2000 Fiscal Year Final Research Report Summary
THE CONTROL OF CA2+ RELEASE CHANNEL EXPRESSION BY THE TET ON-OFF SYSTEMS.
| Project/Area Number |
11670102
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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 | SHOWA UNIVERSITY |
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Principal Investigator |
OYAMADA Hideto SHOWA UNIVERSITY, SCHOOL OF MEDICINE Assistant, 医学部, 助手 (50266160)
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| Co-Investigator(Kenkyū-buntansha) |
OGUCHI Katsuji SHOWA UNIVERSITY, SCHOOL OF MEDICINE Professor, 医学部, 教授 (50129821)
KIUCHI Yuji SHOWA UNIVERSITY, SCHOOL OF PHARMACEUTICAL SCIENCE Professor, 薬学部, 教授 (50204821)
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| Project Period (FY) |
1999 – 2000
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| Keywords | calcium ion / calcium release channel / Tet On-Off / ryanodine receptor / malignant hyperthermia |
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| Research Abstract |
(1) We have hansfected cDNAs of ryanodine receptor (RyR)/Ca2+ release channel and green fluorescent protein (GFP) inserted in a pTRE vector which have tet operator sequence followed by the minimal CMV promoter into the tetracycline-controlled transactivator (tTA) stable expressed CHO cells. Some of the transfected cells showed the GFP signals within the cytoplasm. Only of these GFP expressing cells could be stained with the polyclonal antibodies against RyR.
(2) We found novel RyR mutations in the Japanese malignant hyperthermia (MH) patients. Site-directed mutagenesis of the MH mutations were carried out in the cassettes of the RyR cDNA and expressed by the pTRE vector. One of the MH mutations increased the sensitivity to caffeine similar to the previously reported MH mutations. These results suggested that most of the MH mutations can enhance sensitivities to some drugs such as general anesthetics.
(3) The GFP signals/RyR expressions were suppressed by addition of doxycycline, a tetracycline analogue, in the culture medium. Conversely the signals and the expressions were induced by removing out of the doxycycline from the medium. These results suggested that the expression of the Ca2+ release channles can be controlled by the tetracycline controlled on-off system (Tet On-Off system).
We have now been able to analyze the function of the ryanodine receptor/Ca2+ release channel protein by the Tet On-Off system while the expressed protein levels were monitored by the GFP signals.
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