| Project/Area Number |
03670145
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
SUZUKI Yasuyo Kyushu University, Medical Institute of Bioregulation, Research Associate., 生体防御医学研究所, 助手 (90226564)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Tomokazu Kyushu University, Medical Institute of Bioregulation, Professor., 生体防御医学研究所, 教授 (20028517)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1992: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1991: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Variant transthyretin / Mass screening / Amyloidosis / Familial amyloidotic polyneuropathy / Molecular pathology / ELISA / Reverse phase high performance liquid chromatography / アミロイドーシス / 蛋白単離 / マススクリ-ニング / アミロイド-シス / 家族性アミロイドポリニュ-ロパチ- |
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| Research Abstract |
More than 30 transthyretin(TTR) variants which cause amyloid deposition in different organs and some along peripheral nerves have been described. Neither the mechanism of amyloid formation from variat TTR, nor the reason for different organ distribution is known. To elucidate these problems, we tried to find non-amyloidogenic TTR varians from asymptomatic subjects and to examine the mechanism of amyloid fibril formation by means of protein engineering and molecular pathology.
We already reported a novel diagnostic method for familial amyloidotic polyneuropathy(FAP) based on isolation by reverse phase HPLC of variant TTR. However, this method has a draw back: 5 to 10 ml of plasma is needed. To improve the diagnostic sensitivity, we have developed an ELISA to estimate TTR. This method semimicronized the original diagnostic method, requiring plasma samples of only 0.2 ml. Then we aimed at further improvement of this method in rapidity and simplicity by using syringe pump for chromatography and cartridge columns. The first step involved affinity chromatography on a Hitrap blue (l ml, Pharmacia). The fraction containing TTR was desalted and equilibrated using PD-10(Pharmacia). The second step involved anion exchange chromatography on an Econo-PacQ cartridge(Bio-Rad) with stepwise gradient of 0, 30, 40 and 100% of 0.5 M NaCl in 20mM Tris HCl buffer, pH 7.6. The effluents were monitored by ELISA of TTR. The fraction containing TTR was concentrated and lyophilized. The third step using reverse phase HPLC by Nucleosil 5C18 was the same as the original method. We examined 47 samples of plasma from 28 patients with hypertrophic cardio-myopathy, 9 with colorectal cancer, 1 with amyloidosis, 1 with polycythemia vera, and 1 with liver cirrhosis and 7 ostensibly healthy subjects. Unfortunately we could not detect any variant TTR. We are planning to examine more subjects and to improve the sensitivity by adopting two dimensional electrophoresis.
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