| Project/Area Number |
13470055
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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 |
寄生虫学(含医用動物学)
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| Research Institution | Gunma University |
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Principal Investigator |
KATAKURA Ken Gunma University, Graduate School of Medicine, Associate Professor, 医学部, 助教授 (10130155)
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| Co-Investigator(Kenkyū-buntansha) |
FUJISE Hiroshi Azabu University, Faculty of Veterinary Medicine, Professor, 獣医学部, 教授 (40106232)
TORII Motomi Ehime University, School of Medicine, Professor, 医学部, 教授 (20164072)
HATA Hidekazu Gunma University, Graduate School of Medicine, Associate Professor, 医学部, 助教授 (00110304)
大上 美穂 群馬大学, 医学部, 助手 (40269048)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥10,700,000 (Direct Cost: ¥10,700,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2001: ¥6,300,000 (Direct Cost: ¥6,300,000)
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| Keywords | ABC proteins / anti-peptide antibody / green fluorescent protein / Leishmania amazonensis / lysosome / multidrug resistance (MDR) / subcellular localization / transporters / 間接蛍光抗体法 / Green fluorescent protein / 蛍光顕微鏡 / Leishmania amazonensis / 遺伝子導入 |
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| Research Abstract |
Recent progress in research for ATP-binding cassette (ABC) transporters have shown their important roles in drug resistance in various organisms, including protozoan parasites. We previously isolated two different multidrug resistance (MDR) subfamily genes of the ABC proteins, LaMDR1 and LaMDR2, in Leishmania amazonensis. We found that the LaMDR1 was a homologue to mammalian MDR1, but the LaMDR2 was a novel MDR gene. Overexpression of the LaMDR2 conferred resistance to 5-fluorouracil (5-FU), a fluoropyrimidine used as an anticancer drug, and drug uptake experiments demonstrated a decrease accumulation of 5-FU in transfectants with the LaMDR2. In the present study, we examined whether MDR2 is located in the plasma membrane and extrudes 5-FU out of the cell, or it is localized in the membrane of intracellular organelle and sequesters the drug inside the organelle. Western blot analysis using anti-LaMDR2 peptide antibodies indicated that the expression of LaMDR2 was much higher in the transfectants in the log-phase than the stationary phase. The corresponding protein was detected by RT-PCR in wild-type cells. Indirect immunofluorescence studies showed that the antibodies stained unknown cytoplasmic structures, but not the plasma membrane. Fluorescent and confocal scanning laser microscopic studies revealed that LaMDR2 tagged with green fluorescent protein (GFP) was found in the intracellular tubular, canal or round-shape structure. The GFP signal was most intense in the log-phase cells, but the signal patters varied in individual transfectants. The distribution of the GFP fluorescence was mostly overlapped with that of a lysosome marker, but not with that of a mitochondria marker. These findings suggest that LaMDR2 is located in the membrane of the lysosome and transports 5-FU into the lumen, and that the dug is extruded out of cell by subsequent exocytosis.
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