| Co-Investigator(Kenkyū-buntansha) |
TAKIZAWA Takenori AICHI HUMAN SERVICE CENTER, INSTITUTE FOR DEVELOPMENTAL RESEACH, DEPARTMENT OF NEUROREGULATION, 室長 (40192158)
KOIZUMI Tsutomu FUKUI MEDICAL UNIVERSITY, LABORATORY ANIMAL CENTER, ASSOCIATE PROFESSOR, 医学部, 助教授 (40126579)
KANNO Hitoshi TOKYO WOMEN'S MEDICAL UNIVERSITY, DEPARTMENT OF TRANSFUSION MEDICINE AND CELL PROCESSING, ASSOCIATE PROFESSOR, 医学部, 助教授 (70221207)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
In this study, we screened 73 subjects with non-spherocytic hemolytic anemia of unknown pathogenesis, and diagnosed 5 of pyruvate kinase, 10 of glucose-6-phosphate dehydrogenase, 1 of adenylate kinase and 1 of pyrimidine 5'-nucleotidase (P5N-I) deficient cases.
We identified five novel mutations in nine families with P5N-I deficiency : two missense mutations (425C, 721C), one splicing mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients are homozygous for each mutation.
We expressed mutant P5N-I with 425C and 721C in Cos-7 cells, and examined their intracellular stability. The L124P (425C) showed approximately 20% residual activity and immunoreactive protein of a wild-type control, suggesting that the L124P was an unstable variant. The G241R(721C) had been demonstrated as a kinetic variant with lower affinity for cytidine monophosphate. This mutant was as stable as a control in Cos-7 cells, being compatible with previous results. We could c
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onclude that Gly241 is important for the substrate binding. Haplotype analysis showed that the 721C, which had been identified in five unrelated families, was a founder mutation. Since proteasome inhibitors restored the stability of the L142P, the L142P increases the susceptibility to the degradation by ubiquitin-proteasome pathway.
To examine physiological significance of P5N-I in non-erythroid cells, we established a line of transgenic mouse, which ubiquitously overexpressed human P5N-I. Transgene expression was detected in both liver and kidney. However, expression levels are within 2 times of normal controls. Effects of transgene-derived P5N-I on liver and kidney are being analyzed.
The P5N-I gene can be induced by alpha-interferon, and the expression is augmented in lymphocytes of patients with HIV infection or autoimmune diseases such as SLE. From these observations, the P5N-I has been suggested to have any roles on immune response. We examined effects of the P5N-I on acute immunity of influenza infection ; however, overexpression of the P5N-I did not modify immune responses of infected cells. Further studies will require gaining insights on immunological roles of P5N-I. We thank Takako Hamada and Shin-ichi Okada for their excellent technical supports. Less
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