Increased Aβ 42(43)-plaque deposition in early-onset familial Alzheimer's disease brains with the deletion of exon 9 and the missense point mutation (H163R) in the PS-1 gene

Baculoviruses expressing the human familial Alzheimer's disease presenilin 1 mutation lacking exon 9 increase levels of an amyloid beta-like protein in Sf9 cells

“…The immunoprecipitates underwent electrophoresis using the urea/bicine/bistris/tris/sulfate SDS-PAGE system and, after electrophoretic transfer, the membranes were immunoblotted. As reported previously, 25,26 synthetic Ab peptide was separated into Ab 40 and Ab 42 species (Figure 4b, lanes 4-7). The secreted Ab-like species was present following infection with APP-C99 alone or in combination with PS1DE9 ( Figure 4b, lanes 1 and 2, respectively).…”

“…Overall, the above findings indicate that the secreted Ab-like species may have a C-terminus identical to that of typical Ab 40 . However, the lack of detection of Ab42 in this system may possibly reflect a limitation in the sensitivity of the assays employed in this study.…”

“…Following electrophoresis, the gels were transferred to nitrocellulose membranes and immunoblotted with antibodies WO2 or 21F12, as described. 12 To achieve better separation between the Ab 40 and Ab 42 species, supernatants were subjected to electrophoresis using a 15% bis-bicine SDS-PAGE gel. 25,26 In this method, the immunoprecipitates were resuspended in 0.36 M Bis-Tris, containing 0.16 M Bicine, 1% (w/v) SDS, 15% (w/v) sucrose, 2.5% (v/v) b-mercaptoethanol and 0.004% bromophenol blue, and loaded onto a 15% bis-bicine SDS-PAGE gel.…”

“…Serum-free media were harvested from Sf9 cells infected with APP-C99 at MOI ranging from 1 to 10 ( Figure 2a). The media samples were immunoprecipitated with a mixture of monoclonal antibodies 6E10 (directed against residues 1-17 of Ab) and 29AF (directed against residues 17-24) subjected to SDS-PAGE, and immunoblotted using antibody WO2 (with an epitope spanning residues 5-8 of Ab), revealing a B4 kDa protein (Figure 2a, lanes 1-4, 6) that co-migrated with synthetic Ab 40 and Ab 42 peptides (Figure 2a, lanes 10-12) and with the same Ab peptides when media from bacmid-infected cells were spiked with synthetic peptides (Figure 2a Effects of nicastrin on APP-C99 metabolism Unlike the results following PS1DE9 expression, the addition of baculoviruses containing the nicastrin insert appeared to inhibit the generation of the Ablike species (Figure 3a, compare lanes 8 and 9 vs lanes 12 and 3b). Since the effect of nicastrin viruses on APP-C99 metabolism was strikingly different from the effect of the PS1DE9 viruses, we tested the effect of an unrelated molecule, tropomyosin (TPM3), in order to assess the specificity.…”

The role of the ubiquitin/proteasome system in cellular responses to radiation

“…Cells appear to attempt to protect themselves from toxic intracellular protein overload by activating stress kinases (Meriin et al, 1998;Marcu et al, 2002) and increasing expression of cytosolic heat-shock proteins, which is associated with the acquisition of thermal tolerance (Bush et al, 1997). A number of disease states have as their hallmark accumulation of ubiquitinylated proteins, especially neurodegenerative diseases, such as Parkinson's (Ii et al, 1997), Alzheimer's (Keller et al, 2000), Huntington's , and Angelman's syndrome Ishii et al, 1997).…”