Control of disulphides in the endoplasmic reticulum and in vitro
Application of distance geometry to 3D visualization of prion sequence relationships
Improved NMR structure of Syrian hamster prion
Effectiveness of polyene antibiotics in treatment of TSE
Gamma-secretase enahncement of amyloid precursor protein
Exposure of cryptic epitopes on transthyretin only in amyloid
Experimentally induced BSE did not transmit via goat embryos.
Scrapie in India: louping ill vaccine strikes again
Prionics test: sensitivity and specificity
Detection of prions in collagen and gelatin
Role of germinal centers in prion disease: meeting 1 Aug 99
Maff bibliography: making prion protein in Pichia pastoris, etc.
CJD in 21-year-old in Poland called sporadic
Bioinformatics 1999 Jan;15(1):89-90 Forster M, Heath A, Afzal MThe authors apply distance geometry methods to the three-dimensional visualization of sequence relationships, with prion protein sequences as one example. Sequence-sequence distance measures was obtained from either a multiple sequence alignment or from sets of pairwise alignments. Availabile in C/Perl code or as HTML/VRML files.
Figure 2 shows a virtual reality 3D data set (first download and install VRML plug-in for your web browser and platform) for 41 prion protein sequences showing a clustering that is indicative of species type.
Distance geometry was performed using DGEOM version 42 (Blaney et al. 1984-1990). Methods used are pairwise alignment and Sellers distance and multiple sequence alignment/Gower distances. Pairwise alignments used a simple (1-identity) distance matrix with gap opening, extension penalties of 1.0 and 0.6 respectively. Typically four data sets were derived for each distance set and that with the lowest distance violation selected.
The text of the article presumbably explains the color coding of species groups. Note that virtual reality effects could have been done more easily and informatively accomplished in RasMol or Chime or SwissModel, which have better controls and labelling.
There is a much larger and better data set available now, over twice the size of what the authors used. Note too that repeat region data (except possibly for the first and last repeats) should never be mixed with main chain data: these regions evolve in very different and incommensurate ways.
Protein Data Bank 25 Structures 02-Dec-98 T.L.James et al. 1B10 replaces entry 2PRP. model 1 provides the best coordinatesThis entry provides refined coordinates for a hamster prion fragment. An auxiliary file, AUX1B10.RPT, gives results of diagnostics. PDB has undergone some changes in how it provides quality control and release of data. This entry is a "layer 1" release made without pdb staff intervention; the layer 2 entry will be treated as a correction to this one. An article is apparently in press at Biochemistry. These new coordinates could be used to compare hydrogen-bonding to mouse prion or to re-thread point mutations or thread other species to see what changes in conformation are expected from alleles.
The average backbone atomic rmsd to the mean structure is 0.67A for residues 125-227 of the 25 structures submitted (100 conformers were calculated ). The molecule contains 2778 unambiguous and 306 ambiguous distance restraints. Residues 90-124 are largely unstructured. Secondary structures are indicated below:
helix 1 asp 144 .. asn 153 helix 2 gln 172 .. lys 194 helix 3 glu 200 .. asp 227 sheet 1 met 129 .. gly 131 sheet 2 val 161 .. tyr 163
Journal of Virology, April 1999, p. 3511-3513, Vol. 73, No. 4 Remi Demaimay, Richard Race, and Bruce Chesebro"To date very few drugs have favorably influenced the course of transmissible spongiform encephalopathies. In previous studies, the polyene antibiotics amphotericin B (AmB) and MS-8209 prolonged the incubation time in Syrian hamsters of the 263K strain of scrapie, but AmB had no effect against other scrapie strains in Syrian hamsters.
In the present experiments using transgenic mice expressing Syrian hamster PrP in neurons only, MS-8209 extended the life spans of animals infected with the 263K strain but not the DY strain. AmB was effective against both 263K and DY and prevented death in 18% of DY-infected animals.
The AmB effect against strain 263K was more prominent in mice whose endogenous PrP gene had been inactivated by homologous recombination. It was unclear whether this difference was due to a change in the duration of the disease or to possible interactive effects between the mouse PrP gene and the drugs themselves. The effectiveness of treatment after intracerebral scrapie infection in transgenic mice expressing PrP only in neurons suggested that neurons are important sites of action for these drugs. "
PNAS Vol. 96, Issue 6, 2687-2691, March 16, 1999 Jung-Keun Suh, Lawrence L. Poulsen, Daniel M. Ziegler, and Jon D. RobertusThe flavin-containing monooxygenase from yeast (yFMO or SwissProt) catalyzes the O2- and NADPH-dependent oxidations of biological thiols, including oxidation of glutathione to glutathione disulfide (GSSG). Glutathione and GSSG form the principle redox buffering system in the cell, with the endoplasmic reticulum (ER) being more oxidizing than the cytoplasm. Proper folding of disulfide-bonded proteins in the ER depends on an optimum redox buffer ratio.
yFMO is localized to the cytoplasmic side of the ER membrane. A knockout strain and expression vectors show that yFMO has a major effect on the generation of GSSG transported into the ER. The enzyme is required for the proper folding, in the ER, of test proteins with disulfide bonds, whereas those without disulfide bonds are properly folded independently of yFMO in the ER or in the cytoplasm.
Comment (webmaster): If a comparable situation exists in mammals, this would be a new protein interacting with maturing prion protein. In either prion mutants or FMO alleles, this process could go astray. Humans have at least 5 flavin-containing monooxygenases (9 homolgy matches to the yeast protein are known).
It seems the status of the prion protein disulphide in rogue plaque has never been determined. SDS gels contain dithiothreitol as reducing agent which would reduce a disulphides if present. Standard alkylating agents could be used to trap free sulphydryls but preparation of samples under aerobic conditions or in the presence of cellular glutathione could result in artifacts. It is sometimes argued that cysteines are always reduced inside the cell and that only extra-cellular ones form disulphides, but is prion amyloid found inside cells, outside cells, or inside dead cells and with what exposure to GSSG?
Note in normal prion protein, the sulphydryls are deeply buried, creating conditions where a disulphide could well be favored even under strongly reducing intra-cellular conditions (as happens in other proteins). In rogue prion, if these cysteines become to separated too far to bridge, this would support equilibrium reduction. Reduced sulphydryls could be a temporary thermodynamic barrier between different conformations locked in by a disulphide; recall the mess in trying to reconstitute reduced insulin. But the disease variant Y145stop and the amyloidogenic fragment 105-126 have no cysteines at all. See also:
G. S. Jackson, L. L. P. Hosszu, ... J. Collinge 18 Mar 99 Science
Comment (webmaster): This paper makes a most surprising observation for this late date in the history of the prion protein. They simply reduced the disulphide and lowered the pH on a fragment; both conditions are appropriate for the late endosomal compartment or lysosomal degradation pathway. A soluble monomer resulted with much more beta structure and protease K resistance -- this could represent the long sought conformational change. Ordinary salt precipitation resulted in fibrils. They did think to test these fibrils for congophilic birefringence but the experiment is being repeated. One suppose, given the recent paper by AF Hill et al., that they will soon be testing for infectivity as well. Strain type relations are rather subtle at this point (recombinant protein is not glycosylated).
Article highlights: "Prion propagation involves the conversion of cellular prion protein (PrPC) into a disease-specific isomer, PrPSc, shifting from a predominantly alpha-helical to beta-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native alphaİconformation, characteristic of PrPC, and a compact, highly soluble, monomeric form rich in betaİstructure. The soluble betaİform (beta-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrPSc, and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrPC to beta-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation. ...
Reduction of the disulfide bond in human PrP91-231 and lowering the pH to 4.0,İin a dilute acetate buffer without additives, generated a highly soluble protein that can be concentrated to at least 12İmg/ml. When subjected to gel filtration, it eluted as a monomeric species (Fig. 2). The CD signal in the amide region of the spectrum (Fig. 1A) suggests that this highly soluble, reduced species adopted a different conformation from the native cellular form of PrP (-PrP). The native state is characterized by a strong alpha-helical signal, whereas the reduced form is qualitatively dominated by beta-sheet. This type of secondary structural transition occurs in proteins that go from a soluble monomeric state to an aggregated fibrous (amyloid) form in which beta-structure is stabilized by intermolecular interactions.
However, to our knowledge, it is unprecedented for a protein to undergo such an extensive beta-sheet conversion while remaining in a monomeric state at high protein concentrations and in the absence of denaturants. This is in contrast to the folding intermediates of mouse PrP121-231 and human PrP90-231 (10, 11). We also observe the equilibrium folding intermediate of alpha-PrP described in (10), but in contrast to beta-PrP, this is poorly soluble and has an apparent molecular weight of 40İkD (Fig. 2) indicative of tertiary disorder and expanded molecular volume. In marked contrast, the folded beta-PrP we observe is populated, and therefore stable, in the absence of denaturant with an apparent molecular volume indistinguishable from that of alpha-PrP . ...
The switch from alpha to beta conformation was reversible. When the reduced betaİform was exposed to a higher pH (8.0), the native conformation was restored. However, the rates of conversion in either direction were extremely slow, requiring days for completion (15). This high kinetic barrier, however, could be circumvented by fully denaturing and refolding at the appropriate pH to generate either isoform.
The solubility of the two isoforms was not equivalent. The alpha form of PrP can be titrated with the denaturant guanidine hydrochloride (GuHCl) to determine equilibrium parameters for the folding pathway. However, the betaİform of PrP was also highly soluble in aqueous buffers, whereas titration with GuHCl led to intermolecular associations and a visible precipitate (Fig. 2E). Physiological concentrations of other salts (150İmM NaCl or 150İmM KCl) also caused aggregation, indicating that this was simply an effect of ionic strength. Salt-precipitated material was initially composed of irregular spherical particles (Fig. 3A) that associated over several hours to form fibrils (Fig. 3B) with a diameter of up to 20İnm and lengths up to 500İnm. The morphology and dimensions of these fibrils are within the range described for scrapie-associated fibrils extracted from brains affected by prion disease (16).
As with native PrPC, alpha-PrP was extremely sensitive to digestion with PK (Fig. 3C). However, beta-PrP showed marked protease resistance. This PK resistance was a function of the structural reorganization of the monomeric betaİform, with only a moderate further increase associated with aggregation (Fig. 3C). The different patterns of proteolytic cleavage fragments seen upon PK digestion of alpha-PrP and beta-PrP provide further evidence of a major conformationalrearrangement in beta-PrP. In contrast, the partially structured beta-sheet conformation of reduced hamster PrP90-231 is fully sensitive to PK digestion (17).
Unusually for a protein with a predominantly helical fold, most of the residues in PrP91-231 have a preference for betaİconformation. This suggests that PrPis balanced between radically different folds with a high energy barrier between them--one dictated by local secondary structural propensity (the betaİconformation) and one requiring the precise docking of side chains (the native alphaİconformation).
Such a balance would be influenced by mutations causing inherited prion diseases(18). Individuals homozygous for valine at polymorphicresidue 129İof human PrP (where either methionine or valine canbe encoded) are more susceptible to iatrogenic Creutzfeldt-Jakob disease (19), and valine has a much higher betaİpropensity than methionine. Our results support this hypothesis, because the molecule is capable of slow conversion between alphaİand betaİconformations.
Furthermore, the betaİform can be locked by intermolecular association,thus supplying a plausible mechanism of propagation of a rare conformational state. The precise subcellular localization o fPrPSc propagation remains controversial, although considerable evidence implicates late endosome-like organelles or lysosomes (20-23). This alpha-PrP to beta-PrP conversion, caused by reduction and mild acidification,may be relevant to the conditions that PrPC would encounter within the cell after its internalization during recycling (24). Reduction and acidification within the endosomal pathway is required for activation of diphtheria toxin (25). Such a mechanism could underlie prion propagation and hence could account for the transmitted, sporadic, and inherited etiologies of prion disease. Initiation of a pathogenic self-propagating conversion reaction, with accumulation of aggregated beta-PrP, maybe induced by exposure to a "seed" of aggregated beta-PrP after prion inoculation, or as a rare stochastic conformational change, oras an inevitable consequence of expression of a pathogenic PrPC mutant that is predisposed to form beta-PrP...."
Comment (Roland Heynkes, Aachen): "I looked into my working review and my private database and found something inconclusive.
Exposure of fractions obtained from scrapie-infected mouse brain to Triton X-100 resulted in no change in its infectivity except after sedimentation. Infectivity was reduced after exposure to 2-mercaptoethanol but only in the presence of SDS. These results support the view that protein is an important component of the scrapie agent and that disulfide bonds may play a part in maintaining the structural integrity of the infectious agent. (Somerville,R.A.; Millson,G.C.; Kimberlin,R.H. - Sensitivity of scrapie infectivity to detergents and 2-mercaptoethanol - Intervirology 1980; 13(2): 126-9)
Furthermore an intramolecular disulfide bond, linking Cys 179 and 214, has been found in both, the normal cellular and the disease specific form of the prion protein (Turk,E.; Teplow,D.B.; Hood,L.E.; Prusiner,S.B. - Purification and properties of the cellular and scrapie hamster prion proteins - European Journal of Biochemistry 1988 Sep 1; 176(1): 21-30).
But two years later infectivity measurements revealed a convincing association between infectivity and scrapie amyloid protein, with or without its sugar chains and disulfide bonds (Brown,P.; Liberski,P.P.; Wolff,A.; Gajdusek,D.C. - Conservation of infectivity in purified fibrillary extracts of scrapie- infected hamster brain after sequential enzymatic digestion or polyacrylamide gel electrophoresis - PNAS 1990 Sep; 87(18): 7240-4).
PNAS Vol. 96, Issue 6, 3053-3058, March 16, 1999 Stefan F. Lichtenthaler, ..., and Konrad BeyreutherProteolytic processing of the amyloid precursor protein by beta-secretase yields A4CT (C99), which is cleaved further by the as yet unknown gamma-secretase, yielding the beta-amyloid (A) peptide with 40İ(A40) or 42İresidues (A42). Because the position of gamma-secretase cleavage is crucial for the pathogenesis of Alzheimer's disease, we individually replaced all membrane-domain residues of A4CT outside the A domain with phenylalanine, stably transfected the constructs in COS7 cells, and determined the effect of these mutations on the cleavage specificity of gamma-secretase (A42/A40 ratio). Compared with wild-type A4CT, mutations at Val-44, Ile-47, and Val-50 led to decreased A42/A40 ratios, whereas mutations at Thr-43, Ile-45, Val-46, Leu-49, and Met-51 led to increased A42/A40 ratios. A massive effect was observed for I45F (34-fold increase) making this construct important for the generation of animal models for Alzheimer's disease. Unlike the other mutations, A4CT-V44F was processed mainly to A38, as determined by mass spectrometry. Our data provide a detailed model for the active site of -secretase: gamma-secretase interacts with A4CT by binding to one side of the alpha-helical transmembrane domain of A4CT. Mutations in the transmembrane domain of A4CT interfere with the interaction between gamma-secretase and A4CT and, thus, alter the cleavage specificity of gamma-secretase.
PNAS Kenneth S. KosikHow the beta-amyloid peptide (A) is cleaved from its precursor, the amyloid precursor protein (APP), preoccupies a large portion of the community studying Alzheimer's disease. And for good reason. The A protein, the major component of the amyloid in senile plaques, represents a leading target in the quest for agents to combat Alzheimer's disease. Its formation involves an unusual proteolytic event within a hydrophobic stretch of amino acids predicted to form an alpha-helix within the membrane.
APP is a type I membrane-spanning protein of 770İresidues that secretes its large ectodomain after at least two different types of cleavage. Based on the abundance of the cleavage products, the site most frequently cut is at residue 687,İwhich lies within the A peptide and thus precludes its formation. The activity of the responsible, but elusive, enzyme, dubbed the alpha-secretase, has the property of recognizing an optimal projection length from the membrane more strongly than any specific sequence determinants. The alpha-secretase cleavage occurs in a post-Golgi compartment, possibly including caveolae (2), and may be a glycosyl-phosphatidylinositol-linked aspartyl protease.
A small percentage of APP is not cleaved at the alpha-secretase site, but instead is cleaved at residue 672İof APP (which becomes Asp-1 of A) by an activity dubbed beta-secretase, the first step in Alzheimer's-type amyloidogenesis. Other A species with an elongated or shortened N terminus are known (4). The beta-secretase cleavage, once completed, sets the stage for the next cleavage by an activity referred to as gamma-secretase.
In what is now considered a critical event in the disease pathogenesis, the A peptide is released after cleavage by the gamma-secretase either between residues 40İand 41İor between residues 42İand 43.İNormally, both forms of the A peptideA42 and A40are present, with A40 in great excess of A42. The precise cleavage site of the gamma-secretase, and hence the generation of the shorter or longer A peptide, is widely believed to have important pathologic consequences. Compared with A40, A42 deposits earlier in the disease process (5) and assembles more rapidly into filaments in vitro (6). Explanations of the mechanism of A42 vs. A40 cleavage by the gamma-secretase are problematic because the two proteolytic sites lie on opposite sides of the predicted alpha-helical transmembrane domain of APP.
By systematically mutating to phenylalanine each amino acid through the carboxyl half of the intramembranous segment of APP (or more precisely a fragment called C99 or A4CT which represents the remaining carboxyl-terminal fragment after beta-secretase cleavage), Lichtenthaler et al. (7) noted a remarkable pattern among specific mutations and the exact site of gamma-secretase cleavage. Based on measurements of the ratio of A42/A40, the authors proposed that the side of the alpha-helix to which the -secretase binds determines the relative amounts of each specific A isoform. Thus, mutations can subtly shift the percentage of the gamma-secretase (or gamma-secretases) that binds to one or the other side of the helix. The assumption, of course, is that the APP membrane-spanning domain forms an alpha-helix. ...
Interestingly, the mutation in the APP gene at codon 693,İwhich causes severe amyloid deposition in the cerebral vasculature with much less in the brain parenchyma does not increase either A42 or A40 (16), but instead enhances the aggregation of the mutant peptide into amyloid fibrils.
PNAS Vol. 96, Issue 6, 3108-3113, March 16, 1999 Gundars Goldsteins, ... and Erik Lundgren
Comment (webmaster): This is a bit reminiscent of the Prionics monoclonal antibody 15B3, only in another congophilic disease. They have not been able to block in vitro generation of amyloid using the mAbs because key residues are not exposed in normal conformer; indeed, antibodies might stabilize the rogue conformer.
It is also interesting to note that even though transthyretin is known to much better resolution than the prion protein by xray for both wildtype and amyloidogenic mutant, this has not particularly helped in understanding the structure of the fibrils. Transthyretin has pre-existing beta sheet, but whether this is the same as the cross-beta sheet in the final amyloid is unknown.
Structure of amyloids unfortunately falls through the cracks of modern spectroscopy due to lack of solubility and fibril nature. Another aggravating situation with a different basis exists for determining the copper repeat region structure; here the problem is ambiguity of repeat residues (though this could be side-stepped by isotopic replacement)
Highlights of article: The structural requirements for generation of amyloid from the plasma protein transthyretin (TTR) are not known, although it is assumed that TTR is partly misfolded in amyloid. In a search for structural determinants important for amyloid formation, we generated a TTR mutant with high potential to form amyloid. We demonstrated that the mutant represents an intermediate in a series of conformational changes leading to amyloid.
Two monoclonal antibodies were generated against this mutant; each displayed affinity to ex vivo TTR and TTR mutants with amyloidogenic folding but not to wild-type TTR or mutants exhibiting the wild-type fold. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and which we propose is displaced at the initial phase of amyloid formation, opening up new surfaces necessary for autoaggregation of TTR monomers.
Transthyretin (TTR) is a transport protein in plasma for thyroid hormone and forms a complex with retinol-binding protein. It has a potential to form amyloid fibrils and two major clinical forms are known. Senile systemic amyloidosis affects 25% of the individuals older than 80İyears (1). Most cases of TTR-associated amyloidosis are linked to point mutations, of which more than 50İare known. One of the most common forms has a substitution of valine for methionine at position 30İof the 127-aa-long polypeptide, leading to widespread symptoms in the peripheral nervous system, known as familial amyloidosis with polyneuropathy.
Sixteen other proteins are known to form amyloid. Posttranslational modifications are observed in some cases associated with the formation of amyloid fibrils, including conformational changes and proteolytic cleavage (3). The role for these changes in self-aggregation is only partly understood. Analysis of amyloid fibrils of different origins indicates a common cross-beta-pleated sheet conformation independent of the protein involved (4, 5).
The three-dimensional structure of native TTR is established (6); it is a tetramer with four identical subunits, folding into a globular structure, each monomer having eight beta-strands organized in two sheets. Thus, TTR has a predominance of beta-structure, in contrast to several other amyloid-forming proteins with little beta-structure, which has to be formed before aggregation starts. In the case of TTR-associated amyloid, it is not known whether the original conformation is preserved in the fibrils, although good evidence exists from in vitro experiments that the tetramers need to dissociate into alternatively folded monomers for amyloid to form.
Analysis of the distribution of mutations showed that they occur all along the polypeptide chain, although some areas seem to be spared. We previously described a broad area close to the "edge" of the molecule, i.e., around the beta-strands designated C and D, with more frequent mutations leading to amyloidosis (11). It has been proposed that this area of the molecule is more flexible (12), and a current model proposes that this area bulges out from TTR when amyloid fibrils form (13).
Detailed x-ray diffraction studies with a resolution down to 1.7İİof TTR V30M (TTR with the substitution V30M) has not given information concerning the mechanism for amyloid formation (14, 15). However, recent studies of the clinically aggressive L55P mutant suggested a possible organization of the fibrils based on the packing contacts in the crystal (16). An amyloidogenic intermediate of TTR has been demonstrated, which might occur in a denaturing or degradation pathway (17). Such partly misfolded intermediates were isolated in a previous study from our laboratory (11, 18) by construction of mutants, in which the three amino acids of the D strand were either removed (TTRdel53-55) or substituted (TTR G53S, E54D, L55S, here designated TTRs53-55). These molecules rapidly formed aggregates, which gave a typical cross-beta pattern in x-ray diffraction studies and a positive signal after staining with Congo Red or thioflavine T.İTherefore, these mutants qualify as amyloid precursors in vitro and might carry structural determinants of intermediates in an in vivo pathway leading to amyloid formation.
In the present study we asked whether it would be possible to generate monoclonal antibodies against epitopes expressed only on amyloidogenic TTR mutants. Two such monoclonal antibodies are described here that provide direct biochemical evidence for amyloidogenic conformational changes in TTR and localize them in the edge area of the molecule....
Two cryptic epitopes were detected in ex vivo amyloid, obtained by mild extraction procedures from the vitreous body of the eye in affected individuals. Most importantly, the epitopes are not detectable on wild-type TTR or on two well known mutants, namely, TTR V30M and TTR L55P, the latter giving rise to a clinically aggressive form of amyloidosis. However, after denaturation, all TTR species expose these cryptic epitopes....Generation of amyloid through the activity of so-called pathological chaperones has not been demonstrated in the case of TTR-associated amyloidosis.
We have not been able to block in vitro generation of amyloid by using the mAbs. Therefore, we propose that the cryptic epitopes represent a structural determinant not present in the core of the fibrils but rather exposed on their surface. As pointed out (22), the method used for epitope mapping gives only a minimal estimate of an epitope. mAb 56-61 has specificity for a region at the N-terminal part of the loop connecting the small D strand in the inner sheet with the E strand on the outer loop. In the native configuration, this epitope is buried by the N-terminal end of the peptide (cf. Fig. 4). mAb 39-44 recognizes an epitope, which includes the N-terminal part of the C strand, and the C-terminal part of the loop connecting the B and C strands.
This epitope is more exposed on the external surface but projects toward a valley formed by the D-E loops on both monomers, which might cause a steric hindrance toward antibody binding to this epitope in the native molecule....
The finding of N-terminal truncations (28) and an accessible trypsin cleavage site in amyloid from the vitreous body at position 48/49 in the loop between the C and D strands (18, 23) supports the notion that the edge structures, including the C and D strands, project out from the fibril.
J Gen Virol 1999 Feb;80 (Pt 2):517-24 Foster J, McKelvey W, Fraser H, Chong A, Ross A, Parnham D, Goldmann W, Hunter NGoats are susceptible to experimental challenge with bovine spongiform encephalopathy (BSE). This study set out to investigate whether the transmission of BSE could occur in goats following the transfer of embryos from experimentally infected donor females into uninfected recipient females. The results showed no evidence of transmissible spongiform encephalopathy disease in any of the offspring which developed from embryos from infected donors, nor indeed in any of the recipient females used as surrogate dams. In addition, there was no indication of experimental BSE spreading as either a venereal infection to males used in mating or by maternal transmission to offspring born naturally to experimentally infected donors, although numbers were small.
15 Mar 99 webmaster commentary
Recall the event in the mid-1930's in which England prepared a viral vaccine in scrapie sheep brain and injected this into tens of thousands of sheep, causing a massive outbreak of scrapie. These sheep were then exported all over the place, there being no export ban, bringing the disease with them to new continents. From timing and probabilities alone, this may have been the source of the 1937 scrapie in Canada (which then exported sheep to Michigan and Wisconsin in time for the first scrapie and TME in the US), as well as in Iceland.
Now a fine account of the importation of scrapie into India. [Zlotnik I, Katiyar RD. The occurrence of scrapie disease in sheep of the remote Himalayan foothills. Vet Rec 1961;73:543-4.] concludes that "in spite of the apparent absence of incoordination [and loss of appetite] in the scratching disease of the Himalayan mountain sheep, other clinical symptoms are similar to those of scrapie. However, the histological lesions in the central nervous system of affected animals are identical with those described for natural scrapie in sheep in Britain." (Zlotnik had previously published on scrapie during previous work at Moredun in Edinburgh and did the histopathology on 4 Indian sheep brains.)
Importation took place around 1940. It seems that one Raja Saheb imported a number of Rambouillet rams into the valleys of the Tons and Gangotri and distributed them to farmers in order to improve their stock. The disease spread to 33 villages with incidence varying by flock for 1-10%. It does not say explicity that the Rambouillet rams came from England but given that India was a colony at the time, this seems the likliest scenario.
Interestingly, the natives in both valleys could easily distinguish psoroptic mange ('lodho') from scrapie ('khujali' in Tons, 'mukoo' in Gangotri, both mean scratching) even though both were manifested by rubbing. They further knew how to treat mange (which had been around forever) and that khujali and mukoo (which were new) invariably terminated in death. In 1961, these remote valleys could be reached by taking the train to Rishikesh or Haldwani, then taking an unscheduled local bus, then walking or riding the last 30-80 miles.
Many asinine pronouncements have been made that CJD is holding steady at a one per million rate in "every country in the world" (advocacy subtext: beef and mutton are safe); India by these reckonings should have 1,000 cases per year. Of course, at most 30 cases of CJD were seen in India between 1971 and 1990, ie 19,970 cases were 'missed'. This is not due to flawed ascertainment because actually India has no program for ascertainment. But the data taken at face value actually suggest a huge risk reduction with vegetarianism, contrary to the above-mentioned asinine pronouncements. Even now in the upper Gangotri valley computed tomography and immunohistochemistry are seldom used diagnostically in senile dementia cases and there is completely unsatisfactory baseline data on the incidence of sporadic CJD prior to introduction of scrapie.
SC Arya, an Indian physician, in proposing in 1991 that some Indian CJD may be vaccine-related, notes that the Central Research Institute (also located in the Himalayan foothills) produces 4-5 million doses of rabies vaccine per year in sheep brain. These are needed because 700,000 people per year need post-exposure rabies vaccine annually (there is a horrific problem in Indian cities with street dogs). Here are the precautions taken with the sheep [Arya citing a 1981 WHO document]:
"The existing requirements for production of sheep-brain vaccines for human and veterinary use stipulate an animal quarantine lasting not less than 2 weeks to ascertain freedom from tuberculosis and neural disease other than rabies; there are no recommendations for scrapie surveillance in a sheep flock."
BMJ 1996;313:1405 (30İNovember) Letters Subhash C Aryaİ"Janet Morgan reports that the National Blood Authority in Britain has decided to tighten the donor screening programme to exclude transmission of Creutzfeldt-Jakob disease or its variant through blood donations. Prospective donors will be prevented from donating blood if they have a history of treatment with human growth hormone or if one of their siblings, parents, or grandparents developed the disease. I would point out that similar care should also be taken when immigrants from Asia and Africa offer to donate blood, in case they received rabies vaccine derived from culture of sheep brain cells when they were living in their country of origin.
In many countries in Asia and Africa limited supplies of imported rabies vaccines derived from culture of human cells have been available. Many people continue to be offered indigenously produced sheep brain vaccine after exposure to a rabid animal. Scrapie is known to exist in sheep around many centres where the vaccine is produced. In the mountain sheep of the Kumaon foothills in the Himalayas, for example, scrapie was established more than four decades ago and 1-10% of the flock was reported to have the disease in 1961.
In the Himalayan foothills the Central Research Institute continues to produce four to five million doses of sheep brain vaccine annually. Transmission of abnormal prion protein, PrPsc, in sheep brain vaccine might have occurred in some of the 30 documented cases of Creutzfeldt-Jakob disease in different regions in India. Because Creutzfeldt-Jakob disease has a latency of about 20 years, many recipients of sheep brain rabies vaccine could emigrate to Britain before becoming ill.
Before accepting blood donations from immigrants it would be desirable to ask the potential donors whether they were exposed to a rabid animal and immunised with sheep brain rabies vaccine in their country of origin. Furthermore, indirect assessment should be possible through, for example, assay looking for antibodies specific to rabies."
Neuroepidemiology 1991;10(1):27-32 Satishchandra P, Shankar SKThirty cases including 20 definite and 10 probable cases of Creutzfeldt-Jakob disease (CJD) seen in India between 1971 and 1990 are reported. Demographic analysis has shown similarities to the previously published reports from other parts of the world. Though 21 (70%) of cases were from two centers--Bombay and Bangalore-, suggesting clustering, this seems to be more apparent than real. One subject worked in the medical field, where possibility of iatrogenic transmission could not be ruled out. None of the cases had positive family history of CJD. There is no epidemiological data of CJD from India so far and hence this report is one such pilot study.
Listserve correspondence 10 Mar 99La Commission choisit 4 tests:
Selon une dÈpÍche de l'AFP en date du 2 Mars 1999, l'Union EuropÈenne a choisi 4 parmi la dizaine de tests de l'ESB qui lui avait ÈtÈ soumis en rÈponse ı un appel d'offres lancÈ en Mai 1998. Ces 4 tests seraient ceux d'Enfer Scientific Ltd, EG et G Wallac Ltd (Grande-Bretagne), Prionics Ltd et celui du Commissariat FranÁais ı l'Energie Atomique.
How does the Prionics test compare with respect to sensitivity and specificity with the BSE test systems of European national surveillance units like the Groschup group in Tuebingen? Are there any published or unpublished data that could support or refute the opinion of the German authorities that the Prionics test is less sensitive?
Answer: (Bruno Oesch)
The Swiss validation included a number of BSE or normal control animals, the latter being either normal animals or animals with neurological symptoms unrelated to BSE. Furthermore, we checked the robustness of the procedure by including samples of bad quality. Our recent result with samples from fallen stock (downer cows) indicate that the Prionics Western blot is very robust. In about 1000 animals, 4 cases of BSE were found. These animals were confirmed by immunohistochemistry to be BSE cases.
The best negative gold standard we came up with were animals, old enough to have developed brain lesions and clinical disease in case of infection, from geographic regions at current free of BSE and with now clinical, histopathological >and immunocytochemical signs of the disease (not all individuals in the pool met these inclusion criteria, but all were negative in the standard test procedures).
The best positive gold standard that was available were animals with clinical signs consistant with BSE, histopathological changes and PrP(Sc) accumulation in the brain (IHC).
For these two gold standard populations, the Prionics Western Blot performed very well and we therefore concluded that the sensitivity and specificity of the test is almost 100% (but with the lower limit on the 95% confidence interval below 100% since these are estimates based on samples of limited size).
Regarding specificity: the latest results from the field study of slaughtered cattle (3022 cattle) of which 1 animal was a BSE case. All these animals had been checked and confirmed by the BSE Reference Center with histology and immunohistochemistry. This means no false negatives or positives i.e. a specificity of >99.99%.
I have not seen any data on the sensitivity or specificity of the Groschup method with cattle. I assume that his method is SAF preparation follewed by Western blotting. We have done a comparison of SAF preparation and Prionics Western in collaboration with the VLA (Veterinary Laboratory Agency) in Weybridge. Briefly, the PrionicsCheck was more sensitive than SAF preparation. Whether and how the Groschup method compares with the method used at the VLA is unclear. An extensive publication on the validation and other results will be published.
Internally we have done an analysis on what dilution of a BSE brain homogenate we can still see. Consistently, we still could detect a 100 fold dilution of BSE homogenates. Obviously, animals at defined stages of incubation would be the ideal samples but were unfortunately unavailable to us. When asked by journalists, The German authorities say that the prionics Western blot is not officially validated in Switzerland which is wrong and represents wishfull thinking to get around BSE tests. The Swiss Authorities officially USE the Prionics Western blot for surveillance. We have made the validation report available to EU authorities and authorities of individual EU countries.
Response: (Roland Heynkes)
Is there any scientific reason to think that it might produce false positives and is it really impossible that the Prionics test could detect BSE where the Groschup test finds nothing. I also know that much more than 5% of the BSE infected animals would not produce positive results with any of the today available tests. This does not really mean no false negatives, because even the Swiss BSE Reference Center cannot exclude BSE infections. This only means that there was no detectable difference of sensitivity and specificity between your and the tests of the Reference Center.
Dr. Wiemer of the German ministry of agriculture told me that the test of Dr. Groschup must be more sensitive than the Prionics test, just because it uses an additional purification step by ultracentrifugation. I answered him that in my opinion the prion concentration is not so important, because the Proteinase K digestion of most other proteins is also an effective purification method so that the quality of the antibody and the hybridization conditions are much more important. For this reason I asked him for real data which he does not have. After that he told me that there is a consense among all experts that the Groschup test must be more sensitive than the Prionics test and that I should ask the OIE for data.
I am sure that the British CVL has time series probes. Did you alternatively look for rodent data about the accumulaton of PrPSc within the brain during incubation time? This factor 100 should allow to estimate the point within the incubation time at which the accumulation of PrPSc reaches 1/100 of the final concentration.
The German authorities only say that the Prionics test has not been validated for the EU and that they have to wait for the results of the running tests. The problem is that the tests of the national surveillance units have not been EU-validated either. The political background is that they are not allowed to kill whole herds or to remove bovine products from the market without a EU validated test or a positive result from the national surveillance unit. Therefore they seem to be afraid that they would have to pay too much for such measurements. If now a German journalist would become aware of a positive Prionics test result that did not lead to governmental actions, the minister Baerbel Hoehn would not survive that politically.
Arch Virol 1999;144(1):177-84 Nemoto T, Horiuchi M, Ishiguro N, Shinagawa MWe describe methods for the preparation of collagen and gelatin samples to detect possible prion contaminants using Western blotting of a major component of prions, PrPSc. A commercially available collagen solution containing 2% athero-collagen was spiked with rodent adapted scrapie prion and used as the prion-contaminating collagen. The methods developed center on the enzymatic reduction of the collagen solution viscosity with protease treatments and on the concentration of the prion from the protease-digests with polyethylene glycol-#6000 and NaCl. Recovery of the spiked prion as a partially protease-resistant core fragment of PrPSc fluctuated from 30% to 46% of the input amount.
9 Mar 99 correspondenceThis meeting will take place in Geneva, Switzerland as a satellite conference to a larger immunological meeting. The prion immunology part takes place between 2:00 and 6:00 on 1 Aug 99. There will be four plenary lectures, short oral communications selected from submitted abstracts, and a poster session. obviously this has considerable relevence to early diagnosis and early intervention. Alex Raeber is the person to contact ( tel 1/1 255 2151 or fax 41/1255 4402).
Journal-Of-Investigative-Medicine, 1999, V47, N2, Feb, P A13. Issn:1081-5589. Gharavi, V. and Imperiali B. [Caltech, Div Chem & Chem Engn,Pasadena, CA . ]This one is oddly not yet in Medline, which only has the Jan 99 issue. The MAFF site had this and many other unusual cites. Current UK research projects are also listed
Pichia pastoris is a yeast, about which they say, "Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P. pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P. pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10-100-fold." [J Biomol NMR 1999 Feb;13(2):149-59 ]
Also at the Maff bibliographic section for 1999:
Finckh, U. Mann, U.; Mueller Thomsen T; Eggers, C.; Naber, D.; Nitsch, R. M, and Gal, 28th Annual Meeting of the Society for Neuroscience, Part 1, Los Angeles, California, USA, November 7-12, 1998 Society-For-Neuroscience-Abstracts, 1998, Vol. 24, No. 1-2, P. 758 Issn: 0190-5295.
Molecular-And-Cellular-Biology, 1999, V19, N2, Feb, Pp 1325-1333. Issn: 0270-7306. Newnam, G. P.; Wegrzyn, R. D.; Lindquist, S. L., and Chernoff, Y. O.
Biophysical-Journal, 1999, V76, N2, Feb, Pp 1048-1062. Issn: 0006-3495. Wille, H. and Prusiner, S. B.
Dementia And Geriatric Cognitive Disorders, 1999, Vol/Iss/Pg. 10/1 (47-50), Issn: 1420-8008 Frenkel, Y. R.; Ben Israel J; Korczyn, A. D., and Chapman J.
Blood, 1998, V92, N10, 1, Nov 15, S1, P 2069. Holada, K.; Theisen, P.; MacAuley, C.; Rohwer, R. G., and Vostal, J. G.
Hepatology, Oct., 1998, Vol. 28, No. 4 Part 2, P. 300a Issn: 0270-9139. Kitada, T. ..., and Kuroki, T. Biennial Scientific Meeting of the International Association for the Study of the Liver and the 49th Annual Meeting and Postgraduate Courses of the American Association for the Study of Liver Diseases, Chicago, Illinois, USA, November 4-10, 1998 Sponsored by: International Association for the Study of the Liver.
Folia Neuropathol 1998;36(4):225-8 Kozubski W, Wender M, Szczech J, Lenart-Jankowska D, Liberski PP Department of Neurology, School of Medicine, Poznan.The great concern exists that new variant of CJD (nvCJD) developed as a result of exposure to bovine spongiform encephalopathy (BSE)-infected meat products. Therefore, all cases of CJD in the young, as the one of ours are the matter of interest. The 21-year-old female developed a rapid progression of pyramidal, extrapyramidal and cerebellar signs, visual loss and psychiatric symptoms, leading to death in 16 weeks. The microscopic features were: a neuronal loss accentuated in cerebral cortex with extensive astroglia proliferation and spongiform changes. Immunohistochemical staining, revealed the presence of "synaptic" deposits of PrP in the cerebral cortex and in the cerebellum. No florid amyloid plaques were present. The case was diagnosed as a sporadic CJD, with some features of Heidenhein variant (visual symptoms) and corticostriatocerebellar category. The pathological findings excluded a nv CJD which is linked with BSE.