Bad news on BSE fed to primates
Lethal partial knockout found
Are the longer deletions diseases of normal prion function? Not proven.
Prion repeat copper by mass spec
Prion repeat binds Cu++, tryptophan, glycine carbonyl implicated
Copper binding to extra-cellular domain of Alzheimer APP
Copper chaperones in yeast
Higher repeat properties in cultured cells
Donor pool size and the risk of blood-borne CJD
TSE agents: safe working and the prevention of infection
Advice needed in Argentina 'umbanda' sheep blood CJD
Inhibiting prion conversion with central peptides, 106-141
Amyloid fibril formation by an SH3 domain.
Capillary isoelectric focusing of the scrapie prion protein
C R Acad Sci III 1997 Dec;320(12):971-979 Bons N, Mestre-Frances N, Guiraud I, Charnay Y
|We report on two animals of a non-human primate species Eulemur fulvus mayottensis, housed in the local zoo and fed over a number of years with a food containing cattle meat, that developed serious neurological symptoms associated with prion immunoreactivity in brain and various viscera. Microscopy of the brains showed neuronal vacuolation with patchy/perivacuolar immunolabelling with an abnormal isoform of prion protein (IR-PrP), an important characteristic of spongiform encephalopathy.|
For the first time, we report the presence in the same severely ill animals of IR-PrP in the gastrointestinal tract, detected by immunocytochemistry with mono- and polyclonal antibodies directed against various parts of the PrP. Strong PrP labelling was observed in the epithelial cells lining the pharyngeal and gastrointestinal lumen. The tonsils and the walls of the lymph and blood vessels below the intestinal epithelium were also labelled.
There were no such immunoreactions in healthy lemurians killed as controls, i.e. a younger congener of the same species housed under the same conditions, and others belonging to the smaller species Microcebus murinus, reared in the laboratory and never fed on commercial food products containing cattle meat. These results demonstrate a strong PrP accumulation in the brain, the gastrointestinal tract and underlying lymphoreticular structures in these primates living in a zoological park and suffering from a spongiform encephalopathy.
[This is a prosimian primate species from Madagascar (Mayotte is in the Comorro islands) without prehensile tail; taxonomically Primates; Strepsirhini; Lemuridae; Eulemur; Eulemur fulvus. The prion gene has not been sequenced from any Strepsirhini (prosimian) -- webmaster]
J Neurosci Res 1998 May 1;52(3):260-267 Brown DR, Schmidt B, Kretzschmar HAA fragment of the prion protein (PrP106-126) induces cell death in cultures of wild-type embryonic day (E)16 mouse cortical neurons but not cells derived from mice devoid of cellular PrP(PrPo/o). Two common binding partners for PrP106-126 expressed in both wild-type and PrPo/o mouse brain were isolated and their sequences determined. The two proteins were found to be alpha and beta tubulin.
Further evidence that tubulin binds PrP106-126 within cells comes from cell culture experiments. Colchicine toxicity on PrPo/o mouse cortical cells is enhanced by PrP106-126 and taxol enhances toxicity of PrP106-126 on wild-type mouse cortical cells. Our evidence shows that a fragment of PrP can bind a cellular protein and in so doing, alters the metabolism of cells even when they do not express native PrP. This indicates that PrP106-126 is nontoxic to PrPo/o cells, not because of an inability to interact with these cells but because of the loss of some aspect of a PrP expression-dependent phenotype.
7 May 98 commentary on abstractA most interesting paper in Cell says that prion protein may be (compensatably) essential after all. Recall mice lacking both copies of their prion molecule genes seemed to get along ok, in dire conflict with the slow rate at which evolutionary changes are fixed in prion protein relative, say, to hemoglobin.
Aguzzi/Weissmann previously put forward in Nature News & Views the concept that though the prion gene is essential, knockout mice have sufficient time during development to induce more of a compensatory gene product, as has been exhaustively shown in yeast where essential genes are rare even where function is clearly essential.
Here they are saying something supportive of this: a properly chosen dysfunctional prion protein can be worse than no prion at all, if it can outcompete for ligand or prevent induction of the compensatory protein by virtue of the fragmentary functionality that remains. Interestingly, no effect is seen in heterozygotes (the abstract does not give the time scale used for test animals).
This suggests that the long-sought (non-CJD) human neurological disorders attributable to loss of normal prion function are possible but very rare indeed through the mechanism here -- because of the unusual mutational event needed, possible in utero onset, and autosomal recessive nature. Even if just the right point mutation might accomplish the same end, it would also need to be dominant, which now seems less likely.
As shown in the mouse sequence below, a deletion beginning just distal to the signal region (so the repeat region generator as well as the repeats themselves) up to, or mostly through, the invariant core, give a dramatic effect on pathology and viability. They say that certain shorter deletions from the N-terminal side did not have this lethal effect; similar deletions were looked at earlier unluckily it seems by UCSF.
The reduced protein could plausibly still be properly exported, folded correctly in its globular domain, and be given the GPI insertion moiety. The protein shows up for work but can't get the job done, while signalling the cell that it has.
It should not be concluded that "ataxia and neuronal death" mean that these mice developed TSE. On the contrary, this may be a different disorder altogether, related to loss of normal function.
001 mvkshigswi lvlfvamwsd vglc[signal]kkrpkp gggwntggsr ypgqgspggn ryppqggggw 061 gqphgggwgq phgggwgqph gggwgqphgg gwgqphgggg wgqggthgqw nkpskpktnm 121 khvagaaaag avvgglggym lgsamsrpli hfgsdyedry yrenmhrypn qvyyrpvdqy 181 snqnnfvhdc vnitvkehtv ttttkgenft etdikmmerv veqmcitqyq resqayyqrg 241 asvilfsspp villisflif livg
webmaster 14 May 98: further comments on full text of 17 Apr 98 Cell paper
1. The authors first review the 3 lines of ORF knockout mice and 1 line additionally knocked back 940 bp into intron 2 and 450 bp into 3' untranslated. 2 lines were ok, the third had inconsistent effects depending on research group, and the Sakaguchi line displayed ataxia and Purkinje problems at 70 days. [The abstract at Nature 1996 Apr 11;380(6574):528-531 says Prp was replaced by a drug resistance gene, not deleted]. Del 177-200 and del 201-217 previously gave rise to fatal CNS disorders.
Knockouts do not support scrapie or prion propagation (big news from yesteryear); however, deletions 69-84 and 32-80 [repeat region] did, as did the Fischer gene which lacked the large intron and consisted just of 6kb-ORF-2.2kb [EMBO J. 1996 Mar 15; 15(6): 1255-1264.]. Oddly mRNA was missing specifically in Purkinje cells in the Fischer line despite over-expression in the brain as a whole.
2. The first surprise (to the authors) is that deletions 32-121 and 32-134 but not shorter 32-80, 32-93, or 32-106 caused behavioral disorders, coarse tremor, staggering gait, paresis of hind legs, wasting, ataxaia, astrocytosis within 3 weeks, and cerebellum granular layer degeneration and near-death within 90 days. No Prp-res on histoblot or congophilic birefringence in the longer deletions (data not shown, indecisive in any case).
Do these mice then have a variation of TSE or a new and distinct neurological disorder due to partial disruption of normal prion function? This issue is never addressed head-on.
No transmission to 'indicator' wildype mice was seen 60 weeks after inoculation. However, based on earlier mixed results of Hsiao and Telling with P102L mice, it is absolutely imperative to test for transmission in under-expressors of the cognate deletion. I predict that transmission will be observed.
Somewhere I had gotten the impression that they had constructed reciprocal deletions, of the middle invariant domain but not the repeat. Not so, though UCSF may have (in a differant, non-comparable line of course). They only verified that (an unquantitated amount of) del 32-134 and wt reached the cell surface . They are using whopping gene copy numbers, in the 15-150 range. For some unexplained reason, these only give mRNA excesses of 1.2 to 5x over wildtype control.
The question here is, why are partial deletions worse than complete knockouts?
An alternative answer is obvious (yet still interesting): these mice are getting familial murine TSE either because exactly the longer deletions knock out the second ER signal site or for the same reason maturing point mutations do. Codon 32 is 9 residues past the signal cleavage for ER processing, allowing correct processing there, but topological confusion results when a second expected site is missing. Some protein hangs up, never gets properly modified, partial proteolysis occurs at domain hinges, chaperones try to mend things but drop it down to cross-beta default structure. got essentially the same effect with the point mutation A117V [Hegde et al.]. Knockouts have no protein to begin with, so how are they going to form toxic fibril?
If they extend the set of deletions towards the middle, 107-120, I predict the cross-over in effects will come at the second signal (plus or minus a few residues), around residue 115. I decline to share in the surprise.
3. The second surprise (to the authors) is that one or more wild type gene in trans over-rides these effects completely, as did del 32-93. The supressor effect of wildtype gene did not result from lowered expression of partially deleted protein.
My problem here is that the supporting data in Table 1 show mice were only kept for 30 weeks or so. Ok, a wild type gene slows the effect considerably from the 10 week onset. They might have seen an effect both here and in transmission if they had waited out a 104 week mouse lifespan.
Occam would simply interpret all this as a re-run met/val 129: fibril growth is capped or trapped in heterozygotes. It is really not all that different from P102L to wildtype transmission failure. It is a waste of time to test transmission in multi-copy wildtype mice when a single wildtype gene is already known to suppress the effect.
4. Be this as it may, the authors move on to model-building. They go 'protein x' one step better (or worse) by proposing Prp interacts with a hypothetical ligand L-prp to generate the same signal that L-prp elicits from an even more conjectural protein pi that is similar in function [but not orthologous] to Prp. A string-and ball structural model of Prp is used and laminin receptor is cited as a candidate. They suggest mutations of ligand L-prp might be responsible for various ganule cell degenerative diseases such as infantile cerebello-optic atrophy.
The model goes way beyond the data (which doesn't make it unreasonable or wrong). They need to nail down transmissibility and establish that this is something other than a conventional heterologous effect predicted by the standard model. All in all the paper reminds me a great deal of Langappa's data and theories about A117V, interesting ideas but in the end the old ones probably suffice.
It is quite possible for effects to be conflated, here and elsewhere: mutants could affect both normal prion function and accumulate off-track peptide. The burden of proof, showing that a given mutant with a CNS phenotype is cleanly separated from TSE, is hard to meet. For that reason, vertical genomics may be a much faster, cheaper, and more reliable approach to normal function.
Cell 1998 Apr 17;93(2):203-214 [fulltext online to subscribers] Shmerling D, Hegyi I, ...von Mering C, Hangartner C, Aguzzi A, Weissmann CThe physiological role of prion protein (PrP) remains unknown. Mice devoid of PrP develop normally but are resistant to scrapie; introduction of a PrP transgene restores susceptibility to the disease. To identify the regions of PrP necessary for this activity, we prepared PrP knockout mice expressing PrPs with amino-proximal deletions. Surprisingly, PrP lacking residues 32-121 or 32-134, but not with shorter deletions, caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1-3 months after birth. The defect was completely abolished by introducing one copy of a wild-type PrP gene. We speculate that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand.
Abstracts for prion copper poster papers for the American Society for Mass Spectrometry (ASMS) conference, May 31-June 4, Marriott World Center, Orlando, Florida, $132/night
Two groups have taken a structural look at what is going on with copper and prion protein. This is extremely important in understanding normal prion structure/function. While conclusions weren't exactly divulged in the abstracts, the stoichiometry talk makes it sounds like a single (catalytic) copper is ruled out. A role merely stablilizing secondary structure role would be novel (there are easier ways to build coiled coil) so this could be mixed up with copper scavenging or transport. The number of repeats in mammals has held steady at 5 for 120 million years, with some wobble to 4 or 6; perhaps there is a cooperative explanation for this.
Hopefully, they have rendered some nice imagery of the full structure and substantiated somewhat its physiological relevence (heaven forbid that someone should immunoprecipitate copper-labelled lipase-released native protein). One supposes that NMR failed to distinguish one repeat from the next, never mind all the indistinguishable glycines within a given repeat. Whether structures formed with repeats from different monomer chains would necessarily hold up under ESI MS is unclear.
These results fit the "avian anomaly" better though don't resolve it. In other words, what was the structure/function of the common ancestor to a hexa-repeat and an octa-repeat? Birds have the shorter hexa-repeat yet more iterations that might work out to the same length if copper is structural. My view is that a single functional ancestral repeat was expanded slightly differently from a common ancestor in the various lineages through slippage/repair, just as happens today in repeat-CJD.
The results also fit Blast 2 searching, which invariably tosses back collagen and cuticle structural proteins despite non-homology.
Whittal, Randy; Baldwin, Michael; Ball, Haydn; Prusiner, Stanley; Burlingame, AlmaThe normal physiological function of the prion protein, PrP, is as yet unknown. The mature Syrian hamster prion protein (SHaPrP) contains 209 amino acids, 23-231. By NMR the protein is structured between amino acid 125 and the C-terminus, but the N-terminal region between 23 to 125 is largely unstructured. This is an unusual occurrence and suggests that secondary structure may be induced by some other mechanism. The mature protein contains a highly conserved region with four repeats of the eight amino acids PHGGGWGQ. This has been suggested as a copper (II) binding site, which may induce secondary structure to the protein.
Hornshaw et al.  used MALDI-MS to demonstrate that three and four-octarepeat peptides bind copper. Recent experiments by circular dichroism, fluorescence- spectroscopy, and equilibrium dialysis have showed that recombinant SHa(29-231) selectively binds ~ 2 molecules of copper . However, equilibrium dialysis of PrP(23-98) showed cooperative complexation of 3.4 copper ions with saturation at a Cu(II):peptide ratio of 5.6 .
Recent studies using electrospray ionization mass spectrometry (ESI MS) have shown that solution phase noncovalent protein-metal interactions are maintained in the gas phase, thus solution complexes can be studied directly by mass spectrometry . We have monitored the interaction between the peptides PrP(23-60), PrP(57-91), PrP(73-91), PrP(90-144), PrP(142-177), and PrP(178-231) and metal ions by ESI MS. The results for each peptide will be compared with the recombinant protein, PrP(29-231). The effect of pH, ionic strength, and organic solvent concentration upon complex formation will be studied. The selectivity and stoichiometry of complex formation, observed by ESI MS, will be presented and compared with the results from fluorescence spectroscopy and equilibrium dialysis to evaluate the validity of the mass spectrometric method.
 Hornshaw, M. P.; McDermott, J. R.; Candy, J. M. Biochem. Biophys. Res. Commun. 1995, 207, 621-629.
 Stockel, J.; Safar, J.; Wallace, A.; Cohen, F. E.; Prusiner, S. B. Biochem. in press.
 Brown, D. R. et al. Nature 1997, 390, 684-687.
Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1-23.
Hornshaw, Martin; Welch, William; McKenzie, Debbie; Peltier, JohnPrion diseases such as bovine spongiform encephalopathy, scrapie and CJD share a common feature in that they seem to be caused by the conversion of the normal, alpha-helical rich, cellular prion protein, PrPc to the abnormal, partly beta-sheet, protease-resistant, disease-specific prion protein, PrPSc. It has been hypothesized that an N-terminal region of the prion protein consisting of repeats of an octapeptide PHGGGWGQ, termed the octarepeat region, may be a unique metal binding domain.
Indeed, studies using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) have demonstrated that the N-terminal octarepeat region of the prion is a putative copper-binding domain. PrPC possesses four perfect octarepeats and an imperfect nonapeptide repeat. This domain seems to be lacking in ordered structure except in the presence of copper. In some cases of familial CJD there are additional PHGGGWGQ repeats which seem to be sufficient to cause disease. Here we have investigated the copper-binding of synthetic peptides (PHGGGWGQ)n, where n ranges in number from 4 to 7 using MALDI TOF MS and Electrospray ionization time-of-flight mass spectrometry (ESI TOF MS).
Biochemistry 1998 May 19;37(20):7185-7193 Stockel J, Safar J, Wallace AC, Cohen FE, Prusiner SBThe infectious isoform of the prion protein (PrPSc) is derived from cellular PrP (PrPC) in a conversion reaction involving a dramatic reorganization of secondary and tertiary structure. While our understanding of the pathogenic role of PrPSc has grown, the normal physiologic function of PrPC still remains unclear.
Using recombinant Syrian hamster prion protein [SHaPrP(29-231)], we investigated metal ions as possible ligands of PrP. Near-UV circular dichroism spectroscopy (CD) indicates that the conformation of SHaPrP(29-231) resembles PrPC purified from hamster brain. Here we demonstrate by CD and tryptophan (Trp) fluorescence spectroscopy that copper induces changes to the tertiary structure of SHaPrP(29-231).
Binding of copper quenches the Trp fluorescence emission significantly, shifts the emission spectrum to shorter wavelengths, and also induces changes in the near-UV CD spectrum of SHaPrP(29-231). The binding sites are highly specific for Cu2+, as indicated by the lack of a change in Trp fluorescence emission with Ca2+, Co2+, Mg2+, Mn2+, Ni2+, and Zn2+. [Should have tested cadmium as well. Neuroprotective function at issue here, so important. -- webmaster] Binding of Cu2+ also promotes the conformational shift from a predominantly alpha-helical to a beta-sheet structure. [Not likely: too many glycines and prolines. The CD spectrum may be re-interpretable as a collagen-like left-handed helix instead. --webmaster]
Equilibrium dialysis experiments indicate a binding stoichiometry of approximately 2 copper molecules per PrP molecule at physiologically relevant concentrations, and pH titration of Cu2+ binding suggests a role for histidine as a chelating ligand. NMR spectroscopy has recently demonstrated that the octarepeats (PHGGGWGQ) in SHaPrP(29-231) lack secondary or tertiary structure in the absence of Cu2+. Our results suggest that each Cu2+ binds to a structure defined by two octarepeats (PHGGGWGQ) with one histidine and perhaps one glycine carbonyl chelating the ion. We propose that the binding of two copper ions to four octarepeats induces a more defined structure to this region.
That same issue of Biochemistry also contains:
Biochemistry 19 May 98 37: 7224-7230 Gerd Multhaup, Thomas Ruppert, Andrea Schlicksupp, Lars Hesse, Eckhard Bill, Rüdiger Pipkorn, Colin L. Masters, and Konrad BeyreutherThe extracellular domain of transmembrane Abeta amyloid precursor protein (APP) has a Cu(II) reducing activity upon Cu(II) binding associated with the formation of a new disulfide bridge. The complete assignment of the disulfide bond revealed the involvement of cysteines 144 and 158 around copper-binding histidine residues. The vulnerability of APP-Cu(I) complexes to reactive oxygen species was elaborated as a site-specific and random fragmentation of APP in a time-dependent manner and at low concentrations of H2O2....
Biol Chem 1998 May 22;273(21):12817-12826 Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NM, Romano DM, Hartshorn MA, Tanzi RE, Bush AIThe cortical deposition of Abeta is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Abeta, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Abeta at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Abeta1-40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Abeta1-40 bound three to four Cu2+ ions when precipitated at pH 7.0.
J Biol Chem 1998 Mar 6;273(10):5493-5499 Borza DB, Morgan WTThe middle domain of plasma histidine-proline-rich glycoprotein (HPRG) contains unusual tandem pentapeptide repeats (consensus G(H/P)(H/P)PH) and binds heparin and transition metals. Unlike other proteins that interact with heparin via lysine or arginine residues, HPRG relies exclusively on histidine residues for this interaction. ... There was little binding of HPRG to heparin at physiological pH in the absence of metals, but the interaction was promoted by nanomolar concentrations of free zinc and copper, and its pH dependence was shifted toward alkaline pH by zinc.
We propose that HPRG is a physiological pH sensor, interacting with negatively charged GAGs on cell surfaces only when it acquires a net positive charge by protonation and/or metal binding. This provides a mechanism to regulate the function of HPRG (the local pH) and rationalizes the role of its unique, conserved histidine-proline-rich domain. Thus, under conditions of local acidosis (e.g. ischemia or hypoxia), HPRG can co-immobilize plasminogen at the cell surface as well as compete for heparin with other proteins such as antithrombin.
Biochemistry 19 May 98 37: 7572-7577 Chandra Srinivasan, Matthew C. Posewitz, Graham N. George, and Dennis R. WingeAssembly of functional cytochrome oxidase in yeast requires Cox17, which has been postulated to deliver copper ions to the mitochondrion for insertion into the enzyme. This role for Cox17 is supported by the observation that it binds copper as a binuclear cuprous-thiolate cluster. X-ray absorption spectroscopy, together with UV-visible absorption and emission spectroscopy, indicates the presence of bound cuprous ions, trigonally coordinated by thiolate ligands. Analysis of the EXAFS shows three Cu-S bonds at 2.26 A, plus a short Cu-Cu distance of 2.7 A, indicating a binuclear cluster in Cox17. The cuprous-thiolate cluster in Cox17 is substantially more labile than structurally related clusters in metallothioneins.
J Biol Chem 1997 Sep 19;272(38):23469-23472 Culotta VC, Klomp LW, Strain J, Casareno RL, KremsB, Gitlin JD firstname.lastname@example.orgCopper is distributed to distinct localizations in the cell through diverse pathways. We demonstrate here that the delivery of copper to copper/zinc superoxide dismutase (SOD1) is mediated through a soluble factor identified as Saccharomyces cerevisiae LYS7 and human CCS (copper chaperone for SOD). This factor is specific for SOD1 and does not deliver copper to proteins in the mitochondria, nucleus, or secretory pathway. Yeast cells containing a lys7Delta null mutation have normal levels of SOD1 protein, but fail to incorporate copper into SOD1, which is therefore devoid of superoxide scavenging activity. LYS7 and CCS specifically restore the biosynthesis of holoSOD1 in vivo. Elucidation of the CCS copper delivery pathway may permit development of novel therapeutic approaches to human diseases that involve SOD1, including amyotrophic lateral sclerosis.
J Biol Chem 1998 May 8;273(19):11980-11985 [fulltext online to subscribers] Priola SA, Chesebro BInherited forms of the human transmissible spongiform encephalopathy Creutzfeldt-Jakob disease (CJD) have been associated with mutations in the normal soluble, protease-sensitive form of the host prion protein (PrP-sen). Normal PrP protein contains five copies of a repeating eight-amino acid region, and PrP molecules with six or more copies of this region are associated with disease in familial CJD. It has been hypothesized that these mutations might facilitate spontaneous formation of the abnormal, aggregated protease-resistant PrP isoform, PrP-res, associated with clinical CJD and other transmissible spongiform encephalopathies (TSE). In the present experiments, hamster PrP molecules with 5 (wild-type), 7, 9, or 11 copies of this repeat region were generated and expressed in mouse fibroblast cells or mouse neuroblastoma cells.
In mouse fibroblast cells, mutant hamster PrP molecules expressing 7, 9, and 11 copies of the octapeptide repeat sequence showed altered cell surface expression, but both mutant and wild-type hamster PrP-sen molecules demonstrated abnormal properties of aggregation and increased protease resistance. By contrast in mouse neuroblastoma cells, hamster PrP-sen with 5, 9, and 11 octapeptide repeats were expressed normally on the cell surface, but only PrP-sen molecules with 9 or 11 copies of the repeat motif had abnormal properties of aggregation and increased protease resistance. Overall, regardless of cell type, hamster PrP molecules with greater than 7 octapeptide repeats were more aggregated and more protease-resistant than molecules with 7 repeats or less. However, these abnormal molecules were at least 1000-fold less protease-resistant than bona fide PrP-res derived from TSE-infected brain tissue, and they showed no increased ability to form PrP-res in a cell-free system.
Commentary: they look here at physical properties of a well-chosen series of extra repeats. Longer repeats do have more protease K resistance, but this seems insufficient to account for their CJD-causative properties; perhaps the answer comes later in their interaction with cellular proteases and chaperones. I don't expect cross-beta congophilic structure to come sooner or from extra repeats per se. The MALDI TOF MS people looked at structure for 5 and 7 repeats which happily are two of the lengths considered here.
7 May 98Titles sound great but I can't find "Transfusion" online due to bland name; there are no abstracts at Medline. Jonathan Trouern-Trend of the American Red Cross writes that he publication is put out by the American Association of Blood Banks; he doubts any of it is online. He has a copy of the articles and will post them to BSE listserve as time permits.
Transfusion 1998 Mar;38(3):312-315 Brown P
Transfusion 1998 Mar;38(3):221-223 Dodd RY, Sullivan MT
Thursday 30th April 1998 UK Department Of HealthCopies of the new guidance, Transmissible spongiform encephalopathy agents: safe working and the prevention of infection, ISBN 0-11-322166-5, are available, price 10 pounds sterling, from The Stationery Office. "
The address for orders by post is -- The Stationery Office, Publications Centre, P O Box 276, London SW8 2DR. Payment with order. Cheques should be made out to "The Stationery Office". Press copies only are available from the Department of Health Press Office.
The revised guidance follows a review of earlier advice on laboratory and experimental work, and the management of patients with or suspected to have, or at risk from, CJD and related diseases. The review was prompted by the emergence in 1995 of a new variant of Creutzfeldt-Jakob Disease (nvCJD) and the possibility of a link to Bovine Spongiform Encephalopathy.
The guidance gives advice on safe working practices to prevent the transmission of CJD in healthcare settings, and updates the health and safety requirements for laboratory work with TSE agents. It does not cover incidental exposure, such as on farms, in abattoirs or in the course of other work with animals, which is addressed in separate guidance.
In the clinical context, the guidance stresses that in most cases there is no need for extra precautions for CJD patients beyond those used for other patients. However, because transmission can occur in specific situations associated with clinical interventions, it is important to ensure that special precautions are taken where appropriate.
The guidance makes strict recommendations about the handling of clinical instruments which have been used on patients with symptoms of CJD. It extends previous Department of Health advice, which recommended disposal of instruments that had been in contact with the brain, spinal cord or eye, to apply to all instruments used in the clinical care of this small group of patients. This extra precautionary measure aims to reduce still further the remote possibility of transmitting infection from one patient to another.
The recommendations for patients categorised in the guidance as "at risk" from CJD, but who do not have any symptoms of the disease, remain unchanged from those given in earlier guidance.
The guidance also sets out precautionary measures for laboratory work with BSE, scrapie and other TSE agents. In addition to the general requirements for safe working, it gives more detailed information about experimental work with TSE agents. It includes the new stricter requirements for laboratory work with BSE. This follows the addition of BSE to the 1998 edition of the UK list of pathogenic agents.
A joint working group of experts from the Advisory Committee on Dangerous Pathogens (ACDP) and the Spongiform Encephalopathy Advisory Committee (SEAC) produced the revised guidance which has been endorsed by both committees. They recognised the considerable uncertainties about TSEs, and nvCJD in particular, and stressed the need to review the guidance regularly as more information becomes available.
Flavio Mercado, Dra Guiñazu, and Dr. Torres Neurologist at the San Martin Hospital in Buenos Aires School of Medicine.
"I am a physician, clinic and neurologist. I am making now the teaching course at the university in Buenos Aires and membership of the "Sociedad Neurologica Argentina" (Dr Flavio Mercado). write to show this case that we saw recently get more opinions on it:
I am a neurologist in Argentina and in the past January I had a case of CJD, apparently an sporadic one: The patient was a 55 year-old who developed a subacute dementia , with motoneurone signs, cerebelar syndrome and myoclonus. He died ten month after the onset of the symptoms. No history of familiar neurologic disease or iatrogenic procedures was found. He belonged to a afro-brazilian religion called Umbanda and his wife told us that he had probably drunk sheep blood in religious ceremonies. He would be the second case among umbanda.
I want to know if somebody knows a case like this. We know that blood may be infectious but the infectious titre would be quite low and there has not been any confirmation of transmission of scrapie to humans even among people who eat brain. We would like to know your opinion."
Thank you very much, sincerely Flavio Mercado
J Biol Chem 1998 May 22;273(21):13203-13207 Chabry J, Caughey B, ChesebroThe transmissible spongiform encephalopathies are characterized by the conversion of the protease-sensitive prion protein (PrPsen) into a protease-resistant isoform (PrPres) associated with the neuropathogenic process in vivo. Recently, PrPres has been shown to be capable of directly inducing the conversion of PrPsen to PrPres in a cell-free in vitro system. In the present experiments, various PrP peptides were studied for their ability to enhance or inhibit this cell-free conversion reaction. None of the synthetic peptides was able to confer protease-resistance to the labeled PrPsen molecules on their own.
On the contrary, peptides from the central part of the hamster PrP sequence from 106 to 141 could completely inhibit the conversion induced by preformed PrPres. The presence of residues 119 and 120 from the highly hydrophobic sequence AGAAAAGA (position 113 to 120) was crucial for an efficient inhibitory effect.
Fourier transform infrared spectroscopy analysis indicated that inhibitory peptides formed high beta-sheet aggregates under the conditions of the conversion reaction, but this was also true of certain peptides that were not inhibitory. Thus, the potential to form beta-sheeted aggregates may be necessary, but not sufficient, for peptides to act as inhibitors of PrPres formation. Clearly, the amino acid sequence of the peptide is also important for inhibition. The sequence specificity of the inhibition is consistent with the idea that residues in the vicinity of positions 106-141 of PrPres and/or PrPsen are critically involved in the intermolecular interactions that lead to PrPres formation.
Proc Natl Acad Sci U S A 1998 Apr 14;95(8):4224-4228 Guijarro JI, Sunde M, Jones JA, Campbell ID, Dobson CMThe SH3 domain is a well characterized small protein module with a simple fold found in many proteins. At acid pH, the SH3 domain (PI3-SH3) of the p85alpha subunit of bovine phosphatidylinositol 3-kinase slowly forms a gel that consists of typical amyloid fibrils as assessed by electron microscopy, a Congo red binding assay, and x-ray fiber diffraction. The soluble form of PI3-SH3 at acid pH (the A state by a variety of techniques) from which fibrils are generated has been characterized. Circular dichroism in the far- and near-UV regions and 1H NMR indicate that the A state is substantially unfolded relative to the native protein at neutral pH. NMR diffusion measurements indicate, however, that the effective hydrodynamic radius of the A state is only 23% higher than that of the native protein and is 20% lower than that of the protein denatured in 3.5 M guanidinium chloride.
In addition, the A state binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonic acid, which suggests that SH3 in this state has a partially formed hydrophobic core. These results indicate that the A state is partially folded and support the hypothesis that partially folded states formed in solution are precursors of amyloid deposition. Moreover, that this domain aggregates into amyloid fibrils suggests that the potential for amyloid deposition may be a common property of proteins, and not only of a few proteins associated with disease.
J Chromatogr A 1998 Apr 3;802(1):135-141 Schmerr MJ, Cutlip RC, Jenny APrion diseases or transmissible spongiform encephalopathies belong to a group of neurodegenerative diseases that infect both animals and humans. These diseases are associated with an accumulation of fibrils in the brains of infected individuals. These fibrils are composed of an abnormal isoform of a host-encoded glycoprotein that is characterized by its insolubility and partial resistance to proteases.
Another characteristic of the scrapie prion protein (PrPsc) is the wide range of isoelectric points (pI values) that have been observed on conventional isoelectrofocusing gels. In this study, we explored the use of capillary isoelectric focusing (cIEF) to characterize the pI values for PrPsc isolated from sheep and hamster brain. We used a Beckman 5500 P/ACE using UV detection at 280 nm. A cIEF 3-10 Kit from Beckman Instruments was used to perform the analysis. The PrPsc was solubilized in 0.01 M Tris-HCl, pH 8.00 containing 2 mM EDTA. 5% SDS and 10% hexafluoroisopropanol at 100 degrees C for 10 min. The solubilized PrPsc was placed over a high-performance hydrophilic interaction column. After elution, the peaks were concentrated and assayed for immunoreactivity with specific antisera. The peaks that contained immunoreactivity were then placed on the cIEF capillary. The samples containing PrPsc were solubilized in 1% n-octylglucoside before isoelectric focusing.
The scrapie infected sheep sample had peaks with pI values ranging from 5.2 to 3.00 with a major peak at 3.09. The normal sheep brain had pI values that were higher. The hamster adapted scrapie strain had peaks with pI values ranging from 6.47 to 3.8. These pI values were slightly higher than those obtained for the sheep samples. The use of cIEF to determine the pI values of PrPsc led to the identification of a major species of PrPsc from sheep with a very acidic pI.