BACKGROUNDTo evaluate the influence ultrasonic processing on the aggregation of PrP(Sc) in the brain extracts prepared from the scrapie-infected hamsters, and to seek for the way to prepare lower molecular PrP(Sc) polymer.

METHODSThe extracts of infected brains were prepared with a lysis solution, and treated with ultrasonics at various conditions during the different phases. The distribution and aggregation state of PrP(Sc) were analyzed by proteinase K treated. Western blot, and afterwards, quantitatively calculated with a commercially supplied software Image Totaltech.

RESULTSThe amount of PrP(Sc) in the supernatant of brain homogenates was 1.29-to 1.58-fold increased with appropriate sonication (15 s for 30 times). At the same conditions of ultrasound, the PrP amount in the supernatant prepared from the scrapie-infected hamster brain was significantly increased, whereas that prepared from healthy animal used as normal control showed little change. Comparative analyses of PrP(Sc) pellets prepared by high-speed centrifugation revealed that about 90% PrP(Sc) released into supernatant after ultrasound processing.

CONCLUSIONAppropriate sonication of homogenate of scrapie-infected brain increases the extracted amount of PrP(Sc), being favorable to laboratory diagnosis. Larger molecular PrP(Sc) aggregates can be crashed by ultrasonic processing, engendering lower molecular PrP(Sc) polymers.

Animals ; Blotting, Western ; Brain Chemistry ; Cricetinae ; Image Processing, Computer-Assisted ; PrPSc Proteins ; analysis ; Scrapie ; metabolism ; Sonication

OBJECTIVETo establish a methodology of protein misfolding cyclic amplification (PMCA) and utilize in the detection of PrP(Sc) in brain tissues from prion diseases.

METHODSDifferent amounts of Scrapie 263K agent bulk were mixed with brain homogenates of health hamsters and treated with repeated incubation/sonication for 10 to 15 cycles. The proteinase K-resistant PrP(Sc) was evaluated with Western Blot.

RESULTSIn this experimental situation, 263K agent replicated rapidly in vitro, utilizing hamsters' brains as the medium. With the established PrP(Sc)-PMCA technique, PrP(Sc) signals in the preparations containing less than 10(-5) diluted 263K bulk could be detected. Compared with conveniently used immuno-blot assay, the sensitivity of PrP(Sc)-PMCA for PrP(Sc) was 10(5) to 10(6)-fold increased. It has been also shown that homogenates of cerebellar and brain stem could be used as the medium for PrP(Sc) replication.

CONCLUSIONA rapidly replicating method for PrP(Sc), PrP(Sc)-PMCA, was successfully established, providing a new approach for early diagnosis of prion diseases and research on the biological features of prion.

Animals ; Biochemistry ; methods ; Blotting, Western ; Brain ; metabolism ; pathology ; Cricetinae ; PrPSc Proteins ; analysis ; chemistry ; Prion Diseases ; diagnosis ; metabolism ; Protein Folding ; Reproducibility of Results ; Sensitivity and Specificity

Though the aetiology of transmissible spongiform encephalopathies (TSEs) remains uncertain, proteinase resistant prion protein (PrP-Sc), a converted form of the normal cellular prion protein (PrP-C), accumulates in the lysosome of cells of the nervous systems of animals with TSEs. In this study, clinical and epidemiological examinations of bovine spongiform encephalopathy (BSE) were conducted in Korea. During the investigated period, none of the cattle exhibited typical clinical signs of BSE, such as behavioral disturbances, high sensitivity, and abnormal locomotion. Immunohistochemical analysis and western immunoblotting were established to detect PrP-Sc in the brain tissue using monoclonal antibody (MAb) F89/160.1.5, produced by immunizing mice with a synthetic peptide which corresponds to bovine PrP residues 146-159, NH2-SRPLIHFGSDYEDRC-COOH. Although some BSE-like spongiform changes were observed in bovine brains randomly collected from Korean slaughterhouses from 1996 to 1999, no PrP-Sc was detected in those brains with the established immunohistochemistry and western immunoblotting assay. Also, no positive reaction was observed in bovine brains infected with rabies. These immunohistochemical and western immunoblotting methods using MAbs, specifically reactive with conserved epitopes on ruminant PrP, can be used for postmortem diagnosis of BSE. Further, the method can be applied to antemortem and the preclinical diagnosis of ovine scrapie by detecting PrP-Sc in lymphoid tissues, such as the tonsils, third eyelid or peripheral lymph nodes.
Abattoirs ; Animals ; Blotting, Western ; Brain/*pathology ; Brain Stem/pathology ; Cattle ; Encephalopathy, Bovine Spongiform/*epidemiology/pathology ; Immunohistochemistry ; Korea/epidemiology ; PrPSc Proteins/*analysis ; Sheep ; Sheep Diseases/*epidemiology/pathology

OBJECTIVETo expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrP(Sc) during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrP(Sc) forms.

METHODSHamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrP(Sc) were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayed by electron microscopy analysis (EM) and immuno-golden EM.

RESULTSPrP(Sc) was initially detected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrP(Sc) in brain tissues increased along with the incubation. Several high and low molecular masses of PrP were seen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions.

CONCLUSIONThese results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrP(Sc) may indwell in brain tissues during the infection.

Animals ; Blotting, Western ; Brain ; metabolism ; ultrastructure ; Cricetinae ; Disease Models, Animal ; Female ; Glycosylation ; Immunohistochemistry ; Mesocricetus ; Microscopy, Electron ; PrP 27-30 Protein ; analysis ; metabolism ; PrPC Proteins ; analysis ; metabolism ; PrPSc Proteins ; analysis ; metabolism ; Scrapie ; metabolism ; pathology

OBJECTIVETo report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope.

METHODSA hamster PrP protein (HaPrP) was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrPSc preparation from scrapie strain 263K.

RESULTSProtease-resistant bands were detected after four-day incubation.

CONCLUSIONThe new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Animals ; Biotin ; Blotting, Western ; Cell-Free System ; Chromatography, Affinity ; methods ; Cricetinae ; Escherichia coli ; genetics ; Gene Expression Regulation ; Molecular Weight ; Peptide Hydrolases ; analysis ; metabolism ; PrPSc Proteins ; genetics ; metabolism ; Prion Diseases ; genetics ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Staining and Labeling