| Project/Area Number |
17590375
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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 |
Parasitology (including Sanitary zoology)
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| Research Institution | Osaka City University |
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Principal Investigator |
KIMURA Masatsugu Osaka City University, Graduate School of Medicine, Associate professor (60195378)
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| Project Period (FY) |
2005 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,450,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥150,000)
Fiscal Year 2007: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2006: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2005: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | malaria / artemisimn / calcium pump / pfATP6 / ATPase / antimalarial drug / thapsiearein / SERCA / タプシガギン |
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| Research Abstract |
Artemisinin is are of the most potent antimalarial drugs which has low side effects and is also effective to gametocytes. Recently artemisinin resistant P. falciparum was reported in Africa although it has not been discovered for a long period after clinical use of artemisinin started, and the interest to the action mechanisms of artemisinin becomes higher.
We paid attention to that Artemisinin is sesquiterpene lactone as with thapsigargin, highly specific SERCA inhibitor. From the result that P falciparum endoplasmic reticulum Ca-pump PfATP6 was inhibited highly by artemisinin, we proposed the hypothesis that PfATP6 was the target molecule of artemisinin. However, as the Ca-pump activity was measured by using its ATPase activity, it is necessary to measure the Ca-pump activity by using its Ca-transport potency which has higher selectivity than ATPase activity. To examine it, heterogeneous eukaryote expression system of the PfATP6 gene roast be made since large scale preparation of PfAT
… More
P6 protein is impossible from parasite culture.
A recombinant plasmid clone which contains full length of PfATP6 gene (3684 bases)with a c-myc tag to the C terminus side of the gene was constructed, but it had a mutation of two consecutive nucleotides "ft", instead of"aa" in the tag nucleotide sequence. In order to recover the mutation, a PCR method with digestion by DPN1 restriction enzyme was performed but it was not useful probably because of AT-richness of the P. falciparum gene. Instead a polyclonal antibody to the PfATP6 was made by using an oligopeptide and rabbits.
This recombinant plasmid was transfected to Cos7 cells transiently. Microsome fraction of the transfected cells did not show the enhancement for the ATP-dependent calcium transport ability. PfATP6 protein was expressed in Cos7 cells without showing typical distribution to the endoplasmic reticulum. P falciparum is evolutionally far apart from eukaryote, so it was suggested that the PfATP6 protein might not be buried in the endoplasmic reticulum membrane with exact conformation. Less
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