| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1989: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
Platelet-activating factor(PAF) is a phospholipid synthesized from many cell types involved in inflammatory and allergic reactions, and is thought to be a significant biological mediator involved in many diverse clinical conditions including asthma, thrombosis, circulatory disorders and graft rejection. PAF, which is released from producing cells into blood, is hydrolyzed rapidly by plasma/serum PAF acetylhydrolase PAF-AH) to be inactive lysoPAF. We found some Japanese families with serum PAF-AH deficiency, which appeared to be transmitted by autosomal recessive heredity. The probability of PAF acetylhydrolase deficiency among asthmatic children was significantly higher in groups with severe symptoms (moderate and severe)(P<0.01). From these observations, we proposed that deficiency of serum PAF acetylhydrolase might be one hereditary factor leading to severe respiratory symptoms in as thmatic children.
Furthermore, in order to elucidate whether PAF-AH deficiency results from abnormalit
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y of serum enzyme protein and what kind of cells produces serum PAF-AH, we aimed to develop the immunoassay for PAF-AH enzyme protein. First, we purified PAF-AH from human pooled serum to be a single band on electrophoresis through Phosphotungstic acid precipitation, solubilization with Tween 20, chromatographies using DEAE Cellulofine AM ion exchange column, Q-Sepharose column, TSK-Gel HW-60 HPLC column, Hydroxyapatite HPLC column, Asahipak GS-510 HPLC column and Mono Q FPLC column. Next, we prepared PAF-AH antiserum from rabbits into which above described purified PAF-AH was injected. This PAF-AH antiserum had cross-reactivity with not only purified PAF-AH but also lipoprotein containing no PAF-AH activity on immunoelectrophoresis. Hence, in order to obtain antibody highly specific for PAF-AH, we are in progress to prepare monoclonal antibody using the resulting hybridomas that spleen cells from BALB/C mouse immunized with PAF-AH were fused with myeloma cells.
On the other hand, we elucidated that (1) serum PAF-AH activity in subjects with Kawasakis disease was significantly higher than that in healthy children, (2) that in subjects with systemic lupus erythematosus(SLE ) changed during the course of the sickness, and (3) the onset of water-immersion stress toward rat caused the development of gastric lesions associated with a significant increase in serum PAF-AH activity. Furthermore, although it has been reported that neutrophils apparently retain the PAF they synthesize, we identified the carrier protein in human PAF-AH deficient serum which drew out PAF produced in human neutrophils. Less
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