Objective: To find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric, colon, liver, lung, thyroid, and laryngeal cancers. Methods: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to test the amounts and electrophoretic patterns of total PrP and the tolerance of PK (protease K) digestion among six various cancer tissue types. Results: A mass of PrP signals with a large molecular weight were identified in the homogenates of peripheral tissues. The amounts and electrophoretic patterns of total PrP did not differ significantly between carcinous and pericarcinous tissues. PrPs in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types. Conclusions The study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar. Unlike PrP-specific immunohistochemical assay, evaluation of PrP electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.
Animals ; Blotting, Western ; Brain ; Brain Chemistry ; Cricetinae ; Electrophoresis, Polyacrylamide Gel ; Humans ; Neoplasms/chemistry* ; Prion Proteins/analysis*

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

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