| Project/Area Number |
11670103
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General pharmacology
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| Research Institution | Tokai University |
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Principal Investigator |
KOBAYASHI Hiroyuki Tokai University, School of Medicine, Assistant Professor, 医学部, 講師 (60195807)
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| Co-Investigator(Kenkyū-buntansha) |
MASANOBU Yoshikawa Tokai University, School of Medicine, Assistant Researcher, 医学部, 助手 (90276791)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | μ-opioid receptor / G protein α subunits / antisense oligodeoxynucleotide / morphine-induced analgesia / ribozyme / DNAzyme / RT-PCR / ミューオピオイド受容体 / in situ hybridization / アンチセンスオリゴヌクレオチド |
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| Research Abstract |
To define the molecular identity of the G protein α subunits activated by the μ opioid receptor and assess the oligodeoxynucleotides (AS-ODN), ribozymes, or DNAzymes.
As phosphorothioate ODNs directed against various G protein α subunits (Gi1〜3α, Goα, and Gsα) mRNA were designed, and the changes in G protein α subunit mRNA or protein levels in the periaqueductal gray (PAG) of rat brain were investigated after the microinjection of each ODN into PAG. Each AS-ODN(1.7 nmol) was microinjected into PAG three times at the interval of 48 hours. Twenty four hours after the last injection, the region injected was stained by bromophenol blue for sampling precisely, and the G protein α subunit mRNA or protein levels were determined by RT-PCR or Western blot, respectively. AS-ODN directed against Gi1α suppressed the level of not only Gi1α but also Goα. The target site selected shared 12-bases sequence homology with Goα. Short AS-ODN, of which the homologous sequence was partly deleted, suppressed the level of only Gi1α not Goα This short-type AS-ODNs directed against Gi1α or Goα suppressed morphine-induced analgesia.
To improve the specificity, we designed ribozymes and DNAzymes. Microinjection of ribozymes did not suppress the levels of G protein α subunits mRNA probably due to the instability in the brain. Some DNAzymes modified with O-methy1 group at both ends suppressed the levels of G protein α subunits mRNA after microinjection into brain. The modified DNAzymes directed against Gi1α or Goα suppressed morphine-induced analgesia.
Together, these results suggest that morphine-induced analgesia is partly mediated through Gi1α and Goα.
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