Institut National de la Recherche Agronomique (INRA) Virologie et Immunologie Moléculaires, 78352 Jouy en Josas, France1
Unité de Pharmacologie et dImmunologie, CEA Saclay, Gif/Yvette, France2
Author for correspondence: Mohammed Moudjou. Fax +33 1 34 65 26 21. e-mail moudjou{at}biotec.jouy.inra.fr
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here we report the first quantification of PrPC in different tissues of sheep using a two-site enzyme immunometric assay (EIA). Furthermore, by use of a simple method to immunoprecipitate PrPC from a crude tissue extract of healthy sheep tissue, we have demonstrated that the isoform profile of PrPC is tissue-specific. The most striking PrPC glycoform profile was obtained from skeletal muscle.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue preparation.
Seven 2-year-old ewes (ewe I, PrnP genotype ARQ/ARR at positions 136, 154 and 171; ewe II, ARQ/ARR; ewe III, ARQ/ARR; ewe IV, ARQ/VRQ; ewe V, AHQ/ARR; ewe VI, ARQ/ARR; and ewe VII, ARQ/ARR) in the early stages of pregnancy were sacrificed under controlled conditions and the organs were rapidly removed and placed on ice for a maximum of 30 min. After washing with PBS, each organ was frozen at -80 °C until use.
The genotype at the PrnP locus was determined for codons 136, 154 and 171 at Labogena, as described by Elsen et al. (1999) except that DNA was purified from skeletal muscle tissues.
Tissue extraction.
One sample of each sheep organ was homogenized to a 10% suspension with a Polytron homogenizer (Kinematica) in two different extraction buffers: buffer B (12·5 mM Tris, 12·5 mM MES, pH 6·8, 50 mM NaCl, 1% Zwittergent 3-12); or TL1 buffer (50 mM TrisHCl, pH 7·5, 0·5% Triton X-100, 0·5% deoxycholate). Both buffers contained 1 mM PMSF and 2 µg/ml each of aprotinin, leupeptin and pepstatin (Sigma) as protease inhibitors. After centrifuging the crude extract at 4 °C for 10 min at 4000 g, supernatants were removed and processed.
Quantification of PrPC in different tissues using a two-site EIA.
The solid phase EIA technique used in the present work is described in detail by Rodolfo et al. (2001) . Microtitre plates (Immunoplate Maxisorp, Nunc) were coated with the capture IgG MAbs SAF34 or SAF37. The plates were then saturated with EIA buffer (0·1 M potassium-phosphate buffer, pH 7·4, 0·15 M NaCl, 0·1% BSA) and stored at 4 °C until use. Before use, the plates were washed three times with wash buffer (10 mM phosphate buffer, pH 7·4, containing 0·05% Tween 20) and then processed for the EIA test. Ovine rPrP (VRQ allele), purified as described by Rezaei et al. (2000)
, was used as an internal plate control to produce a standard curve ranging from 10 to 0·078 ng/ml. Different dilutions of extracts corresponding to each tissue were made in EIA buffer containing 0·1% Triton X-100 and processed at the same time as the standard protein. All dilutions were duplicated in each experiment. After 3 h of incubation at room temperature with mild shaking, the microtitre plates were washed three times with wash buffer and incubated overnight at 4 °C with the tracer antibody. The latter consists of Fab' fragments obtained from 12F10 IgGs and coupled to a G4 tetramer of acetylcholinesterase (AchE) (Grassi et al., 1988
). After three washes, AchE activity was assessed by adding Ellman reagent (Ellman et al., 1961
) and measuring absorbance at 414 nm with an automatic reader (LabSystems).
PrPC immunoprecipitation.
Anti-PrP MAbs Pc248 or 4F2 were added to 500 µl (50 mg tissue equivalent) of the different clarified total extracts, obtained as described above. The samples were then incubated for 1 h at room temperature. To spin down the immunocomplexes, protein ASepharose beads (PharmaciaAmersham) were added to the mixtures and incubated for 1 h at room temperature or overnight at 4 °C on a rotating wheel. The beads were then washed four times with the corresponding extraction buffer and once with double-distilled water before dissolving in 50 µl of 8 M urea and 20 mM DTT. The correct volume of 4x Laemmli sample buffer was then added (Laemmli, 1970 ).
Deglycosylation of PrPC.
The immunoprecipitated PrPC present in the urea/Laemmli sample buffer was diluted in deglycosylation buffer (50 mM TrisHCl, pH 8·0, 1% Nonidet-P40) and treated with 0·5 U/ml of N-glycosidase F (Boehringer Mannheim) for 5 h at 37 °C. Samples were precipitated with 10% trichloroacetic acid and washed twice with ethanol before dissolving in Laemmli sample buffer and analysis by Western blotting.
Analytical methods.
SDSPAGE was performed with the Miniprotean II Biorad system. Gel transfer of proteins separated by SDSPAGE was carried out using the Minigel Transblot Cell system (Biorad), according to the manufacturers instructions. Low molecular mass markers (PharmaciaAmersham) were as follows: phosphorylase b, 94 kDa; BSA, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 20 kDa; and -lactalbumin, 14 kDa. After immunoprecipitation with the anti-PrP MAbs Pc248 or 4F2, PrPC was detected using either the purified IgG fraction of MH44 PAb or the biotinylated 4F2 MAb. PrPC was then visualized using the ECL detection technique with specific goat anti-rabbit IgGs coupled to peroxidase or streptavidin-coupled peroxidase (Pierce), respectively. Protein content was determined by the Bradford method.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Tissue-specific isoform distribution of PrPC
To check whether the organs differed only by their content of PrPC or, in addition, by the biochemical signature of the protein, we developed an immunoprecipitation method to concentrate PrPC from whole tissue extracts. All tissues were homogenized to the same wt/vol ratio (10%). We first tried to detect PrPC by classical Western blot using several anti-PrP MAbs and PAbs. Only brain extract showed strong labelling of PrPC (data not shown). However, after lengthy exposure of the nitrocellulose membranes, we detected faint signals of PrPC, but always in the same tissues, in the skeletal muscle, tongue, lungs and heart (data not shown). We then decided to concentrate PrPC by immunoprecipitation directly from each clarified crude tissue extract before its detection by Western blot.
Fig. 3 shows an example of a comparative immunoprecipitation experiment with both MAb Pc248 IgG1 and an anti-PrPC unrelated mouse IgG1 on the same tissue extracts from ewe VII. Only results obtained with extracts prepared in TL1 buffer are shown, as they gave the best results (especially from non-neuronal tissues). The control IgG did not immunoprecipitate PrPC from the different extracts (Fig. 3C
). PrPC was detected after immunoprecipitation with Pc248 using two different probes, PAb MH44 (Fig. 3A
), which was obtained after injection of ovine rPrP into rabbits and had been shown to specifically recognize PrPC in a crude brain homogenate from different species (data not shown), and biotinylated MAb 4F2 (Krasemann et al., 1996
) (Fig. 3B
). In both cases, the same PrPC profile was obtained, although the intensity of PrPC bands differed slightly in a few tissues depending on the primary antibody used. Furthermore, pre-incubation of the primary antibodies used for Western blot with an excess of purified PrP abolished detection of the immunoprecipitated PrPC (data not shown). Altogether, these data showed that the signals obtained with Pc248 were specific for PrPC.
|
|
|
In order to check that the PrPC isoforms detected in some tissues corresponded to glycoforms, we carried out a deglycosylation experiment on PrPC immunoprecipitated from the skeletal muscle, lung and brain (Fig. 5). Treatment of PrPC with N-glycosidase F resulted in simplifying the PrPC profile into one band, which probably corresponds to nonglycosylated PrPC (Fig. 5
). Interestingly the nonglycosylated PrPC isoform obtained from the lung migrated faster than those obtained from the brain and skeletal muscle.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The quantification test used in the present work permitted the PrPC ratio between the brain and the extraneuronal tissues to be determined. The EIA test was able to detect PrPC in several crude non-neuronal tissue extracts, even after a tenfold dilution. We should mention here that the EIA was originally designed to detect PrPres for the post-mortem diagnosis of bovine spongiform encephalopathy. In this context, the EIA was evaluated by the European Commission, supervised by the Directorate XXIV (Moynagh & Schimmel, 1999 ; Moynagh et al., 1999
) and it was concluded that this test was the most sensitive one at that time. In general, there was a good correlation between the results obtained with the EIA and those observed using the immunoprecipitation/Western blot experiment.
The tissue distribution of PrPC in sheep was first studied by Horiushi et al. (1995) . However, Horiushi and co-workers used microsomal preparations from different tissues to enrich PrPC. We have recently shown that the solubilized microsomal PrPC population correspond to 16% of total brain PrPC only (unpublished data). Altogether, these data prompted us to set up a different procedure which could better reflect the true PrPC content of different tissues in both qualitative and quantitative terms. In all cases, PrPC could be detected by loading only 5 mg of tissue equivalent per lane from almost all tissues except the liver. Serial dilutions made from the tissues richest in PrPC content (lungs, skeletal muscle and heart) showed that we could still detect PrPC in an equivalent of 0·5 to 1 mg of tissue (data not shown).
Similar results were obtained from immunoprecipitation/Western blot assays using several combinations of MAbs and PAbs. Furthermore, we have observed that two different anti-PrP PAbs, MH44 and MH48, produced in our laboratory immunoprecipitated an additional faint band of about 66 kDa from skeletal muscle (data not shown). This band could correspond to a dimeric form of PrPC which is present specifically in this tissue. Comparable results from different animals were found in several tissues, such as the lungs, heart, skeletal muscle and tongue. Data from the gastrointestinal tract tissues varied between individual sheep. Given the low number of sheep tested in the present work, we could not determine whether there is any relationship between animal genotype for the PrP locus and the tissue expression of PrPC. Interestingly, in all organs from which PrPC could be immunoprecipitated, bands of either bi- or hyperglycosylated isoforms were clearly detected. Nevertheless, a specific PrPC isoform signature was systematically observed in the skeletal muscle (from either the thigh or the flank): two bands migrating more slowly than the brain mono- and biglycosylated isoforms (Fig. 5). These two bands correspond to PrPC glycoforms, as deglycosylation resulted in one band migrating at the same level as that of the nonglycosylated protein obtained from the brain. However, differences in the composition and length of the oligosaccharide chains present on PrPC could differ in the brain and skeletal muscle, and this could explain the difference in migration observed between the mono- and biglycosylated isoforms from these two tissues. Furthermore, we have shown that the nonglycosylated form obtained from the lungs migrated faster than its equivalents from the brain and skeletal muscle. Whether this is the result of N- or C-terminal truncation is not yet known. Indeed, a PrP isoform truncated at the C-terminal has been observed in mature human and bovine sperm (Shaked et al., 1999
).
We should note here that pieces of skeletal muscle were removed with careful attention to avoid any residual nervous fibres. However, some neuromuscular junctions probably remained. Several arguments indicate that the typical muscular profile described here really belongs to muscle cells. PrPC has indeed been detected in developing mouse muscle cells (Brown et al., 1998 ). Furthermore, PrPC has been immunolocalized at the subsynaptic sarcoplasm of the neuromuscular junction of mammalian muscles (Gohel et al., 1999
; Askanas et al., 1993
). PrPC has also been detected by immunoblotting in skeletal muscle from hamster (Bendheim et al., 1992
) and in quadriceps muscle homogenates from non-transgenic mice (Westaway et al., 1994
). Overexpression of hamster PrPC in homozygote Tg(ShaPrP+/+)7 uninfected older mice resulted in spontaneous degeneration of the central nervous system (CNS) and skeletal muscle neuromyopathy (Westaway et al., 1994
). Altogether, these results underline a potential role of PrPC in skeletal muscle cells.
The tissue distribution and developmental expression of sheep PrP mRNA has been published by Goldmann et al. (1999) . No correlation between mRNA and protein levels can, however, be established. Altogether, these data reflect either a tissue-specific regulation of mRNA initiation, translation and/or stability, or a tissue-specific PrPC catabolism control.
Our finding supports the idea that organs for which infectivity has been demonstrated (tonsil, thymus, intestine and spleen) do not contain more PrPC than tissues shown not to support infection (skeletal muscle and heart) (Hadlow et al., 1979 ; Danner, 1993
). Thus, like the species barrier, a tissue barrier phenomenon for infection might also exist. Several hypotheses could be proposed: (i) it is possible that physical and/or physiological barriers may preclude the infectious agent from reaching some tissues, (ii) the existence of co-factors necessary for the achievement of PrPC conversion in tissues that support infection or the presence of inhibitory molecules in uninfectious organs could also explain this discrepancy, and (iii) a possible relationship between the biochemical features of PrPC (e.g. glycoform diversity among tissues, unidentified tissue-specific PrPC post-translational modifications and tissue differences in PrP clearance) and its susceptibility to be converted into the scrapie form still remains an open question. One of the intriguing phenomena in sheep is the dependence on the PrnP locus genotype for susceptibility to scrapie (Bossers et al., 1996
; Hunter et al., 1996
). It has been demonstrated that animals which are highly susceptible to scrapie (homozygote VRQ/VRQ at positions 136, 154 and 171) accumulate PrPSc in the lymphoid tissue at early stages of the disease (Schreuder et al., 1996
, 1998
; van Keulen et al., 1996
; Andréoletti et al., 2000
). Later on, PrPSc is detected in the CNS. This sequence of PrPSc appearance is not found in sheep that are moderately susceptible to scrapie (heterozygote ARR/VRQ), where PrPSc deposits are observed only in the CNS. These results indicate that the nature of the protein itself might play the role of a physiological bolt to control the conversion events.
In conclusion, the development of such biochemical and quantitative studies for tissue distribution of PrPC might allow important advances in understanding the biochemistry and function of this protein. Furthermore, these studies should be combined with the immunocytochemical localization of PrPC. The latter point will precisely indicate which cell type expresses PrPC in a given organ. It will be interesting then to look at the accumulation of PrPSc at the cellular level during the course of infection. This might aid the development of other pre-clinical diagnoses for TSE.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antoine, N., Cesbron, J. Y., Coumans, B., Jolois, O., Zorzi, W. & Heinen, E. (2000). Differential expression of cellular prion protein on human blood and tonsil lymphocytes. Haematologica 85, 475-480.[Medline]
Askanas, V., Bilak, M., Engel, W. K., Alvarez, R. B., Tomé, F. & Leclerc, A. (1993). Prion protein is abnormally accumulated in inclusion body myositis. Neurology Report 5, 25-28.
Beekes, M. & McBride, P. A. (2000). Early accumulation of pathological PrP in the enteric nervous system and gut-associated lymphoid tissue of hamsters orally infected with scrapie. Neuroscience Letters 278, 181-184.[Medline]
Bendheim, P. E., Brown, H. R., Rudelli, R. D., Scala, L. J., Goller, N. L., Wen, G. Y., Kascsak, R. J., Cashman, N. R. & Bolton, D. C. (1992). Nearly ubiquitous tissue distribution of the scrapie agent precursor protein. Neurology 42, 149-156.[Abstract]
Bons, N., Mestre-Rances, N., Belli, P., Cathala, F., Gajdusek, D. C. & Brown, P. (1999). Natural and experimental infection of nonhuman primates by bovine spongiform encephalopathy agents. Proceedings of the National Academy of Sciences, USA 96, 4046-4051.
Bossers, A., Schreuder, B. E. C., Muileman, I. H., Belt, P. B. G. M. & Smith, M. A. (1996). PrP genotypes contribute to determining survival times of sheep with natural scrapie. Journal of General Virology 77, 2669-2673.[Abstract]
Brown, D. R., Schmidt, B., Groschup, M. H. & Kretzschmar, H. A. (1998). Prion protein expression in muscle cells and toxicity of a prion protein fragment. European Journal of Cell Biology 75, 29-37.[Medline]
Büeler, H. R., Aguzzi, A., Sailer, A., Greiner, R. A., Autenried, P., Aguet, M. & Weissmann, C. (1993). Mice devoid of PrP are resistant to scrapie. Cell 73, 1339-1347.[Medline]
Cashman, N. R., Loertscher, R., Nalbantoglu, J., Shaw, I., Kascsak, R. J., Bolton, D. C. & Bendheim, P. E. (1990). Cellular isoform of the scrapie agent protein participates in lymphocyte activation. Cell 61, 185-192.[Medline]
Danner, K. (1993). BSE: a risk for man through pharmaceutical products? Position and politics of the German pharmaceutical industry. In Transmissible Spongiform Encephalopathies: Impact on Animal and Human Health , pp. 199-205. Edited by F. Brown. Basel:Karger.
DeArmond, S. J., Qiu, Y., Sanchez, H., Spilman, P. R., Ninchak-Casey, A., Alonso, D. & Daggett, V. (1999). PrPC glycoform heterogeneity as a function of brain region: implications for selective targeting of neurons by prion strains. Journal of Neuropathology and Experimental Neurology 58, 1000-1009.[Medline]
Ellman, G., Courteney, K., Andres, V. & Featherstone, R. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88-95.[Medline]
Elsen, J. M., Amigues, Y., Schelcher, F., Ducrocq, V., Andréoletti, O., Eychenne, F., Khang, J. V., Poivey, J. P., Lantier, F. & Laplanche, J. L. (1999). Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov. Archives of Virology 144, 431-445.[Medline]
Gohel, C., Grigoriev, V., Escaig-Haye, F., Lasmezas, C. I., Deslys, J. P., Langeveld, J., Akaaboune, M., Hantai, D. & Fournier, J. G. (1999). Ultrastructural localization of cellular prion protein (PrPC) at the neuromuscular junction. Journal of Neuroscience Research 55, 261-267.[Medline]
Goldmann, W., ONeill, G., Cheung, F., Charleson, F. & Hunter, N. (1999). PrP (prion) gene expression in sheep may be modulated by alternative polyadenylation of its messenger RNA. Journal of General Virology 80, 2275-2283.
Grassi, J., Frobert, Y., Lamourette, P. & Lagoutte, B. (1988). Screening of monoclonal antibodies using antigen labeled with acetylcholinesterase: applications to the peripheral proteins photosystem I. Analytical Biochemistry 168, 436-450.[Medline]
Hadlow, W. J., Kennedy, R. C. & Race, R. E. (1979). Natural infection of Suffolk sheep with scrapie virus. Journal of Infectious Diseases 146, 657-664.
Horiuchi, M., Yamazaki, N., Ikeda, T., Ishiguro, N. & Shinagawa, M. (1995). A cellular form of prion protein (PrPC) exists in many non-neuronal tissues of sheep. Journal of General Virology 76, 2583-2587.[Abstract]
Hunter, N., Foster, J. D., Goldmann, W., Stear, M. J., Hope, J. & Bostock, C. (1996). Natural scrapie in closed flock of Cheviot sheep occurs only in specific PrP genotypes. Archives of Virology 141, 809-824.[Medline]
Jackson, G. S. & Clarke, A. R. (2000). Mammalian prion proteins. Current Opinion in Structural Biology 10, 69-74.[Medline]
Krasemann, S., Groschup, M. H., Haremeyer, S., Hunsmann, G. & Bodemer, W. (1996). Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice. Molecular Medicine 2, 725-734.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227, 680-685.[Medline]
Mabbott, N. A., Brown, K. L., Manson, J. & Bruce, M. E. (1997). T-lymphocyte activation and the cellular form of the prion protein. Immunology 92, 161-165.[Medline]
McBride, P. A., Eikelenboom, P., Kraal, G., Fraser, H. & Bruce, M. E. (1992). PrP protein is associated with follicular dendritic cells of spleens and lymph nodes in uninfected and scrapie-infected mice. Journal of Pathology 168, 413-418.[Medline]
McKinley, M. P., Bolton, D. C. & Prusiner, S. B. (1983). A protease-resistant protein is a structural component of the scrapie prion. Cell 35, 57-62.[Medline]
Maignien, T., Lasmézas, C. I., Beringue, V., Dormont, D. & Deslys, J.-P. (1999). Pathogenesis of the oral route of infection of mice with scrapie and bovine spongiform encephalopathy agents. Journal of General Virology 80, 3035-3042.
Moynagh, J. & Schimmel, H. (1999). Tests for BSE evaluated. Bovine spongiform encephalopathies. Nature 400, 105.[Medline]
Moynagh, J., Schimmel, H. & Kramer, G. N. (1999). The evaluation of tests for the diagnosis of transmissible spongiform encephalopathy in bovines. In Consumer Policy and Consumer Health Protection. General directorate XXIV. Preliminary report by the European Commission. Scientific health opinions: Directorate B.
Pammer, J., Weninger, W. & Tschachler, E. (1998). Human keratinocytes express cellular prion-related protein in vitro and during inflammatory skin diseases. American Journal of Pathology 153, 1353-1358.
Pammer, J., Suchy, A., Rendl, M. & Tschachler, E. (1999). Cellular prion protein expressed by bovine squamous epithelia of skin and upper gastrointestinal tract. Lancet 354, 1702-1703.[Medline]
Prusiner, S. B. (1998). Prions. Proceeding of the National Academy of Sciences, USA 95, 13363-13383.
Prusiner, S. B., Groth, D., Serban, A., Koehler, R., Foster, D., Torchia, M., Burton, D., Yang, S. L. & DeArmond., S. J. (1993). Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proceedings of the National Academy of Sciences, USA 90, 10608-10612.[Abstract]
Race, R., Jenny, A. & Sutton, D. (1998). Scrapie infectivity and proteinase K-resistant prion protein in sheep placenta, brain, spleen, and lymph node: implications for transmission and antemortem diagnosis. Journal of Infectious Diseases 178, 949-953.[Medline]
Rezaei, H., Marc, D., Choiset, Y., Takahashi, M., Hui Bon Hoa, G., Haertlé, T., Grosclaude, J. & Debey, P. (2000). High yield purification and physicochemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility. European Journal of Biochemistry 267, 2833-2839.
Rodolfo, K., Turbica, I., Frobert, Y., Créminon, C., Fretier, P., Demart, S., Comoy, E., Di Giamberardino, L., Rezaei, H., Hunsmann, G., Deslys, J.-P. & Grassi, J. (2001). Quantitative measurement of mammalian cellular prion protein with two-site immunometric assays using specific monoclonal antibodies. Journal of Immunological Methods (in press).
Schreuder, B. E. C., van Keulen, L. J. M., Vromans, M. E. W., Langeveld, J. P. M. & Smits, M. A. (1996). Preclinical test for prion diseases. Nature 381, 536.
Schreuder, B. E. C., van Keulen, L. J. M., Vromans, M. E. W., Langeveld, J. P. M. & Smits, M. (1998). Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie. Veterinary Records 142, 564-568.
Shaked, Y., Rosenmann, H., Talmor, G. & Gabizon, R. (1999). A C-terminal-truncated PrP isoform is present in mature sperm. Journal of Biological Chemistry 274, 32153-32158.
Somerville, R. A. (1999). Host and transmissible spongiform encephalopathy agent strain control glycosylation of PrP. Journal of General Virology 80, 1865-1872.[Abstract]
van Keulen, L. J. M., Schreuder, B. E. C., Meloen, R. H., Mooij-Harkes, G., Vromans, M. E. W. & Langeveld, J. P. (1996). Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. Journal of Clinical Microbiology 34, 1228-1231.[Abstract]
van Keulen, L. J. M., Schreuder, B. E. C., Vromans, M. E. W., Langeveld, J. P. & Smits, M. A. (1999). Scrapie-associated prion protein in the gastrointestinal tract of sheep with natural scrapie. Journal of Comparative Pathology 121, 55-63.[Medline]
Weissmann, C. (1996). Molecular biology of transmissible spongiform encephalopathies. FEBS Letters 389, 3-11.[Medline]
Westaway, D., DeArmond, S. J., Cayetano-Canlas, J., Groth, D., Foster, D., Yang, S.-L., Torchia, M., Carlson, G. A. & Prusiner, S. B. (1994). Degeneration of skeletal muscle, peripheral nerves, and the central nervous system in transgenic mice overexpressing wild-type prion protein. Cell 76, 117-129.[Medline]
Received 3 April 2001;
accepted 8 May 2001.