- Email: [email protected]
Pitfalls in prion research
Medical Hypotheses (2000) 54(5), 698–700 © 2000 Harcourt Publishers Ltd doi: 10.1054/mehy.1999.0929, available online at http://www.idealibrary.com on
Pitfalls in prion research K. E. Peuschel Zürich, Switzerland
Summary Prion research seems to get increasingly enigmatic since the protein only hypothesis has been established as almost the only working hypothesis. This may indicate that the hypothesis could be wrong, and should prompt the search for potential faults in past experiments. In fact some problematic experiments can be pinpointed, for example determination of the N-terminal cleavage site of the prion protein PrP, of the structure of PrP as determined by NMR, some conclusions concerning the function of PrP from gene knockout experiments including potential evidence against the protein only hypothesis, and some aspects of the prion purification procedure. © 2000 Harcourt Publishers Ltd
INTRODUCTION The protein only hypothesis proposed to explain the puzzling phenomenon of prion diseases has not lead to a better understanding, but rather caused increased confusion because of the lack of a comparable disease entity and increasingly complex experiments, although with a correct working hypothesis one would usually expect to be able to explain all aspects of a complex problem with increasing clarity. Therefore former experiments were checked for potential faults that might have lead to misleading conclusions, and interestingly some results worthy to be discussed could in fact be identified including some evidence contradicting the protein only hypothesis. AMINO ACID SEQUENCING Because the prion protein (PrP) is a membrane protein, exact determination of the length of the N- and C-terminal signal peptides is most important for example for subsequent structural investigations. In the special case of the prion protein, a cysteine had to be assigned either to the N-terminal signal peptide or to the prion protein (first
Received 17 February 1999 Accepted 4 June 1999 Correspondence to: K. E. Peuschel MD, Freiestrasse 12, 8032 Zürich, Switzerland. Phone/Fax: +41 79 3202373
698
amino acid), but although it is known that derivatization is required to stabilize a cysteine for Edman-sequencing, a cysteine-derivatization was never done because this cysteine had been predicted to belong to the N-terminal signal peptide by signal peptide specialists (1–6). Moreover the first few amino-terminal amino acids always got lost in peptide-HPLC for mass spectrometry (5,6). Because a cysteine with 2 reactive residues in position 1 can be expected to be more reactive than a cysteine protected by 2 peptide bonds, derivatization would have to be done immediately after opening of disulfide bonds between cysteines to avoid potential organic reactions of cysteine like for example an elimination reaction. Nevertheless prion researchers assigned the cysteine in question to the N-terminal signal peptide (amino acid 22), and thus determination of the structure of the prion protein was bound to lead to a very unusual NMR-structure (12–14) with a completely random N-terminal half of the protein. POSITION OF PrP IN MEMBRANE The fact that the prion protein (PrP) can be released from cell membranes enzymatically with phosphatidylinositol specific phospholipase C has led to the generally accepted conclusion that it is a peripheral membrane protein located at the outside of the membrane (7), although this release does not automatically implicate that PrP is not a transmembrane protein, because for example
Pitfalls in prion research
membrane proteins subject to frequent conformational changes are less stably bound to the membrane and thus may drop out of the membrane if not additionally anchored. Studies to determine the transmembrane orientation of PrP have been done in Xenopus oocytes or cell-free systems in the presence of membranes, for example in rabbit reticulocyte lysates or in the wheat germ cell-free system, and gave results suggesting the existence of 2 populations of PrP, one secretory and the other transmembrane, or of multiple forms in experiments in presence of membranes (8–10). Because of the low expression of PrP in brain final conclusions concerning the transmembrane orientation could not be drawn to date, although with genetically engineered overexpression this would be possible and would be important to know for functional considerations. NMR-STRUCTURE With the amino acid sequence determined as described, the NMR-structure of the prion protein expressed in E. coli was determined and was found to be extremely unusual with a completely random and very poorly soluble N-terminal half and a single-conformation C-term with 3 alpha-helices, 2 beta-sheets and a disulfide bond between cysteines 2 and 3 (13,14). This structure is incompatible with expectations of a protein with 2 conformations, one being PrPc (non infectious), the other one PrPSc (infectious), and the fact that the N-term is completely disoriented may support the idea that the first cysteine attributed to the N-terminal signal peptide could actually be the first amino acid of the prion protein, which would throw over the structure completely because then the prion protein would have 3 instead of 2 cysteines and the disulfide bond could be formed between the first and the second or the third cysteine. Expression of the eukaryotic PrP membrane protein in E. coli instead of in an eukaryotic system does not make the structure more reliable, but the random N-term rather indicates that it may take the unique conditions of a membrane to allow proper folding of the N-term of PrP. PRION PURIFICATION In order to purify the infectious agent found in prion diseases, the so-called prion or PrPSc, brain homogenates were digested in several steps with proteinase K (partial resistance of PrPSc to digestion with proteinase K), DNAse and RNAse to remove contaminating proteins and nucleic acids without destroying prion infectivity (15–17,24), and no nucleic acid of any significance was found in the purified preparations after phenolization, but the purification protocol should have included © 2000 Harcourt Publishers Ltd
699
opening of the disulfide bond of the prion protein to safely release any second molecule involved from the prion protein, because otherwise such a molecule would be moved to the phenol phase together with the prion protein upon phenolization. DNAses and RNAses however would destroy any nucleic acid in the preparation after opening of the disulfide bonds. In fact the possibility that infectivity could have moved to the phenol phase was considered and the phenol phase was tested for the presence of infectivity, but it is very doubtful if uptake of a protein like PrPSc into cells via coated pits would not be critically disturbed after contact with phenol. PHENOTYPE OF PrP KNOCKOUTS Several lines of PrP knockout mice have been produced to find out more about the function of the prion protein. Some of them with remaining PrP sequences (18,19) produced no infectivity, no phenotype, and no histological changes, whereas complete exon knockouts including some of the flanking sequences (21) did not transmit infectivity, but showed a severe phenotype and cerebellar Purkinje cell loss. This should have been very suggestive of a full loss of function, but the somewhat counterintuitive conclusion was that a knockout of the prion protein had no functional and histological consequences, and that the phenotype of the complete exon knockouts was attributed to regulatory elements flanking the PrP exon. Correlation of the phenotype of the complete exon knockouts with a loss of function of the prion protein would have lead to the conclusion that the intact phenotype in the incomplete knockouts could be attributed to a functional rescue by the remaining PrP sequence. Knockout of infectivity but intact PrP function in those incomplete PrP knockout mice (18) could be important evidence against the protein only hypothesis because separation of infectivity and PrP function would argue for the involvement of a second molecule other than PrP in the generation of prion infectivity. Comparison of the knockouts with remaining PrP sequences shows in fact that one of them (18) with electrophysiological changes suggestive of remaining weak PrP activity (weak long term potentiation) contains a fusion PrP mRNA, whereas no PrP mRNA was found in the other knockouts (19,20) with more pronounced electrophysiological changes (short term potentiation). RESISTANCE TO UV, IONIZING RADIATION AND HEAT The unusual resistance of prions to various methods of inactivation has been seen as support of the protein only hypothesis, because UV-inactivation was found to have shifted from 260 nm to 237 nm, and experiments with Medical Hypotheses (2000) 54(5), 698–700
700
Peuschel
ionizing radiation indicated a target size smaller than the target size of any of the smallest known viruses (22,23), whereas the supporters of the protein only hypothesis failed to explain why it takes a higher than average autoclaving-temperature to inactivate a protein only although most proteins would be denatured at much lower temperatures. The possibility was not considered that the physical properties (e.g. optical) of a protein with an abnormally high content of beta-sheets (12) could be significantly different from those of average proteins with a very high percentage of alpha-helices, which could account for the highly unusual physical properties (UVshift and apparent target size). REFERENCES 1. Prusiner S. B., Groth D. F., Bolton C. D., Kent S. B., Hood L. E. Purification and structural studies of a major scrapie prion protein. Cell 1984; 38(1): 127–134. 2. Hope J., Morton L. J., Farquhar C.F., Multhaup G., Beyreuther K., Kimberlin R. H. The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and Nterminal protein sequence as predicted for the normal brain protein (PrP). EMBO J 1986; 5(10): 2591–2597. 3. Turk E., Teplow D. B., Hood L. E., Prusiner S. B. Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem 1988; 176(1): 21–30. 4. Harris D. A., Huber M. T., van Dijken P., Shyng S. L., Chait B. T., Wang R. Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites. Biochemistry US 1993; 32(4): 1009–1016. 5. Safar J., Wang W., Padgett M. P. et al. Molecular mass, biochemical composition and physicochemical behavior of the infectious form of the scrapie precursor protein monomer. Proc Natl Acad Sci USA 1990; 87(16): 6373–6377. 6. Stahl N., Baldwin M. A., Teplow D. B. et al. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry US 1993; 32(8): 1991–2002. 7. Stahl N., Borchelt D. R., Prusiner S. B. Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. Biochemistry US 1990; 29(22): 54045–54012. 8. Hay B., Prusiner S. B., Lingappa V. R. Evidence for a secretory form of the cellular prion protein. Biochemistry US 1987; 26(25): 8110–8115. 9. Hay B., Barry R. A., Lieberburg I., Prusiner S. B., Lingappa V. R. Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein. Mol Cell Biol 1987; 7(2): 914–920.
Medical Hypotheses (2000) 54(5), 698–700
10. Hegde R. S., Mastrianni J. A., Scott M. R. et al. A transmembrane form of the prion protein in neurodegenerative disease. Science 1998; 279(5352): 827–834. 11. Bazan J. F., Fletterick R. J., McKinley M. P., Prusiner S. B. Predicted secondary structure and membrane topology of the scrapie prion protein. Protein Eng 1987; 1(2): 125–135. 12. Baldwin M. A., Pan K. M., Nguyen J. et al. Spectroscopic characterization of conformational differences between PrPC and PrPSc: an alpha-helix to beta-sheet transition. Philos T Roy Soc B 1994; 343(1306): 435–441. 13. Riek R., Hornemann S., Wider G., Glockshuber R., Wuthrich K. NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231). FEBS Lett 1997; 413(2): 262–268. 14. James T. L., Liu H., Ulyanov N. V. et al. Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. Proc Natl Acad Sci USA 1997; 94(19): 10086–10091. 15. Gabizon R., McKinley M. P., Groth D. et al. Immunoaffinity purification and neutralization of scrapie prions. Prog Clin Biol Res 1989; 317: 583–600. 16. Pan K. M., Stahl N., Prusiner S. B. Purification and properties of the cellular prion protein from Syrian hamster brain. Protein Sci 1992; 1(20): 1343–1352. 17. Sklaviadis T., Akowitz A., Manuelidis E. E., Manuelidis L. Nuclease treatment results in high specific purification of Creutzfeld-Jakob disease infectivity with a density characteristic of nucleic acidprotein complexes. Virology 1990; 112(3–4): 215–228. 18. Bueler H., Fischer M., Lang Y. et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992; 356(6370): 577–582. 19. Manson J. C., Clarke A. R., Hooper M. L., Aitchison L., McConnell I., Hope J. 129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal. Mol Neurobiol 1994; 8(2–3): 121–127. 20. Manson J. C., Hope J., Clarke A. R., Johnston A., Black C., MacLeod N. PrP gene dosage and long term potentiation. Neurodegeneration 1995; 4: 113–115. 21. Sakaguchi S., Katamine S., Nishida N. et al. Loss of cerebellar Purkinje cells in aged mice homzygous for a disrupted PrP gene. Nature 1996; 380(6574): 528–531. 22. Bellinger-Kawahara C., Cleaver J. E., Diener T. O., Prusiner S. B. Purified scrapie prions resist inactivation by UV irradiation. J Virol 1987; 61(1): 159–166. 23. Alper T. The scrapie enigma: insights from radiation experiments. Radiat Res 1993; 135(3): 283–292. 24. Prusiner S. B., McKinley M. P. Prions. New York: Academic Press, 1987. 25. Prusiner S. B., Collinge J., Powell J., Anderton B. Prion Diseases of Humans and Animals. Ellis Horwood, 1992. 26. Prusiner S. B. Prions, Prions, Prions. Berlin: Springer, 1996. 27. Baker H. F., Ridley R. M. Prion Diseases. Humana Press, 1996. 28. Collinge J., Palmer M. S. Prion Diseases. Oxford: Oxford University Press, 1997.
© 2000 Harcourt Publishers Ltd
Pitfalls in prion research K. E. Peuschel Zürich, Switzerland
Summary Prion research seems to get increasingly enigmatic since the protein only hypothesis has been established as almost the only working hypothesis. This may indicate that the hypothesis could be wrong, and should prompt the search for potential faults in past experiments. In fact some problematic experiments can be pinpointed, for example determination of the N-terminal cleavage site of the prion protein PrP, of the structure of PrP as determined by NMR, some conclusions concerning the function of PrP from gene knockout experiments including potential evidence against the protein only hypothesis, and some aspects of the prion purification procedure. © 2000 Harcourt Publishers Ltd
INTRODUCTION The protein only hypothesis proposed to explain the puzzling phenomenon of prion diseases has not lead to a better understanding, but rather caused increased confusion because of the lack of a comparable disease entity and increasingly complex experiments, although with a correct working hypothesis one would usually expect to be able to explain all aspects of a complex problem with increasing clarity. Therefore former experiments were checked for potential faults that might have lead to misleading conclusions, and interestingly some results worthy to be discussed could in fact be identified including some evidence contradicting the protein only hypothesis. AMINO ACID SEQUENCING Because the prion protein (PrP) is a membrane protein, exact determination of the length of the N- and C-terminal signal peptides is most important for example for subsequent structural investigations. In the special case of the prion protein, a cysteine had to be assigned either to the N-terminal signal peptide or to the prion protein (first
Received 17 February 1999 Accepted 4 June 1999 Correspondence to: K. E. Peuschel MD, Freiestrasse 12, 8032 Zürich, Switzerland. Phone/Fax: +41 79 3202373
698
amino acid), but although it is known that derivatization is required to stabilize a cysteine for Edman-sequencing, a cysteine-derivatization was never done because this cysteine had been predicted to belong to the N-terminal signal peptide by signal peptide specialists (1–6). Moreover the first few amino-terminal amino acids always got lost in peptide-HPLC for mass spectrometry (5,6). Because a cysteine with 2 reactive residues in position 1 can be expected to be more reactive than a cysteine protected by 2 peptide bonds, derivatization would have to be done immediately after opening of disulfide bonds between cysteines to avoid potential organic reactions of cysteine like for example an elimination reaction. Nevertheless prion researchers assigned the cysteine in question to the N-terminal signal peptide (amino acid 22), and thus determination of the structure of the prion protein was bound to lead to a very unusual NMR-structure (12–14) with a completely random N-terminal half of the protein. POSITION OF PrP IN MEMBRANE The fact that the prion protein (PrP) can be released from cell membranes enzymatically with phosphatidylinositol specific phospholipase C has led to the generally accepted conclusion that it is a peripheral membrane protein located at the outside of the membrane (7), although this release does not automatically implicate that PrP is not a transmembrane protein, because for example
Pitfalls in prion research
membrane proteins subject to frequent conformational changes are less stably bound to the membrane and thus may drop out of the membrane if not additionally anchored. Studies to determine the transmembrane orientation of PrP have been done in Xenopus oocytes or cell-free systems in the presence of membranes, for example in rabbit reticulocyte lysates or in the wheat germ cell-free system, and gave results suggesting the existence of 2 populations of PrP, one secretory and the other transmembrane, or of multiple forms in experiments in presence of membranes (8–10). Because of the low expression of PrP in brain final conclusions concerning the transmembrane orientation could not be drawn to date, although with genetically engineered overexpression this would be possible and would be important to know for functional considerations. NMR-STRUCTURE With the amino acid sequence determined as described, the NMR-structure of the prion protein expressed in E. coli was determined and was found to be extremely unusual with a completely random and very poorly soluble N-terminal half and a single-conformation C-term with 3 alpha-helices, 2 beta-sheets and a disulfide bond between cysteines 2 and 3 (13,14). This structure is incompatible with expectations of a protein with 2 conformations, one being PrPc (non infectious), the other one PrPSc (infectious), and the fact that the N-term is completely disoriented may support the idea that the first cysteine attributed to the N-terminal signal peptide could actually be the first amino acid of the prion protein, which would throw over the structure completely because then the prion protein would have 3 instead of 2 cysteines and the disulfide bond could be formed between the first and the second or the third cysteine. Expression of the eukaryotic PrP membrane protein in E. coli instead of in an eukaryotic system does not make the structure more reliable, but the random N-term rather indicates that it may take the unique conditions of a membrane to allow proper folding of the N-term of PrP. PRION PURIFICATION In order to purify the infectious agent found in prion diseases, the so-called prion or PrPSc, brain homogenates were digested in several steps with proteinase K (partial resistance of PrPSc to digestion with proteinase K), DNAse and RNAse to remove contaminating proteins and nucleic acids without destroying prion infectivity (15–17,24), and no nucleic acid of any significance was found in the purified preparations after phenolization, but the purification protocol should have included © 2000 Harcourt Publishers Ltd
699
opening of the disulfide bond of the prion protein to safely release any second molecule involved from the prion protein, because otherwise such a molecule would be moved to the phenol phase together with the prion protein upon phenolization. DNAses and RNAses however would destroy any nucleic acid in the preparation after opening of the disulfide bonds. In fact the possibility that infectivity could have moved to the phenol phase was considered and the phenol phase was tested for the presence of infectivity, but it is very doubtful if uptake of a protein like PrPSc into cells via coated pits would not be critically disturbed after contact with phenol. PHENOTYPE OF PrP KNOCKOUTS Several lines of PrP knockout mice have been produced to find out more about the function of the prion protein. Some of them with remaining PrP sequences (18,19) produced no infectivity, no phenotype, and no histological changes, whereas complete exon knockouts including some of the flanking sequences (21) did not transmit infectivity, but showed a severe phenotype and cerebellar Purkinje cell loss. This should have been very suggestive of a full loss of function, but the somewhat counterintuitive conclusion was that a knockout of the prion protein had no functional and histological consequences, and that the phenotype of the complete exon knockouts was attributed to regulatory elements flanking the PrP exon. Correlation of the phenotype of the complete exon knockouts with a loss of function of the prion protein would have lead to the conclusion that the intact phenotype in the incomplete knockouts could be attributed to a functional rescue by the remaining PrP sequence. Knockout of infectivity but intact PrP function in those incomplete PrP knockout mice (18) could be important evidence against the protein only hypothesis because separation of infectivity and PrP function would argue for the involvement of a second molecule other than PrP in the generation of prion infectivity. Comparison of the knockouts with remaining PrP sequences shows in fact that one of them (18) with electrophysiological changes suggestive of remaining weak PrP activity (weak long term potentiation) contains a fusion PrP mRNA, whereas no PrP mRNA was found in the other knockouts (19,20) with more pronounced electrophysiological changes (short term potentiation). RESISTANCE TO UV, IONIZING RADIATION AND HEAT The unusual resistance of prions to various methods of inactivation has been seen as support of the protein only hypothesis, because UV-inactivation was found to have shifted from 260 nm to 237 nm, and experiments with Medical Hypotheses (2000) 54(5), 698–700
700
Peuschel
ionizing radiation indicated a target size smaller than the target size of any of the smallest known viruses (22,23), whereas the supporters of the protein only hypothesis failed to explain why it takes a higher than average autoclaving-temperature to inactivate a protein only although most proteins would be denatured at much lower temperatures. The possibility was not considered that the physical properties (e.g. optical) of a protein with an abnormally high content of beta-sheets (12) could be significantly different from those of average proteins with a very high percentage of alpha-helices, which could account for the highly unusual physical properties (UVshift and apparent target size). REFERENCES 1. Prusiner S. B., Groth D. F., Bolton C. D., Kent S. B., Hood L. E. Purification and structural studies of a major scrapie prion protein. Cell 1984; 38(1): 127–134. 2. Hope J., Morton L. J., Farquhar C.F., Multhaup G., Beyreuther K., Kimberlin R. H. The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and Nterminal protein sequence as predicted for the normal brain protein (PrP). EMBO J 1986; 5(10): 2591–2597. 3. Turk E., Teplow D. B., Hood L. E., Prusiner S. B. Purification and properties of the cellular and scrapie hamster prion proteins. Eur J Biochem 1988; 176(1): 21–30. 4. Harris D. A., Huber M. T., van Dijken P., Shyng S. L., Chait B. T., Wang R. Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites. Biochemistry US 1993; 32(4): 1009–1016. 5. Safar J., Wang W., Padgett M. P. et al. Molecular mass, biochemical composition and physicochemical behavior of the infectious form of the scrapie precursor protein monomer. Proc Natl Acad Sci USA 1990; 87(16): 6373–6377. 6. Stahl N., Baldwin M. A., Teplow D. B. et al. Structural studies of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry US 1993; 32(8): 1991–2002. 7. Stahl N., Borchelt D. R., Prusiner S. B. Differential release of cellular and scrapie prion proteins from cellular membranes by phosphatidylinositol-specific phospholipase C. Biochemistry US 1990; 29(22): 54045–54012. 8. Hay B., Prusiner S. B., Lingappa V. R. Evidence for a secretory form of the cellular prion protein. Biochemistry US 1987; 26(25): 8110–8115. 9. Hay B., Barry R. A., Lieberburg I., Prusiner S. B., Lingappa V. R. Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein. Mol Cell Biol 1987; 7(2): 914–920.
Medical Hypotheses (2000) 54(5), 698–700
10. Hegde R. S., Mastrianni J. A., Scott M. R. et al. A transmembrane form of the prion protein in neurodegenerative disease. Science 1998; 279(5352): 827–834. 11. Bazan J. F., Fletterick R. J., McKinley M. P., Prusiner S. B. Predicted secondary structure and membrane topology of the scrapie prion protein. Protein Eng 1987; 1(2): 125–135. 12. Baldwin M. A., Pan K. M., Nguyen J. et al. Spectroscopic characterization of conformational differences between PrPC and PrPSc: an alpha-helix to beta-sheet transition. Philos T Roy Soc B 1994; 343(1306): 435–441. 13. Riek R., Hornemann S., Wider G., Glockshuber R., Wuthrich K. NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231). FEBS Lett 1997; 413(2): 262–268. 14. James T. L., Liu H., Ulyanov N. V. et al. Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. Proc Natl Acad Sci USA 1997; 94(19): 10086–10091. 15. Gabizon R., McKinley M. P., Groth D. et al. Immunoaffinity purification and neutralization of scrapie prions. Prog Clin Biol Res 1989; 317: 583–600. 16. Pan K. M., Stahl N., Prusiner S. B. Purification and properties of the cellular prion protein from Syrian hamster brain. Protein Sci 1992; 1(20): 1343–1352. 17. Sklaviadis T., Akowitz A., Manuelidis E. E., Manuelidis L. Nuclease treatment results in high specific purification of Creutzfeld-Jakob disease infectivity with a density characteristic of nucleic acidprotein complexes. Virology 1990; 112(3–4): 215–228. 18. Bueler H., Fischer M., Lang Y. et al. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992; 356(6370): 577–582. 19. Manson J. C., Clarke A. R., Hooper M. L., Aitchison L., McConnell I., Hope J. 129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal. Mol Neurobiol 1994; 8(2–3): 121–127. 20. Manson J. C., Hope J., Clarke A. R., Johnston A., Black C., MacLeod N. PrP gene dosage and long term potentiation. Neurodegeneration 1995; 4: 113–115. 21. Sakaguchi S., Katamine S., Nishida N. et al. Loss of cerebellar Purkinje cells in aged mice homzygous for a disrupted PrP gene. Nature 1996; 380(6574): 528–531. 22. Bellinger-Kawahara C., Cleaver J. E., Diener T. O., Prusiner S. B. Purified scrapie prions resist inactivation by UV irradiation. J Virol 1987; 61(1): 159–166. 23. Alper T. The scrapie enigma: insights from radiation experiments. Radiat Res 1993; 135(3): 283–292. 24. Prusiner S. B., McKinley M. P. Prions. New York: Academic Press, 1987. 25. Prusiner S. B., Collinge J., Powell J., Anderton B. Prion Diseases of Humans and Animals. Ellis Horwood, 1992. 26. Prusiner S. B. Prions, Prions, Prions. Berlin: Springer, 1996. 27. Baker H. F., Ridley R. M. Prion Diseases. Humana Press, 1996. 28. Collinge J., Palmer M. S. Prion Diseases. Oxford: Oxford University Press, 1997.
© 2000 Harcourt Publishers Ltd