| Project/Area Number |
17590660
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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 |
Gastroenterology
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| Research Institution | University of Miyazaki |
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Principal Investigator |
MONDAL M・S UNIVERSITY OF MIYAZAKI, FACULTY OF MEDICINE, RESEARCHER, 医学部, 研究員 (00398265)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAGUCHI Hideki UNIVERSITY OF MIYAZAKI, FACULTY OF MEDICINE, ASSISTANT PROFESSOR, 医学部, 助手 (10305097)
NAKAZATO Masamitsu UNIVERSITY OF MIYAZAKI, FACULTY OF MEDICINE, PROFESSOR, 医学部, 教授 (10180267)
DATE Yukari UNIVERSITY OF MIYAZAKI, FACULTY OF MEDICINE, PROFESSOR, フロンティア科学実験総合センター, 教授 (70381100)
SHIMBARA Takuya UNIVERSITY OF MIYAZAKI, FACULTY OF MEDICINE, ASSISTANT PROFESSOR, 医学部, 助手 (10398266)
初 春平 宮崎大学, 医学部, 教務補佐員 (90381102)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | gut peptide / peptidomics analysis / orphan receptor / IMACS / tumor cell lines / RIA |
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| Research Abstract |
We hypothesize I that we could utilize a secretopeptidome to identify candidates for novel bioactive peptides, provided we focus on structure of peptides as a hallmark of biologically active peptides. We examined the secretopeptidome prepared from different carcinoma cell lines. Human medullary thyroid carcinoma TT cells actively secretes calcitonin and calcitonin gene-related peptide alpha (CGRPα). We used gel filtration chromatography to obtain a peptide-rich fraction below 10,000 Da in molecular weight, which was subsequently separated into 50 fractions by reverse phase-high performance liquid chromatography. Each fraction was analyzed by off-line nano-ESI MS/MS with a Q-Tof II mass spectrometer (Micromass, Milford, MA) and by MALDI-TOF MS/MS with a Proteomics 4700 mass spectrometer (Applied Biosystems, Foster City, CA). Each MS/MS spectrum was used to probe the NCBI and Swiss-Prot databases with Mascot MS/MS ion search software (Matrix Science, Boston, MA) and also interpreted by SeqMS. Tandem mass spectrometric analysis of column effluent fractions resulted in the identification of 233 peptide sequences, all of which were assigned to known precursors for bioactive peptides or secretory proteins. Of all the peptides identified, we identified novel bioactive peptide using IMACS calcium screening system. The peptide with monoisotopic masses of 2677.4 were derived from a known polypeptide. RT-PCR showed that mRNA of novel peptide was adundantly expressed in gastrointestinal tracts. We established RIA to detect novel bioactive peptide. Among the peripheral tissues, novel bioactive peptide was detected in gastrin-producing cells in the stomach, and in ganglion cells of both intestinal Aueroach and Meissner plexuses, which contain large numbers of additional bioactive peptides that control intestinal motor and secretory functions.
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