Iceland meeting
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Iceland meeting of 22-27 August, 1998
Erice workshop book finally appears
Prion immunoreactivity in appendix
Prion protein expression, superoxide dismutase activity, and iNOS.
High risk sheep alleles found in New Zeeland/Australia
An unusual case of CJD: "parkinson plus"
APO E not correlated with CJD
Is beef ban followed: sensitive test for bovine mDNA
New sheep alleles
Mule Deer sequence update
N. Hunter review of scrapie

Prion protein expression and superoxide dismutase activity.

Biochem J 1998 Sep 1;334( Pt 2):423-429 
Brown DR, Besinger A
The function of the prion protein (PrPc) remains uncertain. It has been suggested that prion protein expression may aid cellular resistance to oxidative stress by influencing the activity of Cu/Zn superoxide dismutase (Cu,Zn SOD). The activity of Cu,Zn SOD was investigated in mice with different levels of PrPc expression. Increasing levels of PrPc expression were linked to increased levels of Cu,Zn SOD activity. Western-blot and Northern-blot analysis indicated that mice either lacking or overexpressing PrPc had levels of Cu,Zn SOD mRNA equivalent to those expressed in wild-type mice. Mice overexpressing the prion protein had lower levels of resistance to oxidative stress but higher expression levels of glutathione peroxidase, probably due to increased levels of hydrogen peroxide produced by increased Cu,Zn SOD activity. When cells were metabolically labelled with radioactive copper, increased radioactivity was immunoprecipitated with Cu,Zn SOD from mice with higher levels of PrPc. In addition, diethyldithiocarbamate, a copper chelator that inactivates Cu,Zn SOD by capturing copper from the molecule, is more able to inactivate Cu,Zn SOD expressed in animals with higher levels of PrPc. As recent studies have suggested that PrPc may regulate some aspect of copper metabolism, it is suggested that PrPc expression may regulate Cu,Zn SOD activity by influencing copper incorporation into the molecule.

Expression of inducible nitric oxide synthase in the brains of scrapie-infected mice.

J Neurovirol 1998 Aug;4(4):445-450 
Ju WK, Park KJ, Choi EK, Kim J, Carp RI, Wisniewski HM, Kim YS
The neuronal cell damage caused by inducible nitric oxide synthase (iNOS) in brain has been reported to be associated, at least in part, with many neurodegenerative diseases including Alzheimer's disease. We recently observed vacuolation and astrocytosis in the brains of ME7 scrapie strain-infected C57BL mice. To investigate if these phenomena might have a relationship to iNOS, the level of iNOS expression was measured immunohistochemically and molecular biologically in the brains of scrapie-infected C57BL mice. The number and size of astrocytes were increased and immunoreactivity of glial fibrillary acidic protein (GFAP) was significantly enhanced. iNOS immunoreactivity was observed in the astrocytes of the scrapie-infected group, but not in the control group. iNOS mRNA levels were increased in scrapie-infected mice compared to the levels in non-infected mice of the same age. Our results suggest that iNOS induction in reactive astrocytes is a part of the neurodegenerative mechanisms in scrapie infection.

Scrapie-free Merino and Poll Dorset sheep from Australia and New Zealand have normal frequencies of scrapie-susceptible PrP genotypes.

J Gen Virol 1998 Aug;79( Pt 8):2079-2082 
Hunter N, Cairns D
As natural scrapie occurs only in sheep of specific PrP genotypes, one proposed aetiology was that scrapie is simply a genetic disease. However, Cheviot and Suffolk sheep of scrapie-susceptible genotypes are found in Australia and New Zealand, both generally accepted to be scrapie-free countries. A study of more common Australia and New Zealand sheep breeds (Merinos and Poll Dorsets) was carried out in order to obtain more generally applicable estimates of Australia and New Zealand sheep PrP genotype frequencies. We have confirmed that animals of highly susceptible PrP genotypes are found in Australia and New Zealand. Interestingly, the Poll Dorset sheep, although born in New Zealand, were brought to the UK as young adult animals and subsequently remained free of clinical scrapie despite 21% of the sheep having scrapie-susceptible genotypes. These results have implications for the genetic control of occurrence of the equivalent human diseases.

An unusual case of Creutzfeldt-Jakob disease.

Neurology 1998 Aug;51(2):617-619 
Vingerhoets FJ, Hegyi I, Aguzzi A, Myers P, Pizzolato G, Landis T
A patient with histopathologically verified sporadic Creutzfeldt-Jakob disease (CJD) presented initially with diplopia, sleep disturbances, and L-dopa-responsive parkinsonism. After more than a year of slow progression, he did not become demented, and failed to fulfill the clinical criteria for possible CJD. No clinical examinations currently proposed to detect CJD showed the disease. CJD should be in the differential diagnosis of "parkinson plus" syndromes until a different etiology has been found or a histopathologic examination performed.

APOE in non-Alzheimer amyloidoses: transmissible spongiform encephalopathies.

Neurology 1998 Aug;51(2):548-553 
Chapman J, Cervenakova L, Petersen RB, Lee HS, Estupinan J, Richardson S, 
Vnencak-Jones CL, Gajdusek DC, Korczyn AD, Brown P, Goldfarb LG
The APOE genotype has been shown to influence the risk of developing sporadic and familial AD. This effect is isoform-dependent, the APOE epsilon4 allele increasing susceptibility and the APOE epsilon2 allele providing protection. Amyloid formation is an important part of the pathogenesis in AD as well as in spongiform encephalopathies; apoE deposition in amyloid plaques has been documented in both conditions. We examined the frequency of the APOE alleles in patients with various forms of transmissible spongiform encephalopathies, or prion diseases, including sporadic and iatrogenic Creutzfeldt-Jakob disease; familial Creutzfeldt-Jakob disease associated with PRNP 178N/129V and 200K/129M point mutations and a 24-nucleotide repeat expansion; fatal familial insomnia caused by the 178N/129M mutation; Gerstmann-Straussler-Scheinker disease associated with 102L/129M mutation; and kuru.

None of the groups we studied had a significant excess of APOE epsilon4 allele when compared with appropriate controls. CONCLUSION: Our results do not support the contention that the APOE epsilon4 allele is a risk factor for developing Creutzfeldt-Jakob disease or related disorders.

Characterization of the sheep apolipoprotein E (ApoE) gene and allelic variations of the ApoE gene in scrapie Suffolk sheep.

Gene 1998 Feb 27;208(2):131-138 
Komatsu Y, Horiuchi M, Ishiguro N, Matsui T, Shinagawa M
Apolipoprotein E (ApoE) plays a central role in lipid transport and is suggested to be involved in neuronal repair. Human ApoE epsilon 4 allele is known as a risk factor for Alzheimer's disease, and an association of the human ApoE genotype with the human prion disease, Creutzfeldt-Jakob disease, is suggested, albeit controversial. We analyzed the sheep ApoE gene to determine whether any association between the sheep ApoE genotype and the sheep prion disease, scrapie, existed. The sheep ApoE cDNA contained an open reading frame (ORF) consisting of 948 base pairs (bp) that encoded 316 amino acids (aa). The sheep ApoE gene was composed of four exons separated by three introns, and the ORF was encoded by three exons, designated exons 2, 3, and 4. Nucleotide sequence analysis also showed the presence of one G/T nucleotide polymorphism in the ORF that resulted in an Ala/Ser amino-acid substitution at codon 258. PCR-restriction fragment length polymorphism analysis of genomic DNA showed the presence of three sheep ApoE genotypes that were the result of the homologous and heterologous combinations of the two alleles. We analyzed the sheep ApoE genotypic and the allelic frequencies in scrapie and control Suffolk sheep, but they did not significantly differ from those in the control sheep, even though PrP genotype-matched populations were compared. The ApoE genotype appeared not to be associated with the progression of the disease when looking at the age at death. These results indicated that in Suffolk sheep, none of the ApoE genotypes was associated with scrapie.

Gerstmann-Straussler-Scheinker disease with A117V mutation in a second French-Alsatian family.

Clin Neuropathol 1998 Jul;17(4):229-234 
Heldt N, Boellaard JW, Brown P, Cervenakova L, Doerr-Schott J, Thomas C, Scherer C, Rohmer F
We report a kindred of French/Alsatian origin with symptoms of Gerstmann-Straussler-Scheinker disease over 3 generations. In the propositus, cerebellar signs and memory disturbance were the presenting features, followed by other neurological manifestations. Biopsy of the cerebral cortex showed numerous multicentric and "kuru"-type amyloid plaques that on immuno-light and electron microscopy stained with antibody to prion protein. Molecular genetic analysis revealed an A117V mutation in the open reading frame of the prion protein gene. Questions as to pathology and spread of this mutation are discussed.

Detection of bovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials.

J Food Prot 1998 May;61(5):513-518 
Tartaglia M, Saulle E, Pestalozza S, Morelli L, Antonucci G, Battaglia PA
A ban on ruminant-derived proteins in ruminant feeds has been introduced as a preventive measure to avoid the spread of bovine spongiform encephalopathy (BSE), as well as to minimize any potential risk of BSE transmission from bovines to humans. In the absence of commercially available efficient methods for identification of bovine-derived proteins in animal feeds, we developed a rapid and sensitive polymerase chain reaction (PCR)-based assay which allows detection and identification of a bovine-specific mitochondrial DNA sequence from feedstuffs. The amplified product encodes for the whole ATPase subunit 8 and the amino-terminal portion of the ATPase subunit 6 proteins, which are known to exhibit a relatively low degree of conservation among vertebrates. The specific amplification of such a bovine mitochondrial sequence from reference feedstuff samples was demonstrated by means of both direct sequencing and single-strand conformational analysis of the PCR product. Specificity was also confirmed by the absence of detectable homologous PCR product when using reference feedstuff samples lacking bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method allows detection of the presence of bovine mitochondrial DNA in feedstuffs containing less than 0.125% of bovine-derived meat and bonemeals. Furthermore, it does not appear to be considerably affected by prolonged heat treatment. DpnII and SspI restriction endonuclease digestions of the unpurified PCR product may be used routinely to confirm the bovine origin of the amplified sequence. Since this method is specific, rapid, and sensitive, it could be successfully utilized as a routine control assay to evaluate the presence of bovine-derived meat and bonemeals in ruminant feeds.

Iceland prion meeting

Scientific Program . Organised by the Institute for Experimental Pathology, University of Iceland, Keldur on August 20-22, 1998 in Reykjavik, Iceland. tel 354 567 4700 Fax 354 567 3979 ... No online abstracts or schedule fora published proceedings -- webmaster wrote on 18 Aug 98 for clarification.

Some highlights from the 'TSE' meeting in Iceland last week [Alex Bossers reporting]:

- B-cells, T-cells?? NO, FDCs (follicle dendritic cells) seem to be the key cells in TSEs. Charles Weismann presented some nice experiments that the FDCs play the major role in replicating TSE, while B-cells in the neighbourhood of FDCs were able to take up but not to replicate the agent. Experiments in the UK also point in the direction of the FDCs. Finally different research groups seem to agree with each other. So far this work was not presented as in press yet.

- John Collinge has plans to screen for nvCJD by tonsillar biopsies. All nvCJD cases analyzed thus far are positive by IHC in the tonsils while the other types of CJD are 'negative' in tonsils. His plans include screening all routinely taken tonsils for nvCJD to get an indication of the nvCJD incedence and the amount of cases still to come.

- Prusiners group also has bovine-PrP expressing mice, however these mice had incubation times after BSE inoculation even longer than the most to BSE susceptible normal mice (R3).

- Prusiners group again: Defining structural differences between TSE strains (same PrP template as basis) using phage-displayed MCA's. MCA's to different regions of PrP-Sc were labelled and incubated with the different strain isolates. Different binding patterns were found for the different strains! ==> more conformations in one (molecule) seed.

Commentary (webmaster):

Prusiner made an interesting comment on unusual aspects of M Bruce's R3 mice strain at the Inquiry hearing, beginning at Day 27 page 22. Basically he doesn't believe the graph on page 500 of Bruce's Nature paper in the fall of 1997: "These R3 mice... behave toward BSE and variant CJD in a way that is very much different than anything that has ever been seen except with transgenic mice.... human, mouse, bovine, feline prion.. quite remarkable, that these curves are superimposable..we are talking about four different amino acid sequences of PrP. ..."

The Iceland result is not surprising per se in that like-like primary sequences in one strain of mouse could have longer incubation times than like-unlike in another strain: there are many possible explanations. However it does point out the impossibility of quantitatively measuring in vivo species-barrier efficiencies -- there will always be some mouse strain out there where it will be still lower.

The monoclonal antibody phage display is a welcome addition. However, different strains do not necessarily have the same underlying covalent structure (or even a unique covalent structure), even though they may have started out with identical covalent primary structure for full-length prion apo-protein (same PrP template as basis?). This is due to different endopeptidase boundaries, ragged N and C exopeptidase termini, differing arginine modification, and the reported 400 different glycosylations.

The prion strain literature repeats this mistake so many times that, just like the giant alligators in the New York City sewer system, it has become an established fact. (More oft-repeated rubbish: the statement that only 1 in 100,000 monomers is infectious when really the '1' means one infectious unit in a particular inefficient 1991 indicator strain bioassay, not that only 1 of the monomers was in an infectious configuration. And how many times have you seen the falsehood that 'the prion protein is the only documented case of an infectious protein.')

If the two strains don't have the same covalent structure, their 3D conformations cannot possibly be the same because (1) they aren't even comparable and (2) the Schroedinger equation forbids it even in stretches where they are identical through boundary conditions. Unless they can show that the final covalent structures are the same globally, their outcome follows from fall-term Chemistry 101.

What they showed instead was that the mAb phage display had the ability to detect differences in the strains. And hopefully they learned some specifics about what stretches were exposed or not exposed and so something about the overall structural pictures. Strain differences are just window-dressing upon a standard underlying architecture.

If there is such a thing as immuno-gold mAb phage display, they might be able to sort out another exceeding vexatious issue in trans-species and heterozygous TSE, namely whether individual congophilic fibrils have identical strain units. Recall that in P102L, A117V, D1787N, F198S, and N217R have amyloid consisting solely of mutant allele, whereas Y145 stop, repeat insertions, E200K, V210I, and sporadic M129V are also able to recruit the 'other' allele [compilation of Cardone and Pocchiari, pg 247, Erice workshop]. I expect in some cases that the individual fibers have sorted themselves out over time through reversible equilibria and are pure monomer of one strain type conformer or the other. So several strains can co-exist with different efficiencies that change over passage, hyper and drowsy in mink etc.

Congophilic fibrils must play an essential role in defining the rogue conformer; GG Glenner had it right 20 years ago. This is implied by very recent results with Sup and Ure in yeast. What are the odds that the only 2 proteins found to have prion-like properties are the only 2 proteins in the genome to have the Asn/Gln-rich domains with prionlike fibril potential? Zero when the organism has 7000 genes.

At the Erice workshop [pg 220] Caughey and Raymond write, "... only ordered aggregates of PrP-res, albeit widely variable in size, can induce the conversion to the protease-resistant form in the cell-free system." They neatly summarize the counter-arguments to worries that not all deposits of Prp_res in vivo are birefringent or have EM-visible amyloid fibrils by (1) short oligomers are not detectable [up to 60], (2) masking by other fibrils or a plethora of other amyloid proteins, and (3) lack of fibril alignment needed for coherent optical rotation. To which I would add, (4) differential fragility to preparative procedures.

Sup was shown to be an infectious protein , with hindsight in 1965. Yeast people view protein infectivity merely as cytoplasmic inheritance associated with cell fusion, which is a very good way of looking at prion infectivity as well. Protein-only is a no-brainer in yeast because researchers have complete control over viruses, transposons, and the like.

Perhaps further taxonomy is needed in the congophilic conformational diseases. The subclasses would consist of the poly-glutamine ladder type (spinocerebral ataxia, sup, ure, ...) and the hairpin formers (Alzheimer, insulin, prion?, ...). Both classes end up as cross-beta fibrils according to fiber diffraction but there may be two structural subclasses that we are not yet distinguishing.

            SCIENTIFIC PROGRAMME Thursday, 20-21 August 1998
[No abstracts are available at this time from conference web site. 
 However, participants evidently received a set of abstracts.  
The webmaster reviews a few of these that were received by fax below:]

              14. The uptake of recombinant prion proteins by human
              and murine cells is mediated by the 37 kDa laminin
              receptor precursor. C.I. Lasm»zas, J.-M. Peyrin, S.
              Ha‘k*, R. Rieger, J-P Deslys, S. Weiss  D. Dormont.

This would be important given the previously reported yeast two-hybrid binding provided 'mediated' 
means something direct.  As I noted just recently, the 37k form is not the usual mature form of this 
protein.  However, the abstract indicates vague associations with no real protein chemistry done (eg 
characterizing cross-linking) and no case is made that this is significant in vivo.

              15.RNA aptamers directed against scrapie associated
              fibrils. C.I. Lasm»zas, S. Weiss

The aptamer selection process is brought to bear against scrapie-associated fibril, 
which they note was found in the French nvCJD case, the idea being that some G-quartet 
among the billions and billions created could provide the necessary specificity. 
This would be like the Prionics monoclonal only with RNA.

            18. Trans-dominant inhibition of PrPSc accumulation
              in scrapie-infected mouse neuroblastoma cells by the
              deletion mutant PrPcD 114-121. C. HĖlscher, H.
              Delius, A. Błrkle. FS 

This goes Shmerling et al one better with a much shorter and more focused deletion than 32-121 or
32-134 .  119MA-GAAAAGAV-VGGLGGYM129 shows the deletion right in the heart of the core region. 
 And in culture too!  Trans-dominant means that the normal allele cannot be recruited by exogenous
 rogue conformer, possibly because the deletion is binding something in the process, perhaps unable 
to join the growing fiber or allow its extension.  Follow-up to J Virol. 72 1153-1159 1998.

              23. Suramin reversibly uncouples PrPc from PrPSc
              biogenesis and generates a prion protein with properties of
              both PrPc and PrPSc. C.Spielhaupter, S. Gilch, I.
              Westner, M. Groschup, H.M. SchĒtzl. 

Suramin is a non-selective P2 -purinoceptor antagonist, a polysulfonated binaphthyl urea that
 competes with heparin and a general growth factor receptor and G protein antagonist.   By 'properties 
of both' they mean 'insolubility, PK sensitivity, and N-terminus retention.'

              56. PrP messenger RNA: Regulation of PrP expression
              through differential RNA processing in sheep tissues and
              implications for scrapie susceptibility. W. Goldmann, G.
              O•Neill, F. Cheung, F. Charlson, K. Donnelly, D. Cairns, N.

This does not relate to whether exon 2 is spliced out or not but rather tissue specific polyadenylation 
(ruminants only), with two speices of mRNA found differing by 2.3kb in length and in translatability.
 "Additional RNA processing in other positions of the untranslated regions has been found resulting in 
a oomplex system of post-transcriptional modulation of PrP expression."  They report high variability 
(3' UTR polymorphisms) within and between breeds possibly associated with scrapie susceptibility. 
These authors have not yet corrected a major sequence screw-up at GenBank concerning the 3' UTR in
 sheep.  There are apparently 82 errors in 3246 bp or 2.5% error rate, in M31313 or AJ223072.  
 D38179 and U67922 agreed completely as independent determinations.

              28. Gene expression in scrapie: cloning of a new scrapie
              responsive gene and the identification of increased levels of
              seven other mRNA transcripts. F. Dandoy-Dron1, F.
              Guillo1, L. Benboudjema, J.-P. Delys, C. Lasm»zas, D.
              Dormont, M.G. Tovey1, M. Dron

Presumably adds to J Biol Chem. 1998 Jul 17; 273(29): 18015-18018.  and J Biol Chem. 1998 Mar 27;
 273(13): 7691-7697. These are  after-the-fact proteins probably not relevent to the core issues but
possibly important therapeutic targets.The new ones ar ScRG-1, preferentially expressed in glial cells,
 of 98 amino acids, a signal peptide and  a known human homologue.

              11. The molecular layer of the human cerebellum in
              Creutzfeldt-Jakob disease: A Golgi and electron
              microscope study. S. Baloyannis, V. Costa. 

              13. New insights into the life cycle of prions. S. Weiss.

              14. The uptake of recombinant prion proteins by human
              and murine cells is mediated by the 37 kDa laminin
              receptor precursor. C.I. Lasm»zas, J.-M. Peyrin, S.
              Ha‘k*, R. Rieger, J-P Deslys, S. Weiss  D. Dormont.

              16. Nucleic acid induced aggregation of mouse recombinant
              prion protein to amyloids and other polymeric structures.
              P.K. Nandi,E. Leclerc

              17. Expression of full length chicken prion protein and
              binding with filamentous phage M13 particles in vitro. H.
              Wang, B.S. Wong, E.A. Gould, I.M. Jones.  

              19. Evidence of a homologous ssDNA in nemavirus is the
              genome of the scrapie agent. H. Narang, Ken Bell
              International, Newcastle-upon-Tyne, UK. 

              20. Unusual characteristics of several scrapie isolates
              derived from hay mites.
              R.I.Carp1, S. Sigurarson2, H.C. Meeker1, R. Rubinstein1,
              R.J. Kascsak1, M. Papini1, P. Kozlowski , H.M.

              21. Studies on the proteinase K sensitivity of cellular
              prion protein (PrPc) of various species. A. Buschmann,
              M.H. Groschup.  

              22. Role of immunogen and immunized species in the
              linear and comformational epitope response to prion
              protein PrP. R.J. Kascsak1, R. Rubenstein1, R. Kascsak1,
              R.I. Carp1, R. Meloen, J. Langeveld . 

              24. Suppression of PrPSc and HIV-1 gp120 induced
              neuronal cell death by sulfated colominic acids. H.C.
              SchrĖder1, H. Ushijima2, S. Perovic1, J. Leuck, P.G.
              Rytik, W.E.G. Młller

              25. MS-8209, an amphotericin B derivative,
              affects both scrapie agent replication and PrPres accumulation in
              Syrian hamster scrapie. K.T. Adjou1, R. Demaimay1,
              J.-P.Deslys1, C.I. Lasm»zas1, V. Beringue1, S. Demart1, F.
              Lamoury1, M. Seman2, D. Dormont

              26. Follicular dendritic cells in the spleen support
              replication in a mouse scrapie model. K.L. Brown, K.
              Stewart, I. McConnell, H. Fraser, M.E. Bruce. 

              27. TSE agents propagation after oral contamination. 
              T.Maignien, C. Lasm»zas, V. Beringue, J.-P. Deslys, D.Dormont. 

              29. Electrophysiological and morphological studies of
              single subicular neurons in brain slices from mice
              infected with scrapie. J.R.T. Greene, V.H. Perry, S.

              45. Severe, early and selective neuronal vulnerability in
              experimental transmissible spongiform encephalopathies.
              M. Guentchev1, M. Groschup2, R. Kordek3, P.P. Liberski3,
              H. Budka 

              46. The Echigo-1 panencephalopathic type of
              Creutzfeldt-Jakob disease passage in hamsters and
              neuropathological characterisation. P.P. Liberski1, J.A.
              Hainfellner2, A. Wali1, R. Kordek1, H. Budka 

              47. Atypical case of sporadic Creutzfeldt-Jakob disease in
              young adult patient. W. Kozubski, M. Wender, P.P.
              Liberski, J. Szczech, D. Lenart-Jankowska.  

              48. Epidemiological determinants of the pattern &
              magnitude of the vCJD epidemic in Great Britain. A. C.
              Ghani, N.M. Ferguson, C.A. Donelly, T.J. Hagenaars, R.M.

              49. Transmissions of "new variant" and sporadic CJD to
              mice. M.E. Bruce, R.G. Will, J. Ironside, I. McConnell,
              D. Drummond, A. Suttie, A. Chree.  

              50. Human susceptibility to Creutzfeldt-Jakob disease
              agents. J.-P. Deslys1, A. Jaegly1, F. Mouthon1, C.
              Lasm»zas1, Villemeur3,4, A. Alp»rovitch2, D.

              51. PrP gene dosage and allelic specificity in the
              transmissible spongiform encephalopathies. J.Manson, R.
              Moore*, P. McBride, J. Hope, J. Selfridge*, D. Melton.
              52. The afferent projections to the mediodorsal and
              anterior thalamic nuclei: possible prion transmission in
              FFI. J.L. Velayos, M. Oliva, F. Alfageme. 

              53. A quantitative study of synapse loss and neuronal
              degeneration in the scrapie-infected murine hippocampus.
              M. Jeffrey1, D.A. Brown2, W.G. Halliday1, J.R. Fraser                

              54. Immunohistochemical characteristics of an antiserum
              to an N-terminal prion protein peptide. E. Monks1, E.D.
              Weavers1, A. Fullam-Church1, M. Rogers   

              57. The influence of the AHQ PrP allele on susceptibility to
              clinical scrapie in British flocks of Swaledale, Welsh
              Mountain and Finn Dorset sheep. M. Dawson, L.J.
              Hoinville, T.C. Martin, C.A. Hoek, A.H. Murray.   

              58. Tonsillar biopsy as a tool for a pre-clinical diagnosis
              of scrapie. B.E.C. Schreuder, L.J.M. van Keulen, M.E.W.
              Vromans, J.P.M. Langeveld, M. Smits.  

              59. Cerebrospinal fluid profile in sheep with scrapie by
              high resolution proton magnetic resonance spectroscopy:
              Possible use as a diagnostic tool ? M. P»pin1, S. Maillet2,
              J. Vion-Dury2, S. Confort-Gouny2, P. Russo1, R. Sanchis1,
              F. Schelcher3, P. Cozzone 

              60. Mathematical models of the epidemiology and control of
              scrapie within a sheep flock. M.E.J. Woolhouse, S.M.
              Stringer, L. Matthews, N. Hunter, R.M. Anderson.  

              61. Transgenic mice expressing the bovine prion protein -
              A useful tool for detection of BSE infectivity. A.
              Buschmann, E. Pfaff, H. Mueller, K. Reifenberg, M.H.

              62. Anti-oxidant status in cattle naturally 
              affected with bovine spongiform encephalopathy. T. Martin, S.
              Cawthraw, M. Dawson.  

              63. Studies on BSE and scrapie agents: Paradoxical
              inactivation data. D.M. Taylor, K. Fernie, P.J. Steele.

              64. Transmission dynamics and the clustering of BSE
              cases. C.A. Donnelly, N.M. Ferguson, A.C. Ghani, T.J.
              Hagenaars, R.M. Anderson.  


Mol Biotechnol 1998 Jun;9(3):225-234 
Hunter N
[Note there is also a good review of BSE in sheep at the Maff Web site from a recent SEAC meeting. -- webmaster]

Scrapie and other transmissible spongiform encephalopathies (TSEs) are characterized by similar pathology, biochemistry and genetics. The PrP protein and its conversion to the disease-related isoform, PrPSC, are crucial for the development of all TSEs. Although scrapie is more often studied in laboratory rodents, it is not a natural disease of these animals, and much can be learned from the normal hosts, sheep. Disease incidence is linked to polymorphisms and mutations of the PrP gene. The complex relationships between PrP genotype and the survival of sheep subjected to scrapie infection are now being investigated in terms of the different structure of the PrP protein molecules produced by each allele. It is these structures and their differing abilities to convert to PrPSC that hold the key to understanding why TSEs occur.

Prion immunoreactivity in appendix

Prion immunoreactivity in appendix before clinical onset of new variant Creutzfeldt-Jakob disease David A Hilton, Edward Fathers, Philip Edwards, James W Ironside, John Zajicek Lancet Volume 352, Number 9129 29 August 1998 Research Letter

John Collinge's group had addressed the subject of the risk from nvCJD appendectomy cases before the case of Tony Barrett surfaced in : Hill et al (1997) "Diagnosis of new variant Creutzfeldt-Jakob disease by tonsil biopsy" The Lancet, 349, 9045, 11January: The following extract from their paper is relevant:

"Recently, we reported that nvCJD is associated with a specific pattern of protease-resistant prion protein (PrP) on Western blot analysis. This marker can already be used to aid differential diagnosis on brain biopsy samples. However, since PrP is widely expressed outside the central nervous system, we investigated whether an alternative and more accessible tissue might be biopsied to allow a diagnosis of nvCJD before death and to avoid brain biopsy. PrP is expressed in the lymphoreticular system and prion replication is known to occur in the spleen and other lymphoreticular tissues in experimental rodent scrapie models; prion infectivity has also been reported in human lymphoreticular tissues.

"We have studied PrP in tonsillar tissue obtained at necropsy using both immunohistochemistry on periodate-lysine-paraformaldehyde and formalin-fixed tissue, and Western blot analysis of frozen tissue. The patient was a 35-year-old woman who died after a 14-month illness with depression at onset followed by ataxia, hyper-reflexia, memory loss and dementia; a diagnosis of nvCJD was made by neuropathology. Abnormal PrP staining was present within tonsillar germinal centres.

Western blot analysis revealed the presence of protease resistant PrP; confirming the diagnosis of prion disease. Furthermore, the sizes and intensity ratios of the three PrP bands (representing diglycosylated, monoglycosylated, and unglycosylated PrP) were similar to those seen in brain from the same patient (designated a type 4 pattern) suggesting that it may be possible to make the specific diagnosis of nvCJD by this technique. However, tonsillar tissues from types 1-3 CJD were not available for comparison. Such study of protease-resistant PrP in lymphoreticular tissue may be particularly relevant in iatrogenic CJD after peripheral prion inoculation (for instance after treatment with human cadaveric pituitary-derived growth hormone where lymphoreticular involvement would be expected.

"In experimental murine and sheep scrapie, prion replication occurs initially in spleen and is only detectable in the central nervous system considerably later in the incubation period. Abnormal PrP immunostaining has been reported in tonsils of experimental scrapie-infected sheep long before the occurrence of clinical signs. It is possible therefore that Western blot analysis of human tonsil material may allow early clinical, or possibly pre-clinical, diagnosis of CJD and nvCJD although extensive prospective studies of suspected cases will be necessary to assess the clinical usefulness of this investigation.

Tonsil tissue can be easily obtained by biopsy under local anaesthetic in most patients and complications are most unusual. Although many adults will have had childhood tonsillectomy, lymphoreticular tissue may still be obtained from the lingual tonsillar remnants. As a result of the extreme resistance of prions to normal sterilisation procedures, current UK recommendations are that neurosurgical instruments from patients with CJD are destroyed. While infective titres of prions in tonsil may be much lower than in brain, similar precautions to avoid cross contamination and iatrogenic transmission of CJD would seem prudent at present. The development of a disposable tonsil-biopsy kit should be feasible. "

New sheep alleles

Alex Bossers reporting:

 'New' natural occuring sheep PrP-ORF polymorphisms. Additionally to the 8
described (at M112T, A136V, M137T, L141F, R154H, Q171R, Q171H, R211Q) reported
     in Cyprus
                Q171K  low incedence, no scrapie
                A136T  low incedence, no scrapie

     in Iceland (and probably also in Norway!?)
                M137T  (finally it has been found outside the Netherlands!)
                S138N  5-9% of Icelandic sheep, no scrapie association yet
          !!!   R151C ! very rare, except for one flock, protective to scrapie?
                        What will happen to the disulfide bridges?????
Comment (webmaster):

For sheep to have 13 amino acid substitutions at 9 positions is quite provocative, given that humans have 17 known point substitutions at 17 positions with *15* clearly associated with CJD and 2 modulating progression (one beneficially); that is, hardly any of the human mutations have been innocuous. Using these odds at face value, which I don't recommend, the sheep alleles mostly cause scrapie. Now humans live a lot longer so small effects show up whereas sheep may get a horizontal jump-start. Given these striking data, the burden of explanation must be on sheep-ologists to tell us why the situation is so turned around in their species.

The curious point is that many thousands of control individuals have been sequenced, not just people from neurological 'flocks' and yet basically no neutral polymorphisms have shown up (other than at 3rd codon position, repeat deletions, and short insertions). Of course, sheep breeding practises make interpretation of gene frequencies impossible, that is, do rare mutations become founder-effect polymorphism? Note sheep have two double and one triple-valued allele -- this is unlike humans where all are single-valued.

Good point on R151C. Disulphides have not yet been specifically disabled in transgenic mice as far as I can recall (but see ref below) Prion protein disulphides are strictly conserved in all species back to at least 310 million years; further, this is the first change to cysteine ever observed in any species in any part of the sequence.

R151C might not be causative if the standard problem is an ER anomaly which these might cross correctly under Glockshuber's views that S-S are always formed outside the cell. [However disulphides can form internally despite the reduced glutathione if the folding protein's 3D environment drives it strongly enough.] Note that there is a disulphide isomerase in the ER; this plus steric considerations and overall energetics could prevent mixed-up disulphides. Which is not to say that R151C is necessarily neutral as it is a highly non-conservative change.

A136V  A136T 
M137T  M137T
Q171R  Q171H  Q171K
(All are single-step genetic code changes)

The importance of the disulfide bond in prion protein conversion.

Herrmann LM, Caughey B
Neuroreport 1998 Aug 3;9(11):2457-2461 
The conversion of normal, protease sensitive prion protein (PrP-sen) to the abnormal protease-resistant form (PrP-res) is of central importance in the pathogenesis of scrapie and other transmissible spongiform encephalopathies. In the present study, the effects of reduction of the disulfide bond on the PrP-sen to PrP-res conversion in a cell-free system were examined. The addition of the disulfide reducing agent dithiothreitol inhibited the cell-free conversion reaction with an IC50 of 2-2.5 mM. Separate pretreatment of either PrP-sen or PrP-res with dithiothreitol and an alkylating agent also inhibited the conversion reaction. Results of this study show that preservation of the disulfide bond is important in the conversion of PrP-sen to PrP-res.

Mule Deer sequence update

28 Aug 98 Correspondent: K. O'Rourke
AF009180, Odocoileus hemionus hemionus prion gene, allele S2, and AF009181, allele S1: These entries have been revalidated. One error was found at base 348 in this truncated sequence. In addition to the coding changes, there were 2 silent changes between N1 and S2, 5 silent changes between N1 and S1, and 5 (not 1) silent changes between S1 and S2. Mule deerwere seen with the N1S2, S2S2 or S1S1 genotypes in a limited sample , no N1N1, no N1S1. A large sample will be studied in the fall.

Alleles 138S2 and 138N1 encode Ser and Asn at codon 138, respectively. Allele 138S1 differs from allele 138S2 by a silent mutation.

Comment [webmaster]: Published and unpublished sequences around the epitope IHFG show that the ser-asn allele occurs separately in many lineages at codon '138' making it doubtful for familial CWD (though allele state could modulate CWD along the lines of codon 129. Curious that mule deer have so many more silent changes than what is seen in humans.

    samsrpl	ihfg	n	dyedryyrenm	canis lupus familiarishallstromii
	samsrpl	ihfg	n	dyedryyrenm	canis lupus familiaris
	samsrpl	ihfg	n	dyedryyrenm	canis lupus canadensis
	samsrpl	ihfg	n	dyedryyrenm	mustela vison
	samsrpl	ihfg	n	dyedryyrenm	mustela putorius
	samsrpl	ihfg	n	dyedryyrenm	equus familiaris caballus
	samsrpl	ihfg	n	dyedryyrenm	equus przewalskii caballus
	samsrpl	ihfg	n	dyedryyrenm	equus quagga boehmi
	samsrpl	ihfg	s	dyedryyrenm	sus scrofa
	samsrpl	ihfg	n	dyedryyrenm	camelus dromedarius
	samsrpl	ihfg	n	dyedryyrenm	lama glama
	samsrpl	ihfg	s	dyedryyrenm	tursiops truncatus
	samsrpl	ihfg	n	dyedryyrenm	gazella subgutturosa
	samnrpl	ihfg	n	dyedryyrenm	bos taurus
	samsrpl	ihfg	n	dyedryyrenm	bos primigenius taurus
	samsrpl	ihfg	s	dyedryyrenm	bos javanicus
	samsrpl	ihfg	s	dyedryyrenm	bos taurus
	samsrpl	ihfg	s	dyedryyrenm	bison bonasus
	samsrpl	ihfg	s	dyedryyrenm	tragelaphus strepsiceros
	samsrpl	thfg	n	dyedhyyrenm	tragelaphus angasi
	samsrpl	ihfg	s	dyedryyrenm	budorcas taxicolor
	samsrpl	ihfg	n	dyedryyrenm	capra hirtus
	samsrpl	ihfg	n	dyedryyrenm	capra ibex nubiana
	samsrpl	ihfg	n	dyedryyrenm	ovibos moschatusmoschatus
	samsrpl	ihfg	n	dyedryyrenm	ovis aries
	samsrpl	ihfg	n	dyedryyrenm	addax nasomaculatus
	samsrpm	ihfg	n	dwedryyrenm	hippotragus niger
	samsspl	ihfg	n	dyedryyrenm	cervus elaphuscanadensis
	samsrpl	ihfg	n	dyedryyrenm	cervus elaphus
	samsrpl	ihfg	s	dyedryyrenm	cervus nippon dybowskii
	samsrpl	ihfg	n	dyedryyrenm	odocoileus hemionus
	samsrpl	ihfg	n	dyedryyrenm	giraffa camelopardalis
	samsrpl	ihfg	n	dyedryyrenm	ateles geoffroyi
	samsrpl	ihfg	n	dyedryyrenm	ateles paniscus
	samsrpl	ihfg	n	dyedryyrenm	cebus apella
	samsrpl	ihfg	n	dyedryyrenm	callithrix jacchus
	samsrpl	ihfg	n	dyedryyrenm	aotus trivirgatus
	samsrpl	ihfg	n	dyedryyrenm	saimiri sciureus
	samsrpl	ihfg	n	dyedryyrenm	cercopithecus mona
	samsrpl	ihfg	n	dyedryyrenm	cercopithecus neglectus
	samsrpl	ihfg	n	dyedryyrenm	cercopithecus patas
	samsrpl	ihfg	n	dyedryyrenm	macaca sylvanus
	samsrpl	ihfg	n	dyedryyrenm	colobus guereza
	samsrpl	ihfg	n	dyedryyrenm	macaca fascicularis
	samsrpl	ihfg	n	dyedryyrenm	cercopithecus dianae
	samsrpl	ihfg	n	dyedryyrenm	callicebus moloch
	samsrpl	ihfg	n	dyedryyrenm	theropithecus gelada
	samsrpl	ihfg	n	dyedryyrenm	cercopithecus aethiops
	samsrpl	ihfg	n	dyedryyrenm	papio hamadryas
	samsrpl	ihfg	n	dyedryyrenm	macaca fuscata
	samsrpl	ihfg	n	dyedryyrenm	presbytis francoisi
	samsrpl	ihfg	n	dyedryyrenm	mandrillus sphinx
	samsrpl	ihfg	n	eyedryyrenm	cercocebus aterrimus
	samsrpl	ihfg	n	dyedryyrenm	macaca nemestrina
	samsrpl	ihfg	n	dyedryyrenm	macaca mulatta
	samsrpl	ihfg	n	dyedryyrenm	cercocebus torquatus atys
	samsrpl	ihfg	n	dyedryyrenm	macaca arctoides
	samsrpi	ihfg	n	dyedryyrenm	pongo pygmaeus
	samsrpi	ihfg	s	dyedryyrenm	pan troglodytes
	samsrpi	ihfg	s	dyedryyrenm	symphalangus syndactylus
	samsrpi	ihfg	s	dyedryyrenm	homo sapiens
	samsrpi	ihfg	s	dyedryyrenm	gorilla gorilla
	samsrpi	ihfg	s	dyedryyrenm	hyalobates lar
	samsrpi	ihfg	s	dyedryyrenm	hylobates syndactylus
	samsrpl	ihfg	n	dyedryyrenm	oryctolagus cuniculus

	samsrpm	ihfg	n	dwedryyrenm	cricetulus migratorius
	samsrpm	ihfg	n	dwedryyrenm	sigmodon fulviventer
	samsrpm	ihfg	n	dwedryyrenm	sigmodon hispiedis
	samsrpm	ihfg	n	dwedryyrenm	meriones unguiculatus
	samsrpm...ihfg	n	dwedryyrenm	rattus rattus
	samsrpm...lhfg	n	dwedryyrenm	cricetulus griseus
	samsrpm...lhfg	n	dwedryyrenm	mus musculus
	samsrpm...lhfg	n	dwedryyrenm	mesocricetus auratus
	samsrpm...mhfg	n	dwedryyrenm	mus musculus

	samsrpv	ihfg	n	eyedryyrenq	trichosurus vulpecula

Erice, Sicily meeting: book available from

The book has _finally_ appeared for August 19-23, 199_6_ Erice workshop. The webmaster will be reporting on the highlights as time permits. The main news is a 'defective' IAP retrovirus found in mouse prion intron 1, a 'mariner' retrotransposon found in sheep [and cattle] 3'UTR, and a mysterious unexpressed 'cryptic' exon 2 homologue in humans. These have no adverse implications whatsever for protein-only but could represent mechanisms for over-production or tissue-specific effects.

Prions and Brain Diseases in Animals and Humans, ed. D. Morrison NATO ISI Series Plenum Press ISBN 0-306-45825-X ... August 19-23, 1996 Erice workshop

Large-scale sequencing of human, mouse, and sheep prion protein genes, pg 59-76

Lee,I.Y., Westaway,D., Smit,A.F., Cooper,C., Yao,H., Prusiner,S.B. and Hood,L.
GenBank entries U29185, U67922, U29186 corresponding to this article refer to an unpublished item called: Structure and Organization of Chromosomal Regions Carrying the Mammalian Prion Gene from Three Species

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