Vet Microbiol. 2007 Apr 7; [Epub ahead of print]
Generation of genuine prion infectivity by serial PMCA.
Weber P, Giese A, Piening N, Mitteregger G, Thomzig A, Beekes M, Kretzschmar HA.
Centre for Neuropathology and Prion Research, Ludwig Maximilians University of Munich, Feodor-Lynen-Strasse 23, 81377 Munich, Germany.
Prions are the causative infectious agents of transmissible spongiform encephalopathies (TSEs). They are thought to arise from misfolding and aggregation of the prion protein (PrP). In serial transmission protein misfolding cyclic amplification (sPMCA) experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the structural conversion of cellular prion protein (PrP(C)) as efficiently as PrP(Sc) from the brain of scrapie-infected (263K) hamsters confirming an autocatalytic misfolding cascade as postulated by the prion hypothesis. However, the fact that PrPres generated in vitro was associated with approximately 10 times less infectivity than an equivalent quantity of brain-derived PrP(Sc) casts doubt on the "protein-only" hypothesis of prion propagation and backs theories that suggest there are additional molecular species of infectious PrP or other agent-associated factors. By combining sPMCA with prion delivery on suitable carrier particles we were able to resolve the apparent discrepancy between the amount of PrPres and infectivity which we were then able to relate to differences in the size distribution of PrP aggregates and consecutive differences in regard to biological clearance. These findings demonstrate that we have designed an experimental set-up yielding in vitro generated prions that are indistinguishable from prions isolated from scrapie-infected hamster brain in terms of proteinase K resistance, autocatalytic conversion activity, and - most notably - specific biological infectivity.
PMID: 17493773
-----Similar analysis using cyclical amplification procedures-----
Novel Metal Clusters Lethal to Cancer Cells
Dr. Vitaly Vodyanoy, Auburn Univ. Alabama
Abstract
Unfolding and subsequent aggregation of proteins is a common phenomenon that is linked to many human disorders. Misfolded hemoglobin is generally manifested in various autoimmune, infectious and inherited diseases. We isolated micrometer and submicrometer parti-cles, termed proteons, from human and animal blood. Proteons lack nucleic acids but contain two major polypeptide populations with homology to the hemoglobin alpha-chain. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers
(PNCs). PNCs are comprised of 1- to 2-nm metallic nanoclusters
containing 40–300 atoms. Each milliliter of human blood contained approximately 7 X 10(13) PNCs and approximately 3 X 10(8) proteons. Exposure of isolated blood plasma to elevated temperatures increased the
number of proteons. When an aliquot of this heated plasma was introduced into untreated plasma that was subsequently heated, the number of proteons further increased, reaching a maximum after a total of three such iterations. Small concentrations of PNCs were lethal to
cultured cancer cells, whereas noncancerous cells were much less affected.
quotes from Vodyanoy's paper:
"These experiments are consistent with a process by which proteons form from preexisting PNCs that serve as nuclei for misfolded protein aggregation, and thereby provide a methodology for the assembly and disassembly of proteons in vitro."
Proteon Nucleating Centers:
When PNCs were removed from blood plasma by filtration through a 5-kDa filter, the amount of protein in the retentate was equivalent to that of the nonfiltered sample, and no protein was found in the filtrate (data not shown). Proteons could not be produced from the retentate
until an aliquot of the filtrate was added back, and proteon production was dependent on the amount of filtrate added (data not shown). Interestingly, proteons could be produced in this manner even after the filtrate was carbonized at 660 ° C, but production was quenched
by 10 m M EDTA, a metal-chelating agent (data not shown)...
TEM showed that the bulk of the dried PNC-containing filtrate was amorphous, and that it contained crystalline metallic nanoparticles of 1–2 nm in diameter.."
(This paper utilizes serial protein misfolding cyclical amplification techniques as well. In the case of a healthy animal, good PNCs (metal nanoclusters) allow for reversible aggregation of misfolded proteins. Dr. Vitaly Vodyanoy calls these "proteons". Prions are a non-reversible version of malformed proteins aggregating around bad (or inappropriate) metals nanoclusters.
Dr. Kretzschmar's team used "suitable carrier particles" in their prion cyclical amplification procedure, thus demonstrating that without the "nucleating center [Vodyanoy's were metal nanoparticles containing 40 - 300 metal atoms] - the prion alone could not cause the disease progression.
So what were Kretzchmar's PNCs made of?