Research activity information

• Neuronal Populations Involved in Motor Function Show Prominent Expression of Sbno1 During Postnatal Brain Development

  Sunjidmaa Zolzaya, Dai Ihara, Munkhsoyol Erkhembaatar, Shinsuke Ochiai, Ayaka Isa, Mariko Nishibe, Jean-Pierre Bellier, Takahiro Shimizu, Satoshi Kikkawa, Ryo Nitta, Yu Katsuyama

  Human genome studies have suggested that strawberry notch homologue 1 (SBNO1) is crucial for normal brain development, with mutations potentially contributing to neurodevelopmental disorders. In a previous study, we observed significant developmental abnormalities in the neocortex of Sbno1 as early as one week after birth. In the present study, we conducted an extensive analysis of Sbno1 postnatal expression in the brain of C57BL/6 mice using a newly developed in-house polyclonal antibody against Sbno1. We found that Sbno1 is expressed in all neurons, with certain neuronal populations exhibiting distinct dynamic changes (both temporal and spatial) in expression level. These findings suggest that the neuronal expression of Sbno1 is developmentally regulated after birth. They also indicate that while Sbno1 may play a general role across all neurons, it may also serve more specialized functions in certain neuronal types and/or for certain cellular activities related to particular neuronal pathways.

  MDPI AG, Jan. 2025, Journal of Developmental Biology, 13(1) (1), 3 - 3

  Scientific journal


• TEAD1 trapping by the Q353R–Lamin A/C causes dilated cardiomyopathy

  Shintaro Yamada, Toshiyuki Ko, Masamichi Ito, Tatsuro Sassa, Seitaro Nomura, Hiromichi Okuma, Mayuko Sato, Tsuyoshi Imasaki, Satoshi Kikkawa, Bo Zhang, Takanobu Yamada, Yuka Seki, Kanna Fujita, Manami Katoh, Masayuki Kubota, Satoshi Hatsuse, Mikako Katagiri, Hiromu Hayashi, Momoko Hamano, Norifumi Takeda, Hiroyuki Morita, Shuji Takada, Masashi Toyoda, Masanobu Uchiyama, Masashi Ikeuchi, Kiminori Toyooka, Akihiro Umezawa, Yoshihiro Yamanishi, Ryo Nitta, Hiroyuki Aburatani, Issei Komuro

  Mutations in the LMNA gene encoding Lamin A and C (Lamin A/C), major components of the nuclear lamina, cause laminopathies including dilated cardiomyopathy (DCM), but the underlying molecular mechanisms have not been fully elucidated. Here, by leveraging single-cell RNA sequencing (RNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), protein array, and electron microscopy analysis, we show that insufficient structural maturation of cardiomyocytes owing to trapping of transcription factor TEA domain transcription factor 1 (TEAD1) by mutant Lamin A/C at the nuclear membrane underlies the pathogenesis of Q353R -LMNA– related DCM. Inhibition of the Hippo pathway rescued the dysregulation of cardiac developmental genes by TEAD1 in LMNA mutant cardiomyocytes. Single-cell RNA-seq of cardiac tissues from patients with DCM with the LMNA mutation confirmed the dysregulated expression of TEAD1 target genes. Our results propose an intervention for transcriptional dysregulation as a potential treatment of LMNA -related DCM.

  American Association for the Advancement of Science (AAAS), Apr. 2023, Science Advances, 9(15) (15), eade7047

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

  Hiromichi Okuma, Yumiko Saijo-Hamano, Hiroshi Yamada, Aalaa Alrahman Sherif, Emi Hashizaki, Naoki Sakai, Takaaki Kato, Tsuyoshi Imasaki, Satoshi Kikkawa, Eriko Nitta, Miwa Sasai, Tadashi Abe, Fuminori Sugihara, Yoshimasa Maniwa, Hidetaka Kosako, Kohji Takei, Daron M Standley, Masahiro Yamamoto, Ryo Nitta

  2023, Genes to Cells, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Structural model of microtubule dynamics inhibition by kinesin-4 from the crystal structure of KLP-12 –tubulin complex

  Shinya Taguchi, Juri Nakano, Tsuyoshi Imasaki, Tomoki Kita, Yumiko Saijo-Hamano, Naoki Sakai, Hideki Shigematsu, Hiromichi Okuma, Takahiro Shimizu, Eriko Nitta, Satoshi Kikkawa, Satoshi Mizobuchi, Shinsuke Niwa, Ryo Nitta

  Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of the inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

  eLife Sciences Publications, Ltd, Sep. 2022, eLife, 11, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre through phase separation

  Tsuyoshi Imasaki, Satoshi Kikkawa, Shinsuke Niwa, Yumiko Saijo-Hamano, Hideki Shigematsu, Kazuhiro Aoyama, Kaoru Mitsuoka, Takahiro Shimizu, Mari Aoki, Ayako Sakamoto, Yuri Tomabechi, Naoki Sakai, Mikako Shirouzu, Shinya Taguchi, Yosuke Yamagishi, Tomiyoshi Setsu, Yoshiaki Sakihama, Eriko Nitta, Masatoshi Takeichi, Ryo Nitta

  Lead, Jun. 2022, eLife, 11, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Structural model of microtubule dynamics inhibition by Kinesin-4 from the crystal structure of KLP-12 –tubulin complex

  Shinya Taguchi, Juri Nakano, Tsuyoshi Imasaki, Tomoki Kita, Yumiko Saijo-Hamano, Naoki Sakai, Hideki Shigematsu, Hiromichi Okuma, Takahiro Shimizu, Eriko Nitta, Satoshi Kikkawa, Satoshi Mizobuchi, Shinsuke Niwa, Ryo Nitta

  Abstract Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.

  Cold Spring Harbor Laboratory, Feb. 2022, bioRxiv, English

  Link to Kobe Univ. Repository (Kernel)


• CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre

  Tsuyoshi Imasaki, Satoshi Kikkawa, Shinsuke Niwa, Yumiko Saijo-Hamano, Hideki Shigematsu, Kazuhiro Aoyama, Kaoru Mitsuoka, Mari Aoki, Ayako Sakamoto, Yuri Tomabechi, Naoki Sakai, Mikako Shirouzu, Shinya Taguchi, Yosuke Yamagishi, Tomiyoshi Setsu, Yoshiaki Sakihama, Takahiro Shimizu, Eriko Nitta, Masatoshi Takeichi, Ryo Nitta

  Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, detachment of a considerable number of microtubules from the centrosome is known to contribute to fundamental processes in cells. Here, we present evidence that minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation from soluble αβ-tubulin independent of γ-tubulin. CAMSAP2 significantly reduces the nucleation barrier close to the critical concentration for microtubule polymerization by stabilizing the longitudinal contacts among αβ-tubulins. CAMSAP2 clusters together with αβ-tubulin to generate nucleation intermediates, from which numerous microtubules radiate, forming aster-like structures. Our findings suggest that CAMSAP2 supports microtubule growth by organizing a nucleation centre as well as by stabilizing microtubule nucleation intermediates.

  Cold Spring Harbor Laboratory, Mar. 2021, bioRxiv


• Constant existence of the sensory branch of the nerve to the pyramidalis distributing to the upper margin of the pubic ramus

  Daijiro Haba, Kenji Emura, Yuko Watanabe, Ikuo Kageyama, Satoshi Kikkawa, Mamoru Uemura, Takamitsu Arakawa

  Sep. 2018, ANATOMICAL SCIENCE INTERNATIONAL, 93(4) (4), 405 - 413, English, Domestic magazine

  [Refereed]

  Scientific journal


• Postnatal Development of the Corticospinal Tract in the Reeler Mouse.

  Tomohiro Namikawa, Satoshi Kikkawa, Go Inokuchi, Toshio Terashima

  Kobe University School of Medicine, Dec. 2015, The Kobe journal of medical sciences, 61(3) (3), E71-81 - E85, English, Domestic magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Expression of Beta Subunit 2 of Ca²+/Calmodulin-Dependent Protein Kinase I in the Developing Rat Retina.

  Ahmad Aulia Jusuf, Hiroyuki Sakagami, Satoshi Kikkawa, Toshio Terashima

  Expression of beta 2 subunit of Ca²+/calmodulin-dependent protein kinase I (CaMKIβ2) of the rat retina during the developmental period and in the adulthood was studied immunohistochemically. The immunoreactivity of CaMKIβ2 was detected in the earliest development of the primordial retina at embryological day (E) 12. The inner neuroblastic layer from which the presumptive ganglion cells are generated showed the ubiquitous CaMKIβ2 immunoreactivity at E15 and persistently expressed at the same level until postnatal day (P) 0 when the inner neuroblastic layer divides into the ganglionic cell layer and the inner plexiform layer. The strong immunoreactivity was detected in the ganglion cell layer and the moderate one in the internal plexiform layer. CaMKIβ2 immunoreactivities were persistantly expressed throughout the postnatal development at the same level. The low level of intensity was first found in the inner nuclear layer at P7, followed by the outer plexiform, outer nuclear and rod-cone cell layers at the age of P12, respectively. The intensities of CaMKIβ2 immunoreactivities in the inner nuclear and rod-cone cell layers were gradually increased to the strong level by P18 and persisted until adulthood. The present study revealed that the expression of CaMKIβ2 in the retina was detected from the earliest development until adulthood, indicating that CaMKIβ2 may be required in both proliferation and differentiation of the retinal precursor cells and subsequent formation of the functional layers. In addition, CaMKIβ2 immunoreactivity in the rod-cone cell layer implies that this protein may be involved in the visual signaling process.

  Mar. 2015, The Kobe journal of medical sciences, 61(4) (4), E115-23 - 123, English, Domestic magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Expression of Beta Subunit 2 of Ca2+/Calmodulin-Dependent Protein Kinase I in the Developing Rat Retina

  JusufAA, SakagamiH, Kikkawa Satoshi, Terashima Toshio

  2015, Kobe J Med Sci, 61(4) (4), E115 - E123, English

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Cytoarchitecture of the olfactory bulb in the laggard mutant mouse

  Sakisaka Toshiaki, Terashima Toshio

  Sep. 2014, Neuroscience, 275, 259 - 71, English

  [Refereed]

  Scientific journal


• Subcortically and callosally projecting neurons are distinct neuronal pools in the motor cortex of the reeler mouse.

  Hideaki Imai, Tatsuro Yamamoto, Yu Katsuyama, Satoshi Kikkawa, Toshio Terashima

  Nov. 2012, The Kobe journal of medical sciences, 58(3) (3), E86-95 - E95, English, Domestic magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Dynamic changes in the gene expression of zebrafish Reelin receptors during embryogenesis and hatching period

  Hideaki Imai, Yoshihiro Oomiya, Satoshi Kikkawa, Wataru Shoji, Masahiko Hibi, Toshio Terashima, Yu Katsuyama

  Feb. 2012, DEVELOPMENT GROWTH & DIFFERENTIATION, 54(2) (2), 253 - 263, English

  [Refereed]

  Scientific journal


• Dynamic changes in the gene expression of zebrafish Reelin receptors during embryogenesis and hatching period

  Kikkawa Satoshi, Terashima Toshio

  Feb. 2012, Development, growth & differentiation, Vol. 54, No. 2, pp. 253-263, English

  [Refereed]

  Scientific journal


• Cortical layer V neurons in the auditory and visual cortices of normal, reeler, and yotari mice.

  Yasuo Yoshihara, Tomiyoshi Setsu, Yu Katsuyama, Satoshi Kikkawa, Toshio Terashima, Kiyoshi Maeda

  Sep. 2010, The Kobe journal of medical sciences, 56(2) (2), E50-9 - E59, English, Domestic magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• GABA modulates development of cerebellar Purkinje cell dendrites under control of endocannabinoid signaling

  Koji Kawaguchi, Tomomi Habara, Toshio Terashima, Satoshi Kikkawa

  Jul. 2010, JOURNAL OF NEUROCHEMISTRY, 114(2) (2), 627 - 638, English

  Scientific journal


• PKH26 is an excellent retrograde and anterograde fluorescent tracer characterized by a small injection site and strong fluorescence emission.

  Koji Kawaguchi, Yu Katsuyama, Satoshi Kikkawa, Tomiyoshi Setsu, Toshio Terashima

  2010, Archives of histology and cytology, 73(2) (2), 65 - 72, English, Domestic magazine

  Scientific journal


• Anterograde labeling of the corticospinal tract in jimpy mutant mice by Di I injection into the motor cortex.

  Riichi Shibata-Iwasaki, Hideyuki Dekimoto, Yu Katsuyama, Satoshi Kikkawa, Toshio Terashima

  Dec. 2007, Archives of histology and cytology, 70(5) (5), 297 - 301, English, Domestic magazine

  [Refereed]

  Scientific journal


• Expression of zebrafish ROR alpha gene in cerebellar-like structures

  Yu Katsuyama, Yoshihiro Oomiya, Hideyuki Dekimoto, Eriko Motooka, Ai Takano, Satoshi Kikkawa, Masahiko Hibi, Toshio Terashima

  Sep. 2007, DEVELOPMENTAL DYNAMICS, 236(9) (9), 2694 - 2701, English

  Scientific journal


• Postnatal development of entorhinodentate projection of the reeler mutant mouse

  Daisuke Muraoka, Yu Katsuyama, Satoshi Kikkawa, Toshio Terashima

  2007, DEVELOPMENTAL NEUROSCIENCE, 29(1-2) (1-2), 59 - 72, English

  [Refereed]

  Scientific journal


• The zebrafish pob gene entodes a novel protein required for survival of red cone photoreceptor cells

  MR Taylor, S Kikkawa, A Diez-Juan, Ramamurthy, V, K Kawakami, P Carmeliet, SE Brockerhoff

  May 2005, GENETICS, 170(1) (1), 263 - 273, English

  [Refereed]

  Scientific journal


• Reelin-expressing neurons in the anterior commissure and corpus callosum of the rat

  K Misaki, S Kikkawa, T Terashima

  Jan. 2004, DEVELOPMENTAL BRAIN RESEARCH, 148(1) (1), 89 - 96, English

  [Refereed]

  Scientific journal


• Missplicing resulting from a short deletion in the reelin gene causes reeler-like neuronal disorders in the mutant shaking rat Kawasaki

  S Kikkawa, T Yamamoto, K Misaki, Y Ikeda, H Okado, M Ogawa, PL Woodhams, T Terashima

  Aug. 2003, JOURNAL OF COMPARATIVE NEUROLOGY, 463(3) (3), 303 - 315, English

  [Refereed]

  Scientific journal


• Apolipoprotein E and Reelin ligands modulate tau phosphorylation through an apolipoprotein E receptor/disabled-1/glycogen synthase kinase-3beta cascade

  TERASHIMA TOSHIO, KIKKAWA SATOSHI

  Feb. 2003, The Faseb Journal, Vol. 17, No. 2, pp. 295-297, English

  [Refereed]

  Scientific journal


• Missplicing due to a short deletion in the reelin gene causes reeler-like neuronal disorders in the mutant shaking rat Kawasaki

  Kikkawa S, Yamamoto T, Misaki K, Ikeda Y, Okado H, Ogawa M, Woodhmas PL, Terashima T

  2003, J Comp Neurol, 463(3) (3), 303 - 315


• Distribution of Ca2+/calmodulin-dependent protein kinase I beta 2 in the central nervous system of the rat

  Rina-Susilowati, Ahmad Aulia Jusuf, Hiroyuki Sakagami, Satoshi Kikkawa, Hisatake Kondo, Yasuhiro Minami, Toshio Terashima

  Elsevier BV, Aug. 2001, Brain Research, 911(1) (1), 1 - 11, English

  [Refereed]

  Scientific journal


• How vertebrate and invertebrate visual pigments differ in their mechanism of photoactivation.

  M Nakagawa, T Iwasa, S Kikkawa, M Tsuda, T G Ebrey

  In vertebrate visual pigments, a glutamic acid serves as a negative counterion to the positively charged chromophore, a protonated Schiff base of retinal. When photoisomerization leads to the Schiff base deprotonating, the anionic glutamic acid becomes protonated, forming a neutral species that activates the visual cascade. We show that in octopus rhodopsin, the glutamic acid has no anionic counterpart. Thus, the "counterion" is already neutral, so no protonated form of an initially anionic group needs to be created to activate. This helps to explain another observation-that the active photoproduct of octopus rhodopsin can be formed without its Schiff base deprotonating. In this sense, the mechanism of light activation of octopus rhodopsin is simpler than for vertebrates, because it eliminates one of the steps required for vertebrate rhodopsins to achieve their activating state.

  May 1999, Proceedings of the National Academy of Sciences of the United States of America, 96(11) (11), 6189 - 92, English, International magazine

  Scientific journal


• A novel photointermediate of octopus rhodopsin activates its G-protein.

  M Nakagawa, S Kikkawa, K Tominaga, N Tsugi, M Tsuda

  The photointermediate of octopus rhodopsin responsible for G-protein activation was examined by a GTPgammaS-binding assay in a reconstituted system with purified rhodopsin and photoreceptor G-protein. When octopus rhodopsin alone was incubated in the dark after illumination, its ability to stimulate GTPgammaS-binding by the G-protein decreased in a time-dependent manner. We associate this decay with the decay of a novel photointermediate, transient acid metarhodopsin, which lies between mesorhodopsin and acid metarhodopsin. Spectroscopic evidence for its existence was suggested by its effects on the turbidity of the vesicles. These results suggest that the transient acid metarhodopsin, not the stable final photoproduct, acid metarhodopsin, activates a G-protein in octopus photoreceptors.

  Oct. 1998, FEBS letters, 436(2) (2), 259 - 62, English, International magazine

  Scientific journal


• A novel rhodopsin kinase in octopus photoreceptor possesses a pleckstrin homology domain and is activated by G protein betagamma-subunits.

  S Kikkawa, N Yoshida, M Nakagawa, T Iwasa, M Tsuda

  G protein-coupled receptor kinases (GRKs) play an important role in stimulus-dependent receptor phosphorylation and desensitization of the receptors. Mammalian rhodopsin kinase (RK) and beta-adrenergic receptor kinase (betaARK) are the most studied members among known GRKs. In this work, we purified RK from octopus photoreceptors for the first time from invertebrate tissues. The molecular mass of the purified enzyme was 80 kDa as estimated by SDS-polyacrylamide gel electrophoresis, and this was 17 kDa larger than that of the vertebrate enzymes. Unlike vertebrate RK, octopus RK (ORK) was directly activated by betagamma-subunits of a photoreceptor G protein. We examined the effects of various known activators and inhibitors of GRKs on the activity of the purified ORK and found that their effects were different from those on either bovine RK or betaARK. To analyze the primary structure of the enzyme, we cloned the cDNA encoding ORK from an octopus retinal cDNA library. The deduced amino acid sequence of the cDNA was highly homologous to betaARK over the entire molecule, including a pleckstrin homology domain located in the C-terminal region, and homology to RK was significantly lower. Furthermore, Western blot analysis of various octopus tissues with an antibody against the purified ORK showed that ORK is expressed solely in the retina, which confirmed the identity of the enzyme as rhodopsin kinase. Thus, ORK appears to represent a unique subgroup in the GRK family, which is distinguished from vertebrate RK.

  Mar. 1998, The Journal of biological chemistry, 273(13) (13), 7441 - 7, English, International magazine

  Scientific journal


• Light-induced protein conformational changes in the photolysis of octopus rhodopsin.

  M Nakagawa, S Kikkawa, T Iwasa, M Tsuda

  Light-induced protein conformational changes in the photolysis of octopus rhodopsin were measured with a highly sensitive time-resolved transient UV absorption spectrophotometer with nanosecond time resolution. A negative band around 280 nm in the lumirhodopsin minus rhodopsin spectra suggests that alteration of the environment of some of the tryptophan residues has taken place before the formation of lumirhodopsin. A small recovery of the absorbance at 280 nm was observed in the transformation of lumirhodopsin to mesorhodopsin. Kinetic parameters suggest that major conformational changes have taken place in the transformation of mesorhodopsin to acid metarhodopsin. In this transformation, drastic changes of amplitude and a shift of a difference absorption band around 280 nm take place, which suggest that some of the tryptophan residues of rhodopsin become exposed to a hydrophilic environment.

  May 1997, Biophysical journal, 72(5) (5), 2320 - 8, English, International magazine

  Scientific journal


• Identification of two palmitoyl groups in octopus rhodopsin.

  M Nakagawa, T Iwasa, S Kikkawa, T Takao, Y Shimonishi, M Tsuda

  We determined the structure and site of fatty acid incorporated in octopus rhodopsin using a combination of fluorescence label and enzymatic cleavage methods in conjunction with fast-atom bombardment (FAB) mass spectrometry. A single peptide containing two adjacent cysteines, Cys337 and Cys338, was successfully isolated using the fluorescence from a dye conjugated to Cys345. The FAB mass spectrometric analysis of the peptide (323Phe-340Phe) revealed that two palmitoyl groups are linked to Cys337 and Cys338 via thioester bonds in octopus rhodopsin as in bovine rhodopsin.

  Jan. 1997, Photochemistry and photobiology, 65(1) (1), 187 - 91, English, International magazine

  Scientific journal


• Simple purification and functional reconstitution of octopus photoreceptor Gq, which couples rhodopsin to phospholipase C.

  S Kikkawa, K Tominaga, M Nakagawa, T Iwasa, M Tsuda

  In invertebrate photoreceptors, illuminated rhodopsin activates multiple G proteins, which are assumed to initiate multiple phototransduction cascades. In this paper, we focused on one of the phototransduction cascades, which utilizes rhodopsin, a Gq-like G protein, and phospholipase C (PLC). A Gq-like G protein from octopus photoreceptors was successfully purified to apparent homogeneity as an active form by simple two-step chromatography. The purified G protein had an alpha beta gamma-trimeric structure consisting of 44-kDa alpha, 37-kDa beta, and 9-kDa gamma subunits. The 44-kDa alpha subunit was assigned to the Gq class by western blot with antiserum against mammalian Gq alpha and by partial amino acid sequencing of its proteolytic fragments. Light-dependent binding of GTP gamma S was observed when the purified octopus Gq was reconstituted with octopus rhodopsin that had been integrated into phospholipid vesicles. Octopus Gq activated PLC beta 1 purified from bovine brain dose-dependently in the presence of A1F4-. Finally, light- and GTP-dependent activation of PLC beta 1 was observed in a reconstitution system consisting of octopus rhodopsin, Gq, and bovine PLC beta 1.

  Dec. 1996, Biochemistry, 35(49) (49), 15857 - 64, English, International magazine

  Scientific journal


• GTP-binding protein couples with metabotropic glutamate receptor in bovine retinal on-bipolar cell.

  S Kikkawa, M Nakagawa, T Iwasa, A Kaneko, M Tsuda

  GTP-binding protein (G protein) linking to metabotropic glutamate receptor of bovine retinal on-bipolar cell was studied by use of pharmacologically selective ligands, 2-amino-4-phosphonobutyric acid (APB) on bacterial toxin-catalyzed ADP-ribosylation and GTP gamma S-binding. In contrast to the electrophysiological findings reported, G protein coupling to APB-sensitive glutamate receptor served as a substrate for pertussis toxin but did not for cholera toxin. Several glutamate analogues effective on on-bipolar cell, as well as APB, increased GTP gamma S binding to retinal membranes devoid of rod outer segments. The enhancement of GTP gamma S binding by APB was completely abolished when the membranes were pretreated with pertussis toxin and NAD. These results suggest that, in retinal on-bipolar cell, the G protein which couples metabotropic glutamate receptor to hyperpolarizing response of the cell is sensitive to pertussis toxin.

  Aug. 1993, Biochemical and biophysical research communications, 195(1) (1), 374 - 9, English, International magazine

  Scientific journal


• Activation of nucleoside diphosphate kinase by mastoparan, a peptide isolated from wasp venom.

  S Kikkawa, K Takahashi, K Takahashi, N Shimada, M Ui, N Kimura, T Katada

  We have previously reported that GDP-bound alpha beta gamma-trimeric GTP-binding (G) proteins can be converted into the active GTP-bound form with nucleoside diphosphate (NDP) kinase and ATP, although its exact activation mechanism still remains to be resolved. In the present study, we investigated whether NDP kinase activity was modified by mastoparan, a wasp venom peptide that is known to activate G proteins as an agonist-receptor complex. The activity of NDP kinase measured by the formation of GTP from ATP and GDP was markedly stimulated, when the kinase was incubated with mastoparan. The concentration of mastoparan required for the activation was much lower than that observed for the peptide-induced activation of G proteins under similar assay conditions. There was also an increase in the phosphorylated intermediate of NDP kinase as well as the catalytic activity upon its incubation with mastoparan. These results suggest that mastoparan not only activates G proteins directly via guanine nucleotide exchange reaction but also stimulates NDP kinase activity.

  Jul. 1992, FEBS letters, 305(3) (3), 237 - 40, English, International magazine

  Scientific journal


• Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins.

  S Kikkawa, K Takahashi, K Takahashi, N Shimada, M Ui, N Kimura, T Katada

  A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.

  Dec. 1990, The Journal of biological chemistry, 265(35) (35), 21536 - 40, English, International magazine

  Scientific journal


• Identification of sites for alkylation by N-ethylmaleimide and pertussis toxin-catalyzed ADP-ribosylation on GTP-binding proteins.

  S Hoshino, S Kikkawa, K Takahashi, H Itoh, Y Kaziro, H Kawasaki, K Suzuki, T Katada, M Ui

  An alpha beta gamma-trimeric GTP-binding protein (Go) serving as the substrate of pertussis toxin-(IAP) catalyzed ADP-ribosylation was purified from rat brain membranes. The constituent alpha-subunit (alpha o) was alkylated with N-ethylmaleimide (NEM), and the functionally important sulfhydryl groups were investigated. There were at least two cysteine residues highly reactive to NEM on the GDP-bound form of alpha o. These alkylations resulted in loss of its ability to be ADP-ribosylated by IAP and to associate with beta gamma, but leaving the GTP-binding site of alpha o intact. The reacted cysteine residues were identified by the sequencing of tryptic fragments of alpha o. One of the alkylation sites was Cys-351, which was four amino acid residues away from the carboxyl-terminus of the molecule. The Cys-351 was proven to be also a site for IAP-catalyzed ADP-ribosylation. Possible roles of cysteine residues on the alpha-subunit of Go are discussed in the functions of the signal transducing protein.

  Dec. 1990, FEBS letters, 276(1-2) (1-2), 227 - 31, English, International magazine

  Scientific journal


• Esterification of chiral secondary alcohols with fatty acid in organic solvents by polyethylene glycol-modified lipase.

  S Kikkawa, K Takahashi, T Katada, Y Inada

  Lipase from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase (PEG-lipase) was soluble and active in organic solvents such as benzene and 1,1,1-trichloroethane. PEG-lipase catalyzed esterification of chiral secondary alcohols with fatty acids in benzene and exhibited preference for R isomers over S isomers. Km and Vmax values for each isomer of various alcohols were obtained by kinetic study of the esterification in benzene. PEG-lipase-catalyzed esterification leads to optical resolution of a racemic alcohol.

  Nov. 1989, Biochemistry international, 19(5) (5), 1125 - 31, English, International magazine

  Scientific journal


• Purification of GTP-binding proteins from bovine brain membranes. Identification of heterogeneity of the alpha-subunit of Go proteins.

  I Kobayashi, H Shibasaki, K Takahashi, S Kikkawa, M Ui, T Katada

  Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with alpha beta gamma-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as alpha-subunit alone and was also ADP-ribosylated by IAP in the presence of beta gamma-subunits. When each alpha-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as Gi-1 and Gi-2, and other 4 appeared to be Go or Go-like G proteins. The alpha-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the alpha-subunit of Go in the brain membranes.

  Oct. 1989, FEBS letters, 257(1) (1), 177 - 80, English, International magazine

  Scientific journal

• MAPs: microtubule associated proteins

  吉川知志, 仁田英里子, 今崎剛, 仁田亮

  (公財)金原一郎記念医学医療振興財団, Aug. 2020, 生体の科学, 71(4) (4), 298 - 303, Japanese


• 中腸相当部に回転異常を示す一例

  渡邊優子, 吉川知志, 荒川高光

  2017, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 122nd


• Dab1遺伝子異常マウスにおけるオリーブ蝸牛束の構築異常の解明

  井之口 豪, 薛 富義, 吉川 知志, 寺島 俊雄, 四宮 瞳, 藤田 岳, 山下 大介, 丹生 健一

  耳鼻咽喉科ニューロサイエンス研究会, May 2016, 耳鼻咽喉科ニューロサイエンス, 30, 20 - 22, Japanese


• 大脳皮質の神経細胞移動制御におけるDab1の新奇作用機序

  吉川知志, 並河知宏, 寺島俊雄

  2016, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 121st


• CRISPR-Cas9システムを用いたReelinノックアウトゼブラフィッシュの作成と中枢神経系の構造解析

  福田晃, 崎浜吉昭, 寺島俊雄, 吉川知志

  2016, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 121st


• 献体登録業務の現況と問題点（補遺）--全国アンケート調査の結果から--

  Terashima Toshio, 薛 富義, 崎浜 吉昭, 吉川 知志

  篤志解剖全国連合会, Mar. 2014, 篤志献体, 56, 71 - 79, Japanese

  [Invited]

  Introduction other


• ゼブラフィッシュdpf1遺伝子の新規転写産物の解析

  鷲見拓哉, 崎浜吉昭, 寺島俊雄, 吉川知志

  2014, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 119th


• 発生期大脳皮質におけるDab1の分子内機能制御機構の解析

  並河知宏, 寺島俊雄, 吉川知志

  2013, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 118th


• ゼブラフィッシュ小脳皮質発生におけるReelinシグナルの機能解析

  吉川知志, 角本貴進, 崎浜吉昭, 寺島俊雄

  2013, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 118th


• エンハンサートラップを用いたdpf1-GFPトランスジェニックゼブラフィッシュの作製と発現解析

  吉川 知志, 角本 貴進, 野々村 達也, 藤本 昌大, 崎浜 吉昭, 寺島 俊雄

  (一社)日本解剖学会, Jun. 2012, 解剖学雑誌, 87(2) (2), 38 - 38, Japanese


• Intramolecular regulation of Dab1 protein function

  Satoshi Kikkawa, Yoshimi Takahashi, Tomohiro Namikawa, Toshio Terashima

  2011, NEUROSCIENCE RESEARCH, 71, E231 - E231, English

  Summary international conference


• ゼブラフィッシュ神経系の発生におけるReelinシグナルの機能解析(Functional analysis of Reelin signaling in the zebrafish neuronal development)

  佐藤 友加里, 寺島 俊雄, 吉川 知志

  日本神経化学会, Aug. 2010, 神経化学, 49(2-3) (2-3), 623 - 623, English


• ゼブラフィッシュの神経系発生におけるReelin(リーリン)シグナル系の機能解析

  吉川知志, 佐藤友加里, 寺島俊雄

  2010, 解剖学雑誌, 85(Supplement) (Supplement)


• Functional analysis of Reelin signaling in the zebrafish neuronal development

  Yukari Sato, Toshio Terashima, Satoshi Kikkawa

  2010, NEUROSCIENCE RESEARCH, 68, E247 - E247, English

  Summary international conference


• ゼブラフィッシュDab1とReelin受容体の相互作用解析(Analysis of interaction between zebra fish Dab1 and Reelin receptors)

  高橋 愛美, 寺島 俊雄, 吉川 知志

  (公社)日本生化学会, Sep. 2009, 日本生化学会大会プログラム・講演要旨集, 82回, 4P - 408, English


• Functional analysis of reelin signaling in the zebrafish neuronal development

  Yukari Sato, Toshio Terashima, Satoshi Kikkawa

  2009, NEUROSCIENCE RESEARCH, 65, S97 - S97, English

  Summary international conference


• Role of endocannabinoid metabolic pathway and CB2 receptor in regulation of cerebellar Purkinje cell dendrogenesis

  Tomomi Habara, Koji Kawaguchi, Toshio Terashima, Satoshi Kikkawa

  2009, NEUROSCIENCE RESEARCH, 65, S97 - S98, English

  Summary international conference


• Analysis of the reelin signaling cascade in the zebrafish central nervous system

  Satoshi Kikkawa, Koji Kawaguchi, Ai Takano, Yu Katsuyama, Toshio Terashima

  2008, NEUROSCIENCE RESEARCH, 61, S164 - S164, English

  Summary international conference


• Reelin/Dab1 signaling is required for formation of superficial layers of the superior colliculus

  Toshio Terashima, Yasuo Yoshihara, Hideyuki Dekimoto, Tomiyoshi Setsu, Yu Katsuyama, Satoshi Kikkawa

  2008, NEUROSCIENCE RESEARCH, 61, S160 - S160, English

  Summary international conference


• ゼブラフィッシュおよびマウス脳におけるER81の発現比較

  大宮 由裕, 出來本 秀行, 吉川 知志, 寺島 俊雄, 勝山 裕

  (一社)日本解剖学会, Sep. 2007, 解剖学雑誌, 82(3) (3), 116 - 116, Japanese


• Comparison of ER81 expression between the brains of zebrafish and mouse

  Hideyuki Dekimoto, Yoshihiro Oomiya, Satoshi Kikkawa, Toshio Terashima, Yu Katsuyama

  2006, NEUROSCIENCE RESEARCH, 55, S238 - S238, English

  Summary international conference


• Abnormal Laminar Structure of the Cerebral Cortex of Shaking Rat Kawasaki (SRK) in Comparison with the Reeler and Yotari Malformation.

  寺島俊雄, 吉川知志

  厚生労働省精神・神経疾患研究班, Mar. 2003, 発達期における高次脳機能障害の病態解明に関する研究総括研究報告書 平成15年, 平成12年度, 107 - 112, Japanese


• 皮質上丘路の形成とリーリンタンパク

  寺島 俊雄, 榊原 俊介, 美崎 佳寿代, 山本 達朗, 薛 富義, 吉川 知志

  (一社)日本解剖学会, Mar. 2002, 解剖学雑誌, 77(Suppl.) (Suppl.), J.A.A.28 - J.A.A.28, Japanese


• 生後発育期ラット脳白質中におけるリーリン免疫陽性細胞の存在

  美崎佳寿代, 吉川知志, 寺島俊雄

  2002, 日本神経科学大会プログラム・抄録集, 25th


• 新生仔マウス脳幹におけるリーリンmRNAの発現

  薜 富義, 奥山 綾子, 山本 達朗, 美崎 佳寿代, 吉川 知志, 寺島 俊雄

  日本神経化学会, Sep. 2001, 神経化学, 40(2-3) (2-3), 460 - 460, Japanese


• 嗅上皮障害後の嗅球におけるリーリン遺伝子発現の変化

  奥山 綾子, 山本 達朗, 吉川 知志, 三木 明徳, 寺島 俊雄

  (一社)日本解剖学会, Feb. 2001, 解剖学雑誌, 76(1) (1), 145 - 145, Japanese


• 新生仔マウス下位脳幹におけるリーリンmRNAの発現

  薛 富義, 山本 達朗, 吉川 知志, 寺島 俊雄

  (一社)日本解剖学会, Feb. 2001, 解剖学雑誌, 76(1) (1), 114 - 114, Japanese


• 生後発育期におけるShaking Rat Kawasaki脳内のリーリン遺伝子発現

  山本 達朗, 吉川 知志, 寺島 俊雄

  (一社)日本解剖学会, Feb. 2001, 解剖学雑誌, 76(1) (1), 114 - 114, Japanese


• 新生仔マウス脳幹におけるリーリンmRNAの発現

  へい富義, 奥山綾子, 山本達朗, 美崎佳寿代, 吉川知志, 寺島俊雄

  2001, 日本神経科学大会プログラム・抄録集, 24th


• リーラーフェノタイプを示す神経奇形ラット・Shaking Rat Kawasaki(SRK)における変異遺伝子リーリンの構造解析

  吉川 知志, 山本 達郎, 寺島 俊雄

  (公社)日本生化学会, Aug. 2000, 生化学, 72(8) (8), 843 - 843, Japanese


• リーラーフェノタイプを示す神経奇形ラット,Shaking rat Kawasaki(SRK)のリーリン遺伝子における変異の解析

  吉川 知志, 寺島 俊雄

  (一社)日本解剖学会, Feb. 2000, 解剖学雑誌, 75(1) (1), 118 - 118, Japanese


• 生後発育期におけるShaking Rat Kawasaki(SRK)脳のリーリン遺伝子の発現

  山本達朗, 吉川知志, せつ富義, 寺島俊雄

  2000, 日本神経科学大会プログラム・抄録集, 23rd


• リーラーフェノタイプを示す神経奇形ラット・Shaking Rat Kawasaki(SRK)におけるリーリン遺伝子の変異解析

  吉川知志, 山本達朗, 寺島俊雄

  2000, 日本神経科学大会プログラム・抄録集, 23rd


• Molecular biology of the normal and abnormal development of the brain. Developmental anomaly of the cerebral cortex with special reference to the reeler mouse.

  寺島俊雄, 吉川知志

  (株)星和書店, Dec. 1999, 脳の科学, 21(12) (12), 1319 - 1324, Japanese


• リーラーフェノタイプを示す神経奇形ラット・Shaking rat Kawasaki(SRK)のリーリン遺伝子の解析

  吉川 知志, 寺島 俊雄

  (一社)日本生物物理学会, Sep. 1999, 生物物理, 39(Suppl.1) (Suppl.1), S56 - S56, Japanese


• リーラーフェノタイプを示す神経奇形ラット・Shaking rat Kawasaki(SRK)のリーリン遺伝子の解析

  吉川 知志, 寺島 俊雄

  (公社)日本生化学会, Aug. 1999, 生化学, 71(8) (8), 1040 - 1040, Japanese


• Cytoarchitectonic Abnormality in the Facial Nucleus of the Reeler Mouse.

  寺島俊雄, せつ富義, 吉川知志, 池田やよい

  (一社)日本解剖学会, Jul. 1999, 解剖学雑誌, 74(4) (4), 411 - 420, Japanese


• ヨタリマウスとリーラーマウスの異所性皮質脊髄路ニューロンの比較

  山本達朗, 吉川知志, 御子柴克彦, 寺島俊雄

  1999, 日本神経科学大会プログラム・抄録集, 22nd


• リーラーフェノタイプを示す新規神経奇形ラット・SRKにおけるリーリンの発現異常

  吉川 知志, 池田 やよい, 岡戸 晴生, 小川 正晴, 寺島 俊雄

  日本神経化学会, Sep. 1998, 神経化学, 37(3) (3), 553 - 553, Japanese


• リーラー様新規神経奇形ラット・SRKの脳におけるリーリンの発現異常

  吉川 知志, 池田 やよい, 岡戸 晴生, 小川 正晴, 寺島 俊雄

  (一社)日本生物物理学会, Sep. 1998, 生物物理, 38(Suppl.2) (Suppl.2), S198 - S198, Japanese


• リーラーフェノタイプを示す新規神経奇形ラット・SRKにおけるリーリンの発現異常

  吉川 知志, 池田 やよい, 岡戸 晴生, 小川 正晴, 寺島 俊雄

  (公社)日本生化学会, Aug. 1998, 生化学, 70(8) (8), 899 - 899, Japanese


• タコ視細胞におけるGo,Gqとロドプシンのカップリング

  進藤英雄, 吉川知志, 中川将司, 岩佐達郎, 津田基之

  1998, 日本生物物理学会年会講演予稿集, 36th


• タコ視細胞におけるロドプシンと共役する複数のG蛋白質

  進藤英雄, 馬場継, 岩佐達郎, 吉川知志, 津田基之

  1997, 日本生物物理学会年会講演予稿集, 35th


• マボヤの光に誘導される配偶子放出と頭部神経節の光レセプター

  大熊真人, 柴田直樹, 吉川知志, 津田基之

  1997, 日本生物物理学会年会講演予稿集, 35th


• ほ乳動物βARK型であるタコロドプシンキナーゼの発現部位は?

  吉田典弘, 吉川知志, 岩佐達郎, 津田基之

  1997, 日本生物物理学会年会講演予稿集, 35th


• Gタンパク質および光受容体キナーゼの多様性

  吉川知志, 中川将司, 岩佐達郎, 津田基之

  1997, 日本生物物理学会年会講演予稿集, 35th


• タコロドプシンの結晶化条件の検索

  井上明美, 大熊真人, 橋本弥生, 吉川知志, 津田基之

  1997, 日本生物物理学会年会講演予稿集, 35th


• Purification of rhodopsin kinase in invertebrate animal (octopus) and activation by G protein .BETA..GAMMA. subunit.

  吉川知志, 吉田典弘, 津田基之

  1996, 日本分子生物学会年会プログラム・講演要旨集, 19th


• タコロドプシンキナーゼ遺伝子のクローニング

  吉川知志, 吉田典弘, 岩佐達郎, 津田基之

  1996, 日本生物物理学会年会講演予稿集, 34th


• タコ視細胞膜で見いだされた新しいタイプのロドプシンキナーゼ

  吉田典弘, 吉川知志, 津田基之

  1996, 日本生物物理学会年会講演予稿集, 34th


• O-linked Carbohydrade of Octopus Rhodopsin.

  宮本貴幸, 中川将司, 岩佐達郎, 吉川知志, 高尾敏文, 下西康嗣, 高崎誠一, 津田基之

  1995, 日本生物物理学会年会講演予稿集, 33rd


• Purification of phospholipase C from octopus photoreceptor.

  吉川知志, 中川将司, 津田基之

  1995, 日本生物物理学会年会講演予稿集, 33rd


• Is the tyrosine residue the retinal Shiff base counter ion in octopus rhodopsin?

  中川将司, 岩佐達郎, 吉川知志, 津田基之

  1995, 日本生物物理学会年会講演予稿集, 33rd


• Activation of G protein by illuminated rhodopsin in octopus photoreceptor membranes.

  冨永佳世, 吉川知志, 津田基之

  1995, 日本生物物理学会年会講演予稿集, 33rd


• A soluble Gq protein in octopus photoreceptor cells.

  吉川知志, 冨永佳世, 氷見政営, 中川将司, 岩佐達郎, 津田基之

  1994, 日本生物物理学会年会講演予稿集, 32nd


• Signal transduction via metabotropic glutamate receptor in retinal ON-bipolar cells.

  吉川知志, 津田基之

  1994, 日本生物物理学会年会講演予稿集, 32nd


• G-protein coupling transduction system in visualk cell and second neuron in retina.

  津田基之, 吉川知志, 岩佐達郎, 中川将司

  1994, 日本分子生物学会年会プログラム・講演要旨集, 17th


• Study of Signal Coupling Proteins in Phototransduction in Invertebrate. III. G-Proteins.

  柳井孝則, 岩佐達郎, 東隆寛, 吉川知志, 中川将司, 津田基之

  1994, 日本生物物理学会年会講演予稿集, 32nd


• Photochemical Intermediates of Octopus Rhodopsin with UV/VIS Time-Resolved Absorption Spectroscopy. (II).

  中川将司, 高原幸司, 藤中智徳, 吉川知志, 岩佐達郎, 津田基之

  1994, 日本生物物理学会年会講演予稿集, 32nd


• GTP-binding proteins coupling to glutamate receptors on bovine retinal membranes.

  S Kikkawa, M Nakagawa, T Iwasa, M Tsuda

  20 Dec. 1993, Annals of the New York Academy of Sciences, 707, 557 - 60, English, International magazine


• Isolation and characterization of two photo-activated G proteins, Gip and Gqp, in octopus photoreceptor.

  吉川知志, 冨永佳世, 土屋敬広, 中川将司, 岩佐達郎, 津田基之

  1993, 日本生物物理学会年会講演予稿集, 31st


• Study of Signal Coupling Proteins in Phototransduction in Iverteb rate. II: G-Proteins.

  岩佐達郎, 柳井孝則, 東隆寛, 吉川知志, 中川将司, 津田基之

  1993, 日本生物物理学会年会講演予稿集, 31st


• Photochemical Intermediates of Octopusrhodopsin with UV/VIS Time-resolved Absorption Spectroscopy.

  中川将司, 赤羽正平, 藤中智徳, 吉川知志, 岩佐達郎, 津田基之

  1993, 日本生物物理学会年会講演予稿集, 31st


• Retrieval of GTP binding protein related with a glutamate receptor which exists in a cattle retina secondary neurone.

  吉川知志, 津田基之

  日本神経化学会, Sep. 1992, 神経化学, 31(1) (1), 56 - 57, Japanese


• ウシ網膜2次ニューロンに存在する,グルタミン酸受容体と連関するGTP結合蛋白質の検索

  吉川知志, 中川将司, 岩佐達郎, 金子章道, 津田基之

  1992, 日本生物物理学会年会講演予稿集, 30th


• 光反応に伴うタコロドプシンの構造変化

  中川将司, 吉川知志, 岩佐達郎, 津田基之

  1992, 日本生物物理学会年会講演予稿集, 30th


• Imaging Today. Function of Rhodopsin-Photosensor.

  津田基之, 岩佐達郎, 中川将司, 吉川知志

  1992, 電子写真学会誌, 31(4) (4)


• ヌクレオシド二リン酸キナーゼによるG蛋白質の活性化

  吉川 知志

  (公社)日本生化学会, Jul. 1990, 生化学, 62(7) (7), 731 - 731, Japanese


• Organic solvent solubilization and magnetization of enzyme proteins. Functional conversion of Lipase by chemical modification.

  大和田きみ子, 吉川知志, 高橋勝宣

  (公社)日本農芸化学会, Jul. 1988, 化学と生物, 26(7) (7), 454 - 455, Japanese


• Chemical modification of proteins to alter and improve thier functions.

  吉川知志, 稲田祐二

  1988, 月刊細胞, 20(7) (7)


• 有機溶媒可溶化酵素(1) 修飾リパーゼによるホスファチジルコリンのエステル交換

  吉川 知志

  (公社)日本生化学会, Aug. 1987, 生化学, 59(8) (8), 795 - 795, Japanese

• A possible novel mode of action for Dab1 in modulation of neuronal migration of neocortical neurons

  Satoshi KIKKAWA, Tomohiro NAMIKAWA, Toshio TERASHIMA

  “Neuroscience 2016” Society for Neuroscience 46th annual meeting, Nov. 2016, English, Society for Neuroscience, San Diego, CA, United States, International conference

  Poster presentation


• New functional role of Dab1 protein in neuronal migration of the cerebral cortex

  Kikkawa Satoshi, 並河知宏, Terashima Toshio

  The 121st Annual Meeting of the Japanese Association of Anatomists, Mar. 2016, Japanese, the Japanese Association of Anatomists, 郡山, Domestic conference

  Poster presentation


• Preparation of reelin-KO zebra fish with an use of CRISPER-Cas9 system

  福田晃, 崎浜吉昭, Terashima Toshio, Kikkawa Satoshi

  The 121st Annual Meeting of the Japanese Association of Anatomists, Mar. 2016, Japanese, the Japanese Association of Anatomists, 郡山, Domestic conference

  Poster presentation


• Dab1遺伝子異常マウスにおけるオリーブ蝸牛束の構築異常の解明

  Inokuchi Gou, 薛富義, Kikkawa Satoshi, Terashima Toshio, 四宮瞳, Yamashita Daisuke, Nibu Ken-ichi

  第33回耳鼻咽喉科ニューロサイエンス研究会, Aug. 2015, Japanese, 神戸大学耳鼻咽喉科, 大阪, Domestic conference

  Oral presentation


• Cytoarchitecture of the cerebellum in the laggard mutant mouse.

  ジュナエヂィ ユヌス, 薛富義, Kikkawa Satoshi, Sakisaka Toshiaki, Terashima Toshio

  The 38th Japan Neuroscience Meeting, Jul. 2015, Japanese, The Japan Neuroscience Association, 神戸, Domestic conference

  Poster presentation


• Intramolecular interaction between amino and carboxyl terminal regions of Disabled-1 (Dab1) modulates its activity in migrating cortical neurons.

  Namikawa Tomohiro, Terashima Toshio, Kikkawa Satoshi

  The 38th Japan Neuroscience Meeting, Jul. 2015, Japanese, The Japan Neuroscience Association, 神戸, Domestic conference

  Poster presentation


• Cytoarchitecture of the cerebellum in the laggard mutant mouse.

  Junaedy Yunus, Tomiyoshi Setsu, Kikkawa Satoshi, Sakisaka Toshiaki, Terashima Toshio

  第120回日本解剖学会総会, Mar. 2015, English, 日本解剖学会, 神戸市, Domestic conference

  Poster presentation


• Functional analysis of the Reelin-Dab1 signal pathway in the developing zebrafish nervous system.

  Kikkawa Satoshi, Takayuki Sumimoto, Takuya Sumi, Yoshiaki Sakihama, Terashima Toshio

  “Neuroscience 2014” Society for Neuroscience 44th annual meeting, Nov. 2014, English, Society for Neuroscience, Washington, DC, USA, International conference

  Poster presentation


• Analysis of the intramolecular regulatory mechanism of Dab1 in the developing neocortex

  並河知宏, Terashima Toshio, Kikkawa Satoshi

  第37回日本神経科学大会, Sep. 2014, Japanese, 日本神経科学会, 横浜, Domestic conference

  Poster presentation


• 発生期大脳皮質におけるDab1 の分子内機能制御機構の解析

  並河 知宏, Terashima Toshio, Kikkawa Satoshi

  第118回日本解剖学会総会, Mar. 2013, Japanese, 日本解剖学会, 高松, Domestic conference

  Poster presentation


• ゼブラフィッシュ小脳皮質発生におけるReelinシグナルの機能解析

  Kikkawa Satoshi, 角本 貴進, 崎浜 吉昭, Terashima Toshio

  第118回日本解剖学会総会, Mar. 2013, Japanese, 日本解剖学会, 高松, Domestic conference

  Poster presentation


• ゼブラフィッシュ中枢神経系の発生におけるReelin-Dab1 シグナル経路の機能的解析

  角本 貴進, 崎浜 吉昭, Terashima Toshio, Kikkawa Satoshi

  第35回日本神経科学大会, Sep. 2012, Japanese, 日本神経科学学会, 名古屋, Domestic conference

  Poster presentation


• Dab1N末端フラグメントの発現は発生期大脳皮質において異所的な細胞集積を誘導する

  並河 知宏, Terashima Toshio, Kikkawa Satoshi

  第35回日本神経科学大会, Sep. 2012, Japanese, 日本神経科学学会, 名古屋, Domestic conference

  Poster presentation


• Analysis of transgenic zebrafish carrying a GFP transgene in the neuron-specific BAF subunit dpf1 locus

  Kikkawa Satoshi, 角本 貴進, 崎浜 吉昭, Terashima Toshio

  第117回日本解剖学会総会全国学術集会, Mar. 2012, Japanese, 日本解剖学会, 西宮, Domestic conference

  Oral presentation


• Intramolecular regulation of Dab1 protein function

  Kikkawa Satoshi, Takahashi Yoshimi, Namikawa Tomohiro, Terashima Toshio

  第34回日本神経科学大会, Sep. 2011, Japanese, 日本神経科学学会, 横浜, Domestic conference

  Poster presentation


• Functional analysis of Reelin signaling in the zebrafish neuronal development

  佐藤 友加里, Terashima Toshio, Kikkawa Satoshi

  第33回日本神経科学大会, Sep. 2010, Japanese, 日本神経科学学会, 神戸, Domestic conference

  Poster presentation


• Functional analysis of Reeling signaling in neurogenesis of zabrafish

  Kikkawa Satoshi, Terashima Toshio

  第115回日本解剖学会総会・全国学術集会, Mar. 2010, Japanese, 日本解剖学会, 盛岡, Domestic conference

  Poster presentation


• Analysis of interaction of zebrafish Dab1 and Reelin receptors

  Terashima Toshio, Kikkawa Satoshi

  第82回日本生化学会大会, Oct. 2009, Japanese, 日本生化学学会, 神戸, Domestic conference

  Poster presentation


• Functional analysis of the Reelin signaling pathway in zebrafish.

  Kikkawa Satoshi, Terashima Toshio

  Construction and Reconstruction of the Brain, Oct. 2009, English, Construction and Reconstruction of the Brain, 淡路, International conference

  Oral presentation


• Role of endocannabinoid metabolic pathway and CB2 receptor in regulation of cerebellar Purkinje cell dendrogenesis)

  Terashima Toshio, Kikkawa Satoshi

  第32回日本神経科学大会, Sep. 2009, Japanese, 日本神経科学学会, 名古屋, Domestic conference

  Poster presentation


• Functional analysis of reelin signaling in the zebrafish neuronal development

  Terashima Toshio, Kikkawa Satoshi

  第32回日本神経科学大会, Sep. 2009, Japanese, 日本神経科学学会, 名古屋, Domestic conference

  Poster presentation


• Reelin/Dab1 signaling is required for formation of superficial layers of the superior colliculus

  Terashima Toshio, Katsuyama Yu, Kikkawa Satoshi

  第33回日本神経科学大会, Jul. 2008, Japanese, 日本神経科学学会, 東京, Domestic conference

  Poster presentation


• Analysis of the reelin signaling cascade in the zebrafish central nervous system

  Kikkawa Satoshi, Katsuyama Yu, Terashima Toshio

  第35回日本神経科学大会, Jul. 2008, Japanese, 日本神経科学学会, 東京, Domestic conference

  Poster presentation


• Endocannabinoids released from cerebellar Purkinje cells regulate their dendrogenesis by retrograde signaling via GABAergic interneurons

  Terashima Toshio, Kikkawa Satoshi

  第34回日本神経科学大会, Jul. 2008, Japanese, 日本神経科学学会, 東京, Domestic conference

  Poster presentation


• Comparison of Er81 expression between the brains of zebrafish and mouse

  KIKKAWA SATOSHI, TERASHIMA TOSHIO, Katsuyama Yu

  第82回日本解剖学会近畿地区学術集会, Nov. 2006, Japanese, 日本解剖学会, 大阪, Domestic conference

  Oral presentation


• Comparison of Er81 expression between the brains of zebrafish and mouse

  KIKKAWA SATOSHI, TERASHIMA TOSHIO, Katsuyama Yu

  第29回日本神経科学大会, Jul. 2006, Japanese, 日本神経科学学会, 京都, Domestic conference

  Poster presentation


• Comparison of ER81 expression between the brains of zebrafish and mouse

  Hideyuki Dekimoto, Yoshihiro Oomiya, Satoshi Kikkawa, Toshio Terashima, Yu Katsuyama

  NEUROSCIENCE RESEARCH, 2006, English, ELSEVIER IRELAND LTD


• 皮質上丘路の形成とリーリンタンパク

  寺島 俊雄, 榊原 俊介, 美崎 佳寿代, 山本 達朗, 薛 富義, 吉川 知志

  解剖学雑誌, Mar. 2002, Japanese, (一社)日本解剖学会


• 生後発育期ラット脳白質中におけるリーリン免疫陽性細胞の存在

  美崎佳寿代, 吉川知志, 寺島俊雄

  日本神経科学大会プログラム・抄録集, 2002


• 新生仔マウス脳幹におけるリーリンmRNAの発現

  薜 富義, 奥山 綾子, 山本 達朗, 美崎 佳寿代, 吉川 知志, 寺島 俊雄

  神経化学, Sep. 2001, Japanese, 日本神経化学会


• 生後発育期におけるShaking Rat Kawasaki脳内のリーリン遺伝子発現

  山本 達朗, 吉川 知志, 寺島 俊雄

  解剖学雑誌, Feb. 2001, Japanese, (一社)日本解剖学会


• 新生仔マウス下位脳幹におけるリーリンmRNAの発現

  薛 富義, 山本 達朗, 吉川 知志, 寺島 俊雄

  解剖学雑誌, Feb. 2001, Japanese, (一社)日本解剖学会


• 嗅上皮障害後の嗅球におけるリーリン遺伝子発現の変化

  奥山 綾子, 山本 達朗, 吉川 知志, 三木 明徳, 寺島 俊雄

  解剖学雑誌, Feb. 2001, Japanese, (一社)日本解剖学会


• 新生仔マウス脳幹におけるリーリンmRNAの発現

  へい富義, 奥山綾子, 山本達朗, 美崎佳寿代, 吉川知志, 寺島俊雄

  日本神経科学大会プログラム・抄録集, 2001


• リーラーフェノタイプを示す神経奇形ラット・Shaking Rat Kawasaki(SRK)における変異遺伝子リーリンの構造解析

  吉川 知志, 山本 達郎, 寺島 俊雄

  生化学, Aug. 2000, Japanese, (公社)日本生化学会


• リーラーフェノタイプを示す神経奇形ラット,Shaking rat Kawasaki(SRK)のリーリン遺伝子における変異の解析

  吉川 知志, 寺島 俊雄

  解剖学雑誌, Feb. 2000, Japanese, (一社)日本解剖学会


• リーラーフェノタイプを示す神経奇形ラット・Shaking Rat Kawasaki(SRK)におけるリーリン遺伝子の変異解析

  吉川知志, 山本達朗, 寺島俊雄

  日本神経科学大会プログラム・抄録集, 2000


• 生後発育期におけるShaking Rat Kawasaki(SRK)脳のリーリン遺伝子の発現

  山本達朗, 吉川知志, せつ富義, 寺島俊雄

  日本神経科学大会プログラム・抄録集, 2000


• リーラーフェノタイプを示す神経奇形ラット・Shaking rat Kawasaki(SRK)のリーリン遺伝子の解析

  吉川 知志, 寺島 俊雄

  生物物理, Sep. 1999, Japanese, (一社)日本生物物理学会


• リーラーフェノタイプを示す神経奇形ラット・Shaking rat Kawasaki(SRK)のリーリン遺伝子の解析

  吉川 知志, 寺島 俊雄

  生化学, Aug. 1999, Japanese, (公社)日本生化学会


• Cytoarchitectonic Abnormality in the Facial Nucleus of the Reeler Mouse.

  寺島俊雄, せつ富義, 吉川知志, 池田やよい

  解剖学雑誌, Jul. 1999, Japanese, (一社)日本解剖学会


• ヨタリマウスとリーラーマウスの異所性皮質脊髄路ニューロンの比較

  山本達朗, 吉川知志, 御子柴克彦, 寺島俊雄

  日本神経科学大会プログラム・抄録集, 1999


• リーラー様新規神経奇形ラット・SRKの脳におけるリーリンの発現異常

  吉川 知志, 池田 やよい, 岡戸 晴生, 小川 正晴, 寺島 俊雄

  生物物理, Sep. 1998, Japanese, (一社)日本生物物理学会


• リーラーフェノタイプを示す新規神経奇形ラット・SRKにおけるリーリンの発現異常

  吉川 知志, 池田 やよい, 岡戸 晴生, 小川 正晴, 寺島 俊雄

  神経化学, Sep. 1998, Japanese, 日本神経化学会


• リーラーフェノタイプを示す新規神経奇形ラット・SRKにおけるリーリンの発現異常

  吉川 知志, 池田 やよい, 岡戸 晴生, 小川 正晴, 寺島 俊雄

  生化学, Aug. 1998, Japanese, (公社)日本生化学会


• タコ視細胞におけるGo,Gqとロドプシンのカップリング

  進藤英雄, 吉川知志, 中川将司, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1998


• タコロドプシンの結晶化条件の検索

  井上明美, 大熊真人, 橋本弥生, 吉川知志, 津田基之

  日本生物物理学会年会講演予稿集, 1997


• Gタンパク質および光受容体キナーゼの多様性

  吉川知志, 中川将司, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1997


• ほ乳動物βARK型であるタコロドプシンキナーゼの発現部位は?

  吉田典弘, 吉川知志, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1997


• マボヤの光に誘導される配偶子放出と頭部神経節の光レセプター

  大熊真人, 柴田直樹, 吉川知志, 津田基之

  日本生物物理学会年会講演予稿集, 1997


• タコ視細胞におけるロドプシンと共役する複数のG蛋白質

  進藤英雄, 馬場継, 岩佐達郎, 吉川知志, 津田基之

  日本生物物理学会年会講演予稿集, 1997


• タコ視細胞膜で見いだされた新しいタイプのロドプシンキナーゼ

  吉田典弘, 吉川知志, 津田基之

  日本生物物理学会年会講演予稿集, 1996


• タコロドプシンキナーゼ遺伝子のクローニング

  吉川知志, 吉田典弘, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1996


• Purification of rhodopsin kinase in invertebrate animal (octopus) and activation by G protein .BETA..GAMMA. subunit.

  吉川知志, 吉田典弘, 津田基之

  日本分子生物学会年会プログラム・講演要旨集, 1996


• Activation of G protein by illuminated rhodopsin in octopus photoreceptor membranes.

  冨永佳世, 吉川知志, 津田基之

  日本生物物理学会年会講演予稿集, 1995


• Is the tyrosine residue the retinal Shiff base counter ion in octopus rhodopsin?

  中川将司, 岩佐達郎, 吉川知志, 津田基之

  日本生物物理学会年会講演予稿集, 1995


• Purification of phospholipase C from octopus photoreceptor.

  吉川知志, 中川将司, 津田基之

  日本生物物理学会年会講演予稿集, 1995


• O-linked Carbohydrade of Octopus Rhodopsin.

  宮本貴幸, 中川将司, 岩佐達郎, 吉川知志, 高尾敏文, 下西康嗣, 高崎誠一, 津田基之

  日本生物物理学会年会講演予稿集, 1995


• Photochemical Intermediates of Octopus Rhodopsin with UV/VIS Time-Resolved Absorption Spectroscopy. (II).

  中川将司, 高原幸司, 藤中智徳, 吉川知志, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1994


• Study of Signal Coupling Proteins in Phototransduction in Invertebrate. III. G-Proteins.

  柳井孝則, 岩佐達郎, 東隆寛, 吉川知志, 中川将司, 津田基之

  日本生物物理学会年会講演予稿集, 1994


• G-protein coupling transduction system in visualk cell and second neuron in retina.

  津田基之, 吉川知志, 岩佐達郎, 中川将司

  日本分子生物学会年会プログラム・講演要旨集, 1994


• Signal transduction via metabotropic glutamate receptor in retinal ON-bipolar cells.

  吉川知志, 津田基之

  日本生物物理学会年会講演予稿集, 1994


• A soluble Gq protein in octopus photoreceptor cells.

  吉川知志, 冨永佳世, 氷見政営, 中川将司, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1994


• GTP-binding proteins coupling to glutamate receptors on bovine retinal membranes.

  S Kikkawa, M Nakagawa, T Iwasa, M Tsuda

  Annals of the New York Academy of Sciences, Dec. 1993, English


• Photochemical Intermediates of Octopusrhodopsin with UV/VIS Time-resolved Absorption Spectroscopy.

  中川将司, 赤羽正平, 藤中智徳, 吉川知志, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1993


• Study of Signal Coupling Proteins in Phototransduction in Iverteb rate. II: G-Proteins.

  岩佐達郎, 柳井孝則, 東隆寛, 吉川知志, 中川将司, 津田基之

  日本生物物理学会年会講演予稿集, 1993


• Isolation and characterization of two photo-activated G proteins, Gip and Gqp, in octopus photoreceptor.

  吉川知志, 冨永佳世, 土屋敬広, 中川将司, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1993


• Retrieval of GTP binding protein related with a glutamate receptor which exists in a cattle retina secondary neurone.

  吉川知志, 津田基之

  神経化学, Sep. 1992, Japanese, 日本神経化学会


• 光反応に伴うタコロドプシンの構造変化

  中川将司, 吉川知志, 岩佐達郎, 津田基之

  日本生物物理学会年会講演予稿集, 1992


• ウシ網膜2次ニューロンに存在する,グルタミン酸受容体と連関するGTP結合蛋白質の検索

  吉川知志, 中川将司, 岩佐達郎, 金子章道, 津田基之

  日本生物物理学会年会講演予稿集, 1992


• ヌクレオシド二リン酸キナーゼによるG蛋白質の活性化

  吉川 知志

  生化学, Jul. 1990, Japanese, (公社)日本生化学会


• 有機溶媒可溶化酵素(1) 修飾リパーゼによるホスファチジルコリンのエステル交換

  吉川 知志

  生化学, Aug. 1987, Japanese, (公社)日本生化学会

• 日本医学教育学会

  - Present


• 日本解剖学会

• Whole-body functional analysis of the BAF complex in the visualized zebrafish nervous system using genome editing

  KIKKAWA Satoshi, TERASHIMMA Toshio, SAKIHAMA Yoshiaki, SUMI Takuya, FUKUDA Koh

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research, Grant-in-Aid for Challenging Exploratory Research, Kobe University, 01 Apr. 2014 - 31 Mar. 2018

  When specialization (differentiation) of each tissue or organ occurs during development of an individual animal, a set of genes are selectively used in each tissue/organ. In order to investigate the role of the BAF complex that is thought to govern the regulation of gene expression in differentiation into nerve tissue, we used a genetically modified zebrafish whose nerve tissue is visualized with fluorescent proteins. Genetic disruption of BAF complex genes by genomic editing technology was conducted on these transgenic fish lines. Several lines of genome edited zebrafish disrupted in the BAF45b gene were generated. These fish lines will be very useful for a functional analysis of the BAF complex in development of the nervous system. We continue to study developmental dysfunction of the nervous system in these fish.


• The barrier structure of glial limitans in the hippocampal fissureis is broken in the normal mouse, but maintained in the reeler.

  TERASHIMA Toshio, KIKKAWA Satoshi, SETSU Tomiyoshi

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research, Grant-in-Aid for Challenging Exploratory Research, Kobe University, 01 Apr. 2012 - 31 Mar. 2015

  In the present study, we injected BDA into the entorhinal cortex of the normal, reeler and yotari mice to examine the course of the entorhinohippocampal fibers, i.e., perforant pathway. BDA-labeled entorhinohippocampal fibers penetrated through the hippocampal fissure in the normal mouse, but they could not penetrate through the hippocampal fissure in the reeler and yotari mice. Next, we placed DiI crystals into the entorhinal cortex of the normal, reeler, and yotari mice during early postnatal weeks. DiI-labeled entorhinohippocampal fibers penetrated through the hippocampal fissure on P1 in the reeler and yotari mice, but no fibers which penetrated through the fissure were identified until P4, and all of DiI-labeled fibers ran in parallel to the fissure instead of perforating it. GFAP-immunopositive astroglia were accumulated along the hippocampal fissure both in the reeler and yotari mice, which may results in obstruction of elongation of entorhinohippocampal projection.


• Studies on the neuron-specific transcriptional regulator Dpf1 using transgenic zebrafish expressing neuronal tracers

  KIKKAWA Satoshi, TERASHIMA Toshio

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research, Grant-in-Aid for Challenging Exploratory Research, Kobe University, 01 Apr. 2012 - 31 Mar. 2015

  We indentified a main dpf1 transcrypt, dpf1-002, in the zebrafish central nervous system and determined the nucleotide sequence and expression pattern in embryos. In addition, in the dpf1-tTA-GFP fish, the GFP transgene was transcrived from the start point different from that of dpf1-002, and the expression of endogenous dpf1-002 was not affected by the expression of the transgene. We intend functional inhibition using morpholino antisense oligonucleotides (MO) for dpf1-002, but unfortunately have not yet observed clear-cut developmental nerve defects in the injected embyros. Examination such as the coinjection of different MOs is necessary. The different gene targeting approach such ad genome editing may also be needed in future.


• Mechanism of laminar formation of cerebral cortex and layer-specific neural circuits

  TERASHIMA Toshio, KIKKAWA Satoshi, KATSUYAMA Yu

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 2008 - 2011

  Previous studies have elucidated many genes that are expressed in specific layers of cortical layers. Among these genes we chose four genes, i. e., mSorLA, ROR-beta, ER81 and Tbr1 and studied phenotypes of cerebral neocortex of the reeler mouse. It has been long considered that the reeler cortex is cytoarchitectually reversed. We have demonstrated that compaction of neuronal components expressing each layer marker is affected in the reeler cortex, which resulted in intermingling of different cortical neurons and blurred cortical layers. The similar abnormalities were also recognized in the dorsal horn of the spinal cord, dorsal cochlear nucleus and cerebellar neocortex of the reeler mouse, suggesting Reelin is essential for compaction of neurons. In the reeler cortex, ROR-beta expressing neurons are not widely scattered, suggesting compaction of ROR-beta expressing neurons-is regulated by molecules other than Reelin protein. In the reeler olfactory bulb, the cytoarchitectural abnormality is also subtle, and therefore formation of this laminar structure is regulated by Reelin-independent molecules. In conclusion, we could not identified reversed laminar structures of the reeler cortex as repeatedly reported by many previous reports, and the reeler malformation is caused by the intriguing outcomes of several mechanisms relating to Reelin protein.

  Competitive research funding


• 海馬裂における軟膜・グリア性境界膜の生理的分解と維持

  寺島 俊雄, 吉川 知志, 勝山 裕, 薛 富義

  日本学術振興会, 科学研究費助成事業 挑戦的萌芽研究, 挑戦的萌芽研究, 神戸大学, 2009 - 2010

  リーラーマウスの嗅内野海馬投射の経路と終末形成が正常であるか否かを明らかにする目的で、成体のリーラーマウスと対照動物の嗅内野の内側部皮質と外側部皮質にBDAを注入し、嗅内野海馬投射路を順行性に標識した。対照動物ではBDA標識嗅内野海馬投射線維は海馬裂を貫通するが、リーラーマウスの嗅内野海馬投射の一部の貫通線維は海馬裂を通過できずに、同所を迂回する線維を発見した。さらにリーラーの嗅内野内側部皮質から起こる嗅内野海馬投射線維終末には野生型では観察されなかった異常な終末が確認された(具体的に書く)。次に、我々は海馬の組織構築を調べる目的で、ラミニン抗体、グリア線維性酸性タンパク(GFAP)抗体を用いて生後0日(Postnatal day 0;P0)、P2、P3、P4および成体(生後6週)のリーラーマウスおよび同齢の対照動物の脳の免疫染色を行った。成体リーラーマウスでは、海馬裂におけるGFAP陽性のアストログリアの異常な集積(塊)が観察された。また、P0～P4正常マウスの海馬裂に、成体リーラーマウスの海馬裂におけるグリア細胞の集積に似たアストログリアの集積が観察された。このアストログリアによる細胞集積は、発生につれてその密度が減少していった。しかし、リーラーマウスではそのようなグリア細胞の集積の減少傾向は見られなかった。カルボシアニン蛍光色素DiIをパラホルムアルデヒド固定脳の嗅内野に注入し、生後発育期における嗅内野海馬投射の形成過程を調べたところ、正常マウスでは貫通線維はP3で始めて海馬裂を横断することが明らかとなった。おそらく、嗅内野海馬投射線維が海馬裂を貫通するためには海馬裂に集積したアストログリア細胞の減少が必須であるということが示唆された。

  Competitive research funding


• Astudy on reulatory mechanisms of glial basement membrane formation and neuronal migration using a fish model

  KIKKAWA Satoshi, TERASHIMA Toshio

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 2006 - 2007

  We cloned and sequenced the complete coding region of the zebrafish fukutin gene. Zebrafish fukutin showed a significant degree of sequence homology to mouse and human fukutin. By whole mount in situ hybridization, fukutin mRNA was detected in a wide variety of tissues, including the brain and the spinal cord, as early as 4 hours post-fertilization. We also examined expression patterns of zebrafish orthologs of other muscular dystrophy genes causing central nervous system defects, namely LARGE and POMGnT1. They were also expressed in a variety of tissues from the early developmental stage, implying that sugar-modifying enzymes play important roles in zebrafish CNS development as well. Next, we analyzed effects of knocking-down Fukutin protein expression on embryogenesis by morpholino antisense oligonucleotide (MO) microinjection into fertilized zebrafish eggs. Injected embryos (morphants) showed a broad range of developmental defects including skeletal and cardiac muscle defects in a dose-dependent manner, reflecting the broad expression of fukutin. We then looked into CNS defects in fukutin morphants using transgenic zebrafish expressing a fluorescent protein either in motor neurons (isl-GFP) or the entire differentiated neuronal pool (HuC-Kaede). Contrary to our expectations, we have not yet been able to find apparent defects of CNS development in both transgenic embryos injected with fukutin MO in spite of gross developmental defects. Further study in more detail is being undertaken for histological analysis.


• 上丘の層形成と視覚性神経回路形成に関するリーリンシグナル伝達系の機能

  寺島 俊雄, 吉川 知志, 薛 富義

  日本学術振興会, 科学研究費助成事業 特定領域研究, 特定領域研究, 神戸大学, 2006 - 2007

  上丘は第1〜3層からなる浅層と第4〜7層からなる深層に区分される。これまでリーラーマウス上丘の組織構築異常を解析した研究があるが(Baba, et. Al., Brain Res. 1140: 205-215,2006)、リーリンの下流遺伝子であるdisabled-1ミュータントであるヨタリマウスの上丘の細胞構築に関する研究はない。そこで,今回、リーラーおよびヨタリマウスを用いてNissl染色,MBP抗体を用いた髄鞘染色,NADPH-d組織化学およびParvalbumin、Calbindin、Calretinin、nNOS、GAD67に対する抗体を用いた免疫組織化学,HRP標識法等を用いてリーラーおよびヨタリ上丘の細胞構築を検討した。Nissl染色により,リーラー,ヨタリともに上丘表層の層構築が乱れ,また髄鞘染色によりこれらのミュータントマウスで髄鞘構築異常が明らかとなった。さらに正常マウスではNADPH-d陽性細胞は上丘の第1層と第2層に層状に分布するが,リーラーでは最表層に,ヨタリでは表層に広く分布していた。抗カルビンディン免疫陽性細胞は,正常マウスでは全層に広く分布したが,特に第1層と第2層に多くの免疫陽性ニューロンが存在した。一方,リーラーでは正常マウスとほぼ同様の陽性像が得られたが,ヨタリでは中間層(4,5層)には陽性ニューロンは存在しなかった。

  Competitive research funding


• Mechanisms of Layer Formation of Cerebral Neocortex and Layer-specific Neural Circuits

  TERASHIMA Toshio, KIKKAWA Satoshi, KATSUYAMA Yu

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 2005 - 2007

  Reeler and yotari mice that are mutant for Reelin or Dab1, respectively, show disorders of cerebral cortical lamination. We injected horseradish peroxidase (HRP)into the upper lumbar enlargement to label corticospinal tract (CST) neurons and wheat germ agglutinin-conjugated HRP (WGA-HRP) into the ventrolateral nucleus of the thalamus to label corticothalamic tract (CTT) neurons in both 19-day-old yotari and reeler mice with the aim of discovering whether or not they show differences in the distribution pattern of layer V or layer VI neurons. Similar injections of tracers were made in normal controls. HRP-labeled CST neurons, which were exclusively distributed in the layer V of the normal cortex, were radially scattered in the cortex of both mutants, but those in reeler were more deeply distributed than in yotari. WGA-labeled CTT neurons, which were mainly located in layer VI in the normal cortex, were superficially distributed just beneath the pia mater in both reeler and yotari cortex. The present quantitative study shows that the distribution pattern of layer V neurons, but not layer VI neurons, differs between reeler and yotari mice, suggesting that the Reelin and Dab1 proteins may play different roles in the migration and cell positioning of layer V neurons.

  Competitive research funding


• Research on mechanisms of development and collapse of the pial and glial limiting membranes, which comprise the cerebrospinal fluid-brain barrier

  KIKKAWA Satoshi, TERASHIMA Toshio

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 2004 - 2005

  We anterogradely labeled entorhinodentate axons in wild type and reeler mice at adult and neonatal stages. The results revealed that pioneer axons of the entrhinodentate tract develop similarly and pass through the hippocampal fissure at postnatal day 1(P1) in wild type and reeler mice. In the reeler mutant, however, follower axons can not pass through the fissure and detour around it, whereas those in wild type mice follow their pioneer and continue to pass through the fissure. Immunohistochemical analysis of postnatal development of the hippocampal area using anti-GFAP antibody demonstrated aberrant accumulation of GFAP-positive astroglia along the hippocampal fissure in the reeler but not wild type brain. This accumulation of astroglia seems to prevent the entorhinohippocampal axons from passing through the hippocampal fissure and forces them to detour along the fissure in the reeler mutant. Next, we double-labeled neocortical neurons in the fukutin-deficient chimeric mouse with antero- or retro-grade axonal tracers and anti-laminin antibody to investigate relationship between collapse of the glial limiting membranes due to fukutin-deficiency and malpositioning of neocortical neurons. The results showed that a population of neocortical neurons positioned ectopically in the area just beneath the disrupted limiting membranes which lack laminin immunoreactivity, suggesting that the glial limiting membranes have certain effects on cortical neuronal migration. Thirdly, we cloned and sequenced the complete coding region of zebrafish fukutin. Zebrafish fukutin showed a significant degree of sequence conservation. Fukutin mRNA was detected a wide variety of tissues including the brain and the spinal cord. We also tried knocking-down fukutin protein expression by antisense morpholino oligonucleotide microinjection into fertilized eggs. Further investigation of effects of fukutin knock-down on development of the central nervous system is currently being continued.


• 嗅球のシナプス回路形成におけるReelinシグナル系の役割の解明

  吉川 知志

  日本学術振興会, 科学研究費助成事業 若手研究(B), 若手研究(B), 神戸大学, 2001 - 2002

  既にクローン化しているreelinおよびdisabled-1(dab1)遺伝子を利用し大腸菌に発現させたReelinおよびDab1部分タンパク質をウサギに免役し、両者に対する抗血清を得た。これらの抗血清は、マウス脳のウェスタンブロットあるいは免疫組織化学において、ReelinおよびDab1タンパク質を認識した。次にin situハイブリダイゼーション(ISH)法により、正常マウス嗅球におけるreelinおよびdab1発現細胞を検討した。reelinメッセンジャーRNA(mRNA)は、僧帽細胞において最も強い発現が見られ、傍糸球細胞などにも発現が認められたが、顆粒細胞では発現はほとんど認められなかった。一方、dab1 mRNAの発現は、顆粒細胞において最も顕著であった。次に、マウス成獣の片側鼻腔内に硫酸亜鉛溶液を投与して嗅上皮および嗅細胞を破壊し嗅球への嗅覚刺激入力を遮断し、reelin遺伝子の発現に与える影響をISH法により検討した。対照標本として処理動物の健常側ならびに未処理動物を用いた比較の結果、硫酸亜鉛処理による嗅球への脱入力により、嗅球僧帽細胞におけるreelin mRNA発現量は著しく減少した。発現の減少は、脱入力直後から検出され、約3週間にわたって減少し続けたが、その後回復し始め、約6週後には正常レベルにまで回復した。reelin発現の減少および回復の過程が嗅神経の消失、再生の過程と一致するかどうかを、硫酸亜鉛処理した動物の嗅神経軸索をトレーサーによる順行性標識法により可視化した。その結果、硫酸亜鉛処理による嗅神経軸索の消失、再生の経過は、先に示したreelin mRNA発現の減少、回復過程と良く一致していた。この結果は、脱入力により神経活動が阻害された僧帽細胞において、何らかの理由でreelin遺伝子の発現がダウンレギュレートされることを示唆する。


• アスペルギルス細胞核の移動とニューロン細胞移動の相似性の証明

  寺島 俊雄, 吉川 知志

  日本学術振興会, 科学研究費助成事業 萌芽的研究, 萌芽的研究, 神戸大学, 2000 - 2001

  アスペルギルスのnud(nuclear distribution)ミュータントは細胞核の移動障害を特徴とする。nud遺伝子にはnudA、nudC、nudF、nudGの4つの遺伝子があり、それぞれ細胞核の移動に関与している。nudFがコードするNUDFタンパクの機能は不明であるが、細胞核移動障害を示すnudC3ミュータントにNUDFを過剰発現させると、核の移動能が回復する。一方、血小板活性化因子PAFを不活化するPAF acetylhydraseは、α1、α2,βの3量体からなる。PAFAH1B1タンパクはNUDFタンパクはとアミノ酸レベルで42%のホモロジーがある。本研究はPAFAH1Bタンパクをコードするpafah1b1遺伝子のcDNA配列をもとにジゴキシゲニン標識cRNAプローブを作成し、生後0日のマウス大脳皮質を用いてin situ hybridizationを行った。pafah1b1の発現は、中間帯および皮質板中の移動ニューロンに認められた。さらに詳細な発現は、PAFAH1Bタンパクに対する抗体を用いて調べる必要があり、今後、ポリクローナル抗体の作成を行う必要がある。これらの研究とは別に、α1とα2をコードするpafah1a1遺伝子とpafah1a2遺伝子のノックアウトマウスの供給を受け、それらの大脳皮質の層構造をHRP(ワサビ過酸化酵素)の逆行性軸索輸送法により検討した。脊髄にHRPを注入し、皮質脊髄路ニューロンを標識すると、pafah1a1、pafaha2ヌルマウスともに標識ニューロンは皮質第5層に分布し、異常を認めなかった。


• Development of gene therapy by the retrograde axonal transport of adenovirus into the reeler mouse

  TERASHIMA Toshio, OKADO Haruo, KIKKAWA Satoshi

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 1999 - 2001

  In this project, we aimed to develop the efficient introduction of exogeneous genes into the central nervous system of the neurological mutant mice, reeler and yatari. The reeler and yotari mouse are auto somal recessive, mutant mice, caused by mutation of reelin and disabled, homolog l (Dab1) gene, respectively. The mutant mouse is recognized by unstable gait and tremor and by \ I early deaths, around at the time of weaning. The cytoarchitectures of cerebeller and cerebral cortices and hippocampal formation of the reeler and yotari mouse are abnormal. In the present study, Lac-Z recombinant adenovirus was injected into the motor crtex of the normal, reeler, and yotari mice to examine how the virus is efficiently incorporated into the nervous system of the experimental animals. In the contralateral cortex, callosal commissure neurons were retrogradely labeled by the lac-Z recombinant adenovirus. The distribution pattern of CG neurons of the yotari was similar to that of the reeler : retrogradely labeled CG neurons were seen throughout all depths of the contralateral Fr1. We clarified that the small volume of the viral solution (〜0.02ul) is enough for the retrograde infection of neurons in the coerebral cortex both in the normal and reeler-phenotype mutants.


• リーラー類似動物大脳皮質運動野の神経回路網形成

  寺島 俊雄, 吉川 知志

  日本学術振興会, 科学研究費助成事業 特定領域研究(A), 特定領域研究(A), 神戸大学, 1999 - 1999

  SRKラットおよびヨタリマウスのテヘロ親を交配し、生後0日より21日までのSRKラットとヨタリマウスを得た。それぞれの同腹の対照動物の脊髄、視床外腹側核、一側大脳皮質にワサビ過酸化酵素(HRP)あるいはデキストランアミンを注入し、運動野の皮質脊髄路ニューロン、視床皮質投射ニューロン、脳梁交連系ニューロンを逆行性に標識した。 標識皮質脊髄路ニューロンは、正常動物では皮質第5層に限局して存在した。一方、SRKラット・ヨタリマウスでもSRKラットと同様に、標識ニューロンは皮質の全層に分布した。標識皮質視床投射ニューロンは、正常動物では運動野の第6層に限局して分布した。SRKラット、ヨタリマウスともに標識ニューロンは運動野の軟膜直下に限局して分布した。標識脳梁交連線維系ニューロンは、正常では皮質の表層(第2/3層)に分布したが、SRKラット・ヨタリマウスともに皮質の深層に脳梁交連線維系ニューロンが逆行性に標識された。さらに正常動物とミュータント動物の脊髄にFast Blue(FB)、視床外側腹側核にDiminido Yellow(DY)を注入して、皮質脊髄路ニューロン、皮質視床投射ニューロンをそれぞれ標識し、相互の位置関係を調べた。すると正常動物では、FBで標識された皮質脊髄路ニューロンは運動野の第5層に、DYで標識された皮質視床投射ニューロンは皮質第6層に分布した。一方、SRKラット・ヨタリマウスではFBで標識された皮質脊髄路ニューロンは運動野の全層に分布するが特に深層に多く分布し、DYで標識された皮質視床投射ニューロンは皮質の表層に分布した。以上より、SRKラットとヨタリマウスの運動野の構造は逆転構造をとることが明らかとなり、リーラーマウスの皮質構築異常に極めて類似することが証明された。


• Abnormal migration of the motor trigeminal, facial, and ambiguus nuclei neurons of the SRK rat

  TERASHIMA Toshio, KIKKAWA Satoshi

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 1997 - 1999

  Reeler mouse is an autosomal recessive mutant mouse, and characterized by ataxic gait and tremor. Since reelin, the gene responsible for the reeler mutation is discovered by D'Arcangelo et al. (Nature 374 : 719-723, 1995), much progresses have occurred in this field. The reelin gene encodes an extracellular protein that is crucial for neuronal migration. During the embryogenesis, reelin is expressed in the cerebral cortex in Cajal-Retzius cells and in the cerebellar cortex in outer granule cells. Although non-laminated structures such as facial nucleus, inferior olivary complex, and dorsal cochlear nucleus are also cytoarchtectually deranged in this mutant, only a few studies have been done to clarify the detailed abnormalities in these non-laminated structures. In this review, we focused the cytoarchitectonic abnormality in the facial nucleus of the reeler mouse. The branchiomotor neurons in the facial nucleus are born in the ventricular zone of the floor of the fourth ventricle, migrate ventrolaterally, and finally settle near by the ventral surface of the hindbrain. Time schedules for the generation, axon formation and migration of facial motoneurons are similar both in normal and reeler mouse, but the reeler phenotype becomes identifiable at the end of neuronal migration. Although the reason why the facial nucleus is cytoarchitecually abnormal in the reeler mouse, such a long migration of the facial motoneurons seems to be more susceptible to Reelin in the reeler mouse than in the normal control. In spite of cytoarcitectual abnormality, retrograde horseradish peroxidase (HRP) study confirmed that musculotopic arrangements within the facial nucleus of the reeler mouse are still preserved, suggesting neuronal migration/rearrangement and target recognition are independently regulated. More recently, other kinds of reeler-like mutants have been known (yotari, scrambler, SRK). Among these, yotari and scrambler mice arises from mutations in mdab1, a mouse gene related to the Drosophila gene disabled (dab). More than 10 years ago, an autosomal recessive rat mutant, shaking rat Kawasaki (SRK), has been described that exhibits a phenotype identical to reeler, but the gene responsible for this rat mutation has remained unknown. Very interestingly, the facial nucleus is cytoarchitectually more deranged in yotari and SRK compared to the normal counterparts. The reason why yotari exhibits a phenotype identical to reeler in the laminated structures but not in non-laminates structures such as the facial nucleus has remained obscure, suggesting mDab1 and Reelin proteins function as signaling molecules in a different way between non-laminated and laminated structures.


• リーラー類似動物(ヨタリ、SRKラット)の大脳皮質運動野の神経回路網

  寺島 俊雄, 吉川 知志

  日本学術振興会, 科学研究費助成事業 特定領域研究(A), 特定領域研究(A), 神戸大学, 1998 - 1998

  SRK(Shaking Rat Kawasaki)ラットは、川崎市の実験動物中央研究所のWistar系統ラットのクローズドコロニーに見つかった自然発症性ミュータントラットで、常染色体劣性遺伝性にその形質が伝えられていく。一方、ヨタリマウスは、inositol-1,4,5-triphosphateに対する受容体遺伝子の標的組換えマウスを作成する過程で生じた奇形マウスである。いづれも歩行失調、振戦等の運動性失調を特徴とする。SRKラットとヨタリマウスはリーラー様の細胞構築異常を呈するミュータント動物である。本研究は、これらのミュータント動物の大脳皮質運動野の神経回路網を明らかにすることを目的とした。 SRKラット、ヨタリマウス、対照動物の上部腰髄にHRPを注入し、皮質脊髄路ニューロンを逆行性に標識したところ、標識ニューロンは、対照動物では運動野下肢領域の第5層に限局して存在したが、SRKラットやヨタリマウスでは運動好下肢領域の皮質全層に分布した。SRKラット、ヨタリマウス、対照動物の運動性視床中継核である外側腹側核にファーストブルーを注入し、皮質視床投射ニューロンを逆行性に標識したところ、対照動物では皮質第6層に標識ニューロシが分布したが、SRKラット、ヨタリマウスでは標識ニューロンは皮質の最表層に分布した。SRKラット、ヨタリマウス、対照動物の反対側大脳皮質にHRPを注入したところ、対照動物では標識ニューロンは皮質の第2+3層に分布したが、SRKラット、ヨタリマウスでは皮質の深層に標識ニューロンは優位に分布した。 以上の結果は、大脳皮質の層特異的神経投射という観点からみて、SRKラット、ヨタリマウスの大脳皮質層構造異常が、リーラーマウスにきわめて類似することを示しているものであり、これらの奇形動物がリーラーフェノタイプ動物であることを改めて証明した。


• ロドプシンキナーゼの特異的基質認識を担う構造要因の検索

  吉川 知志

  日本学術振興会, 科学研究費助成事業 奨励研究(A), 奨励研究(A), 神戸大学, 1997 - 1998

  1. タコ口ドプシンキナーゼの自己リン酸化がロドプシンリン酸化反応に及ぼす影響 タコ口ドプシンキナーゼは、内在性の活性化因子であり、かつ基質であるロドプシンの存在、非存在下に関わらず自己をリン酸化しうる。自己リン酸化されるアミノ酸残基はまだ明らかではないが、ウシロドプシンキナーゼの自己リン酸化部位(488Ser、489Thr)に相当するアミノ酸は酸性アミノ酸(493Asp、496Asp)に置換されている。そこで、タコロドプシンキナーゼの自己リン酸化がロドプシンリン酸化反応に及ぼす影響を検討したところ、タコ口ドプシンキナーゼの自己リン酸化前処理によりロドプシンのリン酸化はほぼ半減した。実験で用いた自己リン酸化口ドプシンキナーゼ標品は非リン酸化キナーゼを含むと考えられ、より詳細な解析には自己リン酸化キナーゼだけを単離する必要があるものの、タコ口ドプシンキナーゼの自己リン酸化によりロドプシンのリン酸化、そしておそらく視細胞の光応答が調節されている可能性が示唆された。 2. タコ口ドプシシリン酸化における"high gain phosphorylation"の可能性の検討 ウシ口ドプシンのリン酸化においては、光活性化されたロドプシンの量以上のリン酸化が観察される、いわゆる"high gain phosphorylation"が報告されている。そこで、種々の量比で活性、不活性のタコ口ドプシンを混合した状態でリン酸化量を定量したところ、リン酸化量は活性化されたロドプシンの混合比に比例していた。これは、光活性化されたロドプシンのみがリン酸化を受けうることを示しており、タコ口ドプシンでは"high gain phosphorylation"は起きないと考えられた。この相違が、各ロドプシンキナーゼの性質によるものか、基質であるロドプシンの構造にも関連があるのかについては、さらに検討が必要である。


• MOLECULAR MECHANISM OF SIGNAL TRANSDUCTION IN VISUAL AND NEURAL SYSTEM OF ASCIDIAN BRAIN

  TSUDA Motoyuki, NAKAGAWA Masashi, IWASA Tatsuo

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A), Grant-in-Aid for Scientific Research (A), HIMEJI INSTITUTE OF TECHNOLOGY, 1995 - 1998

  Ascidian tadpole larvae change swimming behavior during the course of development. The photic behavior of the larvae of Ciona intestinalis was monitored by a computerized cell-tracking system with a time resolution of 0.1 s. Newly hatched larvae swim at an average speed of 1.4 mm/s, but show no response to light stimuli. The larvae were induced to swim more rapidly by a sudden decrease in light intensity 4 h after hatching. During the course of development the maximal speed of swimming behavior increased with time until 8h after hatching and then plateaued. The action spectrum for the step-down photophobic response of the larvae was determined at around 8 h after hatching and was fitted to Dartnall's nomogram with the absorbance maximum of the pigment located at 505 nm. These results suggest retinal proteins in the ocellus of the larvae are the photoreceptors for the photobehavior. Encephalic photoreceptors that are coupled to a biological clock for reproduction drive annual changes in gonadal activity of the preoptic-hypothalamic GnRH system in many animals. Photoreceptor and GnRH system in the cerebral ganglion of ascidians, primitive chordate, were examined and two light-evoked responses were recorded extracellularly from the cerebral ganglion of ascidian, Halocynthia roretzi. A light-evoked slow potential is assumed to originate from the photoreceptor cell and high frequency spontaneous discharges recorded in the dark were completely inhibited by light. Immunohistochemical study shows the cells bearing retinal proteins were found in the peripheral cellular cortex mainly at the dorsal surface. GnRH-immunoreactive cell bodies and fibers were distributed entire part of the cerebral ganglion and the part of those located close to the photoreceptor cells. Since the GnRH neuron exhibits a regular spontaneous beating discharge pattern, these results suggest photoreceptor cell coupled to GnRH neuron and pacemaker signal of GnRH neuron was controlled by light.


• 光情報伝達における受容体-Gタンパク質間の相互作用のリアルタイム測定

  吉川 知志

  日本学術振興会, 科学研究費助成事業 奨励研究(A), 奨励研究(A), 姫路工業大学, 1996 - 1996

  本研究では、EV-R(ダイキン工業社製)ならびにIAsys (Fisions社製)を使用し、タコ視細胞膜より精製したロドプシンと、シグナルトランスデューサーとしてロドプシンと共役するG蛋白質(G_q)との相互作用のリアルタイム測定を試みた。 EV-Rは反応槽に固相化した蛋白質と蛍光標識した蛋白質との相互作用を蛍光法で測定する。本研究ではロドプシンを固相化し、G_qαを蛍光標識する方法をとった。蛍光標識試薬には蛋白質のアミノ基と反応するCy5 (Ex max=649nm、Em max=670nm、Amersham社製)を用いた。標識反応時のG_qαとCy5のモル比は1 : 8、反応条件はHepes (pH8.0)バッファー中で10℃、2時間とした。得られたCy5-G_qα標品は、標識効率が1 : 1、GTPγS結合の残存活性は約40%であった。メーカーによる標準的な条件に従いロドプシンを固相化した反応槽を用いてCy5-G_q添加時の結合シグナルを測定したところ、ロドプシンの固相化量に依存した強度のシグナルが得られた。このシグナルは、Cy5-G_qαを変性させると消失し、また過剰量の比標識G_qαや非固相化ロドプシンの同時添加により競合的に減弱されることより、2蛋白質間の特異的な相互作用を反映するものと考えられる。結合シグナルは至適条件(pH6.5)では3分で飽和に達し、GTPγS結合実験の結果とよく一致した。光照射によるロドプシンの活性化に伴うシグナル変化については予備的実験の段階であり、期待した結果はまだ得られていない。結合シグナルは測定溶液中の界面活性剤や塩に大きく影響されるため、光応答を解析するための至適条件の検索が目下の課題である。 EV-Rとは異なりIAsysは非標識の蛋白質間の相互作用を測定する。EV-Rと同様に反応槽にロドプシンを固相化し、G_qを添加して結合シグナルを測定した。ロドプシンの固相化法や測定溶液組成を始め、測定諸条件の検討を行ったが、反応槽へのG_qの非特異的結合が多く期待した結果を得るには至らなかった。現在はタコ視細胞膜を直接固相化し、膜上での両者の相互作用を測定する方法を検討している。


• 網膜における代謝型グルタミン酸受容体-G蛋白質共役の解析

  吉川 知志

  日本学術振興会, 科学研究費助成事業 奨励研究(A), 奨励研究(A), 姫路工業大学, 1995 - 1995

  1993年に中西らがラット網膜より新たな代謝型グルタミン酸受容体遺伝子、mGluR6を単離し塩基配列を明らかにした。このmGluR6産物は網膜に特異的に発現しており、翻訳産物を発現させた培養細胞はAPBに対して強い応答性を示したことから、mGluR6がオン型双極細胞のグルタミン酸受容体の有力な候補と考えられた。そこで、mGluR6蛋白質の分子的実体を調べる上での有力なツールとして、特異的な抗ペプチド抗体を作製を試みた。C末端の14残基を抗原部位として選び、N端にシステインを加えた合計15アミノ酸よりなる1本鎖ペプチドおよび、キャリアーとしてmultiple antigen peptide(MAP)を直接導入した合成ペプチドを合成した。得られた合成ペプチドをMAPペプチドは直接、1本鎖ペプチドはシステインを介してキャリアー蛋白質と結合させたものを数種類合成し、それぞれ数匹のウサギに免疫した。得られた抗血清を抗原ペプチドを用いたELISAおよびウシ網膜膜標品を用いたウエスタンブロットにより評価した。いずれの抗血清も満足のいく特異性が得られなかったため、さらに抗原固定化カラムによる特異抗体の精製を行った。抗血清の50%飽和硫安沈殿画分を1本鎖ペプチドを結合したセファロース4Bカラムに添加した後、4M塩化マグネシウムにより溶出した画分を精製抗体とした。以上のようにして作成した数種の精製抗体を用いてmGluR6蛋白質の検出を行った。このうち1つの抗体により約100kDaの単一のバンドが検出された。この蛋白質の分子量は遺伝子の塩基配列より推定されるラットのmGluR6受容体の分子量95kDaとほぼ一致しており、本抗体がmGluR6受容体を特異的に認識するものであると考えられた。本抗体を用いた、網膜切片の組織染色や、免疫沈降法によるmGluR6受容体蛋白質の単離などが今後の検討課題となろう。


• 光受容体と共役する複数のG蛋白質;それらの相互作用は如何に調節されているか

  岩佐 達郎, 吉川 知志, 中川 将司

  日本学術振興会, 科学研究費助成事業 一般研究(C), 一般研究(C), 姫路工業大学, 1994 - 1994

  本年度得られた成果と研究の現状について簡単にまとめる。 1:G蛋白質遺伝子の全長のクローニング;ミズダコの眼のcDNAライブラリーの提案されたものがあったので、それを使ってクローニングすべく試みたが、全長を含むクローンは得られてこなかった。それで、新たにタコの眼のcDNAライブラリーを構築した。現在それを使ってポジティブクローンを得たので、解析を進めている。 2:ノーザンブロットにる発現部位の検討;Gi,Go,Gqについて得られた遺伝子断片(700bp)を用いてタコの異なる組織における発現を調べるためにノーザンブロットを行った。その結果、Gqが眼で非常に強く発現していることが判った。ショウジョウバエではGqクラス蛋白質が眼で特異的に発現していることが報告されている。タコでも同様にGqクラスの遺伝子が眼で強く発現していることが示され、無脊椎動物での光情報伝達経路が脊椎動物とは異なったG蛋白質と共役する形で進化してきたことを示唆する結果を得た。Goクラスについては、視葉と脳のみにバンドが観察された。ほとんど中枢神経系のみでGoが発現されていることは脊椎動物の場合と同じであり、Go蛋白質の機能が両者で共通していることを示唆している。 3:βサブユニット遺伝子;タコの網膜と視葉とを各々材料として、イカのβサブユニットのアミノ酸配列を基にして合成したプライマーを用いてPCRを行い、目的サイズのものについて解析した。90bpのPCR断片を計100個のコロニーについて調べたところ、得られてきたβサブユニット遺伝子は一種類であった。次に、得られた720bpの断片をプローブとしてノーザンブロットを行ったところ、調べた全ての組織で2本のバンドが検出された。今後はこの2本のバンドについて、その由来を調べていく計画である。


• MOLECULAR MECHANISM OF HYPERPOLARIZATION OF ON-BIPOLAR CELLS IN RETINA

  TSUDA Motoyuki, NIKKAWA Satoshi, NAKAGAWA Masashi, IWASA Tatsuo

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for General Scientific Research (B), Grant-in-Aid for General Scientific Research (B), HIMEJI INSTITUTE OF TECHNOLOGY, 1993 - 1994

  Light reduce the release of glutamate from photoreceptor cell, resulting in opening of cation channels on on-bipolar cell and subsequent depolarization of the cell. In this respect, retinal onbipolar cell is a novel neuron which hyperpolarizes on contact with neurotransmitter molecules. Electrophysiological studies showed that cGMP act as the second messenger mediating the action of glutamate through the G protein that striking similarities to transduction in photoreceptors. In order to elucidate molecular mechanism of hyperpolarization of on-bipolar cell, we studied signal coupling proteins of on-bipolar cell in bovine retina. GTP-binding protein (G protein) linking to metabotropic glutamate receptor of bovine retinal on-bipolar cell was studied by use of pharmacologically selective ligands, 2-amino-4-phosphonobutyric acid (APB) on bacteria toxin-catalyzed ADP-ribosylation and GTP S-binding. In contrast to the electrophysiological finding reported, G-protein coupling to APB-sensitive glutamate receptor served as a substrate for pertussis toxin but did not for cholera toxin. Several glutamate analogues effective on on-bipolar cell, as well as APB,increased GTP S binding to retinal membranes devoid of rod outer segments. The enhancement of GTP S binding by APB was completely abolished when the membranes were pretreated with pertussis toxin and NAD.These results suggest that, in retinal on-bipolar cell, the G-protein which couples metabotropic glutamate receptor to hyperpolarizing response of the cell is sensitive to pertussis toxin.