to the tripeptide was then obtained by affinity chromatography using formylcellulofine conjugated with Tyr-D-Ala-Phe. On immunospot assay, the best IgG antibody was capable of detecting 125 ng of Tyr-D-Ala-Phe but failed to react even with 2.0 mug of Tyr-L-Ala-Phe or poly L-lysine. Our immunohistochemical examination selectively localized the secretory glands of frog skin. (C) 2000 Elsevier Science Inc. All rights reserved.
  • ラット上頸神経節におけるコリンアセチル基転移酵素含有神経の存在と線条体内移植による変化
    深尾 繁治, 遠山 育夫, 安原 治, 相見 良成, 木村 宏
    神経組織の成長・再生・移植研究会学術集会プログラム・予稿集 15回 38 - 38 神経組織の成長・再生・移植研究会 2000年06月
  • 一次知覚神経における末梢型コリン合成酵素(pChAT)の免疫組織化学
    相見 良成, 遠山 育夫, 安原 治, 木村 宏
    解剖学雑誌 75 1 126 - 126 2000年02月
  • K Nakajima, Tooyama, I, O Yasuhara, Y Aimi, H Kimura
    JOURNAL OF CHEMICAL NEUROANATOMY 18 1-2 31 - 40 2000年02月 [有り][無し]
     研究論文（学術雑誌） 
    Using a recently developed antiserum against a splice variant (pChAT) of choline acetyltransferase, the enzyme which synthesizes acetylcholine, we carried out an immunohistochemical examination in the digestive canal of rats. Positive staining was exclusively localized to neuronal cells and fibers. Positive somata were distributed widely in the intramural ganglia throughout the digestive tract from the esophagus to the rectum. Double staining indicated that, in the rat, virtually all pChAT immunoreactive somata exhibited histochemical activity for acetylcholinesterase but not for NADPH-diaphorase. In the guinea pig, however, there were a few neurons possessing both pChAT and NADPH-diaphorase. We also found a few neuronal somata which were positive for acetylcholinesterase but not for pChAT. The results suggest that pChAT immunohistochemistry is useful for studying the enteric cholinergic system. (C) 2000 Elsevier Science B.V. All rights reserved.
  • M Park, Y Tokunaga, H Kimura, Tooyama, I, T Maeda, TG Renda
    JOURNAL OF CHEMICAL NEUROANATOMY 18 1-2 11 - 22 2000年02月 [有り][無し]
     研究論文（学術雑誌） 
    Foetal rat brain from embryonic day (ED) 12-22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of (D-Ala(2))-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15-17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation. (C) 2000 Published by Elsevier Science B.V. All rights reserved.
  • M. Suzuki, T. Kitanishi, H. Kitano, Y. Yazawa, K. Kitajima, T. Takeda, Y. Tokunaga, T. Maeda, H. Kimura, I. Tooyama
    Hearing Research 139 1-2 51 - 58 2000年01月 [有り][無し]
     研究論文（学術雑誌） 
    C-type natriuretic peptide (CNP) is a member of the atrial natriuretic peptide family (ANP family). The family also includes ANP and brain natriuretic peptide (BNP). These peptides regulate the homeostasis of body fluid and blood pressure as a neuropeptide in the central nervous system as well as a cardiac hormone in the periphery. We have recently reported the expression of CNP mRNA in the inner ear. To assess the possible physiological role of CNP in the inner ear, we investigated the localization of CNP peptide in the rat inner ear by immunohistochemistry at the light and electron microscopic level. CNP-like immunoreactivity was widely distributed in the secretory and the neuronal portion of the inner ear, i.e. the spiral ligament, the dark cell region of the utriculus, the epithelium of the endolymphatic sac, the spiral ganglion cells and the vestibular ganglion cells. The results suggest that CNP may play a role in the homeostasis of the perilymph and endolymph and may also influence nerve activities in the inner ear. Copyright (C) 2000 Elsevier Science B.V.
  • Shun Yu, Tong Zhao, Ming Fan, Ikuo Tooyama, Hiroshi Kimura, Tindaro G. Renda
    Peptides 21 11 1657 - 1662 2000年 [無し][無し]
     研究論文（学術雑誌） 
    A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals. (C) 2000 Elsevier Science Inc.
  • Ikuo Tooyama, Hiromichi Abe, Tindaro G. Renda, Hiroshi Kimura
    Peptides 21 11 1649 - 1655 2000年 [無し][無し]
     研究論文（学術雑誌） 
    Tyr-D-Ala-Phe is a N-terminal sequence commonly found in a peptide family including dermorphin and deltorphin. The tripeptide was synthesized and conjugated with poly L-lysine. Nuclear magnetic resonance (NMR) indicated that approximately 38 molecules of the tripeptide were bound to each molecule of poly L-lysine. The conjugate was used to immunize rabbits, and high titer antisera were obtained. An IgG fraction was purified by protein G affinity chromatography. A specific antibody to the tripeptide was then obtained by affinity chromatography using formylcellulofine conjugated with Tyr-D-Ala-Phe. On immunospot assay, the best IgG antibody was capable of detecting 125 ng of Tyr-D-Ala-Phe but failed to react even with 2.0 μg of Tyr-L-Ala-Phe or poly L-lysine. Our immunohistochemical examination selectively localized the secretory glands of frog skin. (C) 2000 Elsevier Science Inc.
  • Ikuo Tooyama, Hiroshi Kimura
    Journal of Chemical Neuroanatomy 17 4 217 - 226 2000年01月 [無し][無し]
     研究論文（学術雑誌） 
    Central cholinergic systems have been visualized by immunohistochemistry using antibodies to choline acetyltransferase (ChAT). Peripheral cholinergic cells and fibers, however, have been hardly detectable with most of these antibodies. This phenomenon suggests that a different form of ChAT may exist in peripheral tissues. Here we report two types of mRNA for ChAT expressed by alternative splicing in rat pterygopalatine ganglion. One is exactly identical with ChAT mRNA reported in the central nervous system (ChAT of a common type cChAT). The other lacks exons 6, 7, 8 and 9, which was detected only in the pterygopalatine ganglion (ChAT of a peripheral type pChAT). The peculiarity of pChAT in chemical structure, possessing a splice joint of the exons 5 and 10, led us to produce rabbit antisera against a recombinant peptide of 41 amino acids which spans over the splice joint. On Western blots using a successfully obtained antiserum, an intense band of about 50 kDa, corresponding to the expected molecular weight of pChAT, was detected in the pterygopalatine ganglion but not in the brain. Immunohistochemistry using the antiserum failed to reveal positive staining of known brain cholinergic structures, while it permitted us to observe peripheral, probably cholinergic, nerve cells and fibers including those in the pterygopalatine ganglion and enteric nervous system. Copyright (C) 2000 Elsevier Science B.V.
  • Enhancement of progenitor cell division in the dentate gyrus triggered by initial limbic seizures in rat models of epilepsy
    Nakagawa E, Aimi Y, Yasuhara O, Tooyama I, Shimada M, McGeer PL, Kimura H
    Epilepsia 41 10 - 18 2000年 [無し][無し]
     研究論文（学術雑誌）
  • Yu S, Zhao T, Fan M, Tooyama I, Kimura H, Renda TG
    Peptides 21 1657 - 1662 2000年 [無し][無し]
     研究論文（学術雑誌）
  • Ikuo Tooyama, Hiroshi Kimura
    Journal of Chemical Neuroanatomy 17 4 217 - 226 2000年01月 [無し][無し]
     研究論文（学術雑誌） 
    Central cholinergic systems have been visualized by immunohistochemistry using antibodies to choline acetyltransferase (ChAT). Peripheral cholinergic cells and fibers, however, have been hardly detectable with most of these antibodies. This phenomenon suggests that a different form of ChAT may exist in peripheral tissues. Here we report two types of mRNA for ChAT expressed by alternative splicing in rat pterygopalatine ganglion. One is exactly identical with ChAT mRNA reported in the central nervous system (ChAT of a common type cChAT). The other lacks exons 6, 7, 8 and 9, which was detected only in the pterygopalatine ganglion (ChAT of a peripheral type pChAT). The peculiarity of pChAT in chemical structure, possessing a splice joint of the exons 5 and 10, led us to produce rabbit antisera against a recombinant peptide of 41 amino acids which spans over the splice joint. On Western blots using a successfully obtained antiserum, an intense band of about 50 kDa, corresponding to the expected molecular weight of pChAT, was detected in the pterygopalatine ganglion but not in the brain. Immunohistochemistry using the antiserum failed to reveal positive staining of known brain cholinergic structures, while it permitted us to observe peripheral, probably cholinergic, nerve cells and fibers including those in the pterygopalatine ganglion and enteric nervous system. Copyright (C) 2000 Elsevier Science B.V.
  • E Nakagawa, Y Aimi, O Yasuhara, Tooyama, I, M Shimada, PL McGeer, H Kimura
    EPILEPSIA 41 1 10 - 18 2000年01月 [無し][無し]
     研究論文（学術雑誌） 
    Purpose: Mitogenic effects of seizures on granule cell progenitors in the dentate gyrus were studied in two rat models of epilepsy. We investigated which stage of epileptogenesis is critical for eliciting progenitor cell division and whether seizure-induced neuronal degeneration is responsible for the enhancement of progenitor cell division. Methods: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neurogenesis of dentate granule cells was evaluated using the bromodeoxyuridine (BrdU) labeling method, and neuronal degeneration was assessed by in situ DNA fragmentation analysis. Results: After injection of KA, the number of BrdU-positive granule cells began to increase at day 3 after the treatment, peaked at day 5, and returned to baseline at day 10. By day 13, the values were lower than control. After kindling, the number of BrdU-positive cells began to increase after five consecutive experiences of stage I seizures. The increase occurred from day 1 to day 3 after the last electrical stimulation, but returned to baseline by day 7. After generalized seizures were well established, repeated stimulation did not facilitate division of granule cell progenitors. DNA fragmentation was noted in pyramidal neurons in the CA1, CA3, and hilus regions at 18 h after KA injection, but not in the kindling model. Conclusions: These observations indicate that a mechanism in epileptogenesis boosts dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. Pyramidal neuronal degeneration is not necessary for triggering the upregulation. II is suggested that newly born granule cells may play a role in the network reorganization that occurs during epileptogenesis.
  • Yutaka Nakanishi, Ikuo Tooyama, Osamu Yasuhara, Yoshinari Aimi, Kazutomo Kitajima, Hiroshi Kimura
    Journal of Chemical Neuroanatomy 17 1 21 - 32 1999年09月 [無し][無し]
     研究論文（学術雑誌） 
    As shown in the accompanying paper, choline acetyltransferase, so far the best histochemical marker for identifying cholinergic structures, has at least one alternative splice variant. The variant, termed pChAT because of its preferential expression in peripheral organs, encouraged us to study peripheral, probably cholinergic, cells and fibers by immunohistochemistry using an antiserum against a peptide specific for pChAT. We chose the larynx of the rat, since cholinergic innervation in this organ has been well established by physiological studies, but not sufficiently by chemical neuroanatomy. Neuronal somata positive for pChAT were found in the intralaryngeal ganglia. Our double staining study indicated that these somata always possessed acetylcholinesterase activity, while the reverse did not hold true. Nerve fibers positive for pChAT were distributed widely in the intrinsic laryngeal muscles, laryngeal glands, blood vessels and laryngeal mucosa. In the intrinsic laryngeal muscles, pChAT-positive terminals were apposed closely to motor end-plates which were stained positively for acetylcholinesterase activity. Denervation experiments revealed that there were three types of pChAT-positive fibers in the larynx: (1) special visceral efferent fibers to the intrinsic laryngeal muscles, which decreased dramatically in number after vagotomy (2) parasympathetic postganglionic fibers near the laryngeal glands and blood vessels, which appeared unaffected after vagotomy or cervical sympathectomy and (3) afferent fibers innervating the laryngeal mucosa, which reduced markedly in number after vagotomy performed distal, but not proximal, to the nodose ganglion. Such afferent fibers remained unchanged following the neonatal capsaicin treatment, suggesting their independence from those containing substance P. Copyright (C) 1999 Elsevier Science B.V.
  • Y Nakanishi, Tooyama, I, O Yasuhara, Y Aimi, K Kitajima, H Kimura
    JOURNAL OF CHEMICAL NEUROANATOMY 17 1 21 - 32 1999年09月 [無し][無し]
     研究論文（学術雑誌） 
    As shown in the accompanying paper, choline acetyltransferase: so far the best histochemical marker for identifying cholinergic structures, has at least one alternative splice variant. The variant, termed pChAT because of its preferential expression in peripheral organs, encouraged us to study peripheral, probably cholinergic, cells and fibers by immunohistochemistry using an antiserum against a peptide specific for pChAT. We chose the larynx of the rat, since cholinergic innervation in this organ has been well established by physiological studies, but not sufficiently by chemical neuroanatomy. Neuronal somata positive for pChAT were found in the intralaryngeal ganglia. Our double staining study indicated that these somata always possessed acetylcholinesterase activity, while the reverse did not hold true. Nerve fibers positive for pChAT were distributed widely in the intrinsic laryngeal muscles, laryngeal glands, blood vessels and laryngeal mucosa. In the intrinsic laryngeal muscles, pChAT-positive terminals were apposed closely to motor end-plates which were stained positively for acetylcholinesterase activity. Denervation experiments revealed that there were three types of pChAT-positive fibers in the larynx: (1) special visceral efferent fibers to the intrinsic laryngeal muscles, which decreased dramatically in number after vagotomy; (2) parasympathetic postganglionic fibers near the laryngeal glands and blood vessels, which appeared unaffected after vagotomy or cervical sympathectomy; and (3) afferent fibers innervating the laryngeal mucosa, which reduced markedly in number after vagotomy performed distal, but not proximal, to the nodose ganglion. Such afferent fibers remained unchanged following the neonatal capsaicin treatment, suggesting their independence from those containing substance P. (C) 1999 Published by Elsevier Science B.V. All rights reserved.
  • Yasuji Matsuoka, Mitsuhiro Okazaki, Yoshihisa Kitamura, Kazuyuki Takata, Ikuo Tooyama, Hiroshi Kimura, Takashi Taniguchi
    Brain Research 836 1-2 213 - 217 1999年07月 [無し][無し]
     研究論文（学術雑誌） 
    Inducible cyclooxygenase (COX-2) was transiently induced in the neurons throughout the entire hippocampus between 6 and 24 h after injection of kainic acid (KA). The induction of COX-2 correlated more closely with the induction of c-Jun than with that of c-Fos. Phosphorylated c-Jun was induced 6-12 h after in the CA3 pyramidal neurons, which undergo apoptosis. Almost all of neurons with phosphorylated c-Jun were colocalized with COX-2. These results suggest that COX-2 and c-Jun phosphorylation may participate in KA- induced neurodegeneration.
  • Y Matsuoka, M Okazaki, Y Kitamura, K Takata, Tooyama, I, H Kimura, T Taniguchi
    BRAIN RESEARCH 836 1-2 213 - 217 1999年07月 [無し][無し]
     研究論文（学術雑誌） 
    inducible cyclooxygenase (COX-2) was transiently induced in the neurons throughout the entire hippocampus between 6 and 24 h after injection of kainic acid (KA). The induction of COX-2 correlated more closely with the induction of c-Jun than with that of c-Fos. Phosphorylated c-Jun was induced 6-12 h after in the CA3 pyramidal neurons, which undergo apoptosis. Almost all of neurons with phosphorylated c-Jun were colocalized with COX-2. These results suggest that COX-2 and c-Jun phosphorylation may participate in KA-induced neurodegeneration. (C) 1999 Elsevier Science B.V. All rights reserved.
  • K. Sasaki, I. Tooyama, A. J. Li, Y. Oomura, H. Kimura
    Neuroscience 92 4 1287 - 1294 1999年06月 [無し][無し]
     研究論文（学術雑誌） 
    We examined the effects of repeated subcutaneous injections of an acidic fibroblast growth factor fragment analog, [Ala16] acidic fibroblast growth factor (1-29), on learning and memory and on the choline acetyltransferase immunoreactivity of forebrain neurons in senescence-accelerated mice. One group of accelerated senescence-prone mice (accelerated senescence-prone-8) received [Ala16] acidic fibroblast growth factor (1-29), whereas the other group of accelerated senescence-prone-8 mice and a group of accelerated senescence-resistant mice (control) received vehicle solution. Injections began at three weeks after birth and were given weekly for 10 months. In a passive avoidance test, the mean retention latency at three, six and nine months of age was significantly longer in controls (vehicle-treated accelerated senescence-resistant-1) and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than in vehicle-treated accelerated senescence-prone-8 mice, and the latency in acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice was significantly shorter than that in controls only at nine months of age. In the Morris water maze task, the mean latency to climb onto the platform was significantly longer in acidic fibroblast growth factor fragment- and vehicle-treated accelerated senescence-prone-8 mice than in controls. However, the mean latency in the third and fourth trial blocks was significantly shorter for acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than for vehicle-treated accelerated senescence-prone-8 mice. In the probe trials, controls and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice spent significantly more time in the quadrant in which the platform had previously been located than in the other three quadrants. In acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice, the density of medial septum neurons intensely stained for choline acetyltransferase was significantly greater than that in vehicle-treated accelerated senescence- prone-8 mice, but significantly less than that in controls. The results indicate that the beneficial effect of [Ala16] acidic fibroblast growth factor (1-29) on learning and memory function in accelerated senescence- prone-8 mice may be related to a preservation of function in medial septum cholinergic neurons.
  • Tooyama, I, T Nishimura, E Nakagawa, H Morita, S Uemura, Y Aimi, O Yasuhara, H Kimura
    NEUROPATHOLOGY 19 2 203 - 208 1999年06月 [無し][無し]
     研究論文（学術雑誌） 
    Reaction of microglial cells as well as DNA fragmentation in pyramidal cells was investigated using immunohistochemistry and in situ end-labeling method (TUNEL) in the hippocampus of rats after rapid kindling or kainic acid treatment. In intact rats, no or very little DNA fragmentation was detected in the hippocampus. Resting microglia distributed evenly throughout the hippocampus, Neither major histocompatibility complex antigens class I (MHC I) nor class II (MHC II) immunoreactivity was seen in the hippocampus, In the rapid-kindling model, no DNA fragmentation, reactive microglia or MHC antigen-positive cells were present in the hippocampus. In rats given an intraperitoneal injection of kainic acid (12 mg/kg), reactive microglial cells were seen around pyramidal neurons in the CA1 and CA3 field of the hippocampus as well as in the hilus of the dentate gyrus at 3 h, At that point in time, DNA fragmentation was not detected. DNA fragmentation was clearly observed, mainly in the CA1 region of the hippocampus, from 24h to 4 weeks after the kainic acid injection. The number of reactive microglia was quickly increased and reached a maximum at 7 days after the injection, and continued until 8 weeks thereafter, During this period, many reactive microglia expressed MHC I and MHC II. The present study indicates that epileptic seizures do not depend on microglial activation and that microglial activation is closely related to the neuronal death process induced by kainic acid.
  • K. Sasaki, I. Tooyama, A. J. Li, Y. Oomura, H. Kimura
    Neuroscience 92 4 1287 - 1294 1999年06月 [無し][無し]
     研究論文（学術雑誌） 
    We examined the effects of repeated subcutaneous injections of an acidic fibroblast growth factor fragment analog, [Ala16] acidic fibroblast growth factor (1-29), on learning and memory and on the choline acetyltransferase immunoreactivity of forebrain neurons in senescence-accelerated mice. One group of accelerated senescence-prone mice (accelerated senescence-prone-8) received [Ala16] acidic fibroblast growth factor (1-29), whereas the other group of accelerated senescence-prone-8 mice and a group of accelerated senescence-resistant mice (control) received vehicle solution. Injections began at three weeks after birth and were given weekly for 10 months. In a passive avoidance test, the mean retention latency at three, six and nine months of age was significantly longer in controls (vehicle-treated accelerated senescence-resistant-1) and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than in vehicle-treated accelerated senescence-prone-8 mice, and the latency in acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice was significantly shorter than that in controls only at nine months of age. In the Morris water maze task, the mean latency to climb onto the platform was significantly longer in acidic fibroblast growth factor fragment- and vehicle-treated accelerated senescence-prone-8 mice than in controls. However, the mean latency in the third and fourth trial blocks was significantly shorter for acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 than for vehicle-treated accelerated senescence-prone-8 mice. In the probe trials, controls and acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice spent significantly more time in the quadrant in which the platform had previously been located than in the other three quadrants. In acidic fibroblast growth factor fragment-treated accelerated senescence-prone-8 mice, the density of medial septum neurons intensely stained for choline acetyltransferase was significantly greater than that in vehicle-treated accelerated senescence- prone-8 mice, but significantly less than that in controls. The results indicate that the beneficial effect of [Ala16] acidic fibroblast growth factor (1-29) on learning and memory function in accelerated senescence- prone-8 mice may be related to a preservation of function in medial septum cholinergic neurons.
  • Hiroya Kitano, Mikio Suzuki, Tsuyoshi Kitanishi, Yoshiro Yazawa, Kazutomo Kitajima, Takahiro Isono, Taizo Takeda, Hiroshi Kimura, Ikuo Tooyama
    NeuroReport 10 6 1205 - 1207 1999年04月 [無し][無し]
     研究論文（学術雑誌） 
    THE anti-diuretic hormone vasopressin has been shown to be important in regulating inner ear fluid. The diuretic hormone, CNP, and its receptor, ANP- B receptor, may also function in the regulation of inner ear fluid. To determine whether vasopressin directly affects the fluid level, we infused this hormone to rat and assay of V2-AVP receptor mRNA by semiquantitative RT- PCR demonstrated a significantly lower level of this transcript in vasopressin-infused animals than in saline-infused animals. The levels of CNP and ANP-B receptors mRNA, however, were the same in both groups of rats. Results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps by down- regulating the number of vasopressin receptors.
  • H Kitano, M Suzuki, T Kitanishi, Y Yazawa, K Kitajima, T Isono, T Takeda, H Kimura, Tooyama, I
    NEUROREPORT 10 6 1205 - 1207 1999年04月 [無し][無し]
     研究論文（学術雑誌） 
    THE anti-diuretic hormone vasopressin has been shown to be important in regulating inner ear fluid. The diuretic hormone, CNP, and its receptor, ANP-R receptor, may also function in the regulation of inner ear fluid. To determine whether vasopressin directly affects the fluid level, we infused this hormone to rat and assay of V2-AVP receptor mRNA by semiquantitative RT-PCR demonstrated a significantly lower level of this transcript in vasopressin-infused animals than in saline-infused animals. The levels of CNP and ANP-R receptors mRNA, however, were the same in both groups of rats. Results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps by down-regulating the number of vasopressin receptors. (C) 1999 Lippincott Williams & Wilkins.
  • Y Matsuoka, Y Kitamura, H Takahashi, Tooyama, I, H Kimura, PJ Gebicke-Haerter, Y Nomura, T Taniguchi
    NEUROCHEMISTRY INTERNATIONAL 34 2 91 - 99 1999年02月 [無し][無し]
     研究論文（学術雑誌） 
    Interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) induce expression of inducible nitric oxide synthase (iNOS) protein both in cells in vitro and in the brain in vivo. In cultured cells, excessive production of nitric oxide (NO) induces neuronal cell death. However, it is still unclear whether IFN-gamma and LPS might induce neuronal cell death in vivo. In this study, we examined the neuronal cell death and induction of major histocompatibility complex (MHC) antigens after microinjection of IFN-gamma/LPS into thr rat hippocampus. Although microglia appeared morphologically ramified in the normal and vehicle-injected hippocampus, microinjection of IFN-gamma/LPS immediately induced the ameboid type. From days 1-7, iNOS was expressed in ameboid microglia surrounding the site of the microinjection, Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appeared among the granular neurons of the dentate gyrus on day 3 and peaked about 7 days after microinjection. When the NOS inhibitor N-G-nitro-L-arginine (L-NA) was intraperitoneally administered prior to the microinjection, the number of TUN EL-positive neurons decreased in a L-NA dose-dependent manner. These results suggest that IFN-gamma/LPS induces delayed neuronal apoptosis in the hippocampus in vivo, and it possibly involves excessive NO production by iNOS, Thus, this animal model may be one of neurodegenerative with extensive inflammatory activation in the hippocampus. (C) 1999 Elsevier Science Ltd. All rights reserved.
  • Y Matsuoka, Y Kitamura, H Takahashi, Tooyama, I, H Kimura, PJ Gebicke-Haerter, Y Nomura, T Taniguchi
    NEUROCHEMISTRY INTERNATIONAL 34 2 91 - 99 1999年02月 [無し][無し]
     研究論文（学術雑誌） 
    Interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) induce expression of inducible nitric oxide synthase (iNOS) protein both in cells in vitro and in the brain in vivo. In cultured cells, excessive production of nitric oxide (NO) induces neuronal cell death. However, it is still unclear whether IFN-gamma and LPS might induce neuronal cell death in vivo. In this study, we examined the neuronal cell death and induction of major histocompatibility complex (MHC) antigens after microinjection of IFN-gamma/LPS into thr rat hippocampus. Although microglia appeared morphologically ramified in the normal and vehicle-injected hippocampus, microinjection of IFN-gamma/LPS immediately induced the ameboid type. From days 1-7, iNOS was expressed in ameboid microglia surrounding the site of the microinjection, Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appeared among the granular neurons of the dentate gyrus on day 3 and peaked about 7 days after microinjection. When the NOS inhibitor N-G-nitro-L-arginine (L-NA) was intraperitoneally administered prior to the microinjection, the number of TUN EL-positive neurons decreased in a L-NA dose-dependent manner. These results suggest that IFN-gamma/LPS induces delayed neuronal apoptosis in the hippocampus in vivo, and it possibly involves excessive NO production by iNOS, Thus, this animal model may be one of neurodegenerative with extensive inflammatory activation in the hippocampus. (C) 1999 Elsevier Science Ltd. All rights reserved.
  • Yoshihisa Kitamura, Takashi Ota, Yasuji Matsuoka, Ikuo Tooyama, Hiroshi Kimura, Shun Shimohama, Yasuyuki Nomura, Peter J. Gebicke-Haerter, Takashi Taniguchi
    GLIA 25 2 154 - 164 1999年01月 [有り][無し]
     研究論文（学術雑誌） 
    It is now generally accepted that massive neuronal death due to oxidative stress is a regular feature of brains in neurodegenerative diseases. However, much less attention has been given to the death of glial cells. In this study, we examined p53-sensitive apoptosis of cells by using human glioblastoma A172 cells and p53-deficient mouse astrocytes. In human A172 cells, hydrogen peroxide (H2O2) caused cell death in a time- and concentration-dependent manner, accompanied by nucleosomal DNA fragmentation and chromatin condensation. After treatment with H2O2, p53 protein was highly expressed and protein levels of Bak, p21(WAF1/CIP1) and GADD45 were also enhanced. However, the protein levels of Bcl-2 and Bax did not change. On the other hand, primary cultured astrocytes from p53-deficient mouse brain grew faster than wild-type and heterozygous astrocytes. In addition, p53-deficient astrocytes were more resistant to H2O2-induced apoptosis than wild-type and heterozygous astrocytes. These results suggest that glial proliferation and the repair of damaged DNA may be regulated by p53-induced p21(WAF1/CIP1) and GADD45, and that glial apoptosis caused by oxidative stress may be mediated by p53-induced Bak.
  • I. Tooyama, T. Nishimura, E. Nakagawa, H. Morita, S. Uemura, Y. Aimi, O. Yasuhara, H. Kimura
    Neuropathology 19 2 203 - 208 1999年 [無し][無し]
     研究論文（学術雑誌） 
    Reaction of microglial cells as well as DNA fragmentation in pyramidal cells was investigated using immunohistochemistry and in situ end-labeling method (TUNEL) in the hippocampus of rats after rapid kindling or kainic acid treatment. In intact rats, no or very little DNA fragmentation was detected in the hippocampus. Resting microglia distributed evenly throughout the hippocampus. Neither major histocompatibility complex antigens class I (MHC I) nor class II (MHC II) immunoreactivity was seen in the hippocampus. In the rapid-kindling model, no DNA fragmentation, reactive microglia or MHC antigen-positive cells were present in the hippocampus. In rats given an intraperitoneal injection of kainic acid (12 mg/kg), reactive microglial cells were seen around pyramidal neurons in the CA1 and CA3 field of the hippocampus as well as in the hilus of the dentate gyrus at 3 h. At that point in time, DNA fragmentation was not detected. DNA fragmentation was clearly observed, mainly in the CA1 region of the hippocampus, from 24 h to 4 weeks after the kainic acid injection. The number of reactive microglia was quickly increased and reached a maximum at 7 days after the injection, and continued until 8 weeks thereafter. During this period, many reactive microglia expressed MHC I and MHC II. The present study indicates that epileptic seizures do not depend on microglial activation and that microglial activation is closely related to the neuronal death process induced by kainic acid.
  • N. Matsukawa, I. Tooyama, H. Kimura, T. Yamamoto, Y. Tsugu, Y. Oomura, K. Ojika
    Neuroscience 88 1 79 - 92 1999年01月 [無し][無し]
     研究論文（学術雑誌） 
    Hippocampal cholinergic neurostimulating peptide stimulates cholinergic phenotype development by inducing choline acetyltransferase in the rat medial septal nucleus in vitro. Adult senescence-accelerated-prone mice/8, a substrain of the senescence-accelerated-prone mouse, show a remarkable age- accelerated deterioration in learning and memory. We cloned mouse hippocampal cholinergic neurostimulating peptide precursor protein complementary DNA. The deduced amino acid sequence showed that the neurostimulating peptide itself is the same as that found in the rat. In situ hybridization revealed that the highest expression of the precursor protein messenger RNA was in hippocampal pyramidal neurons. Compared with a strain of senescence-accelerated-resistant mouse (control mouse), adult senescence-accelerated-prone mice/8 showed increased expression of both the precursor messenger RNA and the neurostimulating peptide-related immunodeposits in the hippocampal CA1 field. The deposits were intensely and diffusely precipitated in neuropils throughout the strata oriens and radiatum in senescence-accelerated-prone mice/8, but not in control mice. The neurostimulating peptide content in the hippocampus was higher in senescence-accelerated-prone mice/8 than in control mice, while its precursor protein itself was not different between the two strains. Furthermore, our previous and present data show that the medial septal and hippocampal choline acetyltransferase activity was significantly lower in senescence-accelerated-prone mice/8 than in control mice. The data suggest that, in hippocampal neurons in adult senescence-accelerated-prone mice/8, the production of hippocampal cholinergic neurostimulating peptide precursor protein in neuronal somata, which is associated with an increased expression of its messenger RNA in the CA1 field, occurs as a consequence of low activity in their presynaptic cholinergic neurons. This is followed by accelerated processing to generate bioactive peptide and transport to its functional fields. However, certain mechanisms reduce the release of the peptide and lead to its accumulation in the neuropil. These disturbances of the septohippocampal cholinergic system might be the biochemical mechanism underlying the characteristic deterioration of senescence-accelerated-prone mice/8.
  • T Yuasa, T Isono, Tooyama, I, A Seto
    MICROBIOLOGY AND IMMUNOLOGY 43 4 365 - 371 1999年 [無し][無し]
     研究論文（学術雑誌） 
    Inbred rabbits of the B/Jas strain are highly susceptible to herpes simplex virus type-1 (HSV-1) encephalitis, developing seizures of encephalitis after intravenous injection of the KOS strain of the virus. Anti-viral interferon activity became detectable in the serum just prior to or at the onset of seizures, its level being lower in the serum than in the cerebrospinal fluid. The activity was of gamma interferon, as suggested by the acid instability and the inability of Mx protein induction. An immunohistochemical analysis of the brain tissues of encephalitic rabbits showed that MHC class I antigen was expressed on the microglia cells of inflamed lesions but not on these cells in uninflamed areas. These findings were discussed in correlation with the pathogenesis of herpetic encephalitis in the inbred rabbits.
  • Peripheral type choline acetyltransferase: molecular neuroanatomy
    Kimura H, Aste N, Nakajima K, Aimi Y, Yasuhara O, Tooyama I
    Ital J Anat Embryol 104 S1 1999年 [無し][無し]
     研究論文（学術雑誌）
  • Expression of inducible nitric oxide synthase human thyroid papillary carcinoma
    Kitano H, Kitanishi T, Nakanishi Y, Suzuki M, Takeuchi E, Yazawa Y, Kitajima K, Kimura H, Tooyama I
    Thyroid 9 113 - 117 1999年 [無し][無し]
     研究論文（学術雑誌）
  • A. Aisaka, Y. Aimi, O. Yasuhara, I. Tooyama, H. Kimura, M. Shimada
    Neuroscience 90 1 53 - 67 1999年 [無し][無し]
     研究論文（学術雑誌） 
    Although it has been shown that unilateral neonatal cortical ablation induces bilateral corticospinal projections, the explanation for the pathways responsible for this bilateral innervation remains controversial. We hypothesized that such reinnervation may be supplied from newly formed fibers sprouting at the level rostral to, or at, or caudal to the pyramidal decussation. In order to test our hypothesis, we examined the brain and spinal cord of young hamsters which had a unilateral ablution of the right motor cortex at six days postnatally, and then received an injection of an anterograde neuronal lectin tracer, Phaseolus vulgaris-leucoagglutinin, into the hindlimb area of the left motor cortex at 21 days postnatally. For the identification of motoneurons in the lumbar spinal cord, some of these animals also received an injection of cholera toxin subunit B, a retrograde tracer, into the gastrocnemius muscle. A quantitative analysis in the left gray matter of the lumbar spinal cord indicated that the lectin labeling was two to eight times higher in cortically ablated animals than in intact animals. Immunohistochemical detection of the lectin revealed that innervation of the left spinal cord occurred close to targets at lower levels in the spinal cord. Two modes of reinnervation (types I and II) by the intact corticospinal tract were recognized. The type I fibers consisted of recrossing axon collaterals sprouted from the intact dorsal funiculus near their targets, while the type II fibers were recrossing parent axons which entered the intact, right gray matter several levels rostral to their targets, and then changed direction toward the targets. The recrossing at lower spinal levels yielded a large number of ipsilaterally labeled axons and their terminals in the gray matter of the denervated lumbar cord, with a distribution pattern similar to that seen on the intact side. The present results indicate that such ipsilateral innervation may play an important role in the sparing and recovery of function following neonatal hemicortical injury.
  • N Matsukawa, Tooyama, I, H Kimura, T Yamamoto, Y Tsugu, Y Oomura, K Ojika
    NEUROSCIENCE 88 1 79 - 92 1999年01月 [無し][無し]
     研究論文（学術雑誌） 
    Hippocampal cholinergic neurostimulating peptide stimulates cholinergic phenotype development by inducing choline acetyltransferase in the rat medial septal nucleus in vitro. Adult senescence-accelerated-prone mice/8, a substrain of the senescence-accelerated-prone mouse, show a remarkable age-accelerated deterioration in learning and memory. We cloned mouse hippocampal cholinergic neurostimulating peptide precursor protein complementary DNA. The deduced amino acid sequence showed that the neurostimulating peptide itself is the same as that found in the rat. In situ hybridization revealed that the highest expression of the precursor protein messenger RNA was in hippocampal pyramidal neurons. Compared with a strain of senescence-accelerated-resistant mouse (control mouse), adult senescence-accelerated-prone mice/8 showed increased expression of both the precursor messenger RNA and the neurostimulating peptide-related immunodeposits in the hippocampal CAI field. The deposits were intensely and diffusely precipitated in neuropils throughout the strata oriens and radiatum in senescence-accelerated-prone mice/8, but not in control mice. The neurostimulating peptide content in the hippocampus was higher in senescence-accelerated-prone mice/8 than in control mice, while its precursor protein itself was not different between the two strains. Furthermore, our previous and present data show that the medial septal and hippocampal choline acetyltransferase activity was significantly lower in senescence-accelerated-prone mice/8 than in control mice. The data suggest that, in hippocampal neurons in adult senescence-accelerated-prone mice/8, the production of hippocampal cholinergic neurostimulating peptide precursor protein in neuronal somata, which is associated with an increased expression of its messenger RNA in the CA1 held, occurs as a consequence of low activity in their presynaptic cholinergic neurons. This is followed by accelerated processing to generate bioactive peptide and transport to its functional fields. However, certain mechanisms reduce the release of the peptide and lead to its accumulation in the neuropil. These disturbances of the septohippocampal cholinergic system might be the biochemical mechanism underlying the characteristic deterioration of senescence-accelerated-prone mice/8. (C) 1998 IBRO. Published by Elsevier Science Ltd.
  • T Yuasa, T Isono, Tooyama, I, A Seto
    MICROBIOLOGY AND IMMUNOLOGY 43 4 365 - 371 1999年 [無し][無し]
     研究論文（学術雑誌） 
    Inbred rabbits of the B/Jas strain are highly susceptible to herpes simplex virus type-1 (HSV-1) encephalitis, developing seizures of encephalitis after intravenous injection of the KOS strain of the virus. Anti-viral interferon activity became detectable in the serum just prior to or at the onset of seizures, its level being lower in the serum than in the cerebrospinal fluid. The activity was of gamma interferon, as suggested by the acid instability and the inability of Mx protein induction. An immunohistochemical analysis of the brain tissues of encephalitic rabbits showed that MHC class I antigen was expressed on the microglia cells of inflamed lesions but not on these cells in uninflamed areas. These findings were discussed in correlation with the pathogenesis of herpetic encephalitis in the inbred rabbits.
  • Peripheral type choline acetyltransferase: molecular neuroanatomy
    Kimura H, Aste N, Nakajima K, Aimi Y, Yasuhara O, Tooyama I
    Ital J Anat Embryol 104 S1 1999年 [無し][無し]
     研究論文（学術雑誌）
  • Expression of inducible nitric oxide synthase human thyroid papillary carcinoma
    Kitano H, Kitanishi T, Nakanishi Y, Suzuki M, Takeuchi E, Yazawa Y, Kitajima K, Kimura H, Tooyama I
    Thyroid 9 113 - 117 1999年 [無し][無し]
     研究論文（学術雑誌）
  • A. Aisaka, Y. Aimi, O. Yasuhara, I. Tooyama, H. Kimura, M. Shimada
    Neuroscience 90 1 53 - 67 1999年 [無し][無し]
     研究論文（学術雑誌） 
    Although it has been shown that unilateral neonatal cortical ablation induces bilateral corticospinal projections, the explanation for the pathways responsible for this bilateral innervation remains controversial. We hypothesized that such reinnervation may be supplied from newly formed fibers sprouting at the level rostral to, or at, or caudal to the pyramidal decussation. In order to test our hypothesis, we examined the brain and spinal cord of young hamsters which had a unilateral ablution of the right motor cortex at six days postnatally, and then received an injection of an anterograde neuronal lectin tracer, Phaseolus vulgaris-leucoagglutinin, into the hindlimb area of the left motor cortex at 21 days postnatally. For the identification of motoneurons in the lumbar spinal cord, some of these animals also received an injection of cholera toxin subunit B, a retrograde tracer, into the gastrocnemius muscle. A quantitative analysis in the left gray matter of the lumbar spinal cord indicated that the lectin labeling was two to eight times higher in cortically ablated animals than in intact animals. Immunohistochemical detection of the lectin revealed that innervation of the left spinal cord occurred close to targets at lower levels in the spinal cord. Two modes of reinnervation (types I and II) by the intact corticospinal tract were recognized. The type I fibers consisted of recrossing axon collaterals sprouted from the intact dorsal funiculus near their targets, while the type II fibers were recrossing parent axons which entered the intact, right gray matter several levels rostral to their targets, and then changed direction toward the targets. The recrossing at lower spinal levels yielded a large number of ipsilaterally labeled axons and their terminals in the gray matter of the denervated lumbar cord, with a distribution pattern similar to that seen on the intact side. The present results indicate that such ipsilateral innervation may play an important role in the sparing and recovery of function following neonatal hemicortical injury.
  • Hiroshi Kimura, Ikuo Tooyama
    Clinical Neurology 38 12 1005 - 1008 1998年12月 [有り][無し]
     研究論文（国際会議プロシーディングス） 
    Acetylcholine (ACh), the first neurotransmitter to be discovered from the frog heart, is distributed widely in both the central and peripheral nervous systems. In the brain, for example, ACh plays important roles in several higher functions such as memory and learning. In peripheral organs, ACh has been shown to be a neurotransmitter in spinal motoneurons, all autonomic preganglionic neurons, all parasympathetic postganglionic neurons, and an exceptional sympathetic postganglionic neurons innervating the sweat gland. There has been, however, no good method to visualize ACh in tissues and organs. So far, immunohistochemistry for its synthetic enzyme choline acetyltransferase (ChAT E. C. 2. 3. 1. 6) has been used as the most reliable marker for morphological studies of cholinergic neurons. Despite the fact that most antibodies against ChAT clearly stain central cholinergic systems, they have poor ability to detect peripheral cholinergic systems. We found that there are two types of ChAT expressed in the rat pterygopalatine ganglion. One is identical to the ChAT in the brain, and another (pChAT) lacks exons 6,7,8, and 9. The peripheral cholinergic system is now clearly demonstrated by immunohistochemistry using our antiserum raised against the recombinant pChAT.
  • A Nagata, S Nakao, E Miyamoto, T Inada, Tooyama, I, H Kimura, K Shingu
    ANESTHESIA AND ANALGESIA 87 6 1416 - 1420 1998年12月 [無し][無し]
     研究論文（学術雑誌） 
    Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, has psychotomimetic activity. NMDA receptor antagonists cause morphological damage in the posterior cingulate cortex, which may be the brain region responsible for their psychotomimetic effects. Benzodiazepines are effective in preventing these effects through gamma-aminobutyric acid A (GABAA) receptor activation. We investigated the effect of propofol, which has both GABAA receptor-activating and NMDA receptor-suppressing activity, on ketamine-induced c-fos expression in the rat posterior cingulate cortex. Propofol or vehicle was continuously infused IV. Fifteen minutes later, 100 mg/kg ketamine or isotonic sodium chloride solution was injected intraperitoneally. Two hours later, brain sections were prepared, and c-fos expression was detected using immunohistochemical methods. Propofol significantly inhibited ketamine-induced c-fos expression in the posterior cingulate cortex. Propofol itself did not induce c-fos expression in this brain region. We conclude that propofol may be able to inhibit ketamine-induced psychotomimetic activity and neuronal damage. Implications: In the present study, we demonstrated that the clinically relevant dose of propofol significantly inhibited ketamine-induced c-fos expression in the rat posterior cingulate cortex. This finding implies that propofol may inhibit ketamine-induced psychotomimetic activity and neuronal damage.
  • Y Kitamura, T Ota, Y Matsuoka, M Okazaki, J Kakimura, Tooyama, I, H Kimura, S Shimohama, PJ Gebicke-Haerter, Y Nomura, T Taniguchi
    NEUROSCIENCE LETTERS 255 1 57 - 60 1998年10月 [無し][無し]
     研究論文（学術雑誌） 
    We examined kainic acid (KA)-induced neuronal death and changes in glial cells in p53-deficient (p53(-/-)) and wild-type (p53(+/+)) mice which were CBA and C57BL/6 background. The p53(-/-) mouse exhibited a KA-induced loss of CA3 pyramidal neurons similar to that in wild-type mouse. Before neuronal death, c-Jun protein was expressed, phosphorylated and translocated into several nuclei of CA3 pyramidal neurons. In p53(-/-) mouse, microglial activation was slightly faster and more continuous after 1-7 days than that in p53(+/+) mouse. On the other hand, p53(-/-) astrocytes were relatively resistant to KA cytotoxicity, and marked astrocytosis also occurred after 7 days. These observations suggest that p53-null mutation may influence the activation and proliferation of glial cells rather than neuronal death. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.
  • Y Kitamura, T Ota, Y Matsuoka, M Okazaki, J Kakimura, Tooyama, I, H Kimura, S Shimohama, PJ Gebicke-Haerter, Y Nomura, T Taniguchi
    NEUROSCIENCE LETTERS 255 1 57 - 60 1998年10月 [無し][無し]
     研究論文（学術雑誌） 
    We examined kainic acid (KA)-induced neuronal death and changes in glial cells in p53-deficient (p53(-/-)) and wild-type (p53(+/+)) mice which were CBA and C57BL/6 background. The p53(-/-) mouse exhibited a KA-induced loss of CA3 pyramidal neurons similar to that in wild-type mouse. Before neuronal death, c-Jun protein was expressed, phosphorylated and translocated into several nuclei of CA3 pyramidal neurons. In p53(-/-) mouse, microglial activation was slightly faster and more continuous after 1-7 days than that in p53(+/+) mouse. On the other hand, p53(-/-) astrocytes were relatively resistant to KA cytotoxicity, and marked astrocytosis also occurred after 7 days. These observations suggest that p53-null mutation may influence the activation and proliferation of glial cells rather than neuronal death. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.
  • マウス脳内における鉄結合蛋白ラクトフェリンの分布
    近藤 洋一, 岩田 恵美, 宮崎 育子, 相見 良成, 遠山 育夫, 木村 宏, 小川 紀雄
    神経化学 37 3 488 - 488 日本神経化学会 1998年09月
  • AJ Li, Y Oomura, K Sasaki, K Suzuki, Tooyama, I, K Hanai, H Kimura, T Hori
    NEUROSCIENCE 85 3 785 - 794 1998年08月 [無し][無し]
     研究論文（学術雑誌） 
    Effects of a pre-training intraperitoneal glucose injection on learning and memory were tested using two tasks: passive avoidance and Morris water maze. In the former task, mice that had received glucose 2 h prior (but not 1, 3, or 5 h prior) to a trial that combined acquisition with passive avoidance of foot shock showed a significantly increased retention latency when tested 24 h later. Thus, this effect was rime-dependent, and it was also found to be dose-dependent by further experiment. In contrast, 2-deoxy-D-glucose and fructose had no such effect. In the Morris water maze task, glucose injection 2 or 3 h before a block of trials enhanced the spatial memory performance of mice. These glucose-induced memory-facilitation effects were abolished by an intracerebroventricular injection of anti-acidic fibroblast growth factor antibody 30 min before the glucose injection, suggesting a critical role for endogenous acidic fibroblast growth factor in this facilitatory effect. Furthermore, continuous intracerebroventricular infusion of acidic fibroblast growth factor in rats significantly increased retention latency (when tested repeatedly on successive days using a passive avoidance task). Our earlier studies demonstrated that brain acidic fibroblast growth factor is produced in the ependymal cells of the cerebroventricular system, and is released into the cerebrospinal fluid following either a meal or a (intraperitoneal or intracerebroventricular) glucose injection. This released acidic fibroblast growth factor also diffuses into the brain parenchyma, and is taken up by neurons in the hippocampus, hypothalamus, and elsewhere in the brain some 2 h after the meal or glucose injection. These and the present findings indicate (i) that pre-training glucose injection improves memory performance, and (ii) that acidic fibroblast growth factor, especially by its action within the hippocampus, is involved in this enhancement process. (C) 1998 IBRO. Published by Elsevier Science Ltd.
  • Y Matsuoka, Y Kitamura, M Okazaki, J Kakimura, Tooyama, I, H Kimura, T Taniguchi
    NEUROSCIENCE 85 4 1223 - 1233 1998年08月 [無し][無し]
     研究論文（学術雑誌） 
    Heme oxygenase, catalyses oxidation of the heme molecule in concert with NADPH-cytochrome P450 reductase and then specifically cleaves heme into biliverdin, carbon monoxide, and iron. Biliverdin and its product, bilirubin, are known to be strong antioxidants. Kainic acid is a potent neurotoxin, and induces selective neuronal loss in the rat hippocampus. Kainic acid acts on the kainate receptors, and kainic acid neurotoxicity may be in part mediated by oxidative stress. In this study, we examined whether or not heme oxygenase was activated in kainic acid-induced neurotoxicity. After intracerebroventricular injection of kainic acid, the heme oxygenase-l protein level was strongly enhanced, although the constitutive heme oxygenase (heme oxygenase-2) protein level was not changed. One day after treatment, the protein level of heme oxygenase-l reached a maximum and then gradually decreased over a period of three to seven days. In the rat hippocampus, cells expressing heme oxygenase-l in vivo were predominately microglia and only a few astrocytes. In addition, heme oxygenase-l immunoreactivity was predominantly co-localized with major histocompatibility complex class II-, and partly co-localized with class I-immunoreactive microglia. In cultured glial cells in vitro, heme oxygenase-l protein was expressed in the microglia even with the vehicle treatment, and was strongly induced in astrocytes by kainic acid treatment. These results suggest that ameboid microglia, which express both heme oxygenase-l and major histocompatibility complex antigens, may play a key role in a delayed episode of kainic acid-induced microglial activation and neurodegeneration. (C) 1998 IBRO. Published by Elsevier Science Ltd.
  • AJ Li, Y Oomura, K Sasaki, K Suzuki, Tooyama, I, K Hanai, H Kimura, T Hori
    NEUROSCIENCE 85 3 785 - 794 1998年08月 [無し][無し]
     研究論文（学術雑誌） 
    Effects of a pre-training intraperitoneal glucose injection on learning and memory were tested using two tasks: passive avoidance and Morris water maze. In the former task, mice that had received glucose 2 h prior (but not 1, 3, or 5 h prior) to a trial that combined acquisition with passive avoidance of foot shock showed a significantly increased retention latency when tested 24 h later. Thus, this effect was rime-dependent, and it was also found to be dose-dependent by further experiment. In contrast, 2-deoxy-D-glucose and fructose had no such effect. In the Morris water maze task, glucose injection 2 or 3 h before a block of trials enhanced the spatial memory performance of mice. These glucose-induced memory-facilitation effects were abolished by an intracerebroventricular injection of anti-acidic fibroblast growth factor antibody 30 min before the glucose injection, suggesting a critical role for endogenous acidic fibroblast growth factor in this facilitatory effect. Furthermore, continuous intracerebroventricular infusion of acidic fibroblast growth factor in rats significantly increased retention latency (when tested repeatedly on successive days using a passive avoidance task). Our earlier studies demonstrated that brain acidic fibroblast growth factor is produced in the ependymal cells of the cerebroventricular system, and is released into the cerebrospinal fluid following either a meal or a (intraperitoneal or intracerebroventricular) glucose injection. This released acidic fibroblast growth factor also diffuses into the brain parenchyma, and is taken up by neurons in the hippocampus, hypothalamus, and elsewhere in the brain some 2 h after the meal or glucose injection. These and the present findings indicate (i) that pre-training glucose injection improves memory performance, and (ii) that acidic fibroblast growth factor, especially by its action within the hippocampus, is involved in this enhancement process. (C) 1998 IBRO. Published by Elsevier Science Ltd.
  • Y Matsuoka, Y Kitamura, M Okazaki, J Kakimura, Tooyama, I, H Kimura, T Taniguchi
    NEUROSCIENCE 85 4 1223 - 1233 1998年08月 [無し][無し]
     研究論文（学術雑誌） 
    Heme oxygenase, catalyses oxidation of the heme molecule in concert with NADPH-cytochrome P450 reductase and then specifically cleaves heme into biliverdin, carbon monoxide, and iron. Biliverdin and its product, bilirubin, are known to be strong antioxidants. Kainic acid is a potent neurotoxin, and induces selective neuronal loss in the rat hippocampus. Kainic acid acts on the kainate receptors, and kainic acid neurotoxicity may be in part mediated by oxidative stress. In this study, we examined whether or not heme oxygenase was activated in kainic acid-induced neurotoxicity. After intracerebroventricular injection of kainic acid, the heme oxygenase-l protein level was strongly enhanced, although the constitutive heme oxygenase (heme oxygenase-2) protein level was not changed. One day after treatment, the protein level of heme oxygenase-l reached a maximum and then gradually decreased over a period of three to seven days. In the rat hippocampus, cells expressing heme oxygenase-l in vivo were predominately microglia and only a few astrocytes. In addition, heme oxygenase-l immunoreactivity was predominantly co-localized with major histocompatibility complex class II-, and partly co-localized with class I-immunoreactive microglia. In cultured glial cells in vitro, heme oxygenase-l protein was expressed in the microglia even with the vehicle treatment, and was strongly induced in astrocytes by kainic acid treatment. These results suggest that ameboid microglia, which express both heme oxygenase-l and major histocompatibility complex antigens, may play a key role in a delayed episode of kainic acid-induced microglial activation and neurodegeneration. (C) 1998 IBRO. Published by Elsevier Science Ltd.
  • H Kitano, T Takeda, M Suzuki, T Kitanishi, Y Yazawa, K Kitajima, H Kimura, Tooyama, I
    HEARING RESEARCH 121 1-2 109 - 111 1998年07月 [無し][無し]
     研究論文（学術雑誌） 
    Although mechanisms regulating inner ear fluid have not been yet elucidated, control of blood flow has been thought to be of great importance. Vasoactive intestinal polypeptide (VIP) was the first neuropeptide demonstrated in cerebrovascular nerves. To study the possible role of VIP in regulation of inner ear fluid, we investigated the presence of mRNA for VIP and VIP receptor in the rat inner ear using a reverse transcription-polymerase chain reaction (RT-PCR) method. A single band of the size expected for VIP and its receptor was detected in mRNA from the rat inner ear by using primers specific for VIP and the receptor. The nucleotide sequences of the subcloned RT-PCR products were identical to those of rat VIP and the rat lung VIP receptor. These results indicate that both VIP and VIP receptor are expressed in the inner ear of the rat and suggest that VIP may be implicated in regulation of fluid in the inner ear. (C) 1998 Elsevier Science B.V. All rights reserved.
  • H Kitano, T Takeda, M Suzuki, T Kitanishi, Y Yazawa, K Kitajima, H Kimura, Tooyama, I
    HEARING RESEARCH 121 1-2 109 - 111 1998年07月 [無し][無し]
     研究論文（学術雑誌） 
    Although mechanisms regulating inner ear fluid have not been yet elucidated, control of blood flow has been thought to be of great importance. Vasoactive intestinal polypeptide (VIP) was the first neuropeptide demonstrated in cerebrovascular nerves. To study the possible role of VIP in regulation of inner ear fluid, we investigated the presence of mRNA for VIP and VIP receptor in the rat inner ear using a reverse transcription-polymerase chain reaction (RT-PCR) method. A single band of the size expected for VIP and its receptor was detected in mRNA from the rat inner ear by using primers specific for VIP and the receptor. The nucleotide sequences of the subcloned RT-PCR products were identical to those of rat VIP and the rat lung VIP receptor. These results indicate that both VIP and VIP receptor are expressed in the inner ear of the rat and suggest that VIP may be implicated in regulation of fluid in the inner ear. (C) 1998 Elsevier Science B.V. All rights reserved.
  • T Doi, K Terai, Tooyama, I, T Sakata, H Kimura
    HISTOCHEMICAL JOURNAL 30 6 425 - 434 1998年06月 [無し][無し]
     研究論文（学術雑誌） 
    A monoclonal antibody against histamine has been produced. A histamine-haemocyanin conjugate prepared using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling agent was used for immunizing mice. Immunized mice were sacrificed to prepare monoclonal antibody using a hybridoma technique. On immunospot assay, the hybridoma culture supernatant containing a monoclonal antibody was capable of detecting 50 pmol of histamine. Using this antibody, we examined the cellular localization of histamine-like immunoreactivity in the stomach of normal or alpha-fluoromethylhistidine-treated rats and mice. Immunoreactive cells were abundant in the gastric mucosal layer. These positive cells were often located in the basal half of the fundic gland but were rare in the pyloric gland. The cells, small or medium in size, spindle or cone in shape, were intermingled with immunonegative epithelial cells. In the cytoplasm of the positive cells, granular reaction products were densely deposited. In addition, a few positive cells, identified as mast cells by Toluidine Blue staining, were distributed mainly in the submucosal and muscular layer. The antibody preabsorbed with 10 mM histamine gave no positive immunostaining. For pharmacological study, some rats were injected six times with alpha-fluoromethylhistidine every 8 h. in these rats, positive cells except mast cells were no longer detected. In conclusion, the monoclonal antibody produced appears to be highly specific for histamine. Its application in immunohistochemistry should provide a powerful tool for analysing the roles of histamine in enterochromaffin-like or mast cells in the stomach. (C) 1998 Chapman & Hall.
  • M Kage, A Ikemoto, Akiguchi, I, J Kimura, S Matsumoto, H Kimura, Tooyama, I
    NEUROREPORT 9 7 1403 - 1406 1998年05月 [無し][無し]
     研究論文（学術雑誌） 
    RECENT evidence suggests that the growth associated protein GAP-43 is involved in the pathology of amyotrophic lateral sclerosis (ALS). In order to assess the primary structure of the GAP-43 mRNA expressed in the spinal cord of ALS patients, the total coding region of the GAP-43 mRNA was amplified from postmortem human spinal cord specimens using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. GAP-43 amplification products were clearly detected in all of the ALS cases but not in the normal controls. The GAP-43 mRNA and the deduced amino acid sequences from all ALS cases coincided completely with the sequence of human fetal GAP-43. These results suggest that the abnormal expression of GAP-43 mRNA underlying the pathogenesis of ALS is due to a quantitative increase, and that there are no qualitative abnormalities associated with a change in the amino acid sequences. (C) 1998 Rapid Science Ltd.
  • M Kage, A Ikemoto, Akiguchi, I, J Kimura, S Matsumoto, H Kimura, Tooyama, I
    NEUROREPORT 9 7 1403 - 1406 1998年05月 [無し][無し]
     研究論文（学術雑誌） 
    RECENT evidence suggests that the growth associated protein GAP-43 is involved in the pathology of amyotrophic lateral sclerosis (ALS). In order to assess the primary structure of the GAP-43 mRNA expressed in the spinal cord of ALS patients, the total coding region of the GAP-43 mRNA was amplified from postmortem human spinal cord specimens using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. GAP-43 amplification products were clearly detected in all of the ALS cases but not in the normal controls. The GAP-43 mRNA and the deduced amino acid sequences from all ALS cases coincided completely with the sequence of human fetal GAP-43. These results suggest that the abnormal expression of GAP-43 mRNA underlying the pathogenesis of ALS is due to a quantitative increase, and that there are no qualitative abnormalities associated with a change in the amino acid sequences. (C) 1998 Rapid Science Ltd.
  • M Suzuki, H Kitano, T Kitanishi, Y Yazawa, K Kitajima, T Takeda, H Kimura, Tooyama, I
    MOLECULAR BRAIN RESEARCH 55 1 165 - 168 1998年03月 [無し][無し]
     研究論文（学術雑誌） 
    To assess the possible physiological role of the atrial natriuretic peptide (ANP) family, we investigated the expression of mRNA of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and their receptors in rat inner ear using the reverse transcription-polymerase chain reaction method. ANP and CNP message bands were detected in the inner ear, but the BNP message band was not. Amplification products of the expected sizes of ANP-A, ANP-B, and ANP-C receptors were detected in the inner ear. These results suggest that natriuretic peptide family may influence the function of the inner ear through the ANP-A, ANP-B, and ANP-C receptors. (C) 1998 Elsevier Science B.V.
  • M Suzuki, H Kitano, T Kitanishi, Y Yazawa, K Kitajima, T Takeda, H Kimura, Tooyama, I
    MOLECULAR BRAIN RESEARCH 55 1 165 - 168 1998年03月 [無し][無し]
     研究論文（学術雑誌） 
    To assess the possible physiological role of the atrial natriuretic peptide (ANP) family, we investigated the expression of mRNA of ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and their receptors in rat inner ear using the reverse transcription-polymerase chain reaction method. ANP and CNP message bands were detected in the inner ear, but the BNP message band was not. Amplification products of the expected sizes of ANP-A, ANP-B, and ANP-C receptors were detected in the inner ear. These results suggest that natriuretic peptide family may influence the function of the inner ear through the ANP-A, ANP-B, and ANP-C receptors. (C) 1998 Elsevier Science B.V.
  • Yoshihisa Kitamura, Muneki Furukawa, Yasuji Matsuoka, Ikuo Tooyama, Hiroshi Kimura, Yasuyuki Nomura, Takashi Taniguchi
    GLIA 22 2 138 - 148 1998年02月 [無し][無し]
     研究論文（学術雑誌） 
    To determine whether heme oxygenase-1 (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-γ (IFN-γ), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO2- accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as N(G)-nitro-L-arginine (NNA) and N(G)-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO2- accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-1-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-γ, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-γ. These results suggest that endogenous NO production by iNOS in microglia causes autocrine and paracrine induction of HO-1 protein in microglia and astrocytes in vitro and in rat brain.
  • Y Kitamura, M Furukawa, Y Matsuoka, Tooyama, I, H Kimura, Y Nomura, T Taniguchi
    GLIA 22 2 138 - 148 1998年02月 [無し][無し]
     研究論文（学術雑誌） 
    To determine whether heme oxygenase-l (HO-1) protein is induced by endogenous nitric oxide (NO) in rat glial cultures, we examined the effects of lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and NO donors such as S-nitroso-N-acetylpenicillamine (SNAP), in mixed glial cells and in vivo rat hippocampus. In cultured glial cells, treatment with LPS induced the expression of 130-kd inducible NO synthase (iNOS) after 6 h, and NO(2)(-) accumulation and enhancement of the protein level of 33-kd HO-1 after 12 h. In addition, treatment with SNAP induced HO-1 expression after 6 h. Although NOS inhibitors such as N(G)-nitro-L-arginine (NNA) and N(G)-methyl-L-arginine did not change LPS-induced iNOS expression, these inhibitors suppressed both NO(2)(-) accumulation and the enhancement of HO-1. Immunocytochemistry showed that treatment with LPS for 24 h induced iNOS immunoreactivity predominantly in ameboid microglia, while this treatment induced HO-l-immunoreactivity in both microglia and astrocytes. In in vivo rat hippocampus, microinjection of LPS plus IFN-gamma, or SNAP after 24 h also induced HO-1 immunoreactivity in reactive microglia and astrocytes. In addition, intraperitoneal administration of NNA inhibited HO-1 immunoreactivity induced by the microinjection of LPS plus IFN-gamma. These results suggest that endogenous NO production by iNOS in microglia causes autocrine and paracrine induction of HO-1 protein in microglia and astrocytes in vitro and in rat brain. (C) 1998 Wiley-Liss, Inc.
  • K Terai, Tooyama, I, H Kimura
    NEUROSCIENCE 82 3 843 - 852 1998年02月 [無し][無し]
     研究論文（学術雑誌） 
    The localization of GABA(A) receptors was studied by immunohistochemistry in the nucleus tractus solitarii of the rat using a monoclonal antibody (bd17) against the beta-subunit. The pattern of distribution was compared with that of GABA-immunoreactive axons and nerve terminals. Positive staining for GABA(A) receptors was confined to regions near the surface of neuronal somata and their processes. The highest density of positive staining for GABA(A) receptors was seen in the central part of the rostral nucleus tractus solitarii where GABA-positive terminals were also rather dense. At both intermediate and caudal levels of the nucleus tractus solitarii, a moderate density of positive staining for GABA(A) receptors was located in the ventrolateral part, including the ventrolateral subnucleus. In these regions, the density of GABA positive terminals was low. In the medial nucleus tractus solitarii, including the medial subnucleus, very little or no positive staining for GABA(A) receptors was detected, although many GABA-positive terminals were observed. The results suggest that the central part of the rostral nucleus tractus solitarii is controlled by the GABAergic system via GABA(A) receptors, but in the medial subnucleus of the nucleus tractus solitarii the GABA neurons appear to act via receptors that are not detectable by the antibody used. (C) 1997 IBRO.
  • A Aisaka, Y Aimi, O Yasuhara, Tooyama, I, H Kimura, M Shimada
    NEW DEVELOPMENTS IN CHILD NEUROLOGY 17 - 22 1998年 [有り][無し]
     研究論文（国際会議プロシーディングス） 
    We examined the brain and spinal cord of young hamsters which had a unilateral ablation of the right cortex at 6 days postnatally (PD 6), and then received an injection of an anterograde neuronal tracer into the left hindlimb motor cortex at PD 21. A quantitative analysis in the left gray matter of the lumbar spinal cord indicated that the labeling was much higher in ablated than in intact animals. Immunohistochemical detection of the lectin revealed that innervation of the left spinal cord occurred with showing two mode (Type 1 and Type 2) in close to targets. The type I fibers consisted of recrossing axon collaterals sprouted from the intact dorsal funiculus near their targets, while the type ii fibers were recrossing parent axone which entered the intact right gray matter rostral to targets and then changed direction to the targets.
  • AJ Li, Y Oomura, E Sasaki, N Hori, Tooyama, I, Y Nomura, K Hanai, H Kimura, N Yanaihara, H Yoshii, Y Yamazaki, S Take, T Hori
    BRAIN AND BIODEFENCE 21 51 - 61 1998年 [無し][無し]
     研究論文（国際会議プロシーディングス）
  • Atsushi Nagata, Shin-Ichi Nakao, Etsuko Miyamoto, Takefumi Inada, Ikuo Tooyama, Hiroshi Kimura, Koh Shingu
    Anesthesia and Analgesia 87 6 1416 - 1420 1998年 [無し][無し]
     研究論文（学術雑誌） 
    Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, has psychotomimetic activity. NMDA receptor antagonists cause morphological damage in the posterior cingulate cortex, which may be the brain region responsible for their psychotomimetic effects. Benzodiazepines are effective in preventing these effects through γ-aminobutyric acid A (GABAA) receptor activation. We investigated the effect of propofol, which has both GABAA receptor-activating and NMDA receptor-suppressing activity, on ketamine-induced c-fos expression in the rat posterior cingulate cortex. Propofol or vehicle was continuously infused IV. Fifteen minutes later, 100 mg/kg ketamine or isotonic sodium chloride solution was injected intraperitoneally. Two hours later, brain sections were prepared, and c-fos expression was detected using immunohistochemical methods. Propofol significantly inhibited ketamine-induced c-fos expression in the posterior cingulate cortex. Propofol itself did not induce c-fos expression in this brain region. We conclude that propofol may be able to inhibit ketamine- induced psychotomimetic activity and neuronal damage. Implications: In the present study, we demonstrated that the clinically relevant dose of propofol significantly inhibited ketamine-induced c-fos expression in the rat posterior cingulate cortex. This finding implies that propofol may inhibit ketamine-induced psychotomimetic activity and neuronal damage.
  • Takashi Doi, Kazuhiro Terai, Ikuo Tooyama, Toshiie Sakata, Hiroshi Kimura
    Histochemical Journal 30 6 425 - 434 1998年 [無し][無し]
     研究論文（学術雑誌） 
    A monoclonal antibody against histamine has been produced. A histamine- haemocyanin conjugate prepared using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling agent was used for immunizing mice. Immunized mice were sacrificed to prepare monoclonal antibody using a hybridoma technique. On immunospot assay, the hybridoma culture supernatant containing a monoclonal antibody was capable of detecting 50 pmol of histamine. Using this antibody, we examined the cellular localization of histamine-like immunoreactivity in the stomach of normal or α-fluoromethylhistidine- treated rats and mice. Immunoreactive cells were abundant in the gastric mucosal layer. These positive cells were often located in the basal half of the fundic gland but were rare in the pyloric gland. The cells, small or medium in size, spindle or cone in shape, were intermingled with immunonegative epithelial cells. In the cytoplasm of the positive cells, granular reaction products were densely deposited. In addition, a few positive cells, identified as mast cells by Toluidine Blue staining, were distributed mainly in the submucosal and muscular layer. The antibody preabsorbed with 10 mM histamine gave no positive immunostaining. For pharmacological study, some rats were injected six times with α- fluoromethylhistidine every 8 h. In these rats, positive cells except mast cells were no longer detected. In conclusion, the monoclonal antibody produced appears to be highly specific for histamine. Its application in immunohistochemistry should provide a powerful tool for analysing the roles of histamine in enterochromaffin-like or mast cells in the stomach.
  • Evaluation of focal cerebral ischemia in rats by magnetic resonance imaging and immunohistochemical analyses
    Ishii H, Arai T, Morikawa S, Inubushi T, Tooyama I, Kimura H, Mori K
    J Cereb. Blood Flow. Matab. 18 931 - 934 1998年 [無し][無し]
     研究論文（学術雑誌）
  • 遠山育夫, 鈴木幹男, 北野博也
    生体の科学 49 401 - 402 1998年 [無し][無し]
     研究論文（学術雑誌）
  • AJ Li, Y Oomura, E Sasaki, N Hori, Tooyama, I, Y Nomura, K Hanai, H Kimura, N Yanaihara, H Yoshii, Y Yamazaki, S Take, T Hori
    BRAIN AND BIODEFENCE 21 51 - 61 1998年 [無し][無し]
     研究論文（国際会議プロシーディングス）
  • Hisanari Ishii, Toshiyuki Arai, Shigehiro Morikawa, Toshiro Inubushi, Ikuo Tooyama, Hiroshi Kimura, Kenjiro Mori
    Journal of Cerebral Blood Flow and Metabolism 18 9 931 - 934 1998年 [無し][無し]
     研究論文（学術雑誌） 
    Correlation of focal ischemia-induced brain damage evidenced by magnetic resonance imaging (MRI) and by staining with microtubule-associated protein 2 (MAP2) was studied in rats. Ischemia was produced by transient occlusion of the middle cerebral artery (MCAO). The damage was assessed at 6 to 8 hours after MCAO and 1 week later. The area of damage assessed by MRI agreed with that by MAP2 staining at 6 to 8 hours after MCAO, which was smaller (P < 0.001) than that defined by MAP2 staining 1 week after MCAO. Glial staining indicated that glial infiltration affected the signal intensity of MRI in the area of damage.
  • K. Terai, I. Tooyama, H. Kimura
    Neuroscience 82 3 843 - 852 1997年10月 [無し][無し]
     研究論文（学術雑誌） 
    The localization of GABA(A) receptors was studied by immunohistochemistry in the nucleus tractus solitarii of the rat using a monoclonal antibody (bd17) against the β-subunit. The pattern of distribution was compared with that of GABA-immunoreactive axons and nerve terminals. Positive staining for GABA(A) receptors was confined to regions near the surface of neuronal somata and their processes. The highest density of positive staining for GABA(A) receptors was seen in the central part of the rostral nucleus tractus solitarii where GhBA-positive terminals were also rather dense. At both intermediate and caudal levels of the nucleus tractus solitarii, a moderate density of positive staining for GABA(A) receptors was located in the ventrolateral part, including the ventrolateral subnucleus. In these regions, the density of GABA-positive terminals was low. In the medial nucleus tractus solitarii, including the medial subnucleus, very little or no positive staining for GABA(A) receptors was detected, although many GABA- positive terminals were observed. The results suggest that the central part of the rostral nucleus tractus solitarii is controlled by the GABAergic system via GABA(A) receptors, but in the medial subnucleus of the nucleus tractus solitarii the GABA neurons appear to act via receptors that are not detectable by the antibody used.
  • H Kitano, T Takeda, M Suzuki, T Kitanishi, Y Yazawa, K Kitajima, H Kimura, Tooyama, I
    NEUROREPORT 8 9-10 2289 - 2292 1997年07月 [無し][無し]
     研究論文（学術雑誌） 
    THE cause of endolymphatic hydrops, a characteristic finding in Meniere's disease, is not known. To study the possible involvement of the neurohormones vasopressin and oxytocin in this condition, we investigated whether transcripts of the genes encoding the arginine vasopressin (AVP) and oxytocin receptors are expressed in the rat inner ear. Utilizing the reverse transcription-polymerase chain reaction (RT-PCR) method, primers specific for each receptor showed a single message band of the expected size in the rat inner ear. When the PCR products were cloned, the sequences were identical to those of the real-type (V2) AVP receptor and oxytocin receptor transcripts. The finding of vasopressin and oxytocin receptor mRNAs in the inner ear suggests that these neurohypophyseal hormones may have roles in the regulation of inner ear fluid. In particular, the presence of vasopressin receptor mRNA in the inner ear supports the hypothesis of a relationship between high plasma vasopressin levels and endolymphatic hydrops.
  • Y Matsuoka, Y Kitamura, Tooyama, I, H Kimura, T Taniguchi
    EXPERIMENTAL NEUROLOGY 146 1 57 - 66 1997年07月 [無し][無し]
     研究論文（学術雑誌） 
    Although it is well known that brain ischemia is dominantly caused by hypoxia and hypoglycemia, it is still unclear how hypoxia participates in ischemia. We studied the changes in neuronal nitric oxide synthase (nNOS) and the effect of the NOS inhibitor N-G-nitro-L-arginine (NNA) on hypoxia. In vivo hypoxia (5% O-2/95% N-2 for 30 min) induced mild degenerative neuronal changes (shrunken and eosinophilic somata with picnotic nuclei) in neurons of the CA3, the hilus of the dentate gyrus (DG) and the DC;, but not in the CA1. At 3 and 7 days after hypoxia, levels of nNOS protein were significantly enhanced to 153 and 209%, but iNOS protein could not be detected. The numbers of nNOS-immunopositive neurons were significantly enhanced to 145 and 191% in the CA3, 145 and 178% in the hilus of the DG:, and 243 and 387% in the DG after 3 and 7 days, respectively. In contrast, no statistical difference was determined in the CA1. We further examined the effect of NNA administered at 5 min and 3, 6, and 24 h after hypoxia. Administration of MNA (0.1 and 1 mg/kg, ip) significantly decreased the number of damaged neurons in the hilus of the DG and the DG;. However, higher doses of NNA (10 mg/kg, ip) did not prevent damage. These results suggest that hypoxia alone induces enhancement of nNOS protein and nNOS immunoreactivity in neurons of the hippocampus and that NNA has biphasic effects against hypoxia-induced neuronal damage in the hilus of the DG and the DG. (C) 1997 Academic Press.
  • H Kitano, T Takeda, M Suzuki, T Kitanishi, Y Yazawa, K Kitajima, H Kimura, Tooyama, I
    NEUROREPORT 8 9-10 2289 - 2292 1997年07月 [無し][無し]
     研究論文（学術雑誌） 
    THE cause of endolymphatic hydrops, a characteristic finding in Meniere's disease, is not known. To study the possible involvement of the neurohormones vasopressin and oxytocin in this condition, we investigated whether transcripts of the genes encoding the arginine vasopressin (AVP) and oxytocin receptors are expressed in the rat inner ear. Utilizing the reverse transcription-polymerase chain reaction (RT-PCR) method, primers specific for each receptor showed a single message band of the expected size in the rat inner ear. When the PCR products were cloned, the sequences were identical to those of the real-type (V2) AVP receptor and oxytocin receptor transcripts. The finding of vasopressin and oxytocin receptor mRNAs in the inner ear suggests that these neurohypophyseal hormones may have roles in the regulation of inner ear fluid. In particular, the presence of vasopressin receptor mRNA in the inner ear supports the hypothesis of a relationship between high plasma vasopressin levels and endolymphatic hydrops.
  • Y Matsuoka, Y Kitamura, R Fukunaga, S Shimohama, T Nabeshima, Tooyama, I, H Kimura, T Taniguchi
    NEUROCHEMISTRY INTERNATIONAL 30 6 533 - 542 1997年06月 [無し][無し]
     研究論文（学術雑誌） 
    Hypoxia is a major cause of ischaemia-induced neuronal damage. In the present study, we examined the effects of in vivo hypoxia on N-methyl-D-aspartate receptors (NMDAR) in the rat hippocampus. This model of in vivo hypoxia involved placing rats in a hypoxic chamber containing 5% O-2 and 95% N-2 for 30 min. In the hippocampus, neuronal cells in the CA3, the hilus of the dentate gyrus and the dentate gyrus (DG) were damaged. In the CA1, which is known to be vulnerable to ischaemic damage, neuronal cells did not show hypoxia-induced damage. In vivo hypoxia-induced damage caused morphological changes in neuronal cells, such as shrunken, spindle or triangular shapes accompanied by pyknotic nuclei, but did not induce the loss of neuronal cells. On the other hand, the number of binding sites for [H-3]-1-[1-(2-thienyl)cyclohexyl]-3,4-piperidine hydrochloride (TCP) gradually decreased on and after 7 days, and then maximally decreased by 25% at 21 days after hypoxia. The number of NMDAR1-immunopositive cells was decreased by 22% in the DG, but was unchanged in the CA3. Furthermore, we examined the effect of a non-competitive NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b] cyclohepten-5,10-imine hydrogen maleate (MK-801), on against in vivo hypoxia. The administration of MK-801 (3 mg/kg, i.p.), 30 min before hypoxia treatment, partly protected against neuronal damage in the DG, but not in the CA3. These results suggest that hypoxia-induced neuronal damage in the DG involves, in part, the activation of NMDAR. (C) 1997 Elsevier Science Ltd.
  • Y Matsuoka, Y Kitamura, R Fukunaga, S Shimohama, T Nabeshima, Tooyama, I, H Kimura, T Taniguchi
    NEUROCHEMISTRY INTERNATIONAL 30 6 533 - 542 1997年06月 [無し][無し]
     研究論文（学術雑誌） 
    Hypoxia is a major cause of ischaemia-induced neuronal damage. In the present study, we examined the effects of in vivo hypoxia on N-methyl-D-aspartate receptors (NMDAR) in the rat hippocampus. This model of in vivo hypoxia involved placing rats in a hypoxic chamber containing 5% O-2 and 95% N-2 for 30 min. In the hippocampus, neuronal cells in the CA3, the hilus of the dentate gyrus and the dentate gyrus (DG) were damaged. In the CA1, which is known to be vulnerable to ischaemic damage, neuronal cells did not show hypoxia-induced damage. In vivo hypoxia-induced damage caused morphological changes in neuronal cells, such as shrunken, spindle or triangular shapes accompanied by pyknotic nuclei, but did not induce the loss of neuronal cells. On the other hand, the number of binding sites for [H-3]-1-[1-(2-thienyl)cyclohexyl]-3,4-piperidine hydrochloride (TCP) gradually decreased on and after 7 days, and then maximally decreased by 25% at 21 days after hypoxia. The number of NMDAR1-immunopositive cells was decreased by 22% in the DG, but was unchanged in the CA3. Furthermore, we examined the effect of a non-competitive NMDA antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b] cyclohepten-5,10-imine hydrogen maleate (MK-801), on against in vivo hypoxia. The administration of MK-801 (3 mg/kg, i.p.), 30 min before hypoxia treatment, partly protected against neuronal damage in the DG, but not in the CA3. These results suggest that hypoxia-induced neuronal damage in the DG involves, in part, the activation of NMDAR. (C) 1997 Elsevier Science Ltd.
  • Y. Oomura, K. Sasaki, A. Li, H. Yoshii, Y. Fukata, H. Yago, H. Kimura, I. Tooyama, K. Hanai, Y. Nomura, Y. Kitamura, N. Yanaihara
    Japanese Journal of Physiology 47 1 S52 - S54 1997年 [有り][無し]
     研究論文（国際会議プロシーディングス） 
    aFGF injection s.c. once a week into SAMP8 was begun at 3 weeks after birth and continued for 10 months. Saline was injected as a control. Learning and memory and cellular immunological functions in the aFGF group were enhanced significantly, while those of the saline group deteriorated. 1. The number of cholinergic neurons was decreased by less than 20% and ChAT activity in individual neurons in the medial septum which send monosyonaptic terminals to the hippocampus was significantly decreased in the saline group, but not so much in the aFGF group. 2. The respective densities of muscarinic and aFGF receptors, on the hippocampal neurons were significantly higher in the aFGF group than in the saline group. 3. The LTP in hippocampal slice preparations was significantly facilitated in the aFGF group, but not in the saline group. 4. The DTH, (T cell immune response) measured at the end of the 2nd and 7th months were reduced in the 7th month as compared with the 2nd month in the saline group, but aFGF group protected against this reduction. 5. These results show that aFGF provides protection against impairment of not only learning and memory but also the DTH immunoreactivity in SAMP8.
  • T Renda, Erspamer, V, Tooyama, I, H Kimura
    RECENT ADVANCES IN MICROSCOPY OF CELLS TISSUES AND ORGANS 239 - 246 1997年 [有り][無し]
     研究論文（国際会議プロシーディングス） 
    An antiserum raised against [D-Ala(2)]deltorphin-I (DADTI: Sr-D-Ala Phe-Asp-Val-Val-Gly . NH2), a highly selective ligand for delta-opioid receptors, was applied for the immunohistochemical study of positive brain structures during postnatal development and in. the adult rat. On. postnatal day 0, neuronal cell bodies containing DADTI-like immunoreactivity were widely distributed in various brain regions. These included the olfactory tubercle, ventral pallidum, hippocampal CA2 and CA3 subfields, ventral tegmental area, pars compacta of the substantia nigra, supramammillary nucleus and dorsal raphe nucleus. Immunoreactive nerve fibres were observed in the main and accessory olfactory bulbs, olfactory tubercle, prefrontal cortex, cingulate cortex, neostriatum, nucleus accumbens, Lateral septal nucleus, Lateral habenular nucleus, optic tract, lateral geniculate nucleus and superior colliculus. As pups grew, positive cell bodies decreased gradually in both density and intensity, and those in the olfactory tubercle, ventral pallidum and hippocampus had disappeared by postnatal day 14. A distribution pattern resembling that in the adult was reached on postnatal day 21. Positive cell bodies were found only in the ventral midbrain including the pars compacta of the substantia nigra, ventral tegmental area, A8 region and supramammillary nucleus. Intensely stained positive nerve fibres could be traced along the medial forebrain bundle. Dense positive terminals occupied the neostriatum, nucleus accumbens shell, olfactory tubercle, septal areas, cingulate and medial prefrontal cortex. Double immunostaining revealed that, in the substantia nigra, almost all (97.8%) the DADTI-positive neurons co-localized tyrosine hydroxylase (TH) representing about one third (29.1%) of the total population of TH-positive neurons. These DADTI/TH-positive cells showed poor staining for calbindin D-28K The results indicate that [D-Ala(2)]deltorphin-I-like molecule(s) may play an important role in early postnatal development of certain neuronal systems. Furthermore, in the adult, they intervene solely in. certain dopaminergic system functions.
  • Recent advances in neuroscience for Alzheimer's disease
    Kimura H, Yasuhara O, Tooyama I
    Adv. Legal. Med. 3 280 - 283 1997年 [無し][無し]
     研究論文（学術雑誌）
  • Recent Advances in Microscopy of Cells, Tissues and Organs
    Renda T, Erspamer V, Tooyama I, Kimura H
    Antonio Delfino Editore 239 - 246 1997年 [無し][無し]
     研究論文（学術雑誌）
  • Protein expression of fibroblast growth factor receptor-1 in keratinocytes during wound healing in rat skin
    Takenaka H, Kishimoto S, Tooyama I, Kimura H, Yasuno H
    J. Invest. Dermatol. 109 108 - 112 1997年 [無し][無し]
     研究論文（学術雑誌）
  • Matsuoka Y, Kitamura Y, Tooyama I, Kimura H, Taniguchi T
    Exp. Neurol 146 57 - 66 1997年 [無し][無し]
     研究論文（学術雑誌）
  • Tooyama I, Sasaki K, Oomura Y, Li AJ, Kimura H
    Exp. Gerontol 32 171 - 179 1997年 [無し][無し]
     研究論文（学術雑誌）
  • M Suzuki, H Kitano, T Kitanishi, Y Yazawa, K Kitajima, T Takeda, H Kimura, Tooyama, I
    NEUROREPORT 8 2 439 - 443 1997年01月 [無し][無し]
     研究論文（学術雑誌） 
    C-TYPE natriuretic peptide (CNP) is the third member of the natriuretic peptide family which plays an important role in body fluid homeostasis. To determine a possible role of CNP in regulation of an inner ear fluid, we investigated the expression of CNP and atrial natriuretic peptide B receptor (ANP-B receptor) mRNAs in rat inner ear using a reverse transcription polymerase chain reaction (RT-PCR) method. Amplification products with sizes expected for CNP and ANP-B were detected in the inner ear. After cloning and analysis, the sequences for PCR products were identical to those of CNP or ANP-B receptor in the brain. These results indicate that both CNP and ANP-B receptor are expressed in the inner ear of the rat and suggest that CNP may play a role in inner ear function (such as regulation of inner ear fluid) in an autocrine and/or paracrine manner.
  • Recent advances in neuroscience for Alzheimer's disease
    Kimura H, Yasuhara O, Tooyama I
    Adv. Legal. Med. 3 280 - 283 1997年 [無し][無し]
     研究論文（学術雑誌）
  • Recent Advances in Microscopy of Cells, Tissues and Organs
    Renda T, Erspamer V, Tooyama I, Kimura H
    Antonio Delfino Editore 239 - 246 1997年 [無し][無し]
     研究論文（学術雑誌）
  • Hideya Takenaka, Saburo Kishimoto, Ikuo Tooyama, Hiroshi Kimura, Hirokazu Yasuno
    Journal of Investigative Dermatology 109 1 108 - 112 1997年 [無し][無し]
     研究論文（学術雑誌） 
    Fibroblast growth factors have been shown to play important roles in wound healing. To define their sites of action, we examined the expression of fibroblast growth factor receptor-1 (FGFR-1) during burn wound healing in rat skin by immunohistochemistry and western blot analysis. In cryostat sections of intact skin, little or no staining was observed. After a burn, however, staining for FGFR-1 was found in newly forming epidermis. The suprabasal layer of such epidermis, composed mostly of regenerating keratinocytes, was stained intensely, whereas keratinocytes in newly forming hair follicles were devoid of staining. Staining gradually decreased week by week after wound closure and was hardly visible 10 weeks after the burn, when the thickness of the epidermis had returned to the normal level. Staining was also found in small blood vessels and capillaries of granulation tissues of the dermis. Western blot analysis using the same antiserum was performed in the newly forming epidermis 10 d after the burn. A single band was detected with an apparent molecular weight of 120 kDa, corresponding to the short membrane- bound form of rat FGFR-1. Our study indicates that FGFR-1 is expressed during wound healing, mainly in regenerating epidermis and to some extent in blood vessels of the dermis. Fibroblast growth factors may affect the proliferation and differentiation of epidermal keratinocytes as well as angiogenesis in the dermis via the FGFR-1 expressed during wound healing.
  • Ikuo Tooyama, Kazuo Sasaki, Yutaka Oomura, Li Ai-Jun, Hiroshi Kimura
    Experimental Gerontology 32 1-2 171 - 179 1997年01月 [無し][無し]
     研究論文（国際会議プロシーディングス） 
    We examined the effects of chronic administration of acidic fibroblast growth factor (aFGF) on memory and choline acetyltransferase (ChAT) immunoreactivity in the forebrain of senescence-accelerated mice (SAMP8 strain). Subcutaneous injection of aFGF (aFGF group) or saline vehicle (saline group) once a week into SAMP8 was begun at three weeks after birth and continued for nine months. In the passive avoidance test, the retained latency was significantly longer in the aFGF group than in the saline group. In the Morris test, the mean latency to climb on a platform was significantly shorter in the aFGF group than in the saline group. The number of ChAT- positive neurons in the forebrain septum was greater in the aFGF group than in the saline group, and was at the level of that in the control mouse strain (SAMR1). The intensity of ChAT staining in the aFGF group appeared slightly weaker than in SAMR1 but significantly stronger than in the saline group. The results indicate that the effect of aFGF on memory function in SAMP8 may be related to the preservation of function in septal cholinergic cells.
  • M Suzuki, H Kitano, T Kitanishi, Y Yazawa, K Kitajima, T Takeda, H Kimura, Tooyama, I
    NEUROREPORT 8 2 439 - 443 1997年01月 [無し][無し]
     研究論文（学術雑誌） 
    C-TYPE natriuretic peptide (CNP) is the third member of the natriuretic peptide family which plays an important role in body fluid homeostasis. To determine a possible role of CNP in regulation of an inner ear fluid, we investigated the expression of CNP and atrial natriuretic peptide B receptor (ANP-B receptor) mRNAs in rat inner ear using a reverse transcription polymerase chain reaction (RT-PCR) method. Amplification products with sizes expected for CNP and ANP-B were detected in the inner ear. After cloning and analysis, the sequences for PCR products were identical to those of CNP or ANP-B receptor in the brain. These results indicate that both CNP and ANP-B receptor are expressed in the inner ear of the rat and suggest that CNP may play a role in inner ear function (such as regulation of inner ear fluid) in an autocrine and/or paracrine manner.
  • Yoshihisa Kitamura, Hideaki Takahashi, Yasuji Matsuoka, Ikuo Tooyama, Hiroshi Kimura, Yasuyuki Nomura, Takashi Taniguchi
    GLIA 18 3 233 - 243 1996年11月 [無し][無し]
     研究論文（学術雑誌） 
    To clarify whether the inducible nitric oxide synthase (iNOS) protein can be induced in in vivo brain, we examined the influence of direct intrahippocampal injection with interferon-γ (IFN-γ) plus lipopolysaccharide (LPS) in the rat. In the area surrounding the microinjection site, NOS activity (NO2 accumulation) was enhanced 24 h after injection with IFN-γ plus LPS. Although the level of 160-kDa nNOS protein was not changed, the 130-kDa iNOS protein was induced 12 h after the injection. On the other hand, iNOS mRNA could be detected at 6 and 12 h but not at 24 h. iNOS immunoreactivity was observed in CD11b-immunopositive microglia in close proximity to the injection site, but the immunoreactivity was not colocalized with glial fibrillary acidic protein-immunopositive astrocytes. Although CD11b-immunopositive microglia were of the ramified type even after injection with vehicle after 24 h, injection with IFN-γ plus LPS caused numerous microglia to change to the ameboid type and to express major histocompatibility complex (MHC) class II antigens. In some of these ameboidal microglia, iNOS immunoreactivity was observed. These results suggest that intrahippocampal injection with IFN-γ plus LPS induced iNOS mRNA after 6 h and iNOS protein after 12 h in some of the ameboidal microglia that expressed MHC class II antigens in in vivo rat brain.
  • Y Kitamura, H Takahashi, Y Matsuoka, Tooyama, I, H Kimura, Y Nomura, T Taniguchi
    GLIA 18 3 233 - 243 1996年11月 [無し][無し]
     研究論文（学術雑誌） 
    To clarify whether the inducible nitric oxide synthase (iNOS) protein can be induced in in vivo brain, we examined the influence of direct intrahippocampal injection with interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS) in the rat. In the area surrounding the microinjection site, NOS activity (NO2- accumulation) was enhanced 24 h after injection with IFN-gamma plus LPS. Although the level of 160-kDa nNOS protein was not changed, the 130-kDa iNOS protein was induced 12 h after the injection. On the other hand, iNOS mRNA could be detected at 6 and 12 h but not at 24 h. iNOS immunoreactivity was observed in CD11b-immunopositive microglia in close proximity to the injection site, but the immunoreactivity was not colocalized with glial fibrillary acidic protein-immunopositive astrocytes. Although CD11b-immunopositive microglia were of the ramified type even after injection with vehicle after 24 h, injection with IFN-gamma plus LPS caused numerous microglia to change to the ameboid type and to express major histocompatibility complex (MHC) class II antigens. In some of these ameboidal microglia, iNOS immunoreactivity was observed. These results suggest that intrahippocampal injection with IFN-gamma plus LPS induced iNOS mRNA after 6 h and iNOS protein after 12 h in some of the ameboidal microglia that expressed MHC class II antigens in in vivo rat brain. (C) 1996 Wiley-Liss, Inc.
  • S Nakao, T Adachi, M Murakawa, T Shinomura, J Kurata, T Shichino, M Shibata, Tooyama, I, H Kimura, K Mori
    ANESTHESIOLOGY 85 4 874 - 882 1996年10月 [無し][無し]
     研究論文（学術雑誌） 
    Background: Ketamine, a noncompetitive N-methyl-D-aspartate antagonist, has psychotomimetic side effects. Recent studies have shown that noncompetitive N-methyl-D-aspartate antagonists cause morphologic damage to the cingulate and retrosplenial cortices and induce c-fos protein (c-Fos) in the same regions. Although benzodiazepines are effective in preventing these side effects, the neural basis of the drug interactions has not been established. Methods: The effects of diazepam and halothane on c-Fos expression induced by ketamine were studied. Diazepam (1 and 5 mg/kg) or vehicle were administered subcutaneously, followed 7 min later by 100 mg/kg ketamine given intraperitoneally. Halothane (1.0 and 1.80%), was administered continuously from 10 min before ketamine administration until brain fixation. Two hours after ketamine injection, rats were perfused and their brains fixed and extracted. Brain sections were prepared in a cryostat and c-Fos expression was detected using immunohistochemical methods. Results: Ketamine induced c-Fos-like immunoreactivity in the cingulate and retrosplenial cortices, thalamus, and neocortex. Diazepam suppressed the ketamine-induced c-Fos-like immunoreactivity in the cingulate and retrosplenial cortices in a dose-dependent manner, leaving the thalamus and neocortex less affected. Halothane suppressed the ketamine-induced c-Fos-Like immunoreactivity in the cingulate and retrosplenial cortices and the neocortex in a dose-dependent manner, leaving the thalamus relatively unaffected. Conclusion: Halothane and diazepam inhibited ketamine induced c-Fos expression in the cingulate and retrosplenial cortices, leaving the thalamus relatively unaffected.
  • Tooyama, I, S Uemura, Y Kubota, Y Seino, TG Beach, WB Woodhurst, PL McGeer, H Kimura
    EPILEPSIA 37 64 - 64 1996年07月 [有り][無し]
     研究論文（学術雑誌）
  • T Nishimura, S Uemura, Tooyama, I, Y Kitamura, S Takeo, H Kimura
    EPILEPSIA 37 97 - 98 1996年07月 [有り][無し]
     研究論文（学術雑誌）
  • Up Date痴呆 Alzheimer型痴呆を中心に 成因と病態 発症機構 神経成長因子
    遠山 育夫, 相見 良成, 木村 宏
    現代医療 28 6 1335 - 1339 (株)現代医療社 1996年06月
  • Mitsuo Takahashi, Tatsuo Yamada, Ikuo Tooyama, Iku Moroo, Hiroshi Kimura, Takayuki Yamamoto, Hidechika Okada
    Neuroscience Letters 204 3 201 - 204 1996年02月 [無し][無し]
     研究論文（学術雑誌） 
    Neurotrophic effects resulting from the insulin/insulin receptor system have been recognized as important in determining the etiological basis of neurodegenerative disorders. In Parkinson's disease, selective neuronal loss in the substantia nigra is accompanied by decreased immunoreactivity of the insulin receptor as determined using immunohistochemical studies. We performed semiquantitative mRNA analysis by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for human insulin receptor exon 22, which encodes a region of the beta subunit of the receptor serving as a tyrosine kinase domain. The relative levels of mRNA in the substantia nigra from Parkinson's brain tissues showed a marked depression compared with those of normal controls. Further investigations are needed to decide whether this is a primary, disease-specific alteration of gene expression or merely a secondary process.
  • AJ Li, Y Oomura, T Hori, S Aou, K Sasaki, H Kimura, Tooyama, I
    EXPERIMENTAL NEUROLOGY 137 2 318 - 323 1996年02月 [有り][無し]
     研究論文（学術雑誌） 
    Previous studies have shown that acidic and basic fibroblast growth factor (aFGF and bFGF) and certain fragments of the aFGF N-terminal suppress food intake in rats due to their inhibitory actions on the glucose-sensitive neurons in the lateral hypothalamic area (LHA). The present study was planned to determine the role of FGF receptor-1 (FGFR-1), which was found in the LHA neurons of rats, on feeding regulation. The structure-activity relationship of aFGF fragments in feeding suppression was also investigated. An injection of anti-FGFR-1 antibody (250 and 350 ng) into the bilateral LHA significantly increased food intake. Synthesized aFGF fragments were infused into the III ventricle to elucidate the structure-activity relationship on the inhibition of feeding. Although aFGF-(1-29) did not affect food intake, [Ser(16)]aFGF-(1-29) (400 ng) and [Glu(16)]aFGF-(1-29) (400 ng), in which the cysteine residue at position 16 of aFGF-(1-29) was replaced with structurally similar serine and glutamic acid, were observed to significantly inhibit food intake. These findings suggest that endogenous FGFR-1 in the LHA plays an important role in FGF-induced feeding suppression, while, in addition, the dissolving disulfide bond formation in aFGF fragments enhances their inhibitory effects on feeding. (C) 1996 Academic Press, Inc.
  • M Suzuki, H Kitano, Y Yazawa, K Kitajima, Tooyama, I, H Kimura, T Takeda
    SYDNEY '97 - XVI WORLD CONGRESS OF OTORHINOLARYNGOLOGY HEAD AND NECK SURGERY, TOMES 1 AND 2 1333 - 1337 1996年 [有り][無し]
     研究論文（国際会議プロシーディングス） 
    C-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family which plays an important role in body fluid homeostasis. To determine a possible role of CNP in regulation of an inner ear fluid, we investigated the expression of CNP and atrial natriuretic peptide B receptor (ANP-B receptor) mRNAs in rat inner ear using a reverse transcription polymerase chain reaction method. Amplification products with sizes expected for CNP and ANP-B were detected in the inner ear. After cloning and analysis, the sequences for PCR products were identical to those of CNP or ANP-B receptor in the brain. These results indicate that both CNP and ANP-B are expressed in the inner ear of the rat and suggest that CNP may play a role in inner ear functions (such as regulation of inner ear fluid) in a paracrine and/or autocrine manner.
  • Memory facillitation by an endogenous satiety substance, acidic fibroblast growth factor (aFGF)
    Oomura Y, Sasaki K, Li AJ, Kimura H, Tooyama I, Hanai K, Nomura Y, Kitamura Y, Yanaihara N, Yago H
    Prog. Obes. Res. 7 433 - 438 1996年 [無し][無し]
     研究論文（学術雑誌）
  • Shin Ichi Nakao, Takehiko Adachi, Masahiro Murakawa, Tetsutaro Shinomura, Jiro Kurata, Tsutomu Shichino, Masatoshi Shibata, Ikuo Tooyama, Hiroshi Kimura, Kenjiro Mori
    Anesthesiology 85 4 874 - 882 1996年 [無し][無し]
     研究論文（学術雑誌） 
    Background: Ketamine, a noncompetitive N-methyl-D-aspartate antagonist, has psychotomimetic side effects. Recent studies have shown that noncompetitive N-methyl-D-aspartate antagonists cause morphologic damage to the cingulate and retrosplenial cortices and induce c-fos protein (c-Fos) in the same regions. Although benzodiazepines are effective in preventing these side effects, the neural basis of the drug interactions has not been established. Methods: The effects of diazepam and halothane on c-Fos expression induced by ketamine were studied. Diazepam (1 and 5 mg/kg) or vehicle were administered subcutaneously, followed 7 min later by 100 mg/kg ketamine given intraperitoneally. Halothane (1.0 and 1.8%), was administered continuously from 10 min before ketamine administration until brain fixation. Two hours after ketamine injection, rats were perfused and their brains fixed and extracted. Brain sections were prepared in a cryostat and c-Fos expression was detected using immunohistochemical methods. Results: Ketamine induced c-Fos-like immunoreactivity in the cingulate and retrosplenial cortices, thalamus, and neocortex. Diazepam suppressed the ketamine-induced c-Fos-like immunoreactivity in the cingulate and retrosplenial cortices in a dose-dependent manner, leaving the thalamus and neocortex less affected. Halothane suppressed the ketamine-induced c-Fos-like immunoreactivity in the cingulate and retrosplenial cortices and the neocortex in a dose-dependent manner, leaving the thalamus relatively unaffected. Conclusion: Halothane and diazepam inhibited ketamine-induced c-Fos expression in the cingulate and retrosplenial cortices, leaving the thalamus relatively unaffected.
  • Basic FGF partially prevents degeneration of paraventricular vasopressin neurons after hypophysectomy
    Takeichi Y, Nakasu Y, Tooyama I, Kimura H
    Restr. Neurol. Neurosci 10 161 - 166 1996年 [無し][無し]
     研究論文（学術雑誌）
  • Immunohistochemical demonstration of GABA-B receptors in the rat brain
    Kimura H, Terai K, Tooyama I, Kuriyama K
    Pharmacol. Rev. Comm 8 167  1996年 [無し][無し]
     研究論文（学術雑誌）
  • Molecular biological approaches to the GABA-B receptor
    Hirouchi M, Tooyama I, Kimura H, Kuriyama K
    Pharmacol. Rev. Comm 8 151  1996年 [無し][無し]
     研究論文（学術雑誌）
  • Kyoji Nakajima, Ikuo Tooyama, Kinya Kuriyama, Hiroshi Kimura
    Neurochemical Research 21 2 211 - 215 1996年 [無し][無し]
     研究論文（学術雑誌） 
    Immunohistochemical localization of GABAB-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABAB-receptor. Immunoreactive staining for GABAB-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABAB-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throughout the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABAB-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABAB-receptors. © 1996 Plenum Publishing Corporation.
  • Y Oomura, K Sasaki, A Li, H Yoshii, Y Fukata, H Yago, H Kimura, I Tooyama, K Hanai, Y Nomura, N Yanaihara
    PHARMACOLOGICAL INTERVENTION IN AGING AND AGE-ASSOCIATED DISORDERS 786 337 - 347 1996年 [無し][無し]
     研究論文（学術雑誌）
  • Takahashi M, Yamada T, Tooyama I, Morro I, Kimura H, Yamamoto T, Okada H
    Neurosci. Lett 204 201 - 204 1996年 [無し][無し]
     研究論文（学術雑誌）
  • T Hase, T Tani, T Oka, T Yokota, M Kodama, H Kimura, Tooyama, I
    IMMUNE CONSEQUENCES OF TRAUMA, SHOCK AND SEPSIS - MECHANISMS AND THERAPEUTIC APPROACHES, VOL II, PTS 1 AND 2 2 1 353 - 358 1996年 [無し][無し]
     研究論文（国際会議プロシーディングス）
  • Memory facillitation by an endogenous satiety substance, acidic fibroblast growth factor (aFGF)
    Oomura Y, Sasaki K, Li AJ, Kimura H, Tooyama I, Hanai K, Nomura Y, Kitamura Y, Yanaihara N, Yago H
    Prog. Obes. Res. 7 433 - 438 1996年 [無し][無し]
     研究論文（学術雑誌）
  • Basic FGF partially prevents degeneration of paraventricular vasopressin neurons after hypophysectomy
    Takeichi Y, Nakasu Y, Tooyama I, Kimura H
    Restr. Neurol. Neurosci 10 161 - 166 1996年 [無し][無し]
     研究論文（学術雑誌）
  • Immunohistochemical demonstration of GABA-B receptors in the rat brain
    Kimura H, Terai K, Tooyama I, Kuriyama K
    Pharmacol. Rev. Comm 8 167  1996年 [無し][無し]
     研究論文（学術雑誌）
  • Molecular biological approaches to the GABA-B receptor
    Hirouchi M, Tooyama I, Kimura H, Kuriyama K
    Pharmacol. Rev. Comm 8 151  1996年 [無し][無し]
     研究論文（学術雑誌）
  • Kyoji Nakajima, Ikuo Tooyama, Kinya Kuriyama, Hiroshi Kimura
    Neurochemical Research 21 2 211 - 215 1996年 [無し][無し]
     研究論文（学術雑誌） 
    Immunohistochemical localization of GABAB-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABAB-receptor. Immunoreactive staining for GABAB-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABAB-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throughout the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABAB-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABAB-receptors. © 1996 Plenum Publishing Corporation.
  • Y Oomura, K Sasaki, A Li, H Yoshii, Y Fukata, H Yago, H Kimura, I Tooyama, K Hanai, Y Nomura, N Yanaihara
    PHARMACOLOGICAL INTERVENTION IN AGING AND AGE-ASSOCIATED DISORDERS 786 337 - 347 1996年 [無し][無し]
     研究論文（学術雑誌）
  • T Hase, T Tani, T Oka, T Yokota, M Kodama, H Kimura, Tooyama, I
    IMMUNE CONSEQUENCES OF TRAUMA, SHOCK AND SEPSIS - MECHANISMS AND THERAPEUTIC APPROACHES, VOL II, PTS 1 AND 2 2 1 353 - 358 1996年 [無し][無し]
     研究論文（国際会議プロシーディングス）
  • S Yu, Tooyama, I, WG Ding, H Kitasato, H Kimura
    OBESITY RESEARCH 3 S753 - S760 1995年12月 [無し][無し]
     研究論文（学術雑誌） 
    Immunohistochemical localization of the glucose transporters was studied in the rat hypothalamus by using a specific rabbit antiserum raised against either the isoform 1 (GLUT1) or the isoform 3 (GLUT3). Immunoreactive staining for GLUT1 was found in glia cells and capillaries, whereas positive staining for GLUT3 occurred mainly in neurons and partly in ependymal cells, Double immunostaining indicated that a small population of GLUT1-positive cells were reactive for glial fibrillary acidic protein, a marker for astrocytes, Another doubly stained section showed that GLUT1-positive glia cells were never stained with an OX42 antibody, a marker for microglia cells, Neurons staining positively for GLUT3, often large in cell size, were confined mainly in the lateral hypothalamic area and partly in the dorsomedial and periventricular hypothalamic nuclei. Possible significance of these two glucose transporters in the hypothalamus is briefly discussed.
  • Y MATSUOKA, Y KITAMURA, T TSUKAHARA, K TERAI, TOOYAMA, I, H KIMURA, T TANIGUCHI
    NEUROREPORT 6 16 2205 - 2208 1995年11月 [無し][無し]
     研究論文（学術雑誌） 
    MODELS of cerebral ischaemia were used for analysis of mechanism of neuronal cell death and/or damage. Ischaemia is caused dominantly by severe hypoxia and hypoglycaemia: in the present study, we examined the influence of severe in vivo hypoxia (5% O-2/95% N-2 for 30 min at 22 degrees C). After hypoxia, neuronal damage was observed in the CA3 and dentate gyrus (DG) after 3 and 21 days of survival, but not in the CA1. 2,3-Dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), an antagonist for AMPA/kainate receptors, showed neuroprotective effects in the CA3 and DG. These results suggest that hypoxia may induce neuronal damage in the CA3 and DG through activation of AMPA/kainate receptors.
  • Y MATSUOKA, Y KITAMURA, T TSUKAHARA, K TERAI, TOOYAMA, I, H KIMURA, T TANIGUCHI
    NEUROREPORT 6 16 2205 - 2208 1995年11月 [無し][無し]
     研究論文（学術雑誌） 
    MODELS of cerebral ischaemia were used for analysis of mechanism of neuronal cell death and/or damage. Ischaemia is caused dominantly by severe hypoxia and hypoglycaemia: in the present study, we examined the influence of severe in vivo hypoxia (5% O-2/95% N-2 for 30 min at 22 degrees C). After hypoxia, neuronal damage was observed in the CA3 and dentate gyrus (DG) after 3 and 21 days of survival, but not in the CA1. 2,3-Dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), an antagonist for AMPA/kainate receptors, showed neuroprotective effects in the CA3 and DG. These results suggest that hypoxia may induce neuronal damage in the CA3 and DG through activation of AMPA/kainate receptors.
  • TOOYAMA, I, T KAWAMATA, H AKIYAMA, H KIMURA, SK MOESTRUP, J GLIEMANN, A MATSUO, PL MCGEER
    BRAIN RESEARCH 691 1-2 235 - 238 1995年09月 [無し][無し]
     研究論文（学術雑誌） 
    The subcellular localization of the alpha(2)-macroglobulin receptor, also known as the low density lipoprotein receptor-related protein (LRP), was studied in postmortem human brain tissue by light and electron microscopic immunocytochemistry. A specific monoclonal antibody (A2MR2) against the extracellular alpha-chain of the molecule was utilized. Light microscopically, LRP was detected strongly in neurons, weakly in some glial cells and discontinuously along capillary membranes. At the electron microscopic level, positive reaction products were found to be associated with plasma membranes, ribosomes, lysosomes and lipofuscin granules of neurons, glial cells and pericytes. The results suggest that LRP may have a function, particularly in neurons, of receptor-mediated endocytosis with subsequent lysosomal uptake and degradation of ligands such as alpha(2)-macroglobulin proteinase complexes and apolipoprotein E.
  • TOOYAMA, I, T KAWAMATA, H AKIYAMA, H KIMURA, SK MOESTRUP, J GLIEMANN, A MATSUO, PL MCGEER
    BRAIN RESEARCH 691 1-2 235 - 238 1995年09月 [無し][無し]
     研究論文（学術雑誌） 
    The subcellular localization of the alpha(2)-macroglobulin receptor, also known as the low density lipoprotein receptor-related protein (LRP), was studied in postmortem human brain tissue by light and electron microscopic immunocytochemistry. A specific monoclonal antibody (A2MR2) against the extracellular alpha-chain of the molecule was utilized. Light microscopically, LRP was detected strongly in neurons, weakly in some glial cells and discontinuously along capillary membranes. At the electron microscopic level, positive reaction products were found to be associated with plasma membranes, ribosomes, lysosomes and lipofuscin granules of neurons, glial cells and pericytes. The results suggest that LRP may have a function, particularly in neurons, of receptor-mediated endocytosis with subsequent lysosomal uptake and degradation of ligands such as alpha(2)-macroglobulin proteinase complexes and apolipoprotein E.
  • Osamu Yasuhara, Akinori Matsuo, Ikuo Tooyama, Hiroshi Kimura, Edith G. McGeer, Patrick L. McGeer
    Acta Neuropathologica 89 4 322 - 330 1995年04月 [無し][無し]
     研究論文（学術雑誌） 
    Pick's disease (PD) brains were examined immunohistochemically for the expression of antigens known to be associated with Alzheimer's disease (AD) lesions. Most antibodies which label intracellular neurofibrillary tangles (NFTs) in AD were found to stain Pick bodies (PBs). Among them was the monoclonal antibody A2B5, which is known to recognize neuronal surface gangliosides. This result indicates that membrane proteins are probably incorporated into PBs as into NFTs. However, PBs, in contrast to NFTs, showed a paucity of staining for heparan sulfate glycosaminoglycan and basic fibroblast growth factor (bFGF). Staining for midline, seen in senile plaques in AD, was not seen in PD. The relative lack of staining for these two neurotrophic factors in PD brain may reflect underlying mechanisms which are distinct from those in AD. We also describe two glial abnormalities in PD: glial fibrillary tangles and clusters of granules positive for the complement protein C4d in the hippocampal dentate fascia. These are presumably related to complement-activated oligodendroglia, and both pathological structures are more abundant in advanced cases, suggesting that they may be hallmarks of the disease progression. © 1995 Springer-Verlag.
  • Osamu Yasuhara, Akinori Matsuo, Ikuo Tooyama, Hiroshi Kimura, Edith G. McGeer, Patrick L. McGeer
    Acta Neuropathologica 89 4 322 - 330 1995年04月 [無し][無し]
     研究論文（学術雑誌） 
    Pick's disease (PD) brains were examined immunohistochemically for the expression of antigens known to be associated with Alzheimer's disease (AD) lesions. Most antibodies which label intracellular neurofibrillary tangles (NFTs) in AD were found to stain Pick bodies (PBs). Among them was the monoclonal antibody A2B5, which is known to recognize neuronal surface gangliosides. This result indicates that membrane proteins are probably incorporated into PBs as into NFTs. However, PBs, in contrast to NFTs, showed a paucity of staining for heparan sulfate glycosaminoglycan and basic fibroblast growth factor (bFGF). Staining for midline, seen in senile plaques in AD, was not seen in PD. The relative lack of staining for these two neurotrophic factors in PD brain may reflect underlying mechanisms which are distinct from those in AD. We also describe two glial abnormalities in PD: glial fibrillary tangles and clusters of granules positive for the complement protein C4d in the hippocampal dentate fascia. These are presumably related to complement-activated oligodendroglia, and both pathological structures are more abundant in advanced cases, suggesting that they may be hallmarks of the disease progression. © 1995 Springer-Verlag.
  • N IWAI, K HANAI, TOOYAMA, I, Y KITAMURA, M KINOSHITA
    HYPERTENSION 25 3 431 - 436 1995年03月 [無し][無し]
     研究論文（学術雑誌） 
    Neuronal nitric oxide synthase (nNOS) has been suggested to be involved in cardiovascular homeostasis. We studied the regulation of nNOS expression, determining nNOS mRNA expression levels in various tissues in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We also investigated the effects of antihypertensive treatment with the angiotensin II antagonist hydralazine or reserpine on nNOS mRNA expression. The expression levels of nNOS mRNA and nNOS protein were determined by Northern and Western blot analysis, respectively. NADPH-diaphorase histochemistry was used to identify cells in the adrenal medulla that expressed nNOS. No significant differences in expression levels in SHR and WKY were observed in the cerebellum and brain stem. nNOS mRNA expression levels in the decapsular portion of the adrenal gland were developmentally modulated and in a 24-week-old WKY were 2.5 times higher than in an age-matched SHR. This reduced expression of nNOS mRNA in the decapsular portion of the adrenal gland of SHR seemed to be result of hypertension in the SHR, because administration of either an angiotensin II antagonist (TCV-116) or hydralazine upregulated nNOS mRNA expression in both SHR and WKY. Marked augmentation of nNOS mRNA expression in the decapsular portion of the adrenal gland by reserpine treatment suggested an intimate relation between nNOS in the decapsular portion of the adrenal gland and the sympathoadrenal system. Reserpine treatment also increased the expression of nNOS protein; however, reserpine treatment did not affect the distribution pattern of nNOS-positive cells (NADPH-diaphorase-positive cells) in the adrenal medulla. The present results suggest that nNOS gene expression in the decapsular portion of the adrenal gland is not constitutive and that an intimate relation may exist between nNOS in the decapsular portion of the adrenal gland and the sympathoadrenal system.
  • N IWAI, K HANAI, TOOYAMA, I, Y KITAMURA, M KINOSHITA
    HYPERTENSION 25 3 431 - 436 1995年03月 [無し][無し]
     研究論文（学術雑誌） 
    Neuronal nitric oxide synthase (nNOS) has been suggested to be involved in cardiovascular homeostasis. We studied the regulation of nNOS expression, determining nNOS mRNA expression levels in various tissues in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). We also investigated the effects of antihypertensive treatment with the angiotensin II antagonist hydralazine or reserpine on nNOS mRNA expression. The expression levels of nNOS mRNA and nNOS protein were determined by Northern and Western blot analysis, respectively. NADPH-diaphorase histochemistry was used to identify cells in the adrenal medulla that expressed nNOS. No significant differences in expression levels in SHR and WKY were observed in the cerebellum and brain stem. nNOS mRNA expression levels in the decapsular portion of the adrenal gland were developmentally modulated and in a 24-week-old WKY were 2.5 times higher than in an age-matched SHR. This reduced expression of nNOS mRNA in the decapsular portion of the adrenal gland of SHR seemed to be result of hypertension in the SHR, because administration of either an angiotensin II antagonist (TCV-116) or hydralazine upregulated nNOS mRNA expression in both SHR and WKY. Marked augmentation of nNOS mRNA expression in the decapsular portion of the adrenal gland by reserpine treatment suggested an intimate relation between nNOS in the decapsular portion of the adrenal gland and the sympathoadrenal system. Reserpine treatment also increased the expression of nNOS protein; however, reserpine treatment did not affect the distribution pattern of nNOS-positive cells (NADPH-diaphorase-positive cells) in the adrenal medulla. The present results suggest that nNOS gene expression in the decapsular portion of the adrenal gland is not constitutive and that an intimate relation may exist between nNOS in the decapsular portion of the adrenal gland and the sympathoadrenal system.
  • Yoshihiko Wakabayashi, Tadao Tomoyoshi, Ikuo Tooyama, Kunio Kitahama, Seung U. Kim, Toshihiro Maeda
    Neuroscience Letters 186 1 9 - 12 1995年02月 [有り][無し]
     研究論文（学術雑誌） 
    The localization of low-affinity nerve growth factor receptor (LNGFR) in the human urinary bladder was examined immunohistochemically using the mouse monoclonal antibody (ME20-4) against human LNGFR. LNGFR immunoreactivity was present in the human urinary bladder. The distribution of LNGFR-positive fibers was more abundant in the mucosa than in the muscle layer. Results also showed that some LNGFR-positive fiber bundles contained tyrosine hydroxylase immunoreactivity. Electron microscopic examination revealed that LNGFR immunoreactivity was located on the surface of Schwann cells, and frequently on the interface of axons and Schwann cells. © 1995.
  • Y. Oomura, K. Sasaki, A. Li, H. Yoshii, Y. Fukata, H. Yago, H. Kimura, I. Tooyama, K. Hanai, Y. Nomura, Y. Kitamura, N. Yanaihara
    Neurobiology 3 3-4 371 - 380 1995年 [有り][無し]
     研究論文（国際会議プロシーディングス） 
    Subcutaneous injection of aFGF once per a week into senescence accelerated mice (SAM)P8 was begun at 3 weeks after birth and continued for 10 months. Saline was injected as a control. Learning and memory and cellular immunological functions in the aFGF (F) group were enhanced significantly and while those of the saline (S) group deteriorated. The number of cholinergic neurons was decreased slightly and choline acetyltransferase activity in individual neurons in the medial septum which send monosynaptic terminals to the hippocampus was significantly decreased in the S group, but were more spared in the F group. The MAO-B activity was significantly lower in the F group than in the S group. The respective densities of muscarinic and NMDA receptors and the aFGF receptor, i.e. FGFR-1 in the hippocampus were also significantly higher in the F group than in the S group. The delayed type hypersensitivity reactions (DTH) in the footpad caused by challenge with trinitrophenyl or sheep red blood cells as measured at the end of the 2nd and 7th months, indicated the T cell immune response. Both types of DTHs were reduced in the 7th month as compared with the 2nd month in the S group. However, aFGF administration protected against this reduction in response with age. These results show that aFGF provides protection against impairment of not only learning and memory but also the DTH immunoreactivity in SAMP8, indicating thereby a close relationship between learning-memory and T cell immune function.
  • Kazuo Sasaki, Yutaka Oomura, Ai‐Jun Li, Kazumitsu Hanai, Ikuo Tooyama, Hiroshi Kimura, Noboru Yanaihara, Tetsuro Hori
    Obesity Research 3 5 S 697 - 706 1995年 [有り][無し]
     研究論文（学術雑誌） 
    Acidic fibroblast growth factor (aFGF) has suppressive effects on food intake. In the present study, the effect of aFGF fragments on food intake were investigated in rats. Infusion of a carboxyl‐terminal fragment of aFGF, aFGF‐(1 14–140), did not affect food intake, whereas an amino‐terminal fragment of aFGF, aFGF‐(1–15), was significantly inhibitory. Other amino‐terminal fragments, aFGF‐(1–20), aFGF‐(1–29) and aFGF‐(9–29), did not affect food intake. However, [Ala16]aFGF‐(1–29) and [Ser16]aFGF‐(1–29) in which the cysteine residue at position 16 was replaced with alanine and serine, respectively, had significant suppressive effects on food intake. Infusion of a functional antagonist for FGF receptor, anti‐FGFR‐1 antibody, into the lateral hypothalamus (LHA) significantly increased food intake. The results suggest that: the amino‐terminal portion of aFGF is active in food intake suppression the replacement of cysteine residue by alanine or serine is important in some amino‐terminal aFGF fragments and the LHA is involved in feeding suppression actions by aFGF and some fragments. 1995 North American Association for the Study of Obesity (NAASO)
  • PL MCGEER, TOOYAMA, I, H KIMURA, EG MCGEER
    AGE-RELATED DOPAMINE-DEPENDENT DISORDERS 14 235 - 243 1995年 [有り][無し]
     研究論文（国際会議プロシーディングス）
  • Shun Yu, Ikuo Tooyama, Wei‐Guang Ding, Hiroshi Kitasato, Hiroshi Kimura
    Obesity Research 3 5 S 753 - 760 1995年 [無し][無し]
     研究論文（学術雑誌） 
    Immunohistochemical localization of the glucose transporters was studied in the rat hypothalamus by using a specific rabbit antiserum raised against either the isoform 1 (GLUT1) or the isoform 3 (GLUT3). Immunoreactive staining for GLUT1 was found in glia cells and capillaries, whereas positive staining for GLUT3 occurred mainly in neurons and partly in ependymal cells. Double immunostaining indicated that a small population of GLUT1‐positive cells were reactive for glial fibrillary acidic protein, a marker for astrocytes. Another doubly stained section showed that GLUT1‐positive glia cells were never stained with an OX42 antibody, a marker for microglia cells. Neurons staining positively for GLUT3, often large in cell size, were confined mainly in the lateral hypothalamic area and partly in the dorsomedial and periventricular hypothalamic nuclei. Possible significance of these two glucose transporters in the hypothalamus is briefly discussed. 1995 North American Association for the Study of Obesity (NAASO)
  • Demonstration of fibroblast growth factor receptor-1 in human prostate by polymerase chain reaction and immunohistochemistry
    Hamaguchi A, Tooyama I, Yoshiki T, Kimura H
    Prostate 27 141 - 147 1995年 [無し][無し]
     研究論文（学術雑誌）
  • Akikazu Hamaguchi, Ikuo Tooyama, Tatsuhiro Yoshiki, Hiroshi Kimura
    The Prostate 27 3 141 - 147 1995年 [無し][無し]
     研究論文（学術雑誌） 
    The expression and localization of fibroblast growth factor receptor‐1 were investigated in human prostatic tissues with or without benign hyperplasia. Using a polymerase chain reaction method, we were able to demonstrate that prostatic tissues with benign hyperplasia expressed a significantly higher level of fibroblast growth factor receptor‐1 mRNA than normal prostatic tissues (P < 0.01 by Anova). Western blot analysis using an antiserum against the receptor gave 2 bands with molecular weights of about 140 kDa and 80 kDa these correspond to the expected sizes of the long and secreted forms of the fibroblast growth factor receptor‐1, respectively. An immunohistochemical study using the same antiserum further demonstrated that the immunoreactive staining occurred mainly in the basal cells of the glandular epithelium and occasionally in the stromal cells. These results suggest that fibroblast growth factors may influence, at least in part, the proliferation of the epithelial cells seen in benign hyperplasia of human prostate. © 1995 Wiley‐Liss, Inc. Copyright © 1995 Wiley‐Liss, Inc., A Wiley Company
  • S NAKAO, J KURATA, T ARAI, M MURAKAWA, T ADACHI, MN AVRAMOV, K MORI, O YASUHARA, TOOYAMA, I, H KIMURA
    ACTA ANAESTHESIOLOGICA SCANDINAVICA 38 8 845 - 851 1994年11月 [無し][無し]
     研究論文（学術雑誌） 
    Recent studies have shown that proto-oncogene c-fos mRNA is induced in the central nervous system by a variety of stimuli including generalised convulsions. In this study, the expression of c-fos protein (c-Fos) following lignocaine-induced convulsions was examined and Compared with that following convulsions induced by non-anesthetic convulsants, such as pentylenetetrazol, kainic acid and electroconvulsive shocks, in rat brain. Administration of 120 mg.kg(-1) lignocaine by the intraperitoneal route induced generalised convulsions in all rats examined within 10 min. C-Fos was markedly induced in the piriform cortex and amygdala, and slightly induced in the neocortex and thalamus, while no c-Fos expression was observed in the hippocampus. In contrast, c-Fos expression following generalised convulsions induced by non-anaesthetic convulsants was very marked in the hippocampal region, piriform cortex and amygdala, and extended to the thalamus and neocortex. These results contradict these of previously reported local cerebral metabolic studies using 2-deoxyglucose as a metabolic marker, and suggest that lignocaine-induced convulsions, unlike those induced by nonanaesthetic convulsants, may not cause severe sequelae (plastic changes) in the hippocampus.
  • K NONOMURA, TOOYAMA, I, H ABE, T RENDA, ERSPAMER, V, M SHIMADA, H KIMURA
    JOURNAL OF COMPARATIVE NEUROLOGY 349 2 223 - 243 1994年11月 [無し][無し]
     研究論文（学術雑誌） 
    [D-Ala(2)]deltorphin-I, a highly selective ligand for delta opioid receptors, is a heptapeptide originally purified from frog skin. Previous immunohistochemical studies indicate that [D-Ala(2)]deltorphin-I-like molecule(s) may be present in adult rat brain, including specific neuronal cells and fibers partially overlapping with the mesocortical and nigrostriatal dopaminergic systems. Here, we examined the developmental aspect of such immunoreactive brain structures in early postnatal rats. In newborn to al-day-old rats, positive staining in the brain occurred mainly in subpopulations of neurons and occasionally in tanycytes. On postnatal day 0, neuronal cell bodies containing [D-Ala(2)]deltorphin-I-like immunoreactivity were found in various brain regions, including the olfactory tubercle, ventral pallidum, hippocampus, ventral tegmental area, pars compacta of the substantia nigra, supramammillary nucleus, and dorsal raphe nucleus. Immunoreactive nerve fibers were observed in the main and accessory olfactory bulbs, olfactory tubercle, prelimbic area, anterior cingulate cortex, neostriatum, accumbens, lateral septal nucleus, lateral habenular nucleus, and superior colliculus. As pups grew, positive staining of cell bodies decreased gradually in both density acid intensity, and those in the olfactory tubercle and ventral pallidum were no longer visible on postnatal day 14. On postnatal day 21, positive cells were found only in the ventral midbrain, including the pars compacta of the substantia nigra, ventral tegmental area, A8 region, and supramammillary nucleus. Positive fibers also decreased in density with age except in the accessory olfactory bulb, olfactory tubercle, prelimbic area, and anterior cingulate cortex. (C) 1994 Wiley-Liss, Inc.
  • H KIMURA, TOOYAMA, I, PL MCGEER
    TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE 174 3 279 - 293 1994年11月 [無し][無し]
     研究論文（学術雑誌） 
    Immunohistochemical examination of postmortem brain tissue of Alzheimer's disease revealed that acidic fibroblast growth factor (aFGF) was specifically expressed in a subpopulation of reactive astrocytes which were congregated at the margin of the senile plaque. Double immunostaining indicated that such upregulation of aFGF expression might be related to the presence of reactive microglia rather than p-amyloid protein deposits. Although, on the other hand, immunohistochemical staining for fibroblast growth factor receptor-1 occurred in some cortical neurons of Alzheimer's disease, the staining pattern did not differ from that in age-matched controls. Possible significance of aFGF-positive astrocytes in the surroundings of the senile plaque will be discussed in relation receptor mediated or non-mediated mechanisms.
  • Y HARA, TOOYAMA, I, O YASUHARA, H AKIYAMA, PL MCGEER, J HANDA, H KIMURA
    BRAIN RESEARCH 664 1-2 101 - 107 1994年11月 [無し][無し]
     研究論文（学術雑誌） 
    Expression of acidic fibroblast growth factor (aFGF) was studied by immunocytochemistry in the rat brain with or without cerebral infarction. In a model of infarction produced by occlusion of the middle cerebral artery, aFGF-positive staining that was not seen in controls appeared in both neurons and macrophages. Positive neurons were, distributed mainly in the periventricular region of the hypothalamus. The neuronal aFGF began to be detectable about 24 h after the occlusion. The staining intensity for such neuronal aFGF increased until about 14 days after the surgery, but decreased gradually and was undetectable after more than 30 days. Positive macrophages first appeared on the 3rd day after the occlusion and were persistently seen up to the 7th day, but were no longer visible after 14 days. The present results suggest that aFGF may be involved in the repair processes following brain infarction.
  • S. NAKAO, J. KURATA, T. ARAI, M. MURAKAWA, T. ADACHI, M. N. AVRAMOV, K. MORI, O. YASUHARA, I. TOOYAMA, H. KIMURA
    Acta Anaesthesiologica Scandinavica 38 8 845 - 851 1994年11月 [無し][無し]
     研究論文（学術雑誌） 
    Recent studies have shown that proto–oncogene c–fos mRNA is induced in the central nervous system by a variety of stimuli including generalised convulsions. In this study, the expression of c–fos protein (c–Fos) following lignocaine–induced convulsions was examined and compared with that following convulsions induced by non–anesthetic convulsants, such as pentylenetetrazol, kainic acid and electroconvulsive shocks, in rat brain. Administration of 120 mg kg‐1 lignocaine by the intraperitoneal route induced generalised convulsions in all rats examined within 10 min. C–Fos was markedly induced in the piriform cortex and amygdala, and slightly induced in the neocortex and thalamus, while no c–Fos expression was observed in the hippocampus. In contrast, c–Fos expression following generalised convulsions induced by non–anaesthetic convulsants was very marked in the hippocampal region, piriform cortex and amygdala, and extended to the thalamus and neocortex. These results contradict those of previously reported local cerebral metabolic studies using 2–deoxyglucose as a metabolic marker, and suggest that lignocaine–induced convulsions, unlike those induced by non–anaesthetic convulsants, may not cause severe sequelae (plastic changes) in the hippocampus. Copyright © 1994 Acta Anaesthesiol Scand
  • K NONOMURA, TOOYAMA, I, H ABE, T RENDA, ERSPAMER, V, M SHIMADA, H KIMURA
    JOURNAL OF COMPARATIVE NEUROLOGY 349 2 223 - 243 1994年11月 [無し][無し]
     研究論文（学術雑誌） 
    [D-Ala(2)]deltorphin-I, a highly selective ligand for delta opioid receptors, is a heptapeptide originally purified from frog skin. Previous immunohistochemical studies indicate that [D-Ala(2)]deltorphin-I-like molecule(s) may be present in adult rat brain, including specific neuronal cells and fibers partially overlapping with the mesocortical and nigrostriatal dopaminergic systems. Here, we examined the developmental aspect of such immunoreactive brain structures in early postnatal rats. In newborn to al-day-old rats, positive staining in the brain occurred mainly in subpopulations of neurons and occasionally in tanycytes. On postnatal day 0, neuronal cell bodies containing [D-Ala(2)]deltorphin-I-like immunoreactivity were found in various brain regions, including the olfactory tubercle, ventral pallidum, hippocampus, ventral tegmental area, pars compacta of the substantia nigra, supramammillary nucleus, and dorsal raphe nucleus. Immunoreactive nerve fibers were observed in the main and accessory olfactory bulbs, olfactory tubercle, prelimbic area, anterior cingulate cortex, neostriatum, accumbens, lateral septal nucleus, lateral habenular nucleus, and superior colliculus. As pups grew, positive staining of cell bodies decreased gradually in both density acid intensity, and those in the olfactory tubercle and ventral pallidum were no longer visible on postnatal day 14. On postnatal day 21, positive cells were found only in the ventral midbrain, including the pars compacta of the substantia nigra, ventral tegmental area, A8 region, and supramammillary nucleus. Positive fibers also decreased in density with age except in the accessory olfactory bulb, olfactory tubercle, prelimbic area, and anterior cingulate cortex. (C) 1994 Wiley-Liss, Inc.
  • TOOYAMA, I, EG MCGEER, T KAWAMATA, H KIMURA, PL MCGEER
    BRAIN RESEARCH 656 1 165 - 168 1994年09月 [無し][無し]
     研究論文（学術雑誌） 
    A count of pigmented neurons per mm(3) in sections of the substantia nigra at the level where the oculomotor nerve emerges in 11 neurologically normal controls aged 15-82 showed the expected slow loss of such neurons with age. Most (82 +/- 3.8%) of the pigmented neurons showed immunoreactivity for basic fibroblast growth factor (bFGF) and this percentage was unaffected by age. This is in marked contrast to the case in Parkinson's disease where only same 12.7 +/- 2.6% of the remaining dopaminergic neurons showed bFGF-like immunoreactivity, providing further evidence against the hypothesis that Parkinson's disease is due to some early insult followed by age-related attrition of the remaining neurons.
  • K SASAKI, AJ LI, Y OOMURA, T MUTO, K HANAI, TOOYAMA, I, H KIMURA, N YANAIHARA, H YAGI, T HORI
    PHYSIOLOGY & BEHAVIOR 56 2 211 - 218 1994年08月 [無し][無し]
     研究論文（学術雑誌） 
    The effects of acidic fibroblast growth factor (aFGF), basic FGF (bFGF), and related peptides, such as aFGF fragments, on food and water intake were investigated. Infusion of aFGF and bFGF into the third cerebral ventricle significantly suppressed food intake. The potency of aFGF was 1.5 that of bFGF in food intake inhibition. Both FGFs also suppressed water intake. Infusion of a carboxyl-terminal fragment of aFGF, aFGF-(114-140), did not affect food intake, whereas an amino-terminal fragment of aFGF, aFGF-(1-5), was significantly inhibitory. Other amino-terminal fragments, aFGF-(1-20) and aFGF-(1-29), did not affect food intake. However, [Ala(16)]aFGF-(1-29), in which the cysteine residue at position 16 was replaced with alanine, significantly suppressed food intake. Infusions of functional antagonists for FGFs, anti-aFGF, anti-bFGF, and anti-aFGF-(1-15) IgGs, into the lateral hypothalamus significantly increased food intake. The results suggest that: aFGF, bFGF, and some amino-terminal peptides of aFGF participate in the central regulation of food intake; the lateral hypothalamus is involved in their feeding suppression actions; and these peptides may function as physiologically relevant substances in the adult central nervous system, other than as neurotrophic factors.
  • K SASAKI, AJ LI, Y OOMURA, T MUTO, K HANAI, TOOYAMA, I, H KIMURA, N YANAIHARA, H YAGI, T HORI
    PHYSIOLOGY & BEHAVIOR 56 2 211 - 218 1994年08月 [無し][無し]
     研究論文（学術雑誌） 
    The effects of acidic fibroblast growth factor (aFGF), basic FGF (bFGF), and related peptides, such as aFGF fragments, on food and water intake were investigated. Infusion of aFGF and bFGF into the third cerebral ventricle significantly suppressed food intake. The potency of aFGF was 1.5 that of bFGF in food intake inhibition. Both FGFs also suppressed water intake. Infusion of a carboxyl-terminal fragment of aFGF, aFGF-(114-140), did not affect food intake, whereas an amino-terminal fragment of aFGF, aFGF-(1-5), was significantly inhibitory. Other amino-terminal fragments, aFGF-(1-20) and aFGF-(1-29), did not affect food intake. However, [Ala(16)]aFGF-(1-29), in which the cysteine residue at position 16 was replaced with alanine, significantly suppressed food intake. Infusions of functional antagonists for FGFs, anti-aFGF, anti-bFGF, and anti-aFGF-(1-15) IgGs, into the lateral hypothalamus significantly increased food intake. The results suggest that: aFGF, bFGF, and some amino-terminal peptides of aFGF participate in the central regulation of food intake; the lateral hypothalamus is involved in their feeding suppression actions; and these peptides may function as physiologically relevant substances in the adult central nervous system, other than as neurotrophic factors.
  • A MATSUO, TOOYAMA, I, S ISOBE, Y OOMURA, AKIGUCHI, I, K HANAI, J KIMURA, H KIMURA
    NEUROSCIENCE 60 1 49 - 66 1994年05月 [有り][無し]
     研究論文（学術雑誌） 
    The localization of fibroblast growth factor receptor-1 was investigated in rat brain by immunohistochemistry using a polyclonal antibody against an acidic peptide sequence of chicken fibroblast growth factor receptor-1 For raising the antisera in rabbits, we synthesized the oligopeptide EDDDDEDDSSSEEKEAD which is a highly acidic region of chicken fibroblast growth factor receptor-1 The oligopeptide was used as a haptenic antigen by conjugating with poly-L-glutamate as a carrier protein. On immunospot assay, the best antiserum was capable of detecting 15.7 pmols of both the chicken and its analogous human oligopeptides but failed to react even with up to 1 nmol of poly-L-glutamate, When rat brain homogenate was examined by Western blots, the antiserum revealed two bands with molecular weights of 145,000 and 75,000 corresponding to known sizes of the membrane-bound and secreted forms of the rat receptor, respectively. Immunohistochemistry in rat brain demonstrated that putative fibroblast growth factor receptor-1 immunoreactivity sites were present mainly in neurons but also in tanycytes and ependymal cells. Positive neurons were distributed widely in various brain regions, but were particularly abundant in such regions as the lateral hypothalamus, substantia nigra, locus coeruleus and raphe nuclei. The present study suggests that fibroblast growth factor receptor-1 is expressed preferentially in certain neuronal systems that appear to be under the influence of fibroblast growth factors in the normal brain. The result should facilitate study of the functional significance of fibroblast growth factors in these brain neurons.
  • W. G. Ding, I. Tooyama, H. Kitasato, M. Fujimura, H. Kimura
    The Histochemical Journal 26 5 453 - 459 1994年05月 [無し][無し]
     研究論文（学術雑誌） 
    The distribution of neuropeptide Y was investigated by light and electron microscopic immunohistochemistry in the liver of various vertebrates including the eel, carp, bullfrog, turtle, chicken, mouse, rat, guinea-pig, dog, monkey and human. The ontogenetic development of neuropeptide Y was also studied in the mouse liver. In all species examined except the eel, neuropeptide Y-like immunoreactivity was detected in nerve fibres. In the carp, bullfrog, turtle, chicken, mouse and rat, positive fibres were distributed around the wall of hepatic vessels and the bile duct of the Glisson's sheath. The density of the positive fibres increased with evolution. On the other hand, in the guinea-pig, dog, monkey and human, numerous neuropeptide Y-positive fibres were observed not only in the Glisson's sheath but also in the liver parenchyma. Positive fibres formed a dense network to surround hepatocytes. The present immunoelectron microscopic study has confirmed that neuropeptide Y-positive terminals are closely apposing to hepatocytes. Ontogenetically, neuropeptide Y-positive fibres were first found in embryonic liver of 19-day-old mice. Positive fibres increased with age and the highest peak was seen one week after birth. This ontogenetic pattern has suggested that neuropeptide Y plays a certain role in developing liver. © 1994 Chapman & Hall.
  • WG DING, TOOYAMA, I, H KITASATO, M FUJIMURA, H KIMURA
    HISTOCHEMICAL JOURNAL 26 5 453 - 459 1994年05月 [無し][無し]
     研究論文（学術雑誌） 
    The distribution of neuropeptide Y was investigated by light and electron microscopic immunohistochemistry in the liver of various vertebrates including the eel, carp, bullfrog, turtle, chicken, mouse, rat, guinea-pig, dog, monkey and human. The ontogenetic development of neuropeptide Y was also studied in the mouse liver. In all species examined except the eel, neuropeptide Y-like immunoreactivity was detected in nerve fibres. In the carp, bullfrog, turtle, chicken, mouse and rat, positive fibres were distributed around the wall of hepatic vessels and the bile duct of the Glisson's sheath. The density of the positive fibres increased with evolution. On the other hand, in the guinea-pig, dog, monkey and human, numerous neuropeptide Y-positive fibres were observed not only in the Glisson's sheath but also in the liver parenchyma. Positive fibres formed a dense network to surround hepatocytes. The present immunoelectron microscopic study has confirmed that neuropeptide Y-positive terminals are closely apposing to hepatocytes. Ontogenetically, neuropeptide Y-positive fibres were first found in embryonic liver of 19-day-old mice. Positive fibres increased with age and the highest peak was seen one week after birth. This ontogenetic pattern has suggested that neuropeptide Y plays a certain role in developing liver.
  • T ARAI, K WATANABE, S NAKAO, H MORI, M MURAKAWA, K MORI, TOOYAMA, I, H KIMURA, S KOJIMA
    NEUROSCIENCE LETTERS 173 1-2 107 - 110 1994年05月 [無し][無し]
     研究論文（学術雑誌） 
    The effects of neopterin on ischemic neuronal damage were examined. Cerebral ischemia was produced in the gerbil by bilateral common carotid occlusion for 8 min or unilateral occlusion for 30 min. which resulted in delayed neuronal death in the CA1 region of the hippocampus. However, preischemic treatment with neopterin (3 mg/kg i.p.) markedly reduced hippocampal neuronal damages in both cases, Since neopterin serves both as an antioxidant and as an oxidant depending on its redox state, these findings indicate that neopterin attenuates the ischemic neuronal injury by scavenging oxygen free radicals and/or by inhibiting their generation.
  • MOROO, I, T YAMADA, H MAKINO, TOOYAMA, I, PL MCGEER, EG MCGEER, K HIRAYAMA
    ACTA NEUROPATHOLOGICA 87 4 343 - 348 1994年04月 [無し][無し]
     研究論文（学術雑誌） 
    Immunohistochemistry using both a newly developed polyclonal, and a commercially available monoclonal, anti-insulin receptor antibody was done on the midbrain from cases of idiopathic Parkinson's disease (PD), Alzheimer's disease, amyotrophic lateral sclerosis, vascular parkinsonism and non-neurological controls. Both antibodies gave identical patterns of neuronal staining. The neurons of the oculomotor nucleus were immunopositive in all the brains. However, the neurons in the pars compacta of the substantia nigra, paranigral nucleus, parabrachial pigmental nucleus, tegmental pedunculopontine nucleus, supratrocheal nucleus, cuneiform nucleus, subcuneiform nucleus and lemniscus medialis, which were positive in other diseases and in non-neurological controls, were not stained by these antibodies in PD brains. These results suggest that, in PD, a dysfunction of the insulin/insulin receptor system may precede death of the dopaminergic neurons.
  • MOROO, I, T YAMADA, H MAKINO, TOOYAMA, I, PL MCGEER, EG MCGEER, K HIRAYAMA
    ACTA NEUROPATHOLOGICA 87 4 343 - 348 1994年04月 [無し][無し]
     研究論文（学術雑誌） 
    Immunohistochemistry using both a newly developed polyclonal, and a commercially available monoclonal, anti-insulin receptor antibody was done on the midbrain from cases of idiopathic Parkinson's disease (PD), Alzheimer's disease, amyotrophic lateral sclerosis, vascular parkinsonism and non-neurological controls. Both antibodies gave identical patterns of neuronal staining. The neurons of the oculomotor nucleus were immunopositive in all the brains. However, the neurons in the pars compacta of the substantia nigra, paranigral nucleus, parabrachial pigmental nucleus, tegmental pedunculopontine nucleus, supratrocheal nucleus, cuneiform nucleus, subcuneiform nucleus and lemniscus medialis, which were positive in other diseases and in non-neurological controls, were not stained by these antibodies in PD brains. These results suggest that, in PD, a dysfunction of the insulin/insulin receptor system may precede death of the dopaminergic neurons.
  • T TANIGUCHI, R FUKUNAGA, Y MATSUOKA, K TERAI, TOOYAMA, I, H KIMURA
    BRAIN RESEARCH 640 1-2 119 - 125 1994年03月 [有り][無し]
     研究論文（学術雑誌） 
    The time course of c-fos protein expression after hypoxia was examined in rat hippocampus and cerebral cortex using an immunohistochemical method. The rats were exposed to in vivo hypoxia for 30 min in a chamber containing 5% O2 and 95% N2. Immediately after the treatment, c-fos protein-like immunoreactivity was observed in the granule cell layer of the dentate gyrus. The change was transient, and the density of immunoreactive cells returned quickly to a control level 3 h after the exposure. However, the density of positive cells was again increased 1 day after hypoxia and reached the maximum 7 days after. In the cerebral cortex, on the other hand, no change was detected in the pattern of staining at any time, with an exception on 21 days after hypoxia. At this period, positively stained neurons were significantly increased in both density and intensity throughout the entire extent of the cerebral cortex including the cingulate gyrus. These results clearly indicate that hypoxia induces different patterns of c-fos protein expression among various regions of the brain. The biphasic pattern seen in the dentate gyrus as well as the delayed expression in the cerebral cortex may be related to delayed neuronal damages induced by hypoxia.
  • Y OOMURA, K SASAKI, A LI, H KIMURA, TOOYAMA, I, K HANAI, Y NOMURA, Y KITAMURA, N YANAIHARA, H YAGO
    SAM MODEL OF SENESCENCE 1062 415 - 418 1994年 [有り][無し]
     研究論文（国際会議プロシーディングス）
  • T KAWAMATA, S NAKAMURA, AKIGUCHI, I, TOOYAMA, I, N SERIU, M TAKEMURA, J KIMURA, H KIMURA, EG MCGEER, PL MCGEER, K HIGUCHI, M HOSOKAWA, T TAKEDA
    SAM MODEL OF SENESCENCE 1062 347 - 350 1994年 [有り][無し]
     研究論文（国際会議プロシーディングス）
  • Effects of neopterin on ischemic neuronal damage in gerbils
    Arai T, Watanabe K, Nakao S, Mori H, Murakawa M, Mori M, Tooyama I, Kimura H, Kojima S
    Neuroscience Letters 166 131 - 134 1994年 [有り][無し]
     研究論文（学術雑誌）
  • Neuropeptide Y-containing nerves in the mammalian liver and liver disease
    Ding W-G, Kitasato H, Fujimura M, Tooyama I, Kimura H
    Biomed.Res 15(supple 2) 283 - 287 1994年 [無し][無し]
     研究論文（学術雑誌）
  • Immunohistochemical localization of taurine-conjugated bile acids in the liver of mouse, rat, monkey and human
    Ding WG, Tooyama I, Kimura H, Kuriyama K
    Adv. Exp. Med. Biol 359 91 - 98 1994年 [無し][無し]
     研究論文（学術雑誌）
  • Kimura H, Tooyama I, McGeer PL
    Tohoku J. Exp. Med 174 279 - 293 1994年 [無し][無し]
     研究論文（学術雑誌）
  • Acidic fibroblast growth factor-like immunoreactivity in rat brain following cerebral infarction
    Hara Y, Tooyama I, Yasuhara O, Akiyama H, McGeer PL, Hande J, Kimura H
    Brain Res 664 101 - 107 1994年 [無し][無し]
     研究論文（学術雑誌）
  • Tooyama I, Kawamata T, Kimura H, McGeer PL, McGeer EG
    Brain Res 656 165 - 168 1994年 [無し][無し]
     研究論文（学術雑誌）
  • Effects of neopterin on ischemic neuronal damage in gerbils
    Arai T, Watanabe K, Nakao S, Mori H, Mori K, Tooyama I, Kimura H, Kojima S
    Neurosci. Lett 173 107 - 110 1994年 [無し][無し]
     研究論文（学術雑誌）
  • H ABE, TOOYAMA, I, T RENDA, ERSPAMER, V, H KIMURA
    PEPTIDES 15 1 49 - 54 1994年01月 [無し][無し]
     研究論文（学術雑誌） 
    In an immunohistochemical study, a specific antiserum raised against [D-Ala(2)]deltorphin-I (DADTI), a highly selective ligand for delta opioid receptors, was used to demonstrate immunoreactive structures in the rat retina. [D-Ala(2)]Deltorphin-I immunoreactivity occurred in a subpopulation of the retinal amacrine cells situated in the inner nuclear layer. Their stained professes were mainly distributed in the sublaminae 1 and 3 of the inner plexiform layer. A few positive cells, probably displaced amacrine cells, were also seen in the ganglion cell layer. Double immunostaining revealed that 12.8% of DADTI-immunoreactive cells costored GABA and 27.7% costored neuropeptide Y, whereas only few DADTI-positive cells colocalized tyrosine hydroxylase (0.3%) and almost no other peptides. These findings suggest that some retinal amacrine cells possess DADTI-like molecule(s), possibly acting as neurotransmitter(s) or neuromodulator(s).
  • H AKIYAMA, TOOYAMA, I, H KONDO, K IKEDA, H KIMURA, EG MCGEER, PL MCGEER
    BRAIN RESEARCH 635 1-2 257 - 268 1994年01月 [無し][無し]
     研究論文（学術雑誌） 
    We investigated the early response of microglia with complement and other proteins in well controlled rat central nervous system lesions. A selective neuronal degeneration in the hippocampal CA3 region was induced without direct tissue damage by an intraventricular injection of a small amount of kainic acid. As early as 1 h post injection, complement proteins Clq, C4, and C3 and immunoglobulin(Ig)G were found in the lesioned area. After 2 h, non-specific leakage of other plasma proteins occurred. By 3 h, reactive microglia gathered around the injured pyramidal neurons. Areas surrounding the lesions were depleted, on the other hand, indicating that these reactive microglia had originally resided in and migrated from such vacant areas. Upregulation of ICAM-1 expression by vascular endothelial cells commenced after 6 h. LFA-1-positive leucocytes were, then, accumulated in the vasculature, which was followed by an infiltration of leucocytes into the lesioned brain parenchyma. These results indicate that, following an acute neuronal injury, the response of the humoral factors such as complement proteins and IgG precedes the microglial reaction. Activation of vascular endothelial cells and subsequent infiltration of blood leucocytes occurs much later than the activation and migration of brain resident microglia. The origin of complement proteins and IgG in the lesioned brain parenchyma remains to be determined, although the production of complement proteins by microglia is suggested.
  • Neuropeptide Y-containing nerves in the mammalian liver and liver disease
    Ding W-G, Kitasato H, Fujimura M, Tooyama I, Kimura H
    Biomed.Res 15(supple 2) 283 - 287 1994年 [無し][無し]
     研究論文（学術雑誌）
  • WG DING, TOOYAMA, I, H KIMURA, K KURIYAMA
    TAURINE IN HEALTH AND DISEASE 359 91 - 98 1994年 [無し][無し]
     研究論文（国際会議プロシーディングス）
  • H ABE, TOOYAMA, I, T RENDA, ERSPAMER, V, H KIMURA
    PEPTIDES 15 1 49 - 54 1994年01月 [無し][無し]
     研究論文（学術雑誌） 
    In an immunohistochemical study, a specific antiserum raised against [D-Ala(2)]deltorphin-I (DADTI), a highly selective ligand for delta opioid receptors, was used to demonstrate immunoreactive structures in the rat retina. [D-Ala(2)]Deltorphin-I immunoreactivity occurred in a subpopulation of the retinal amacrine cells situated in the inner nuclear layer. Their stained professes were mainly distributed in the sublaminae 1 and 3 of the inner plexiform layer. A few positive cells, probably displaced amacrine cells, were also seen in the ganglion cell layer. Double immunostaining revealed that 12.8% of DADTI-immunoreactive cells costored GABA and 27.7% costored neuropeptide Y, whereas only few DADTI-positive cells colocalized tyrosine hydroxylase (0.3%) and almost no other peptides. These findings suggest that some retinal amacrine cells possess DADTI-like molecule(s), possibly acting as neurotransmitter(s) or neuromodulator(s).
  • H AKIYAMA, TOOYAMA, I, H KONDO, K IKEDA, H KIMURA, EG MCGEER, PL MCGEER
    BRAIN RESEARCH 635 1-2 257 - 268 1994年01月 [無し][無し]
     研究論文（学術雑誌） 
    We investigated the early response of microglia with complement and other proteins in well controlled rat central nervous system lesions. A selective neuronal degeneration in the hippocampal CA3 region was induced without direct tissue damage by an intraventricular injection of a small amount of kainic acid. As early as 1 h post injection, complement proteins Clq, C4, and C3 and immunoglobulin(Ig)G were found in the lesioned area. After 2 h, non-specific leakage of other plasma proteins occurred. By 3 h, reactive microglia gathered around the injured pyramidal neurons. Areas surrounding the lesions were depleted, on the other hand, indicating that these reactive microglia had originally resided in and migrated from such vacant areas. Upregulation of ICAM-1 expression by vascular endothelial cells commenced after 6 h. LFA-1-positive leucocytes were, then, accumulated in the vasculature, which was followed by an infiltration of leucocytes into the lesioned brain parenchyma. These results indicate that, following an acute neuronal injury, the response of the humoral factors such as complement proteins and IgG precedes the microglial reaction. Activation of vascular endothelial cells and subsequent infiltration of blood leucocytes occurs much later than the activation and migration of brain resident microglia. The origin of complement proteins and IgG in the lesioned brain parenchyma remains to be determined, although the production of complement proteins by microglia is suggested.
  • H AKIYAMA, TOOYAMA, I, T KAWAMATA, K IKEDA, PL MCGEER
    BRAIN RESEARCH 632 1-2 249 - 259 1993年12月 [無し][無し]
     研究論文（学術雑誌） 
    The localization of CD44 was investigated immunohistochemically in postmortem human brain tissue of control subjects and patients with Alzheimer's disease. CD44 is a multifunctional cell surface glycoprotein that serves as a receptor for hyaluronic acid, collagen types I and VI, and mucosal vascular addressin. In gray matter, it was found to be associated with some astrocytes of both protoplasmic and fibrous morphology. These positively stained astrocytes were most frequently observed in association with blood vessels, and had morphologies that were highly comparable to those described with the Golgi technique. Double immunostaining for CD44 and glial fibrillary acidic protein (GFAP) revealed that a significant number of these astrocytes were positive for both antigens. However, GFAP staining was mostly confined to the cell somata and proximal processes, while CD44 staining extended to a rich and extensive array of processes. Occasional CD44 positive cells of spherical morphology with a few thin varicose processes were observed. Their processes formed thick terminations on blood vessels, suggesting that these cells are a special class of astrocyte. In Alzheimer's disease brain, the number of CD44 positive astrocytes increased dramatically. These data suggest that astrocytes have very extensive branching patterns, which are reflected by CD44 staining patterns. CD44 may be an important adhesion molecule for these astrocytic processes.
  • H AKIYAMA, TOOYAMA, I, T KAWAMATA, K IKEDA, PL MCGEER
    BRAIN RESEARCH 632 1-2 249 - 259 1993年12月 [無し][無し]
     研究論文（学術雑誌） 
    The localization of CD44 was investigated immunohistochemically in postmortem human brain tissue of control subjects and patients with Alzheimer's disease. CD44 is a multifunctional cell surface glycoprotein that serves as a receptor for hyaluronic acid, collagen types I and VI, and mucosal vascular addressin. In gray matter, it was found to be associated with some astrocytes of both protoplasmic and fibrous morphology. These positively stained astrocytes were most frequently observed in association with blood vessels, and had morphologies that were highly comparable to those described with the Golgi technique. Double immunostaining for CD44 and glial fibrillary acidic protein (GFAP) revealed that a significant number of these astrocytes were positive for both antigens. However, GFAP staining was mostly confined to the cell somata and proximal processes, while CD44 staining extended to a rich and extensive array of processes. Occasional CD44 positive cells of spherical morphology with a few thin varicose processes were observed. Their processes formed thick terminations on blood vessels, suggesting that these cells are a special class of astrocyte. In Alzheimer's disease brain, the number of CD44 positive astrocytes increased dramatically. These data suggest that astrocytes have very extensive branching patterns, which are reflected by CD44 staining patterns. CD44 may be an important adhesion molecule for these astrocytic processes.
  • Ikuo Tooyama, Hiromichi Abe, Tindaro Renda, Vittorio Erspamer, Hiroshi Kimura
    Proceedings of the National Academy of Sciences of the United States of America 90 20 9635 - 9639 1993年10月 [無し][無し]
     研究論文（学術雑誌） 
    Using a specific antiserum recently raised against [D-Ala2]deltorphin I (DADTI: Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2), a highly selective ligand for δ-opioid receptors, we have previously demonstrated the occurrence of positive immunostaining in several structures of mouse brain. We describe here the neuroanatomical distribution patterns of DADTI-immunoreactive neuronal bodies, axons, and tanycytes in rat brain. Positive neuronal somata were localized mainly in the ventral mesencephalon, including the ventral tegmental area and the pars compacta of the substantia nigra. A minor population of positive somata was found in the pars reticulate and pars lateralis of the substantia nigra, raphe nuclei, supramammillary nucleus, and retrorubral reticular nucleus. All these regions, except for the supramammillary nucleus, contain dopamine cell bodies. Intensely stained positive nerve fibers could be traced along the medial forebrain bundle. Dense positive terminals were seen in the neostriatum, nucleus accumbens shell, olfactory tubercle, septal areas, cingulate, and medial prefrontal cortex. Double-immunostaining study revealed that, in the substantia nigra, almost all (97.8%) DADTI-positive neurons colocalized with tyrosine hydroxylase (TH), and the doubly stained cells occupied about one-third (29.1%) of the total population of TH-positive neurons. Only a few DADTI/TH-positive cells also stained for 28-kDa calbindin D, although many neurons double-stained for 28-kDa calbindin D and TH. In contrast, the supramammillary nucleus contained a number of DADTI-positive cells, which nearly always stained positively for 28-kDa calbindin D but did not stain for TH. The association of DADTI-like immunoreactivity with certain dopaminergic pathways seems of particular interest. A small population of DADTI-immunostained tanycytes was present in the ventral part of the third ventricle wall.
  • TOOYAMA, I, H ABE, T RENDA, ERSPAMER, V, H KIMURA
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 90 20 9635 - 9639 1993年10月 [無し][無し]
     研究論文（学術雑誌） 
    Using a specific antiserum recently raised against [D-Ala2]deltorphin I (DADTI: Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2), a highly selective ligand for delta-opioid receptors, we have previously demonstrated the occurrence of positive immunostaining in several structures of mouse brain. We describe here the neuroanatomical distribution patterns of DADTI-immunoreactive neuronal bodies, axons, and tanycytes in rat brain. Positive neuronal somata were localized mainly in the ventral mesencephalon, including the ventral tegmental area and the pars compacta of the substantia nigra. A minor population of positive somata was found in the pars reticulata and pars lateralis of the substantia nigra, raphe nuclei, supramammillary nucleus, and retrorubral reticular nucleus. All these regions, except for the supramammillary nucleus, contain dopamine cell bodies. Intensely stained positive nerve fibers could be traced along the medial forebrain bundle. Dense positive terminals were seen in the neostriatum, nucleus accumbens shell, olfactory tubercle, septal areas, cingulate, and medial prefrontal cortex. Double-immunostaining study revealed that, in the substantia nigra, almost all (97.8%) DADTI-positive neurons colocalized with tyrosine hydroxylase (TH), and the doubly stained cells occupied about one-third (29.1 %) of the total population of TH-positive neurons. Only a few DADTI/TH-positive cells also stained for 28-kDa calbindin D, although many neurons double-stained for 28-kDa calbindin D and TH. In contrast, the supramammillary nucleus contained a number of DADTI-positive cells, which nearly always stained positively for 28-kDa calbindin D but did not stain for TH. The association of DADTI-like immunoreactivity with certain dopaminergic pathways seems of particular interest. A small population of DADTI-immunostained tanycytes was present in the ventral part of the third ventricle wall.
  • T RENDA, L NEGRI, TOOYAMA, I, C CASU, P MELCHIORRI
    NEUROREPORT 4 10 1143 - 1146 1993年09月 [無し][無し]
     研究論文（学術雑誌） 
    PREVIOUS biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a H-3-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.
  • T RENDA, L NEGRI, TOOYAMA, I, C CASU, P MELCHIORRI
    NEUROREPORT 4 10 1143 - 1146 1993年09月 [無し][無し]
     研究論文（学術雑誌） 
    PREVIOUS biochemical and pharmacological studies have shown that [D-Ala2]-deltorphin-I (DADTI) has a high affinity and selectivity for delta-opioid receptors. In this study, designed to provide morphological details, the distribution of DADTI binding sites was examined by autoradiography on coronal, sagittal and horizontal frozen sections of adult rat brain. The sections were incubated with tritiated DADTI solution and exposed for 12 weeks to a H-3-sensitive film. DADTI labelling clearly demonstrated selective and high affinity binding sites of delta-opioid type in several brain regions, including olfactory system, neostriatum, nucleus accumbens, and cortical layers I-II and V-VI.
  • BJ SNOW, TOOYAMA, I, EG MCGEER, T YAMADA, DB CALNE, H TAKAHASHI, H KIMURA
    ANNALS OF NEUROLOGY 34 3 324 - 330 1993年09月 [無し][無し]
     研究論文（学術雑誌） 
    Postmortem counts of dopaminergic cell densities in the substantia nigra (5 subjects) and striatal levels of dopamine (DA) and its metabolites (6 subjects) were determined on 1 parkinsonian (PD), 3 progressive supranuclear palsy (PSP), 1 amyotrophic lateral sclerosis, and 1 Alzheimer's case who had been positron emission tomography scanned with 6-[F-18]fluorodopa during life. [F-18]Fluorodopa uptake rate constants, which presumably depend on the number of functioning striatal DA terminals, were strictly proportional to cell densities (significant correlation with zero intercept) and also correlated significantly with striatal DA levels but with an intercept indicating greater losses of DA than of terminals in PSP and PD. Postmortem data on 6 PD, 1 PSP, and 9 neuronally normal controls substantiated the significant correlation between cell counts and DA levels, with the latter being the more depressed in pathological cases.
  • H AKIYAMA, T KAWAMATA, T YAMADA, TOOYAMA, I, T ISHII, PL MCGEER
    ACTA NEUROPATHOLOGICA 85 6 628 - 634 1993年05月 [無し][無し]
     研究論文（学術雑誌） 
    Intercellular adhesion molecule-1 (ICAM-1) was localized immunohistochemically in postmortem brain tissue of Alzheimer's disease (AD), progressive supranuclear palsy, amyotrophic lateral sclerosis, Pick's disease. and controls. In controls, only capillaries were stained for ICAM-1. In affected areas of neurologically diseased brains, a subset of reactive astrocytes was also strongly stained. In addition, there were irregular, diffuse patches of positive staining in the tissue matrix. In AD. many of these patches had dense cores which corresponded with senile plaques. Double immunostaining for glial fibrillary acidic protein and ICAM-1 indicated that some reactive astrocytes at the periphery of senile plaques were positive for ICAM-1. Within such plaques, microglial aggregates were stained intensely for leukocyte function-associated antigen-1 (LFA-t), the adhesion molecule for ICAM-1. The LFA-1/ICAM-1 system appears to play an important role in the interaction of astrocytes and microglia in several neurological diseases.
  • T KAWAMATA, TOOYAMA, I, T YAMADA, DG WALKER, PL MCGEER
    AMERICAN JOURNAL OF PATHOLOGY 142 5 1574 - 1585 1993年05月 [無し][無し]
     研究論文（学術雑誌） 
    Lactotransferrin (LF) expression was investigated immunocytochemically in postmortem brain tissues of normal controls and patients with Alzheimer's disease (AD). The antibody to LF stained some neurons weakly in young adult brains, but it stained many neurons as well as the glia of all types in elderly brains. LF expression was greatly up-regulated in both neurons and glia in affected AD tissue. It was very strongly associated with such extracellular pathological entities as diffuse and consolidated amyloid deposits and extracellular neurofibrillary tangles. In addition, it was identified in a minority of intracellular neurofibrillary tangles, neuropil threads, and degenerative neurites. LF is an iron scavenger and a complement inhibitor. Up-regulation may be a defense mechanism in AD-affected brain tissue.
  • H AKIYAMA, T KAWAMATA, T YAMADA, TOOYAMA, I, T ISHII, PL MCGEER
    ACTA NEUROPATHOLOGICA 85 6 628 - 634 1993年05月 [無し][無し]
     研究論文（学術雑誌） 
    Intercellular adhesion molecule-1 (ICAM-1) was localized immunohistochemically in postmortem brain tissue of Alzheimer's disease (AD), progressive supranuclear palsy, amyotrophic lateral sclerosis, Pick's disease. and controls. In controls, only capillaries were stained for ICAM-1. In affected areas of neurologically diseased brains, a subset of reactive astrocytes was also strongly stained. In addition, there were irregular, diffuse patches of positive staining in the tissue matrix. In AD. many of these patches had dense cores which corresponded with senile plaques. Double immunostaining for glial fibrillary acidic protein and ICAM-1 indicated that some reactive astrocytes at the periphery of senile plaques were positive for ICAM-1. Within such plaques, microglial aggregates were stained intensely for leukocyte function-associated antigen-1 (LFA-t), the adhesion molecule for ICAM-1. The LFA-1/ICAM-1 system appears to play an important role in the interaction of astrocytes and microglia in several neurological diseases.
  • T KAWAMATA, TOOYAMA, I, T YAMADA, DG WALKER, PL MCGEER
    AMERICAN JOURNAL OF PATHOLOGY 142 5 1574 - 1585 1993年05月 [無し][無し]
     研究論文（学術雑誌） 
    Lactotransferrin (LF) expression was investigated immunocytochemically in postmortem brain tissues of normal controls and patients with Alzheimer's disease (AD). The antibody to LF stained some neurons weakly in young adult brains, but it stained many neurons as well as the glia of all types in elderly brains. LF expression was greatly up-regulated in both neurons and glia in affected AD tissue. It was very strongly associated with such extracellular pathological entities as diffuse and consolidated amyloid deposits and extracellular neurofibrillary tangles. In addition, it was identified in a minority of intracellular neurofibrillary tangles, neuropil threads, and degenerative neurites. LF is an iron scavenger and a complement inhibitor. Up-regulation may be a defense mechanism in AD-affected brain tissue.
  • W. G. Ding, I. Tooyama, H. Kimura, K. Kuriyama, J. Ochi
    The Histochemical Journal 25 5 376 - 383 1993年05月 [無し][無し]
     研究論文（学術雑誌） 
    The ontogenic pattern of development of taurine-like immunoreactivity (TLI) was studied in the mouse liver. The effect on adult mice of carbon tetrachloride or phenobarbital treatment was also examined. Light-microscopically, granules of TLI were first found in the liver from 17-day-old embryos, diffusely distributed throughout the lobules. These positive granules increased with age, were most numerous in the two-week-old mouse, and were notably decreased in the central region of some lobules in the three-week-old mouse. In mature mice, hepatocytes containing TLI-positive granules were distributed unevenly in each liver lobule, and were located predominantly in the peripheral region. Electron-microscopically, TLI was observed in small vesicles in the cytoplasm of hepatocytes and was found mainly in the cisternal lumen of smooth-surfaced endoplasmic reticulum. Some taurine-positive vesicles surrounding the reticulum seemed to associate with the protoplasm. Similar positive vesicles were often located near the bile canaliculi. In carbon tetrachloride-intoxicated mature mice, TLI was no longer limited to the peripheral region of lobules hepatocytes situated in the central region of lobules also contained intense TLI. In mice injected with a small and repeated dose of phenobarbital, the distribution pattern of TLI was similar to that in the untreated group. However, in mice injected with a large dose of phenobarbital, TLI was markedly increased, especially in the central region of lobules. The results demonstrate that the distribution pattern of TLI in mouse liver changes during development, and that the pattern in mature mice is affected by intoxication with carbon tetrachloride or a toxic dose of phenobarbital. © 1993 Chapman & Hall.
  • TOOYAMA, I, HPH KREMER, MR HAYDEN, H KIMURA, EG MCGEER, PL MCGEER
    BRAIN RESEARCH 610 1 1 - 7 1993年04月 [無し][無し]
     研究論文（学術雑誌） 
    The immunohistochemical localizations of acidic and basic fibroblast growth factor (aFGF and bFGF) were investigated in the striatum and midbrain of Huntington's disease (HD) and control cases using specific antibodies. In the striatum of control cases, the ependymal cell layer was stained for aFGF and bFGF. In addition, a few subependymal astrocytes were positive for aFGF, and some neurons stained weakly for bFGF. In HD striatum, many astrocytes and remaining neurons were strongly stained for aFGF. aFGF-positive astrocytes were particularly conspicuous in the subependymal region of the caudate but appeared throughout the caudate and putamen. The number of bFGF-positive astrocytes was slightly increased. In contrast to the caudate/putamen, the globus pallidus, nucleus of the oculomotor nerve and substantia nigra showed very similar patterns for both aFGF and bFGF in control and most HD brains. Reports that FGF can protect against glutamate neurotoxicity, and that the FGF receptor (FGFR3), with its gene located in the HD region on chromosome 4, appears in striatal neurons. make it tempting to speculate on a possibly important role for FGF-FGFR3 interactions in HD pathology.
  • TOOYAMA, I, HPH KREMER, MR HAYDEN, H KIMURA, EG MCGEER, PL MCGEER
    BRAIN RESEARCH 610 1 1 - 7 1993年04月 [無し][無し]
     研究論文（学術雑誌） 
    The immunohistochemical localizations of acidic and basic fibroblast growth factor (aFGF and bFGF) were investigated in the striatum and midbrain of Huntington's disease (HD) and control cases using specific antibodies. In the striatum of control cases, the ependymal cell layer was stained for aFGF and bFGF. In addition, a few subependymal astrocytes were positive for aFGF, and some neurons stained weakly for bFGF. In HD striatum, many astrocytes and remaining neurons were strongly stained for aFGF. aFGF-positive astrocytes were particularly conspicuous in the subependymal region of the caudate but appeared throughout the caudate and putamen. The number of bFGF-positive astrocytes was slightly increased. In contrast to the caudate/putamen, the globus pallidus, nucleus of the oculomotor nerve and substantia nigra showed very similar patterns for both aFGF and bFGF in control and most HD brains. Reports that FGF can protect against glutamate neurotoxicity, and that the FGF receptor (FGFR3), with its gene located in the HD region on chromosome 4, appears in striatal neurons. make it tempting to speculate on a possibly important role for FGF-FGFR3 interactions in HD pathology.
  • Shin-ichi Nakao, Toshiyuki Arai, Kenjiro Mori, Osamu Yasuhara, Ikuo Tooyama, Hiroshi Kimura
    Neuroscience Letters 151 1 33 - 36 1993年03月 [無し][無し]
     研究論文（学術雑誌） 
    The effects of high-dose ketamine on the c-fos protein (c-Fos) expression were investigated in rat by an immunohistochemical technique. The administration of 100 mg/kg ketamine i.p. induced seizure-like activity (limbic seizure). No c-Fos immunoreactivity was observed in hippocampus, piriform cortex and amygdala, while it was observed in neocortex and thalamus. These findings disagree with the reports that ketamine depresses the neuronal function of the neocortex and thalamus, while it stimulates the limbic system. © 1993.
  • H AKIYAMA, T YAMADA, PL MCGEER, T KAWAMATA, TOOYAMA, I, T ISHII
    ACTA NEUROPATHOLOGICA 85 4 400 - 403 1993年03月 [有り][無し]
     研究論文（学術雑誌） 
    The spatial pattern of beta-amyloid protein (BAP) deposits in Alzheimer's disease cerebral cortex was investigated. In cortical areas where the accumulation of BAP was relatively sparse, the deposits tended to accumulate vertically in a columnar arrangement. Typically, these aggregates consisted of both consolidated and diffuse deposits approximately 200 to 600 mum in width. Blood vessels running perpendicularly to the pial surface were sometimes observed penetrating the center of these columns, but this was not a consistent finding. These BAP extracellular aggregates might be related to the columnar organization of the cerebral cortex.
  • TOOYAMA, I, T KAWAMATA, D WALKER, T YAMADA, K HANAI, H KIMURA, M IWANE, K IGARASHI, EG MCGEER, PL MCGEER
    NEUROLOGY 43 2 372 - 376 1993年02月 [無し][無し]
     研究論文（学術雑誌） 
    Basic fibroblast growth factor (bFGF) has a neurotrophic effect on mesencephalic dopaminergic neurons in vitro and in vivo. To explore whether an abnormality in bFGF expression occurs in Parkinson's disease (PD), we examined the substantia nigra (SN) of six PD and eight control cases immunohistochemically using a monoclonal antibody to bFGF. The mean number of melanin-positive neurons in sections of PD SN was 30.3% of the control mean, but the number of bFGF-immunopositive neurons was only 4.7% of the control mean. bFGF-immunoreactivity was present in only 8.2% of PD, but in 93.7% of control melanin-positive neurons. These results suggest a profound depletion of bFGF in surviving dopaminergic neurons of the SN in PD, and this depletion may be related to the disease process.
  • TOOYAMA, I, T KAWAMATA, D WALKER, T YAMADA, K HANAI, H KIMURA, M IWANE, K IGARASHI, EG MCGEER, PL MCGEER
    NEUROLOGY 43 2 372 - 376 1993年02月 [無し][無し]
     研究論文（学術雑誌） 
    Basic fibroblast growth factor (bFGF) has a neurotrophic effect on mesencephalic dopaminergic neurons in vitro and in vivo. To explore whether an abnormality in bFGF expression occurs in Parkinson's disease (PD), we examined the substantia nigra (SN) of six PD and eight control cases immunohistochemically using a monoclonal antibody to bFGF. The mean number of melanin-positive neurons in sections of PD SN was 30.3% of the control mean, but the number of bFGF-immunopositive neurons was only 4.7% of the control mean. bFGF-immunoreactivity was present in only 8.2% of PD, but in 93.7% of control melanin-positive neurons. These results suggest a profound depletion of bFGF in surviving dopaminergic neurons of the SN in PD, and this depletion may be related to the disease process.
  • I. Tooyama
    Clinical Neurology 33 12 1270 - 1274 1993年 [有り][無し]
     研究論文（国際会議プロシーディングス） 
    Fibroblast growth factors (FGFs) are a family consisting of at least seven members, and the FGF family will continue to expand. The best studied members of this family are acidic and basic FGF (aFGF and bFGF). They are richly concentrated in brain, and have potent trophic effects for neurons and glia. I now report that expressions of aFGF and bFGF are altered in several neurodegenerative disorders such as Alzheimer disease (AD), Huntington disease (HD) and Parkinson disease (PD). In AD aFGF was upregulated in reactive astrocytes in severely affected areas. The bFGF-positive astrocytes were also increased. In addition, bFGF was detected in senile plaques, neurofibrillary tangles and neuropil threads. In HD, aFGF and bFGF showed a similar alteration pattern. A large number of aFGF- or bFGF-positive astrocytes were observed in the striatum. Some remaining neurons were strongly stained for aFGF or bFGF. In PD, marked loss of bFGF was observed on midbrain dopaminergic neurons. The depletion of bFGF preceded the loss of tyrosine hydroxylase. Since bFGF has a potent trophic effect on midbrain dopaminergic neurons, this depletion may be related to the disease process.
  • PL MCGEER, T KAWAMATA, DG WALKER, H AKIYAMA, TOOYAMA, I, EG MCGEER
    GLIA 7 1 84 - 92 1993年01月 [無し][無し]
     研究論文（学術雑誌） 
    Microglia express many leukocyte surface antigens which are upregulated in such chronic degenerative neurological diseases as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). These surface antigens include leukocyte common antigen, immunoglobulin Fc receptors, MHC class I and class II glycoproteins, beta2-integrins, and the vitronectin receptor. Ligands for these receptors are also found. They include immunoglobulins, complement proteins of the classical pathway, T lymphocytes of the cytotoxic/suppressor and helper/inducer classes, and vitronectin. T lymphocytes marginate along capillary venules, with some penetrating into the tissue matrix. Immunoglobulins and complement proteins are synthesized locally in brain, although they may also come from the bloodstream if the blood-brain barrier is compromised. The membrane attack complex, which is formed from C5b-9, the terminal components of complement, has been identified in AD and multiple sclerosis brain tissue. In addition, proteins designed to defend against bystander lysis caused by the membrane attack complex, including protectin, C8 binding protein, clusterin, and vitronectin, are associated with damaged neuronal processes in AD. Autodestruction may play a prominent part in these 2 diseases.
  • Columnar arrangement of
    Akiyama H, Yamada T, McGeer PL, Kawamata T, Tooyama I, Ishii T
    Acta Nueropathol. 85 400 - 403 1993年 [無し][無し]
     研究論文（学術雑誌）
  • TOOYAMA, I, T KAWAMATA, H AKIYAMA, SK MOESTRUP, J GLIEMANN, PL MCGEER
    MOLECULAR AND CHEMICAL NEUROPATHOLOGY 18 1-2 153 - 160 1993年01月 [無し][無し]
     研究論文（学術雑誌） 
    Localization of the alpha2, macroglobulin receptor (alpha2MR) was studied in postmortem human brain tissue of Alzheimer disease (AD) and age-matched control cases with a monoclonal antibody (A2MRalpha2) to the receptor. In control cases alpha2MR was detected in neurons, glia, and some capillaries. Neuronal staining was most conspicuous in the hippocampus and entorhinal cortex. In AD, alpha2MR immunoreactivity was enhanced. The staining intensity of some neurons was increased, as was the number of positive glial cells. In addition, senile plaques, tangles, and dystrophic neurites were strongly stained. These results suggest that alpha2MR is involved in AD pathology.
  • Phylogenetic Study of Neuropeptide Y-Containing Nerves in the Liver.
    Fujimura M, Ding WG, Kitasato H, Tooyama I, Kimura H
    The International Conference on Gut Hormone (GUT HORMONE 1993年 [無し][無し]
  • Human positron emission tomographic [18F]fluorodopa studies correlate with dopamine cell counts and levels
    Snow BJ, Tooyama I, Mcgeer EG, Yamada T, Calne DB, Takahashi H, Kimura H
    Ann. Neurol 34 324 - 330 1993年 [無し][無し]
     研究論文（学術雑誌）
  • High-dose ketamine does not induce c-Fos protein expression in rat hippocampus
    Nakao S, Arai T, Mori K, Yasuhara O, Tooyama I, Kimura H
    Neurosci. Lett 151 33 - 36 1993年 [無し][無し]
     研究論文（学術雑誌）
  • Distribution of taurine-like immunoreactivity in the mouse liver during ontogeny and after carbon tetrachloride or phenobarbital intoxication.
    Ding WG, Tooyama I, Kimura H, Kuriyama K, Ochi J
    The Histochemical Journal 25 376 - 383 1993年 [無し][無し]
     研究論文（学術雑誌）
  • PL MCGEER, T KAWAMATA, DG WALKER, H AKIYAMA, TOOYAMA, I, EG MCGEER
    GLIA 7 1 84 - 92 1993年01月 [無し][無し]
     研究論文（学術雑誌） 
    Microglia express many leukocyte surface antigens which are upregulated in such chronic degenerative neurological diseases as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). These surface antigens include leukocyte common antigen, immunoglobulin Fc receptors, MHC class I and class II glycoproteins, beta2-integrins, and the vitronectin receptor. Ligands for these receptors are also found. They include immunoglobulins, complement proteins of the classical pathway, T lymphocytes of the cytotoxic/suppressor and helper/inducer classes, and vitronectin. T lymphocytes marginate along capillary venules, with some penetrating into the tissue matrix. Immunoglobulins and complement proteins are synthesized locally in brain, although they may also come from the bloodstream if the blood-brain barrier is compromised. The membrane attack complex, which is formed from C5b-9, the terminal components of complement, has been identified in AD and multiple sclerosis brain tissue. In addition, proteins designed to defend against bystander lysis caused by the membrane attack complex, including protectin, C8 binding protein, clusterin, and vitronectin, are associated with damaged neuronal processes in AD. Autodestruction may play a prominent part in these 2 diseases.
  • Columnar arrangement of
    Akiyama H, Yamada T, McGeer PL, Kawamata T, Tooyama I, Ishii T
    Acta Nueropathol. 85 400 - 403 1993年 [無し][無し]
     研究論文（学術雑誌）
  • TOOYAMA, I, T KAWAMATA, H AKIYAMA, SK MOESTRUP, J GLIEMANN, PL MCGEER
    MOLECULAR AND CHEMICAL NEUROPATHOLOGY 18 1-2 153 - 160 1993年01月 [無し][無し]
     研究論文（学術雑誌） 
    Localization of the alpha2, macroglobulin receptor (alpha2MR) was studied in postmortem human brain tissue of Alzheimer disease (AD) and age-matched control cases with a monoclonal antibody (A2MRalpha2) to the receptor. In control cases alpha2MR was detected in neurons, glia, and some capillaries. Neuronal staining was most conspicuous in the hippocampus and entorhinal cortex. In AD, alpha2MR immunoreactivity was enhanced. The staining intensity of some neurons was increased, as was the number of positive glial cells. In addition, senile plaques, tangles, and dystrophic neurites were strongly stained. These results suggest that alpha2MR is involved in AD pathology.
  • Phylogenetic Study of Neuropeptide Y-Containing Nerves in the Liver.
    Fujimura M, Ding WG, Kitasato H, Tooyama I, Kimura H
    The International Conference on Gut Hormone (GUT HORMONE 1993年 [無し][無し]
  • I. Tooyama, D. Walker, T. Yamada, K. Hanai, H. Kimura, E. G. McGeer, P. L. McGeer
    Brain Research 593 2 274 - 280 1992年10月 [無し][無し]
     研究論文（学術雑誌） 
    A rabbit antiserum (R917) was raised to a purified fraction of bovine brain basic fibroblast growth factor (bFGF). On Western blots of rat midbrain extract, the antiserum did not recognize low molecular weight forms of bFGF. Instead, it recognized a single band of 27-28 kDa. Immunohistochemically, the antiserum preferentially stained a subpopulation of calbindin-negative mesencephalic dopaminergic neurons. The positive somata were mainly packed in a ventral portion of the tegmentum including the A10 region, the ventral tegmental area and the pars compacta of the medial substantia nigra, but were also scattered in both the pars compacta and reticulata portions of the lateral substantia nigra. Processes of dendrites and axons were clearly visible. Terminal fields were located in striosomes, the dorsolateral rim of the neostriatum, the anterodorsal aspect of the nucleus accumbens shell, the infralimbic cortex, and the medial prefrontal cortex. These results suggest that trophic specialization in subpopulations may occur in all three of these dopaminergic projection systems, i.e. the nigrostriatal, mesolimbic and mesocortical pathways. © 1992.
  • TOOYAMA, I, D WALKER, T YAMADA, K HANAI, H KIMURA, EG MCGEER, PL MCGEER
    BRAIN RESEARCH 593 2 274 - 280 1992年10月 [無し][無し]
     研究論文（学術雑誌） 
    A rabbit antiserum (R917) was raised to a purified fraction of bovine brain basic fibroblast growth factor (bFGF). On Western blots of rat midbrain extract, the antiserum did not recognize low molecular weight forms of bFGF. Instead, it recognized a single band of 27-28 kDa. Immunohistochemically, the antiserum preferentially stained a subpopulation of calbindin-negative mesencephalic dopaminergic neurons. The positive somata were mainly packed in a ventral portion of the tegmentum including the A10 region, the ventral tegmental area and the pars compacta of the medial substantia nigra, but were also scattered in both the pars compacta and reticulata portions of the lateral substantia nigra. Processes of dendrites and axons were clearly visible. Terminal fields were located in striosomes, the dorsolateral rim of the neostriatum, the anterodorsal aspect of the nucleus accumbens shell, the infralimbic cortex, and the medial prefrontal cortex. These results suggest that trophic specialization in subpopulations may occur in all three of these dopaminergic projection systems, i.e. the nigrostriatal, mesolimbic and mesocortical pathways.
  • H ABE, TOOYAMA, I, T RENDA, ERSPARMER, V, H KIMURA
    NEUROREPORT 3 8 669 - 672 1992年08月 [無し][無し]
     研究論文（学術雑誌） 
    ANTISERUM to haptenic [D-Ala2]deltorphin I (DADTI: Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2), a highly selective ligand for delta-opioid receptors, was produced in rabbits. By immunospot assay, the antiserum recognized 62.5 pmol DADTI but failed to react even with 4 nmol carrier protein of the immunogen. Although the antiserum reacted equally with an isomer [L-Ala2]deltorphin I, virtually no cross-reaction occurred with other analogues such as [D-Ala2]deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2) and similar peptides lacking the C-terminal glycine amide. Therefore, the major epitope for immunorecognition appeared to be in the C-terminal region which is known to be the specific domain for delta-opioid receptor selectivity. Immunohistochemical study using this antiserum revealed positive neuronal structures in some specific systems of the mouse brain.
  • TOOYAMA, I, T YAMADA, SU KIM, PL MCGEER
    NEUROSCIENCE LETTERS 136 1 91 - 94 1992年02月 [有り][無し]
     研究論文（学術雑誌） 
    Localization of gangliosides positively stained by the monoclonal antibody A2B5 was investigated in postmortem brain tissue of Alzheimer disease, amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP) and control cases. In control cases, A2B5-staining was granular, appearing in selective neuronal populations. In the neocortex, the A2B5-positive neurons were distributed mainly in deep cortical layers. In the cerebellum, A2B5-positive structures were detected in processes extending from the Purkinje cell layer into the molecular layer. In Alzheimer cases, many neurofibrillary tangles, neuropile threads and dystrophic neurites were strongly A2B5-positive. In addition, aggregations of A2B5-positive granules were detected in some neurons lacking neurofibrillary tangles. Alterations of A2B5-positive gangliosides were also detected in ALS and PSP cases. In ALS cases, A2B5-positive granules were aggregated in Betz cells of the precentral gyrus. In PSP cases, globose-type neurofibrillary tangles were also strongly A2B5-positive. The results indicate that A2B5-positive gangliosides are widely but selectively distributed in human brain and may be involved in several neuropathological processes.
  • Y OOMURA, K SASAKI, K SUZUKI, T MUTO, AJ LI, ZI OGITA, K HANAI, TOOYAMA, I, H KIMURA, N YANAIHARA
    AMERICAN JOURNAL OF CLINICAL NUTRITION 55 1 278 - 282 1992年01月 [無し][無し]
     研究論文（学術雑誌） 
    The concentration of fibroblast growth factor (FGF), which is found in cerebrospinal fluid (CSF), markedly increases after the start of feeding. Food intake was dose-dependently suppressed by picomole doses of FGF and facilitated by anti-FGF antibody. This suppression was caused by activation of protein kinase C in glucose-sensitive neurons in the lateral hypothalamus. In situ hybridization by use of cDNA showed that acidic (a)FGF was produced in ependymal cells. The ependymal cells released aFGF by responding to glucose increase in CSF after feeding. Released aFGF diffused into the brain parenchyma and was taken by neurons. Passive avoidance was significantly more reliable after aFGF infusion into CSF. Clamping cerebral arteries in the gerbil induced ischemia, which damaged neurons in the CA1 layer of the hippocampus. Pretreatment with aFGF prevented this damage. Thus, aFGF is not only the most potent substance yet found for the suppression of feeding, but it is also extremely effective as a neurotrophic and memory facilitating substance.
  • Production of antiserum to [D-Ala2]deltorphin I and its immunohistochemical application to the mouse brain.
    Abe H, Tooyama I, Renda T, Ersparmer V, Kimura H
    Neuroreport 3 669 - 672 1992年 [無し][無し]
     研究論文（学術雑誌）
  • Y OOMURA, K SASAKI, K SUZUKI, T MUTO, AJ LI, ZI OGITA, K HANAI, TOOYAMA, I, H KIMURA, N YANAIHARA
    AMERICAN JOURNAL OF CLINICAL NUTRITION 55 1 278 - 282 1992年01月 [無し][無し]
     研究論文（学術雑誌） 
    The concentration of fibroblast growth factor (FGF), which is found in cerebrospinal fluid (CSF), markedly increases after the start of feeding. Food intake was dose-dependently suppressed by picomole doses of FGF and facilitated by anti-FGF antibody. This suppression was caused by activation of protein kinase C in glucose-sensitive neurons in the lateral hypothalamus. In situ hybridization by use of cDNA showed that acidic (a)FGF was produced in ependymal cells. The ependymal cells released aFGF by responding to glucose increase in CSF after feeding. Released aFGF diffused into the brain parenchyma and was taken by neurons. Passive avoidance was significantly more reliable after aFGF infusion into CSF. Clamping cerebral arteries in the gerbil induced ischemia, which damaged neurons in the CA1 layer of the hippocampus. Pretreatment with aFGF prevented this damage. Thus, aFGF is not only the most potent substance yet found for the suppression of feeding, but it is also extremely effective as a neurotrophic and memory facilitating substance.
  • WG DING, M FUJIMURA, A MORI, TOOYAMA, I, H KIMURA
    GASTROENTEROLOGY 101 4 1054 - 1059 1991年10月 [有り][無し]
     研究論文（学術雑誌）
  • Yukiko Uehara-Kunugi, Kazuhiro Terai, Takashi Taniguchi, Ikuo Tooyama, Hiroshi Kimura
    Developmental Brain Research 59 2 157 - 162 1991年04月 [有り][無し]
     研究論文（学術雑誌） 
    The time course of NADPH-diaphorase expression was examined in primary cultures of rat central nervous system and in embryonic or neonatal rat brains using a histochemical method. In cerebral and brainstem cultures from 17-day-old embryonic rats, neuronal cells moderately expressing NADPH-diaphorase were first detected on about the 5th to 7th day of culturing. Both the density of positive cells and the staining intensity increased with age of cultures. The density of positive cells, calculated as a percent of the total number of cells, increased up to day 21 in cultures from both the cerebrum and the brainstem, indicating that NADPH-diaphorase is preferentially expressed in neurons with longer viability. On the other hand, virtually no intensely positive cells were detectable in cerebellar cultures at any period examined up to 21 days. In the in vivo study, moderately stained NADPH-diaphorase-positive neurons were first detected, mainly in the laterodorsal-pedunculopontine tegmental nuclei complex and partly in the striatum, in 16-day-old embryonic rat brain. At 2 days postnatal, intensely stained neurons were detectable in the cerebral cortex as well as in the tegmental nuclei complex and the striatum, indicating some delay in the in vitro, as compared to the in vivo, expression of neuronal NADPH-diaphorase. © 1991.
  • WG DING, M FUJIMURA, TOOYAMA, I, H KIMURA
    CELL AND TISSUE RESEARCH 263 2 237 - 243 1991年02月 [有り][無し]
     研究論文（学術雑誌） 
    The distribution pattern of serotonin (5HT) in the pancreas was studied immunohistochemically by using a 5HT monoclonal antibody in various vertebrates including the eel, bullfrog, South African clawed toad, turtle, chicken, mouse, rat, guinea-pig, cat, dog and human. In all species examined, except the bullfrog, 5HT-like immunoreactivity was observed in nerve fibers, in endocrine cells, or in both. Positive nerve fibers were found in the eel, turtle, mouse, rat and guinea-pig. These fibers ran mainly along the blood vessels and partly through the gap between the exocrine glands. In the eel and guinea-pig, positive fibers invaded the pancreatic islet. Occasionally, these positive fibers were found adjacent to the surface of both exocrine and endocrine cells, suggesting a regulatory role of 5HT in pancreatic function. 5HT-positive endocrine cells were observed in the pancreas of all species except for the bullfrog and rat. In the eel and in mammals such as the mouse, guinea-pig, cat, dog and human, 5HT-positive cells were mainly observed within the pancreatic islet. In the South African clawed toad, turtle and chicken, the positive cells were mainly in the exocrine region. The present study indicates that the distribution patterns of 5HT in the pancreas varies considerably among different species.
  • Kazuo Sasaki, Yutaka Oomura, Kenji Suzuki, Tadashi Muto, Kazumitsu Hanai, Ikuo Tooyama, Hiroshi Kimura, Noboru Yanaihara
    Brain Research Bulletin 27 3-4 327 - 332 1991年 [有り][無し]
     研究論文（学術雑誌） 
    In the present study, the relations between acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), platelet-derived growth factor (PDGF), and food intake were studied. When aFGF-, bFGF-, and PDGF-like activity in cerebrospinal fluid (CSF) was examined by bioassay, the activity of those factors significantly increased in postfeeding CSF, compared to prefeeding CSF. Injections of aFGF, bFGF, aFGF1-15 (synthetic amino-terminal peptide of aFGF), and PDGF into the third cerebral ventricle decreased food intake, and injections of anti-aFGF, anti-bFGF, and anti-aFGF1-15 antibodies into the lateral hypothalamus (LHA) increased food intake. The activity of LHA glucose-sensitive neurons was inhibited by electrophoretic application of aFGF. These results suggest that aFGF, bFGF and PDGF have in vivo physiological roles in the central nervous system, distinct from those as mitogens. © 1991.
  • A. Matsuo, I. Tooyama, I. Akiguchi, J. Kimura, M. Kameyama
    Clinical Neurology 31 7 742 - 745 1991年 [有り][無し]
     研究論文（学術雑誌）
  • Osamu Yasuhara, Ikuo Tooyama, Haruhiko Akiyama, Ichiro Akiguchi, Jun Kimura, Patrick L. McGeer, Yasushi Hara, Hiroshi Kimura
    Dementia and Geriatric Cognitive Disorders 2 2 64 - 70 1991年 [有り][無し]
     研究論文（学術雑誌） 
    Localization of acidic fibroblast growth factor (aFGF) was investigated immunohistochemically in postmortem brain tissue of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and age-matched control cases using a specific rabbit antiserum to aFGF. Small numbers of glial cells were faintly stained in white but not gray matter of control cases. Larger numbers of reactive astrocytes were stained intensely for aFGF in severely affected areas of AD cases and in the primary motor cortex of ALS cases. In AD, aFGF-positive astrocytes congregated at the margin of senile plaques. Double immunostaining indicated that such upregulation of aFGF expression might be related to the presence of reactive microglia rather than [3-amyloid protein deposits. © 1991 S. Karger AG, Basel.
  • TOOYAMA, I, H AKIYAMA, PL MCGEER, Y HARA, O YASUHARA, H KIMURA
    NEUROSCIENCE LETTERS 121 1-2 155 - 158 1991年01月 [有り][無し]
     研究論文（学術雑誌） 
    Localization of acidic fibroblast growth factor (aFGF)-like immunoreactivity was examined in postmortem human brain tissue of Alzheimer and age-matched control cases using a rabbit polyclonal antibody specific for aFGF. In control cases, a small number of glial cells were stained very weakly in white but not in gray matter. In Alzheimer cases, some astrocytes were strongly stained for aFGF in both gray and white matter. These intensely staining cells were frequently observed surrounding senile plaques, but represented a small proportion of the total astrocytic population. The present study suggest that aFGF may be upregulated in areas of Alzheimer pathology.
  • TOOYAMA, I, Y HARA, O YASUHARA, Y OOMURA, K SASAKI, T MUTO, K SUZUKI, K HANAI, H KIMURA
    NEUROSCIENCE 40 3 769 - 779 1991年 [有り][無し]
     研究論文（学術雑誌） 
    Antisera against acidic fibroblast growth factor purified from bovine brain were produced in rabbits and used for immunohistochemical study of the rat brain. When examined in an immunospot assay using a nitrocellulose membrane, the best antibody was capable of detecting 80 fmol of acidic fibroblast growth factor but failed to react even with up to 5 pmol of basic fibroblast growth factor. Using this antiserum, the immunohistochemical distribution of acidic fibroblast growth factor was examined in rat brain. Acidic fibroblast growth factor-like immunoreactivity was localized mainly in a subpopulation of ependymal cells and tanycytes, as well as in some glial cells. Positive ependymal cells were observed throughout the walls of ventricles, including the third ventricle and cerebral aqueduct. Immunoreactive processes of tanycytes were found extending from the ventral wall of the third ventricle to the brain parenchyma and surface. The most intense immunostaining was observed in circumventricular organs such as the organum vasculosum laminae terminalis and the subfornical organ. Particularly in the latter organ, there was an extremely dense plexus of immunoreactive fibers and processes around the wall of capillaries. The present results suggest that the effects of acidic fibroblast growth factor on brain functions may be exerted through the circumventricular organs and/or ependymal cells.
  • TOOYAMA, I, H KIMURA, H AKIYAMA, PL MCGEER
    BRAIN RESEARCH 523 2 273 - 280 1990年07月 [有り][無し]
     研究論文（学術雑誌）
  • Ko Nakamura, Kunihiko Higashiura, Naoharu Nishimura, Atsushi Yamamoto, Ikuo Tooyama, Hiroshi Kimura, Kazuharu Ienaga
    Journal of Neurochemistry 55 3 1064 - 1066 1990年 [有り][無し]
     研究論文（学術雑誌） 
    Abstract: We isolated a glutamyltaurine from bovine brains and determined its structure as γ‐glutamyltaurine (γ‐Glu‐Tau glutaurine) by use of a new mass spectrometric technique [B/E linked scan sputtered ion mass spectrometry (SIMS)], which we have recently shown to be useful for distinguishing the γ‐ from the α‐isomer of glutamyl‐dipeptides. Neither the α‐isomer of glutamyltaurine nor any aspartyltaurines could be detected in bovine brain. Copyright © 1990, Wiley Blackwell. All rights reserved

書籍

  • 分子遺伝学の基礎 神経内科テキスト（改訂第２版）
    ()
    南江堂 2005年
  • 神経内科学テキスト
    ()
    南江堂 2000年
  • The Bioligy of Nitric Oxide
    ()
    1998年
  • 神経伝達物質update-基礎から臨床まで
    ()
    中外医学社 1998年
  • The Bioligy of Nitric Oxide
    ()
    1998年

MISC

受賞

  • 2010年 第10回バイオビジネスコンペJAPAN奨励賞
  • 2006年 日本脳科学会研究奨励賞
     JPN
  • 2006年 加藤記念難病研究基金平成１８年度研究奨励賞
     JPN

共同研究・競争的資金等の研究課題

  • 鼻粘膜中の病因タンパクのプリオン活性を検出するAI技術の開発と認知症診断への応用
    日本学術振興会：科学研究費助成事業 挑戦的研究(開拓)
    研究期間 : 2020年07月 -2024年03月 
    代表者 : 遠山 育夫
  • フッ素MR画像法によるAβオリゴマー、Aβフィブリル、リン酸化タウ蛋白の同時解析
    日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2021年03月 
    代表者 : 遠山 育夫, 赤津 裕康
     
    アルツハイマー病の病態は、ベータアミロイドペプチ（Aβ）オリゴマー形成、老人斑の形成、神経原線維変化の形成と進んでいくと考えられている。しかしながら、それぞれの異常蛋白相互の関連については良く解っていない。これらの異常蛋白相互の関連を明らかにするためには、複数の異常蛋白をin vivoで同時に画像化する技術が不可欠である。我々はこれまで、超高磁場MR画像装置を用い、フッ素MR画像法による画像化技術の開発に取り組み、最近、試薬の出す19F-NMR信号のケミカルシフトの違いを利用して、複数の脳内異常蛋白を同時画像化（多重フッ素MR画像法）することに成功した。本研究では、Aβオリゴマー、老人斑、神経原線維変化、の３つの標的に的を絞り、多重フッ素MR画像法を用いて同時解析を行う。アルツハイマー病の遺伝子改変モデルマウスに対して、経時的に多重フッ素MR画像を試み、Aβオリゴマーや老人斑、神経原線維変化がどのように脳内で形成されて伝搬していくか、in vivoで解析する. アルツハイマー病は、本来、この３つの主要病変が相互作用しながら進展しており、ひとつの病変（たとえばアミロイド病理）を標的にした薬であっても、３病変に対して影響を及ぼしていることは、十分に考えられる。したがって、動物を生かしたまま複数の病変を解析できる技術は、治療薬開発にとっても極めて重要と考える。そこで、多重フッ素ＭＲ画像法を治療薬開発研究に応用する。とともに、治療薬の効果を検証することも研究計画に加える。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 遠山 育夫, 椎野 顯彦, 田口 弘康
     
    我々は新しいクルクミン誘導体であるf-methyl-curcumin1 (FMeC1, Shiga-Y5 と FMeC2(Shiga-Y6)を新規に合成した。水晶発信子マイクロバランス法による解析では、両Shiga-Y化合物は線維化AβとAβオリゴマーの両方に結合した。Aβ凝集体にのみ結合するShiga-X22を組み合わせた二重フッ素MR画像法を用いて、アルツハイマー病モデルマウス脳のAβオリゴマーの画像を得ることができた。ついでShiga-Y5を6ヶ月間アルツハイマー病モデルマウスに経口投与したところ、Shiga-Y5が認知機能の低下を防ぎ、脳内アミロイド病変を軽減することを確認した。
  • ケミカルシフトの違いを利用したフッ素MR画像による複数の脳内異常蛋白の同時解析
    日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 遠山 育夫
     
    本研究では、クルクミンの構造を一部変えた新規の化合物を多種類合成して、アルツハイマー病の老人斑を検出する MR 画像診断用試薬の開発に取り組んできた。今回、それら化合物のひとつである Shiga-Y5 が、認知症の治療効果を持つことをアルツハイマー病のモデルマウスを用いて明らかにした。Shiga-Y5は、アルツハイマー病の画像診断薬としても治療薬としても役立つ可能性を示唆している.。この成果を国際学術誌に掲載・発表した（Neurobiol Aging 36: 201-210 2015)。また、本研究で開発した40種類のクルクミン誘導体のアミロイド結合能や細胞毒性抑制作用などをまとめてデータベース化し、論文発表した（ Biochem Biophys Report 4: 357-368, 2015）。 さらに、フッ素MR画像法を用いると非放射性同位元素である19FのもつNMR信号を利用して、PETと同様な機能画像を得ることができることから、「脳内環境」で開発した技術やMR画像診断薬について総説としてまとめて報告した。なかでも試薬の出す19F-NMR信号のケミカルシフトの違いを利用した二重フッ素画像法を利用して、世界で初めてアミロイドオリゴマーの候補画像を得ることに成功し、国際学術誌に報告した（Ageing Research Reviews, 2016 doi: 10.1016/j.arr.2015.12.008）。
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 遠山 育夫, 赤津 裕康, 井之上 浩一, 清水 志乃, 道川 誠, 松川 則之
     
    アルツハイマー病（AD）の早期診断法として、PETによるアミロイドイメージングや髄液中のβアミロイドペプチド（Aβ）の測定が提唱されているが、もっと簡便で侵襲が少なく安価な診断法の開発が期待されている。我々は、血液を介さずに脳内との間で物質の移動が行われる鼻腔・鼻粘膜に着目し、鼻粘膜に存在する認知症バイオマーカーの同定と測定技術の確立を目的に本研究を行った。その結果、ヒト鼻粘膜中にAβ、タウ、リン酸化タウが存在することを明らかにした。さらに、ヒト鼻粘膜サンプルをからAβ42を定量測定する方法を開発して学術誌に発表した。開発した方法を用いてAD患者、対照例を対象に臨床研究を実施した。
  • フッ素ＭＲ画像法と光画像法によるアミロイドオリゴマーのｉｎ ｖｉｖｏ病態解析
    日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 遠山 育夫
     
    アルツハイマー病などの神経変性疾患に共通する脳内環境変化として異常蛋白の蓄積があり、その形成や伝搬機構の解明が診断治療法の開発にとって重要な鍵となる。アルツハイマー病で最も早期に起こる病理変化はAβ凝集体の出現であり、とくに神経毒性の強い可溶性Aβ凝集体がアルツハイマー病の発症に強く関与すると考えられている。我々は、これまで高磁場MR画像装置を用いてフッ素ＭＲ画像法によるアミロイドイメージング試薬の開発を推進してきた。その中でクルクミンを骨格とする化合物が、オリゴマーを含む可溶性Aβ凝集体に強く結合して強い蛍光を発することを発見した。本研究では、この技術を発展させ、フッ素ＭＲ画像法と光画像技術を組み合わせて可溶性Aβ凝集体をIn vivoで画像化する試薬と技術を開発することを試みた。まずAβ 凝集体のみならず Aβ オリゴマーにも結合して画像化ができるShiga-Y5とAβ 凝集体のみに強く結合するShiga-X22を開発した。ついでShiga-Y5とShiga-X22の混合液をAPP/PS1マウスに投与した。それぞれの化合物に固有のフッ素ケミカルシフト値を用いることで、2つの化合物それぞれのMR画像を得た。また、両者の画像の差分を算出することにより、Aβ オリゴマーの候補画像を作成することに成功した。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2010年04月 -2014年03月 
    代表者 : 遠山 育夫, 田口 弘康, 犬伏 俊郎
     
    本研究の目的は、アルツハイマー病の遺伝子改変モデルマウスを用いて、フッ素MR画像法を用いたアミロイドイメージング法を開発することである。まず我々は、クルクミンを基本骨格とするフッ素MR画像診断候補薬であるShiga-Y系化合物を新規合成し、それらがベータアミロイドペプチド（Aβ）凝集体やAβオリゴマーに結合することを明らかにした。このうち、Shiga-Y5(FMeC1)が、脳内で最も強くフッ素NMR信号を出した。そこで、Shiga-Y5をアルツハイマー病遺伝子改変モデルマウスの尾静脈から投与して、7テスラMR画像装置で測定したところ、50分の測定でＭＲによるアミロイドイメージングに成功した。
  • 日本学術振興会：科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2011年 -2012年 
    代表者 : 遠山 育夫
     
    我々はAβ凝集体が存在するとケト型からエノール型になり老人斑に結合するクルクミン誘導体(Shiga-Y5)を開発した。本研究ではShiga-Y5の性質を利用することで老人斑の量を測定する血清診断法の開発を行った。その結果、遺伝子組換えマウスの血液中にShiga-Y5 を注射すると脳に入って老人斑と結合し、血清への排泄が遅延すること、Shiga-Y5とFACSを組み合わせ、液中のAβ凝集体量を測定できることを示した。
  • ES細胞におけるナトリウム利尿ペプチドの役割の解明
    日本学術振興会：科学研究費助成事業 特別研究員奨励費
    研究期間 : 2009年 -2011年 
    代表者 : 遠山 育夫, ABDELALIMM Essam, ABDELALIM M. Essam, ESSAM Abdelalim M.
     
    B型ナトリウム利尿ペプチド(BNP)は心筋細胞から分泌されるペプチドホルモンであるが、我々は未分化なES細胞がBNPを産生していることを見出した。これはBNPが心筋モルモンとしてのみならずES細胞の増殖や分化に関与していることを示している。本研究の目的は、ES細胞が分泌するナトリウム利尿ペプチドの機能的意義の解明を通して「ナトリウム利尿ペプチドはES細胞ホルモンである」という作業仮説を検証することである。平成22年度はES細胞におけるナトリウム利尿ペプチドの受容体NPR-Aの役割について検討した。平成23年度はES細胞におけるナトリウム利尿ペプチドの受容体NPR-Cの役割について検討した。その結果、1)NPR-Cは未分化なES細胞に強く発現しており、ES細胞が分化するとNPR-Cの発現は減少する。2)RNA干渉法を用いてES細胞のNPR-Cの発現をノックアウトすると、ES細胞はアポートシスによる細胞死に至る。3)その際に、p53の遺伝子発現が増加する。4)p53の遺伝子発現を抑制すると、NPR-Cの発現をノックアウトしたときに誘導されるES細胞のアポートシスが抑制される。5)次にNPR-Cの拮抗薬であるcANF(4-23)を投与すると、p53の遺伝子発現はブロックされる。以上の結果は、NPR-Cはp53を介するES細胞のアポトーシスを制御していることを示している。 これらの成果を国際学術誌Cell Death and Diseaseなど3報に掲載するとともに国際学会で報告した。また、ドイツで行われたcGMAに関する国際会議で招待講演を行った。
  • 日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2007年 -2009年 
    代表者 : 遠山 育夫, 田口 弘康, 犬伏 俊郎
     
    本研究では、7テスラ高磁場MR装置を用い非放射性フッ素(^<19>F)を標的にしたフッ素核磁気共鳴画像法(^<19>F-MRI)によるパーキンソン病の画像診断法の開発を行った。まず^<19>F-DOPAおよび^<19>Fを含むドパミン神経関連化合物について検討した結果、^<19>F-DOPAで最も高いS/N比が得られた。次に、培養細胞実験を行いPC12細胞内に取り込まれた^<19>F-DOPAの画像化に成功した。さらに動物実験を行い、6-OHDA投与パーキンソンモデルラットの腹腔内に^<19>F-DOPA後、線条体に取り込まれた^<19>F-DOPAの画像化に成功した。本研究の結果は、高磁場MR装置を用いたパーキンソン病の画像診断法開発の重要な基礎データを提供している。
  • ES細胞をししたアルツハイマー病モデルサルの作成
    日本学術振興会：科学研究費助成事業 萌芽研究
    研究期間 : 2006年 -2007年 
    代表者 : 遠山 育夫, 鳥居 隆三, 高田 達之
     
    本実験の目的は,ES細胞を利用してアルツハイマー病の遺伝子改変モデルサル作成を試みることである。昨年度は,最適なコンストラクトの作製と,サルES細胞への遺伝子導入技術の確立を行った。本年度は,まず,ヒトのアミロイド前駆体タンパク(APP)遺伝子プロモーターと変異APP遺伝子より構成されるコンストラクトを作製した。そして,昨年度の研究で最も良い成績を収めたリポフェクタミン法により,サルES細胞に遺伝子導入した。っいで,抗生物質G-418を用いて,ヒトAPP遺伝子の導入されたサルES細胞を選別した結果,安定してAPPタンパクを発現するES細胞株を2株樹立した。 一方,共同研究者の鳥居は,核移植法の確立を試みて実験を行い,サルの核移植クローン胚の作製に成功した。そこで,現在,上記のES細胞株を用いて,核移植法によるアルツハイマー病モデルサルの作製とキメラサル作製に着手した。なお,神経難病の原因遺伝子を導入したカニクイザルES細胞の核をカニクイザルの除核した未受精卵に核移植し,活性化刺激を与え2〜4細胞期胚になったことを確認後,これを排卵2-3日目のレシピエントのカニクイザルの子宮あるいは卵管に移植する予定である。 サルは齧歯類に比べ妊娠期間も長いので,本研究計画が順調に推移したとしても,実際の誕生はまだ先になる。本研究の成果の一部を国際学術誌で報告した。なお,本研究計画は,半数の外部委員を加えた動物生命科学研究倫理委員会で審査を受けて,承認されている。
  • ヒトおよびサル脳の新規アセチルコリン合成酵素pChATの遺伝子構造と脳機能回路
    日本学術振興会：科学研究費助成事業 基盤研究(B)
    研究期間 : 2004年 -2006年 
    代表者 : 遠山 育夫, 木村 宏, 松尾 明典
     
    コリンアセチル基転移酵素(ChAT)は、コリン作動性神経の神経伝達物質であるアセチルコリンの合成酵素である。ChATは、これまで1種類であると考えられていた。しかし、多くのChAT抗体が末梢コリン神経を検出できないという事実をヒントに、我々は、ラットの翼口蓋神経節から新しいChATサブタイプを発見し、pChATと命名した(Tooyama and Kimura,2000)。当初、pChATは末梢神経系にのみ発現していると予想されたが、pChAT抗体を作製してラット脳を検索したところ、視床下部の隆起乳頭体核に細胞体を持ち大脳皮質に投射するニューロンがpChATを含有することを見い出した(Neuroscience 2003)。すなわち哺乳動物の脳にもChATサブタイプが複数個存在する可能性が高い。そこで本研究では、ヒトおよび霊長類を対象に、ChATサブタイプの遺伝子構造を明らかにするとともに、それぞれのサブタイプの解剖学的局在と機能的意義について研究を行った。その結果、1)サルおよびヒトのChATサブタイプの遺伝子構造、2)ChATサブタイプの解剖学的局在(とくにサル脳において)、3)ChATサブタイプの機能的意義の一端を解明した。本研究の結果、ヒトやサルにもChATバリアントが存在することが明らかになった。さらに、ChATバリアントはアセチルコリンを合成するだけでなく、核内において転写因子と相互作用するなど、新たな機能をもつ可能性が示唆された。脳ではコリン神経系は学習・記憶に深く関わっていることが知られており、今後、アルツハイマー病をはじめとするヒト疾患で、ChATバリアントがどのような働きをしているのか、検討していく予定である。研究成果は、11編の国際学術誌に報告するとともに(業績欄参照)、2つの国際学会で発表を行った。
  • アルツハイマー病における新しいコリン作動性神経路に関する研究
    日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2001年 -2002年 
    代表者 : 遠山 育夫, 松尾 明典
     
    本件究補助金が授与された2年間にpChATをアセチルコリン合成酵素とする新しいコリン作動性神経回路について、以下の研究成果を得た。 新しいコリン作動性神経回路の発見 ラット脳でのpChAT局在を詳細に検討した結果、新しいコリン作動性神経回路として、視床下部の隆起乳頭体に細胞体を持ち大脳皮質に投射するニューロンがpChATを含有することを見いだした。視床下部の隆起乳頭体のpChAT陽性神経は、軸索輸送を阻害するコルヒチン投与により、より明瞭に検出される。逆行性トレーサーのコレラトキシンBを大脳皮質に注射したところ、pChAT陽性神経にコレラトキシンBを検出したことから、視床下部の隆起乳頭体のpChAT陽性神経が大脳皮質に神経投射することを確認できた。 ヒトpChATの構造 魚類から哺乳動物まで、pChAT mRNAとその産物についてスクリーニングしたところ、ラット、ネコ、ウシ、サルなどの哺乳動物に広く存在することが判明した。pChATにはスプライシングの違いにより、少なくとも2種類存在する。ひとつは、エクソン6、7、8、9を欠くもので、我々がラット翼口蓋神経節から最初のクローニングしたpChATに相当する。もうひとつは、エクソン7、8を欠くタイプで、これをpChAT2と命名した。しかし、ヒトでは今の所、pChATやpChAT2に相当するcDNAは検出されず、変異スプライシングの機構に違いがあると考えられた。ヒト神経節を検索した結果、エクソン7、8、9を欠くと同時にエクソン6の一部も決失した遺伝子を検出した(HpChATと略す)。 アルツハイマー例におけるpChAT発現 アルツハイマー例および対照例に剖検脳を用いて、pCHATやHpChATの発現を検討した。その結果、既知の「無名質-大脳皮質コリン神経回路」には、いずれにおいてもpChATの存在は確認できなかった。
  • 神経細胞の核内転写因子の人工制御による脳機構の解明
    日本学術振興会：科学研究費助成事業 萌芽的研究
    研究期間 : 1998年 -2000年 
    代表者 : 遠山 育夫, 木村 宏
     
    本研究の目的は、FGFのN端に存在する核移行ペプチドを利用して、哺乳動物の細胞核内にペプチドやオリゴヌクレオチドを送り込む方法を開発し、特定の遺伝子の核内転写因子の人工制御することにより、脳機能を解析することである。3年間の研究により、次の成果をあげた。 1)FGF-1のN端に存在する核移行ペプチドに、蛍光物質ペプチド(GFP)や自然界に存在しないと推測される取りペプチドマーカーTyr-D-A1a-Pheを結合させたいわゆる核内運び野ペプチドを作成した。 2)この運び野ペプチドを哺乳動物の培養細胞(CHO細胞)に投与することにより、マーカーペプチドが核内に侵入することを確認した。 3)この運び野ペプチドとアンチセンスプローブとの複合体をラットに投与するIn Vivo実験を行った。Targetとしては、我々が解析方法に熟知しているグルタメートのトランスポーターとiNOS遺伝子を選んだ。核移行ペプチドとアンチセンスプローブ(PNA)との複合体をラットの脳室内に投与し、グルタメートのトランスポーターとiNOS遺伝子の発現抑制効果を検討したが、今のところ期待されたような結果を観察することができなかった。 以上のように、核内にペプチドやアンチセンスプローブを送り込む運び野ペプチドの作成することができた。少なくとも培養細胞では一定の成果を上げることができた。投与濃度、投与方法などの最適条件が決定されれば、in vivoへの投与も可能となり、新しい研究手法を切り開くものと期待される。なお、成果の一部を論文発表した。
  • 副腎における酸性線維芽細胞成長因子の局在と機能
    日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 1995年 -1996年 
    代表者 : 遠山 育夫
     
    線維芽細胞成長因子(FGF)は、中胚葉および神経性外胚葉由来の細胞群に対する栄養因子ファミリーである。なかでも酸性線維芽細胞成長因子(aFGF, FGF-1)は、一部の組織を除けば脳や網膜という中枢神経系に局在することから、神経系の栄養因子と考えられており、これまでaFGFの末梢組織における役割についてはほとんどわかっていなかった。しかし本研究によって、副腎の髄質においてaFGFのmRNAの発現が多く認められること、髄質のアドレナリン分泌細胞にaFGFが局在していることが明らかになった。さらにFGF 1型受容体が副腎皮質に存在することも明らかとなった。aFGF投与によって副腎皮質ホルモンの分泌が亢進することから、aFGFは副腎皮質ホルモンの分泌調整という局所ホルモン的な働きを有している可能性を示唆する。こうした成果は、aFGFが中枢神経系の栄養因子であるという従来の概念を越えており、栄養因子のホルモン的機能という新しい学問的概念を提示するものである。 さらに末梢投与したaFGFが、おそらく脳血液関門を欠く脳室周囲器官などから髄液中に入り、脳内のコリン神経に作用することも明らかとなった。加えて、視床下部外側野の摂食中枢を抑制して食行動を調節するなど、aFGFのホルモン様作用は副腎という局所にとどまらないことも示された。こうした研究成果から、aFGFは中枢神経系にとどまらず末梢神経・内分泌系機能をも調節していることが考えられる。この研究成果を基に、栄養因子のホルモン的機能という新しい視点を持った研究が、更に展開していくものと考えられる。
  • ラット脳およびヒト剖検脳における酸性線維芽細胞成長因子の免疫組織化学的研究
    日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1990年 -1990年 
    代表者 : 遠山 育夫
  • 脳内セロトニン神経系の機能形態学的研究
    日本学術振興会：科学研究費助成事業 奨励研究(A)
    研究期間 : 1989年 -1989年 
    代表者 : 遠山 育夫
  • アルツハイマー病の診断・治療法に関する研究
  • 認知症の神経病理学的研究
  • コリン神経の分子科学
  • Study on Alzheimer
  • Neuropathological study on dementia
  • Molecular Neurosuence in Cholinergic neuron

委員歴

  • 2016年06月 - 現在   日本神経病理学会   評議員
  • 2003年 - 現在   日本脳科学会   理事   日本脳科学会
  • 2000年 - 現在   日本認知症学会   理事   日本認知症学会
  • 1998年 - 現在   日本解剖学会   評議員   日本解剖学会
  • 1995年 - 現在   日本組織細胞化学会   評議員   日本組織細胞化学会
  • 2017年01月 - 2019年12月   日本組織細胞化学会   監事