Bovine spongiform encephalopathy in the United States.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R F Marsh
Publications and source records attributed to R F Marsh.
Presence of mitochondrial D-loop DNA in scrapie-infected brain preparations enriched for the prion protein.
The prion preparation has, in recent years, been the focal point of scrapie research. The inability to identify agent-specific nucleic acids in this sample has led to the formulation of the infectious protein or prion hypothesis. In this study, we analyzed three different prion protein-enriched preparations and found all to contain significant amounts of mitochondrial nucleic acid. Southern blot analyses indicated that they are enriched for a specific component of the mitochondrial genome, the single-stranded displacement loop fragment. Our results suggest that if mitochondrial nucleic acids are involved in scrapie infection, it is the displacement loop fragment that is specifically responsible.
Animals↗
The search for scrapie agent nucleic acid.
Despite decades of research, the identity of the scrapie agent has remained elusive. Recent studies have discovered much about the influence of the host genome upon scrapie infection, yet relatively little is known about the causative agent itself. The predominant hypothesis in the scrapie field (the prion hypothesis) argues that the disease is the result of an infectious protein and that nucleic acid is not required for infection. Biological studies of the scrapie agent, however, suggest that a nucleic acid may be involved in the disease. Sensitive molecular biology techniques have yet to identify this putative nucleic acid.
Animals↗
Evidence of mitochondrial involvement in scrapie infection.
Two cDNA libraries were constructed from brain membrane and cytoskeletal preparations purified from scrapie-infected hamster brains. Four recombinants strongly preferential to the scrapie cytoskeletal preparation were identified by the differential hybridization of 7,000 recombinants. These clones were not, however, preferential to total nucleic acids extracted from scrapie-infected hamster brains. DNA sequence analysis revealed all four clones to have significant sequence similarities to the mouse mitochondrial genome. This correlation led us to consider a mitochondrial association with scrapie infectivity. Brain mitochondria were purified by sucrose gradient density centrifugation and found to contain high infectivity. Removal of mitochondrial outer membranes by osmotic shock or digitonin treatment resulted in no detectable loss of titer.
Animals↗
Photoreceptor degeneration in experimental transmissible mink encephalopathy of hamsters.
Hamsters were inoculated intracerebrally with the agent of transmissible mink encephalopathy and developed clerical signs of encephalopathy. Photoreceptor degeneration occurred in all animals examined histologically. The changes were similar to those in scrapie, although less extensive. The findings suggest that either transmissible mink encephalopathy is a mink-adapted form of scrapie or, in rodents, photoreceptor degeneration is a characteristic of infection with agents of the spongiform encephalopathies.
Animals↗
Photoreceptor degeneration during infection with various strains of the scrapie agent in hamsters.
Hamsters were inoculated intracerebrally with the 22C, 79A, and ME7 strains of the scrapie agent to compare the effects on the retina with those caused by strain 263K. The animals developed clinical signs of encephalopathy. Photoreceptor degeneration occurred in all experimental animals. The changes were similar to those seen in animals infected with the 263K strain of scrapie although somewhat more variable and less extensive.
Animals↗
Effects of different methods of purification on aggregation of scrapie infectivity.
High levels of scrapie infectivity were found in detergent-insoluble residues of hamster brain purified by either repeated pelleting in 10% NaCl or by separation in Nycodenz gradients. Titres determined by the method of incubation interval assay were 100-fold higher than titres measured by endpoint dilution assay. The protein profiles and end-labelled RNA examined by one-dimensional polyacrylamide gel electrophoresis were not different from samples prepared from uninfected brain. Preparations produced by repeated pelleting were treated with RNase A and/or 7 M-urea with no loss of scrapie infectivity. However, the infectivity of samples prepared by gradient centrifugation in Nycodenz were reduced by 2 to 3 log10 LD50 by treatment with RNase A alone but not in combination with SDS. These results suggest that the scrapie agent may be aggregated by methods of purification employing pelleting in high concentrations of salt, or by adding polycations to disaggregated samples.
Animals↗
Immunochemical characterization of proteins from scrapie-infected hamster brain, using immunoblot analysis.
Preparations of brain plasma membrane from scrapie-infected or noninfected hamsters were extracted with a solvent and were used to inoculate rabbits. Antisera evaluated by immunoblot analysis revealed a protein of 45 kD in scrapie-infected hamster brain that had a greater signal compared with proteins of comparable relative mass in noninfected brain. This 45-kD protein was not increased in scrapie-infected mouse, sheep, or goat brain. Seemingly, the 45-kD protein may be a degradation product of glial fibrillary acidic protein.
Animals↗
Effect of bacterial flora and mouse genotype (euthymic or athymic) on scrapie pathogenesis.
Euthymic and athymic female BALB/c mice, reared under either germfree or defined flora conditions, were used to investigate the pathogenesis of scrapie after intracerebral or intraperitoneal inoculation. Time in days to onset of clinical signs (Stage I), to endstage (Stage II), and the time interval between Stage I and Stage II were compared among groups. In addition, scrapie agent titers in spleen were determined at 28 and 90 days after infection, as were agent titers in spleen and brain at Stage II. Three-way analysis of variance indicated that the bacterial flora, the presence or absence of a thymus, and the route of agent inoculation interact to produce significant differences in the pathogenesis of disease. The three factors in the experimental design also influenced the spleen titers of scrapie infectivity. The variation in scrapie pathogenesis among the groups of mice is likely to be mediated by differences in their reticuloendothelial systems. These differences may alter the agent's adsorption in spleen and/or route of transport from spleen to brain.
Animals↗
Asymmetry of retinal lesions in experimental scrapie after intracerebral inoculation of hamsters.
Hamsters injected into the right cerebral hemisphere with the scrapie agent developed retinal lesions to a greater extent in the contralateral than in the ipsilateral eye. This asymmetry was evident during the incubation period as well as during the clinical encephalopathy. The results explain much of the variation in the degree of retinal disease seen earlier at specific times after inoculation. Moreover, they strengthen the hypothesis that scrapie spreads via neurons.
Animals↗
Retinal degeneration in experimental scrapie after intraperitoneal or subcutaneous inoculation of hamsters.
Hamsters injected intraperitoneally or subcutaneously with the scrapie agent developed photoreceptor degeneration. The degree of degeneration did not correlate well with infectivity titers of retinal tissue or stage of clinical encephalopathy, and was not as great as seen in intracerebrally injected animals. We conclude that retinal degeneration is universal in hamsters experimentally inoculated with the scrapie agent regardless of the route of inoculation.
Animals↗
Comparison of RNA from healthy and scrapie-infected hamster brain.
Density gradient fractions prepared from healthy or scrapie-infected hamster brain tissue enriched in plasma membrane vesicles were treated with nucleases prior to phenol extraction and ethanol precipitation. The recovered nucleic acids were 3' end-labelled and run on one-dimensional polyacrylamide gels. Autoradiography revealed the presence of low molecular weight RNAs (4S) in both healthy and scrapie samples. Two-dimensional fingerprint analysis indicated that the RNAs isolated from scrapie-infected hamsters contained oligonucleotides that were not present in RNAs isolated from healthy hamsters.
Animals↗
Inactivation of the scrapie agent by ultraviolet irradiation in the presence of chlorpromazine.
The sensitivity of the scrapie agent to u.v. inactivation was found to be related to the purity of the tissue preparation. Scrapie infectivity associated with membrane vesicles was unaffected when irradiated with 10(4) J/m2. Irradiation of more highly purified preparations from detergent-extracted CsCl gradient fractions reduced scrapie infectivity from 10(7.8) log10 LD50 per ml to as low as 10(4.5). Sensitivity of membrane-associated scrapie infectivity to inactivation by u.v. irradiation could be increased by addition of chlorpromazine, a phenthiazine antipsychotic which penetrates lipid bilayers and induces single-strand breaks in nucleic acids under irradiation. Chlorpromazine without irradiation, and a semiquinone protein-binding radical of chlorpromazine, failed to decrease scrapie infectivity by themselves. A closely related phenthiazine antipsychotic, trifluoperazine, which does not bind to nucleic acids, did not reduce scrapie infectivity. These findings suggest that the target of u.v. radiation for inactivation of scrapie infectivity in the presence of chlorpromazine is an essential nucleic acid.
Chlorpromazine↗
Characterization of proteins in membrane vesicles from scrapie-infected hamster brain.
Previous studies have shown that the scrapie agent is highly membrane-associated. We examined the protein composition of gradient fractions enriched for large membrane vesicles prepared from scrapie-infected and uninfected hamster brain using various methods to extract membrane proteins. We also examined proteins in detergent-extracted membrane vesicles fractionated on CsCl gradients. No qualitative differences in protein composition were seen comparing scrapie-infected and uninfected samples by one-dimensional gel electrophoresis. Extraction of proteins from membrane vesicles by phenol, pyridine, perchloric acid or lithium diiodosalicylate also failed to reveal any unique proteins in scrapie-infected hamster brain. Attempts to solubilize hydrophobic proteins (proteolipids) from CsCl gradient fractions into organic solvents were unsuccessful. These findings indicate that any hydrophobic protein associated with the scrapie agent is not a proteolipid, and that the ability of solvents to reduce scrapie infectivity is not a result of extraction of a proteolipid.
Animals↗
Characterization of lipids in membrane vesicles from scrapie-infected hamster brain.
The lipid compositions of membrane vesicles from scrapie-infected and uninfected hamster brains were examined before and after detergent extraction. No differences were observed in polar lipids, glycolipids, gangliosides or neutral lipids examined by thin-layer chromatography. Analysis of detergent-extracted CsCl gradient fractions with high scrapie infectivity failed to reveal any glycerolphosphatides, although neutral lipids were demonstrated. The major neutral lipid associated with detergent-extracted membrane vesicles from both infected and uninfected brain was an unidentified lipid which was found to absorb u.v. radiation strongly from 250 to 300 nm wavelengths. Membrane neutral lipids that strongly absorb u.v. radiation at wavelengths normally used to inactivate viruses may protect a small nucleic acid essential for scrapie infectivity.
Animals↗
Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain.
This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed.
Animals↗
Equilibrium density gradient centrifugation of the scrapie agent in Nycodenz.
Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains were detergent-extracted, then separated by equilibrium density centrifugation in continuous Nycodenz gradients. The highest level of infectivity was always associated with the insoluble residue which sedimented through 40% Nycodenz. The degree of aggregation in these insoluble complexes varied depending upon treatment. Centrifugation in gradients containing 2 M- to 8 M-urea resulted in the formation of large insoluble aggregates which seemed to retain a high level of infectivity when measured by the method of incubation interval assay. However, measurement of infectivity in these same samples by endpoint titration of tenfold dilutions resulted in values a thousand times lower. These observations reinforce previous findings that scrapie infectivity exists as a macromolecular complex and, furthermore, they emphasize the necessity for using non-denaturing conditions for purification of the scrapie agent.
Animals↗
Purification of the scrapie agent by density gradient centrifugation.
Plasma membrane-enriched preparations from scrapie-infected and healthy hamster brains, as well as preparations of neural retina, were sonicated, then separated by rate-zonal sedimentation in 10 to 25% Nycodenz gradients. Gradient fractions were extracted with 0.5% Triton X-100 and re-fractionated by equilibrium density centrifugation in linear 25 to 40% CsCl gradients. Infectivity was highest in a fraction having a density of 1.280 g/ml and which contained a visible band of material. Digestion of the Nycodenz fractions with proteinase K before detergent extraction and CsCl fractionation resulted in a shift in the visible band to a density of 1.235 g/ml with most of the scrapie infectivity remaining at 1.280 g/ml. When labelled with 125I after 40-fold concentration, this 1.280 g/ml CsCl fraction from the proteinase K-treated gradients contained only a single band of protein(s) having a mol. wt. near 30 000. No differences were seen between proteins in healthy or scrapie-infected preparations.
Animals↗