1992 Fiscal Year Final Research Report Summary
Screening and Development of New Immnomodulatory Drugs using PAF Receptor Gene as a Probe
| Project/Area Number |
03557017
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
SHIMIZU Takao The University of Tokyo, Faculty of Medicine, Professor, 医学部(医), 教授 (80127092)
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| Co-Investigator(Kenkyū-buntansha) |
TERAO Shinji Takeda Chemical Institute, Director, 化学研究所, 所長
HONDA Zenichiro The University of Tokyo, Health Service Center, Assistant, 保健センター, 助手 (70238814)
SEYAMA Yousuke The University of Tokyo, Faculty of Medicine, Professor, 医学部(医), 教授 (90010082)
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| Project Period (FY) |
1991 – 1992
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| Keywords | PAF / Receptor / Endotoxin / Phospholipases / Transformants / Antagonists |
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| Research Abstract |
Platelet-activating factor (PAF) is a potent mediator of inflammation, immunological disorders, and endotoxin shock. We have recently isolated cDNA clones of human and guinea pig PAF receptors. Using these clones as probe, we have obtained following results.
1. Isolation and structure of PAF receptor gene.
PAF receptor gene is consisted of three exons which are separated by >20kb introns. All exons are localized on chromosome 1. By alternative splicing, the leukocyte- and heart-type transcripts are produced. They have differentially regulated by distinct promoters.
2. Elucidation of signal transduction through a cloned PAF receptor.
A stable transfectant cell (CHO) was established carrying a cloned PAF receptor. By the application of PAF, phospholipase C and A_2, and MAP (mitogen-associated protein) are all activated, while adenylate cyclase was inhibited. These cellular events appear to be regulated by different types of G-proteins. Furthermore, we found that endotoxin. LPS, stimulated PAF receptor and yielded a Ca increase in human platelets as well as CHO transfectants. Thus, a part of LPS signaling, especially that related to platelet-aggregation might be due to the direct activation of PAF receptor.
3. Development of Screening System of PAF Receptor Agonists and Antagonists.
PAF receptor was expressed in Xenopus oocytes and a sensitive assay system of PAF receptor agonists/antagonists was made. Furthermore, COS cells/CHO cells carrying an excess amount of PAF receptors were constructed. By using membrane fractions of these cells, the highly specific screening system of PAF receptor modulator (either receptor antagonists or uncoupler of receptor-G-protein) was established. Through screening of naturally-occurring and synthetic compounds, several candidate chemicals have been obtained, which have potentiality for immunomodulatory drugs.
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[Publications] Honda Z., Nakamura, M., Miki, I., Minami, M., Watanabe, T., Okado, H., Toh, H., Ito, K., Miyamoto, T. and Shimizu, T.: "Cloning by functional expression of guinea-pig lung platelet-activating factor receptor." Nature. 349. 342-346 (1991)
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[Publications] Nakamura, M., Honda, Z., Izumiz, T., Sakanaka, C., Mutoh, H., Minami, M., Bito, H., Seyama, Y., Matsumoto, T. Noma, M., and Shimizu, T.: "Molecular cloning and expression of human platelet-activating factor receptor." J. Biol. Chem.266. 20400-20405 (1991)
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[Publications] Takano, T., Honda, Z., Watanabe, T., Uchida, S., Shimizu, T. and Kurokawa, K.: "Demonstration of platelet-activating factor receptor in guinea pig kidney." Biochem. Biophys. Res. Commun.177. 54-60 (1991)
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[Publications] Bito, H., Nakamura, M., Honda, Z., Izumi, T., Iwatsubo, T. Seyama, Y., Ogura, A., Kudo, H., and Shimizu, T.: "Platelet activating factor receptor in rat brain; PAF mobilizes intracellular Ca in hippocampal neuron" Neuron. 9. 285-294 (1992)
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[Publications] Sakanaka; C., Izumi, T., Nakamura, M., Honda, Z., Watanabe, T., Minami, M., Mutoh, H., Bito, H., Seyama, Y. Ui, M., and Shimizu, T.: "Three types of Gi proteins of the guinea pig lung; cDNA cloning and tissue distribution." Biochim. Biophys.Acta 1175. 61-66 (1992)
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