|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1991 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1990 : ¥1,700,000 (Direct Cost : ¥1,700,000)
For the purpose of investigating the molecular organization of the desmosome, quantitative analysis of the immunoelecton microscopy (IEM) using immuno-gold method with anti-D mono/polyclonal antibodies (Abs) was carried out. According to the analysis, such useful tool as position of peaks, shape of curves obtained from length/frequency curves which were specific to the DG and DP molecules are recognized in the first year. However, theoretical meanings were remained to be uncertain. From the second year, computer aided distributions were investigated as the simulated models of immunoelectron microscopy. By applying fitting analysis to the histogram, a combined curve of two normal distribution curves with the different value of half width and the same value of peak position were appeared as a least square curve and from this combined curve, the resolution was estimated as 60 A. Basing on the theoretical analysis and the quantitative immunoelectron microscopy of desmosome, a three dimensional model of cytoskeletal-D proteins complex at the desmosome was proposed.
A two dimensional architecture of D proteins and E-cadherin (ECAD) was also analyzed by IEM using isolated D pre-treated with 6 M guanidine HCl (G-HCl). At the proper region of D, density of DG1, DP1/2, DG2/3 was decreased in this order forming a random distribution. The considerable amount of DP3 was extracted by the G-HCl treatment. Only a few amount of E-CAD was detected at the peripheral region of D. The difference of the localization pattern between the D molecules and E-CAD was also confirmed in cultured keratinocytes. Though the break-down of cell-to-cell adhesion of the epitherial cells was observed under the presence of anti-DG1 and/or DG2/3 Abs for 27 hs incubation, non of the effect of pepstatin was observed.