| Project/Area Number |
12670613
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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 |
Neurology
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| Research Institution | Kagoshima University |
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Principal Investigator |
MORITOYO Takashi Kagoshima University, Faculty of Medicine, Assistant Professor, 医学部, 講師 (90305110)
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| Co-Investigator(Kenkyū-buntansha) |
SORIMACHI Masaru Kagoshima University, Faculty of Medicine, Professor, 医学部, 教授 (70036440)
OSAME Mitsuhiro Kagoshima University, Faculty of Medicine, Professor, 医学部, 教授 (10041435)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | HTLV-I / HAM / cerebrospinal fluid cells / in situ hybridization / immunohistochemistry / Laser Scanning Cytometer / HTLV-I |
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| Research Abstract |
We tried to develop the simultaneous and quantitative detection system of human T-lymphotropic virus type I (HTLV-I) proviral DNA and transcripts in cerebrospinal fluid cells from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The fluorescence detection system of HTLV-I proviral DNA, mRNA and viral proteins were established. In addition to the highly sensitive detection system of HTLV-I mRNA and viral proteins that we developed, a novel technique to visualize a single copy of HTLV-I proviral DNA was developed. HTLV-I proviral DNA was successfully detected by fluorescence in situ hybridization (FISH) using refined highly sensitive probes. FISH allowed us to count the number of copies of proviral DNA from the number of signals on chromosomes within a metaphase spread and the interphase nucleus. We used Laser Scanning Cytometer (LSC) to measure the total amount of fluorescence of proviral DNA, mRNA or viral proteins from an individual cell quantitatively. But LSC did not have enough sensitivity to detect the proviral DNA. LSC was needed to be more sensitive to detect the lower intensity of fluorescence. We examined the condition of simultaneous detection of proviral DNA and mRNA or viral proteins. The best fixative condition was different from each other, but we found the appropriate condition to detect simultaneously. LSC was used to detect the two different fluorescence from double-labeled samples. LSC was good to detect the same intensity of different fluorescence, but did not have enough ability to detect the different amount of fluorescence quantitatively. We started to collect and stock the samples of cerebrospinal fluid cells from patients with HAM/TSP and HTLV-I-associated diseases, and tried to test the new some techniques that had been available. These techniques were also applied to investigate the other samples including different diseases from HAM/TSP or HTLV-I-associated diseases.
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