2001 Fiscal Year Final Research Report Summary
Molecular biological studies of Ten-m2 gene function for the olfactory sensory neuron projections
| Project/Area Number |
12470356
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Otorhinolaryngology
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| Research Institution | Okayama University |
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Principal Investigator |
OOHASHI Toshitaka OKAYAMA UNIVERSITY, GRADUATE SCHOOL OF MEDICINE AND DENTISTRY, LECTURER, 大学院・医歯学総合研究科, 講師 (50194262)
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| Co-Investigator(Kenkyū-buntansha) |
YONEZAWA Tomoko OKAYAMA UNIVERSITY, GRADUATE SCHOOL OF MEDICINE AND DENTISTRY, ASSISTANT, 大学院・医歯学総合研究科, 助手 (30304299)
NINOMIYA Yoshifumi OKAYAMA UNIVERSITY, GRADUATE SCHOOL OF MEDICINE AND DENTISTRY, PROFESSOR, 大学院・医歯学総合研究科, 教授 (70126241)
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| Project Period (FY) |
2000 – 2001
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| Keywords | ten-m2 / neurestin / knockout mouse / brain / olfactory sensory neurons / International collabolation (Germany, Switzerland) |
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| Research Abstract |
To investigate the function of ten-m2/Neurestin gene for the olfactory sensory neuron projection, we have done the following experiments and got some data.
Ten-m2 specific antibodies were raised using the C-terminal unique amino acid sequence among the ten-m family. The specific staining was confirmed in the mouse section and confirmed its embryonic expression in combination with in situ hybridization.
Detection of Ten-m2 ligand: The extracellular domain of Ten-m2 protein was expressed in HEK293 cell culture system. We could detect specific protein band in the SDS-PAGE which bound to the ecto-domain. We are now identifying the protein.
Chromosomal mapping of human ten-m2 gene. The ten-m2, 3, and 4 genes were mapped to the chromosome 5, 4and 11, respectively. There was no genetic disorder reported to the locus.
Phenotypic analysis of the ten-m2 knockout mouse: By introducing a neomycin expression cassette in to the transmembrane exon of type II transmembrane protein, ten-m2, ten-m2 null mice were generated. There was no obvious phenotype so far. Ten-m2 null mice were viable and fertile, had a normal life span. There could be some compensation by another ten-m family members. The olfactory neuronal axon is now stained with MBP, marker of myelin and observed carefully.
Another approach to look for the ligand for ten-m2: The ten-m protein seems to be quite unique for its protein structure. Structural analysis will become a very important information to search the ligands. Ultrastructural analysis of recombinants revealed the dimerfomation through the disufide-bond. The dimer could be formed between the different members of ten-m family in our reconstitution experiments. Since CSPGs are known to act for the axon guidance, and the versican is expressed in the olfactory bulb, we tried to clone some related genes. The Brain link protein is cloned and the expression pattern was analyzed. The data for the novel link protein is published (see references).
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[Publications] Hirakawa, S., Oohashi. T., Su, W-D., Yoshioka, H., Murakami, T., Arata, J., and Ninomiya, Y.: "The brain link protein-1 (BRAL1): cDNA cloning, genomic structure, and characterization as a novel link protein expressed in adult brain."Biochem. Biophy. Res. Commun.. 276(3). 982-989 (2000)
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[Publications] Oohashi T, Hirakawa S, Bekku Y, Rauch U, Zimmermann DR, Su W-D, Ohtsuka A, Murakami T, and Ninomiya Y: "Brall, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult mouse central nervous systems."Mol. Cell Nourosci.. 19. 43-57 (2002)
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