KATO Goro Yamanashi Med.Univ., Dept.of Biochemistry, Research Associ., 医学部, 助手 (60177441)
MABUCHI Tadashi Yamanashi Med.Univ., Dept.of Biochemistry, Research Associ., 医学部, 助手 (80150308)
|Budget Amount *help
¥8,100,000 (Direct Cost: ¥8,100,000)
Fiscal Year 1998: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥3,200,000 (Direct Cost: ¥3,200,000)
1) Every type of amyloid deposit examined contained serum amyloid P component (SAP). Using the SAP-deficient mice, we showed that, although not essential in the deposition of AA amyloid, SAP significantly promotes the amyloid deposition. Thus, SAP enhances the induction of murine AA amyloidosis and may play an important role in the pathogenesis of human amyloidoses. However, no enhancement in the rate of regression of splenic AA amyloid was observed in the SAP-deficient mice relative to wild-type mice. These results suggest that dissociation of bound SAP from AA amyloid deposits would not significantly accelerate regression of the deposits in vivo.
2) To elucidate the role of transthyretin (TTR) and SAP in the Aβ amyloid deposition, we generated mouse lines carrying a null mutation either at the endogenous ttr or sap locus and human mutant amyloid precursor protein (app) gene, responsible for familial Alzheimer's disease, as a transgene. We are currently examining whether there are any
significant differences in the onset, progression, and tissue distribution of amyloid deposition between the ttr^<-/-> or sap^<-/-> and control wild-type transgenic mice expressing the human mutant app gene.
3) To assess the ultrastructure of in situ AA amyloid fibrils, we examined the ultrastructure of splenic AA amyloid fibrils in SAP-deficient and wild-type mice. Ultrastructural analysis by quick-freezing and deep-etching method revealed significant differences in the structure of amyloid fibrils in situ between the two types of mice. The light and electron microscopic immunohistochemical analyses, following trypsin treatment, suggested that AA filaments were on the exterior surface of heparan sulfate proteoglycan (HSPG), and that SAP bound to the most exterior surface of the fibrils.
4) We generated mice carrying a point mutation (Val 30 Met) in the endogenous ttr gene with the use of a novel gene targeting procedure. We suggest that this procedure may be used to introduce subtle mutations efficiently into most genes in mice. Amyloid deposits were detected in the heart, liver, kidney, stomach, small intestine, large intestine, and spleen in 2 out of 22 to 25 month-old 16 heterozygous mutant mice examined. On the other hand, amyloid deposits were not detected in any of the age-matched 16 homozygous mutant and 5 wild-type mice. Less