The binding efficiencies of MoPrP under reducing conditions were not significantly different from those under nonreducing conditions Selleckchem Ferroptosis inhibitor in the four prion strains and Chandler (Fig. 3a, open and solid columns). The efficiencies of conversion of 79A, ME7, and Obihiro and Chandler under reducing conditions were not significantly different
from those under nonreducing conditions (Fig. 3b, open and solid columns). However, the efficiency of the mBSE strain was about three times greater under reducing conditions (P < 0.01). To determine whether the disulfide bond or thiol groups of MoPrP were involved in binding with PrPSc and conversion into PrPres, binding and conversion assays using Cys-less mutants (C178, 213S) were performed. No significant differences were observed in binding efficiencies under nonreducing conditions among C178, 213S, and MoPrP, although the binding efficiencies of C178, 213S with ME7, and Obihiro PrPSc were about half those of MoPrP
(Fig. 3a, c, open columns). The efficiency of mBSE-seeded conversion of C178, 213S was not different from that of MoPrP, but the efficiencies of the other strains were lower than Src inhibitor that of MoPrP. Furthermore, the difference in the efficiencies of Chandler and 79A were significant (Fig. 3b, d, open columns). Thus, the findings suggested that the effects of Cys to Ser substitutions on binding and conversion were different for each prion strain, and that the presence of Cys or thiol groups was especially important for conversion into PrPres in Chandler and 79A strains. The binding efficiencies of C178, 213S in each prion strain under reducing conditions were not significantly different from those under nonreducing conditions (Fig. 3c, open and solid columns). The conversion efficiencies of C178,
213S in the Chandler, 79A, ME7, and Obihiro strains under reducing conditions were not significantly Dipeptidyl peptidase different from those under nonreducing conditions, although a significant increase in the conversion efficiency of mBSE was observed under reducing conditions (Fig. 3d, open and solid columns). Therefore, the effect of reducing conditions on the binding and conversion of Cys-less mutants was similar to those of MoPrP, suggesting that neither Cys residues nor thiol groups are involved in the acceleration of mBSE-seeded conversion under reducing conditions. Immunohistochemical and HE staining of brain tissue from mice infected with each strain were performed (Fig. 4a). In mice inoculated with Chandler and 79A strains, diffuse synaptic deposits were found throughout the brain, and the PrPSc accumulation patterns of both strains were very similar. In contrast, ME7 and Obihiro strains produced PrPSc accumulation throughout the neuropil in most areas of the brain, although some areas were predictably severely affected. Large numbers of PrP-aggregates were also detected, but these tended to be small and had less obvious amyloid cores.