NR AXUT
AU Price,J.C.; Deering,C.; Wille,H.; Safar,J.G.; Prusiner,S.B.
TI Mechanistic Insights into in Vitro Amplification of Prions
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.04
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB
Background: The in vitro technique PMCA (Protein Misfolding Cyclic Amplification) uses bursts of ultra sound to drive the conversion of PrPc to PrPsc in cell free brain homogenates (Soto et al. FEBS Letters 2005, Nishina et al. Biochemistry 2006). Although the in vitro produced PrPsc has been shown to retain infectivity, little is known about the mechanism of PrPsc amplification.
Objective: To elucidate the mechanism of in vitro PrPc to PrPsc conversion, by investigating the composition of the rate limiting step in the conversion process.
Methods: The fidelity of the Sc237 strain characteristics were monitored over multiple rounds of PMCA amplification, using Western blots, conformation-dependent immunoassay (CDI), and bioassay. We also assess the effects of changing reactant concentrations upon the kinetics of amplification.
Results: We show that Syrian hamster Sc237 prions retain their strain characteristics after many rounds of PMCA. Although the conformational state of the product seems to be conserved, we find that the kinetics of the system are subject to large variations and difficult to quantify. Measuring the final concentration of the in vitro generated prions in saturated samples allowed us to assess the effect of the changing substrate concentrations.
Conclusion: The results suggest that a well-defined mechanism for PrPc to PrPsc conversion is operating during the cycles of high energy sonication. Although PMCA in its current form is not able to provide appreciable kinetic information, we show that the rate limiting step of the amplification is sensitive to the composition of the amplification mixture.
AD J.C. Price, C. Deering, H. Wille, J.G. Safar, S.B. Prusiner, University of California San Francisco, Institute for Neurodegenerative Disease, USA
SP englisch
PO Schottland
EA pdf-Datei und Poster (Titel: Investigation of the in vitro amplification of rPrPsc; Autorenliste ohne C. Deering und H. Wille)