No abstract

Prions, infectious agents causing transmissible spongiform encephalopathy (TSE), are composed primarily of the pathogenic form (PrP Sc) of the host-encoded prion protein. Although very low levels of infectivity have been detected in urine from scrapie-infected rodents, no reports of urinary PrP Sc have been substantiated. Studies on the dynamics of urinary PrP Sc during infection are needed to ensure the safety of urine-derived biopharmaceuticals and to assess the possible horizontal transmission of prion diseases. Using the protein misfolding cyclic amplification technique, a time-course study of urinary excretion and blood levels of PrP Sc was performed in Sc237-infected hamsters and a high rate of PrP Sc excretion was found during the terminal stage of the disease. Following oral administration, PrP Sc was present in all buffy coat samples examined; it was also present in most of the plasma samples obtained from hamsters in the symptomatic stage. PrP Sc was excreted in urine for a few days after oral administration; subsequently, urinary PrP Sc was not detected until the terminal disease stage. These results represent the first biochemical detection of PrP Sc in urine from TSE-infected animals.

Several lines of evidence suggest that microglia have important roles in the pathogenesis of prion diseases. Here, we establish a novel microglial cell line (MG20) from neonatal tga20 mice that overexpress murine prion protein. After exposure to Chandler scrapie, we observed the replication and accumulation of disease-associated forms of the prion protein in MG20 cells up to the 15th passage. Furthermore, MG20 cells were susceptible to ME7, Obihiro scrapie, and bovine spongiform encephalopathy agents. Thus, MG20 cell lines persistently infected with various murine prion strains provide a useful model for the study of the pathogenesis of prion diseases.

Asp. niger a-glucosidase wascrystallized in ammonium sulfate solution after chromatographies on DEAE-Sepharose CL-6Band TOYOPEARL HW-55columns. The crystalline a-glucosidase, which was a glycoprotein containing 25.5% carbohydrate as glucose, gave a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be about 12.5 x 104 by SDS-disc gel electrophoresis. However, the crystalline enzyme consists of two components (M.W. about 3.3 x 104 and 9.8 x 104, respectively) separable only by reverse-phase HPLCcausing irreversible inactivation. The optimumpH was 4.3. The enzymealso hydrolyzed a-glucans such as soluble starch and amylose. The analyses of the primary structure and the catalytic groups of the crystalline a-glucosidase are under way in our laboratory. This paper describes the crystallization, the substrate specificity, and the subsite affinities of Asp. niger a-glucosidase.

ABSTRACT. Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzymelinked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP Sc ) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP Sc detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP Sc was observed in WB, even when performed after 4 days of incubation at 37°C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP Sc due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses. KEY WORDS: BSE, ELISA, PrP Sc , Western blot.