Copurification of Sp33-37 and Scrapie Agent from Hamster Brain Prior to Detectable Histopathology and Clinical Disease

Abstract

Studies were conducted to determine whether accumulation of the scrapie agent protein Sp33-37 in brain correlated with the appearance of the scrapie agent or with pathology. The concentrations of the scrapie agent and Sp33-37 were measured in purified fraction P5 isolated from hamster brains at weekly intervals after inoculation. The scrapie agent concentration in fraction P5 was approximately 10 -1 LDs0/g brain 1 day post-inoculation and increased to 10 9.4 LDJg at day 77. Sp33-37 was first detected in P5 at day 21, when the agent titre was 10 3.9 LDs0/g. Sp33-37 concentration increased in concert with the scrapie agent concentration, although the apparent rate of increase was somewhat lower for the protein than for the agent. The histopathological evidence of disease, consisting of mild vacuolation and gliosis, was first seen at 35 days, but was not conspicuous until 49 to 56 days postinoculation. Vacuolation and gliosis increased until termination of the experiment at day 77. Amyloid plaques were first detected at 56 days and were widespread at day 77. Clinical disease was first seen in these animals at day 66, with an average onset at day 71. Control animals inoculated with buffer alone showed some mild gliosis, but were otherwise normal. The fact that Sp33-37 purified with the scrapie agent isolated from brain 14 days prior to detectable (light microscopic) pathology supports the theory that Sp33-37 is the major structural component of the scrapie agent and not solely a product of the pathology.

Molecular biology of transmissible spongiform encephalopathies

“…No chemical differences have so far been found between PrP c and PrP sc [21]. However, because the ratio of infectious units to PrP s~ molecules is only about 1 : 100 000 [22], the structure of the PrP molecule actually associated with infectivity cannot be definitively inferred. For this reason and because specific infectivity can vary considerably, the PrP species responsible for infectivity is presently better designated as PrP* [23]; it may or may not be identical with PrP so, the major species that has been characterized chemically and physicochemically.…”

Bovine spongiform encephalopathy (BSE): Its context in New Zealand and new tests

“…Cyclic amplification ("PCMA") of abnormal prion protein (analogous to the polymerase chain reaction for DNA) was described recently (Saborio et al 2001) as possibly capable of detecting PrP Sc at such low levels, i.e., c. 12 pg or 0.4 fmol. This compares with a sensitivity of 1000 pg for older immunoblot techniques (Bolton et al 1991). An extension of the PCMA work described the ability of PCMA to detect P r PSc i n hamsters not more than 2 weeks after inoculation and well before clinical signs, exemplifying the earlier promise of a highly sensitive ante mortem test .…”

“…Infectious units in brains are about 10 5 less in numbers than the PrP Sc molecules present (from fig. 4 in Bolton et al 1991) and accumulate at a greater rate. Thus, it is unlikely the infectious unit is individual PrP Sc of fibril molecules already in the steric zippers.…”

“…Infectivity was first hypothesised to be a property of proteins in the 1960s (Alper et al 1967;Griffith 1967;Pattison & Jones 1967), and the suggestion was met with widespread skepticism. Rigorous studies later demonstrated PrP Sc transmits and titres with infectivity (Bolton et al 1982(Bolton et al , 1991, but did not formally prove that protein was the infectious agent. Unconventional though it was, the case for protein infectivity was strong and led to the proposition of the "Prion Hypothesis" (Prusiner 1982(Prusiner , 1998.…”

Prion Propagation: The Role of Protein Dynamics

“…60,70,72,73 Indirect evidence points to the importance of fragmentation of PrP complexes in vivo. For example, PrP Sc accumulation is exponential following infection, [74][75][76] and only mathematical models that consider fragmentation can accurately describe these kinetics. 63,77 Moreover, prion infectivity greatly increases upon partial disruption of ex vivo preparations of PrP by denaturation, 69 liposome dispersion, 67,78 homogenization 79 and sonication, 68,80 highlighting the potent effect of fragmentation on prion titre.…”