Analysis of Creutzfeldt-Jakob disease infectious fractions by gel permeation chromatography and sedimentation field flow fractionation

Nucleic acid binding proteins in highly purified Creutzfeldt-Jakob disease preparations.

“…Clearly reproducible filtration and physical studies indicate a viruslike core structure of significant size (7,8,10). The present demonstration of nucleic acid binding proteins is further evidence for protected nucleic acid-protein complexes in highly purified infectious fractions.…”

“…Human CJD brain, which cannot be titered directly but which had been verified by rodent transmission and/or neuropathology, was used as a species control. Analytical studies of colloidal gold stained proteins, Gp34 detected with antibodies, and 32P-labeled nucleic acids showed human CJD gradient profiles were comparable to those previously described for hamster CJD (8,10 VSV, were simultaneously included to assess sensitivity and sequence specificity of different binding proteins in Northwestern blots.…”

“…The supernatant was centrifuged on a linear 10-30% sucrose gradient to resolve the infectious 120S peak, yielding an '=100,000-fold purification of infectivity with respect to starting nucleic acids (8) and >11,000-fold with respect to protein (7). The 120S peak sucrose fractions were diluted =3-fold and were concentrated by pelleting at 215,000 x g for 2 hr with no loss of titer (10). Initial homogenization releases only =20% ofthe brain infectivity, almost all ofwhich is recovered in the final concentrates-i.e., 1.5-3 x 107 IU/g (7,8,10 RNA (8).…”

“…The 120S peak sucrose fractions were diluted =3-fold and were concentrated by pelleting at 215,000 x g for 2 hr with no loss of titer (10). Initial homogenization releases only =20% ofthe brain infectivity, almost all ofwhich is recovered in the final concentrates-i.e., 1.5-3 x 107 IU/g (7,8,10 RNA (8). This RNA was reverse transcribed by using random primers, the resultant cDNAs were converted to double-stranded DNA molecules, and then designed PCR adaptor/primers were blunt-end ligated to the cDNA for sequence-independent amplification (12).…”

Transcytosis of Murine-Adapted Bovine Spongiform Encephalopathy Agents in anIn VitroBovine M Cell Model

“…It is well known that the detergent-insoluble and relatively proteinase K (PK)-resistant prion protein (PrP-res) is detectable in many kinds of TSE-infected tissues, including the brain. Although some studies have revealed that PrP-res does not correlate with infectivity levels in animal tissues as well as in subcellular fractions (37,40), PrP-res is a useful surrogate marker for TSE infection.…”