In reply to whether or not dogs were ever inoculated the answer is yes. In l970 Dr. Gajdusek and I inoculated dogs with two cases of kuru and two cases of CJD intracerebrally and intravenously. Both cases of kuru and both cases of CJD had caused spongiform encephalopathy in chimpanzees and several sspecies of old world and new world monkeys.
The dogs remained alive and well for seven years at which time they were electively killed; their brains were examined for evidence of PrP and evidence of neuropathological lesions. All tests were negative. I should also point out that similar results were obtained in mini pigs inoculated with 5 cases of kuru and 4 cases of CJD: 9 years post inoculation no PrP; No pathology; no transmission to mice.
The PrP-knockout mice produced in our laboratory  do not show neurological abnormalities at any time and we have never reported such an effect. Moreover, no structural neuronal abnormalities were found in an independent line of PrP-knockout mice generated by Manson et al. .
The phenomenon referred to by Lansbury and Caughey was observed only by Sakaguchi et al.  in a line of mice in which not only the PrP coding region but also part of the large intron was deleted. It is not possible to attribute the observed phenotype to ablation of PrP because correction of the phenotype through introduction of a PrP-expressing cDNA has not been carried out. Electrophysiological changes were reported   for two different lines of PrP-null mice, ours and those of Manson et al., but these were not reflected in neurological deficits.
1.Lansbury PT, Jr, Caughey B: The double life of the prion protein. Curr Biol 6 : 914-916. 2.B¸eler H, Fischer M, Lang Y, Bluethmann H, Lipp HP, DeArmond SJ, et al.: Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 1992, 356 : 577-582. 3.Manson JC, Clarke AR, Hooper ML, Aitchinson 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 : 121-127. 4.Sakaguchi S, Katamine S, Nishida N, Moriuchi R, Shigematsu K, Sugimoto T, et al.: Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature 1996, 380 : 528-531. 5.Whittington MA, Sidle KCL, Gowland I, Meads J, Hill AF, Pulman MA, et al.: Rescue of neurophysiological phenotype seen in PrP null mice by transgene encoding human prion protein. Nature Genet 1995, 9 : 197-201. 6.Manson JC, Hope J, Clark AR, Johnston A, Black C, Macleod N: PrP gene dosage and long term potentiation. Neurodegeneration 1995, 4 : 113-115.See also:
Mice devoid of PrPC (Prnpo/o) are resistant to scrapie and do not allow propagation of the infectious agent (prion). PrPC-expressing neuroectodermal tissue grafted into Prnpo/o brains but not the surrounding tissue consistently exhibits scrapie-specific pathology and allows prion replication after inoculation. Scrapie prions administered intraocularly into wild-type mice spread efficiently to the central nervous system within 16 weeks. To determine whether PrPC is required for scrapie spread, we inoculated prions intraocularly into Prnpo/o mice containing a PrP-overexpressing neurograft. Neither encephalopathy nor protease-resistant PrP (PrPSc) were detected in the grafts for up to 66 weeks. Because grafted PrP-expressing cells elicited an immune response that might have interfered with prion spread, we generated Prnpo/o mice immunotolerant to PrP and engrafted them with PrP-producing neuroectodermal tissue. Again, intraocular inoculation did not lead to disease in the PrP-producing graft. These results demonstrate that PrP is necessary for prion spread along neural pathways.
Ann. Neurol. 39: 767-778 (1996) P. Parchi, R. Castellani, S. Capellari, et al Division of Neuropathology, Institute of Pathology, Case Western Reserve University,We sequenced the prion protein gene and studied the biochemical characteristics and the intracerebral distribution of protease-resistant prion protein with Western blot and immunohistochemistry in 19 cases of sporadic Creutzfeldt-Jakob disease. We identified four groups of subjects defined by the genotype at codon 129 of the prion protein gene, the site of a common methionine/valine polymorphism, and two types of protease-resistant prion proteins that differed in size and glycosylation. The four Creutzfeldt-Jakob disease groups showed distinct clinicopathological features that corresponded to previously described variants. The typical Creutzfeldt-Jakob disease phenotype or myoclonic variant and the Heidenhain variant were linked to methionine homozygosity at codon 129 and to "type 1" protease-resistant prion protein. The atypical and rarer variants such as that with dementia of long duration, the variant, and the variant with kuru plaques were linked to different genotypes at codon 129 and shared the "type 2" protease-resistant prion protein. Our data indicate that the sporadic form of Creutzfeldt-Jakob disease comprises a limited number of variants. The methionine/valine polymorphism at codon 129 of the prion protein gene and two types of protease-resistant prion proteins are the major determinants of these variants. These findings suggest the existence of prion strains in humans and provide the molecular basis for a novel classification of sporadic Creutzfeldt-Jakob disease.
|variant||codon 129||prion 'type'|
|long duration||other||type 2|
Am. J. Pathol. 148: 361-366 (1996) R. Vidal, F. Garzuly, H. Budka, M. Lalowski, R. P. Linke, F. Brittig, B. Frangione,Wisniewski Department of Pathology, New York University Medical Center, NY 10016, USA.We describe a novel transthyretin mutation at codon 18 where Asp is replaced by Gly (D18G) in a Hungarian kindred. This mutation is associated with meningocerebrovascular amyloidosis, producing dementia, ataxia, and spasticity. Fifty different transthyretin mutations are related to amyloid deposition, typically producing a peripheral neuropathy or cardiac dysfunction. These symptoms are absent in this family. Up to now, amyloid-beta (A beta), cystatin C, and prion proteins have been known to be deposited as amyloid in the brain, leading to stroke or dementia. With this report we establish that transthyretin amyloid deposition can also produce central nervous system dysfunction as the major clinical symptom.
Med. Hypotheses 46: 225-228 (1996) R. T. Radulescu & C. KorthPrion diseases are transmissible, neurodegenerative disorders associated with as yet incompletely defined isoforms of a cellular protein termed prion protein (PrP). We have now identified in PrP structural information compatible with nucleotide- and nucleic acid-binding. As such, PrP contains a putative nicotinamide adenine dinucleotide (NADH)-binding site. Moreover, the PrP octarepeats reveal homology to the nucleic acid-binding and strand-annealing octarepeats of mammalian heterogeneous ribonucleoprotein (RNP) A1. Therefore, PrP may have NADH-dependent oxidoreductase activity as well as A1-like functions such as nucleic acid annealing and splicing. Moreover, we propose that infectious prions are propagated through a dynamic molecular symbiosis between a ribozyme-like nucleic acid and a conformational isomer of the RNP-like prion protein. Thus, our model has important implications for the understanding and treatment of prion diseases.
It is very common for protein search engines such as BLASTp to return borderline, unpersuasive homologies that can be subject to misinterpretation -- webmaster.
Purdey Mark High Barn Farm, Somerset, UK. Med Hypotheses 46: 429-43 (1996)It is proposed that exposure of the bovine embryo to specific high-dose lipophilic formulations of organophosphate insecticide (containing phthalimide) applied exclusively in the UK during the 1980s/early 1990s was the primary trigger that initiated the UK's bovine spongioform encephalopathy epidemic. Multi-site binding organophosphate toxic metabolites penetrate the fetus, covalently binding with, phosphorylating and ageing serine, tyrosine or histidine active sites on fetal central nervous system prion protein. An abnormal negative charge corrupts prion protein molecular surface, which blocks both proteases and chaperones from accessing their cleavage/bonding sites. This impairs normal degradation and folding of prion protein respectively. Once the abnormally phosphorylated abnormal prion protein isoform agent is initiated, any stress event ensuing in adult life induces a nerve growth factor-mediated synthesis of normal cellular prion protein isoform that aggregates to abnormally phosphorylated abnormal prion protein isoform, thereby becoming 'infected'/transformed into the same; due to the vicious circle of positive feedback invoked by the blocking of a prion protein-specific kinase. Prion protein could therefore serve as a hitherto unrecognized critical link in a chain of delayed neuroexcitotoxic proteins that are triggered off by chronic exposure to specific classes of chemical/metal that 'hit and run' during the vulnerable in utero period, producing spongioform encephalopathy disease years later.
PROTEIN MARKERS IN CEREBROSPINAL FLUID FROM BSE AFFECTED CATTLE JONES V ET AL THE VETERINARY RECORD, VOLUME 139 NO 15, OCTOBER 12 1996 360- ACCELERATED SLAUGHTER SCHEME (LETTER) HOWIE NM ET AL THE VETERINARY RECORD, VOLUME 139 NO 9, AUGUST 31 1996 219 DISPOSAL OF RENDERED SPECIFIED OFFAL (LETTER) THE VETERINARY RECORD, VOLUME 139 NO 9, AUGUST 31 1996 SCRIMGEOUR EM ET AL RISK OF MATERNAL TRANSMISSION OF BSE (LETTER) NEWEY R ET AL THE VETERINARY RECORD, VOLUME 139 NO 7, AUGUST 17 1996 171 BSE AND RABIES POLICIES: SCIENCE OR SOPHISTRY? (LETTER) DONE J THE VETERINARY RECORD, VOLUME 139 NO 6, AUGUST 10 1996 148
Consider how selenocysteine is coded: in procaryotes, it is directed by local neighbors of distinguished UGA codon, however, in eukaryotes the particular essential stem-loop structure is located in the 3' untranslated region and not in the immediate neighbourhood of the selenocysteine codon J. Mol. Biol. (1996) 263, 8‚19
This raises a question about whether Helix C found conserved in the mRNA overlays might have this or some analogous specialized role, rather than merely impacting rate or frequency of translation. Might the function of the octapeptide repeats lie not in the amino acids (which hardly seem capable of folding) but rather in the DNA or mRNA. This could explain why CJD-cauksing mutations in this region are repeat-length and never point mutations.
In other words, the octapeptide repeat region has no particular role as protein; instead, it functions solely a the level of DNA or mRNA in a regulatory or control manner. Thus hairpins and loops could have a role in transcription initiation or translation control. Possible only a single repeat should be used in homology searches, four repeats could cause poor alignment results if an ancient weak homologue has but one.
There will be a need to further refine the prion types [codon 129 ± level of glycosylation] as the scattergram blurs with more situations tested from more species. Strain-typing will have a lot of ramifications, if it is accepted. The big push will be on cow-to-sheep, where it wouldn't necessarily be like other scrapie strains any more. It seems that the species barrier may depend on the glycosylation and passage history of the infecting agent, not just on the primary prion amino acid sequence.
The problem in the UK is that they just took sheep heads off the market last month (spinal cord still sold), thinking BSE-to-sheep-to-people wasn't a worry (along the lines of BSE was just scrapie in cows). If they have been selling high-titre BSE-sheep into the market for 12 years, human impacts could be worse than from the cows - nvCJD could actually be from the sheep, with cows just being the amplification and modifying agent.
For CJD infectivity in lymphatic tissue see:
Infection Control 1982; 3(3): 238-9 or this site.
The tonsil test of van Keulen et al. was succesful with human tissue but this seems to be unpublished. The cellular prion protein has allso been found on the surface of human
Cell 1990; 61(1): 185-92).
Infectivity has also been detected in lymphoid tissue of CJD infected mice:
J. Virol. 1991; 65(11): 6292-5.
Infectivity has been detected in lymphatic tissue of sheep, mice and mink"
J.Virology 1987: 61(10): 3235-40
A growing number of membrane proteins are being identified as tethered to the membrane by a glycosylphosphatidylinositol (GPI) anchor, rather than by a hydrophobic part of the protein. A range of proteins have been established to be GPI-anchored. These include acetylcholinesterases, 5'-nucleotidase and other cell surface hydrolases, protozoan coat proteins, activation antigens of the immune system, adhesion molecules, scrapie prion protein, and the carcinoembryonic antigen, a human tumor marker
. Because of the broad diversity of these proteins, the functional importance of their GPI anchors is not yet clear. Of significance is the increased lateral mobility of GPI anchors compared to that of hydrophobic polypeptide anchors, the regulated release by GPI cleavage, the role in cellular distribution of proteins (e.g. apical targeting of proteins in polarized cells), the exclusion from clathrin-coated pits, uptake of small molecules involving caveolae, and the possible link to signal transduction. At least one disease, paroxysmal nocturnal hemoglobinurea (PNG), is thought to be linked to a deficiency in GPI synthesis.
The protein carboxy terminus forms an amide bond to ethanolamine-phospho-6Man(12)Man(16)Man(14)GlcN which in turn is glycosidically linked to the 6 hydroxyl group of PI. Variations in the structure of GPIs occur as additions to the glycan moiety, as well as differences in the lipid composition of PI. Bacterial PI-PLCs cleave the GPI anchor to release diacylglycerol (DAG) and the water-soluble portion of the GPI attached to the protein. In some cases, additional fatty acids are found esterified to 2-OH of myo-inositol, hindering cleavage by B. cereus PI-PLC and other bacterial PI-PLCs.