Prion glycan: bittersweet situation
30% scrapie in a closed Romanov flock
TSE symposium in Tubingen Germany: 23-25 September 1999
French E200K cluster
A French new list: ESB-liste
High risk scrapie alleles found in New Zealand
US SEAC meets June 2, 1999: FDA blood deferals
Guinea pig prion sequence: PCR errors claimed
Plasma fractionation processes and causative agents of TSE
10 May 99 webmaster reviewTthe mouse prion glycan paper by Alma L Burlingame and coworkers, Biochemisty 38: 4885-95 99, is an excellent paper that investigates, for the first time, both codon 180 and 196 asparagine-linked carbohydrates, so much discussed in human CJD strain types.
The authors omitted a legend for Figure 6, which sums up their final structural determinations of structural variants (over 60) and do not offer a glossary of terms for the generic prion scientist who has never worked on glycoproteins but might want to read this paper. It is also assumed that the reader has one eye on Biochemistry 32: 1991-2002 93, their earlier technical paper on hamster position 181.
Figure 6 symbols and terms explained:
... bond to peptide is at right (reducing end) ... reducing end: terminal free carbonyl in an oligosaccharide that can reduce ferric or cupric ion. ... alpha or beta: the anomer formed at the carbonyl carbon as a monosaccharide condenses to a ring. ... L or D: mirror pair of sugar stereoisomers, all D here except L fucose, not determinable by mass spec. ... ellipse: sialic acid = NeuAc = NeuNAc = N-acetyl neuraminic acid = 9 carbon derivative of mannosamine. ... open circle: mannose = C2 epimer of glucose ... solid circle: galactose = C4 epimer of glucose = Gal, terminally as b1-4 linkage. ... triangle: fucose = 6 deoxy mannose ... square: N-acetyl glucosamine = GlcNAc ... antennae: oligosaccharides branching off a central core mannose ... braces: substitutents could not be unambiguously allocated to antennae ... hex: hexose = mannose + galactose (same mol wt in mass spec) ... Lewisx: branched antennae trisaccharide, GlcNAc with a1-3 fucose and b1-4 Gal substitutents ... sialyl L:ewis x: tetra-saccharide with terminal sialic acid attached to Gal of Lewisx ... bisecting: a GlcNAc between two antennae, ie at position 3 if antennae are linked at 2 and 4 to core beta mannose ... chitobiose: unbranched core oligosaccharide similar to chitin (b1-4 GlcNAc x 3), initial a1-6 fucose here.
They purified PrP-Sc from a single mouse strain infected with scrapie strain ME7 of undescribed passage history. Their data thus does not directly address what carbohydrates might be found on native prion protein released from the cell surface by cleavage of its GPI anchor. The issue here is that PrP-Sc is partially recycled already in the peptide chain, so possibly some carbohydrate has been lost as well, relative to the cell surface precursor.
Next, they used trypsin to cleave between the two asparagines, in order to chromatographically separate the two substituents: 180:NITIK/QHTVTTTTK/GEN:196. Here they ran into two aggravations: pyro-glu forming from the glutamine and incomplete cleavage -- despite 5% trypsin overnight at 37C on reduced and alkylated urea-denatured prion -- at K193 because of steric hindrance by the bulky substituent at 196. K184 was not hindered. In effect, the first glycan became easier to analyze than the broad peak containing the second.
Trypsin cleavage in Collinge strain-typing could resolve the mono-substituted cases; in this paper they do report that either site may be completely unoccupied. If humans are like mice, Colling/Gambetti strain-types are a huge over-simplification. The combinatorics suggest tens of thousands of different covalent structures. The fibrils probably do not sort this out fully, making them quasi- one dimensional crystals (ie, have microheterogenity in the unit cells). However, I predict that the carbohydrates will be on the external surface of the fibril in all strain types and a minor influence on the cross-beta structure, which will depend more on clipping of the peptide ends. Y145stop has no carbohydrate but still causes familial CJD.
The technology used is impressive and only new developments in mass spectroscopy enabled the idenficiation of very small amounts of individual products. They coupled various rounds of MS with specific enzyme cleavages to work out the various structures involved in a way too complex to describe here but well-presented in the paper. Some residual ambiguity remains but the bottom line (Fig 6) is likely correct. Asp180 is significantly less complicated than Asp196, being mostly bi- and tri-antennary structures. Hamster and mouse have the same substituents but in different proportions, insofar as hamster was determinable by 1993 methods.
The sad state of affairs concerning glycoprotein biosynthetic enzymes is discussed: some ER enzymes are known with the needed capabilities but no connection has been made with any actual role. Assuming that the results here reflect no catabolism, it seems that the fucosylated core is probably added in a single step and the antennae, sialic acids, and bisecting GlcNAc are added (or not added) in independent processes that proceed to varying levels of completeness. Note however whole brains were used, so no differentiation between brain regions was possible.
The bittersweet news here is that 60 structures represents extreme complexity, yet here and in hamster, all variants can be taken intermediates aspiring to (but never quite attaining) a single master platonic ideal structural design: tetra-antennerary, fully sialylated, and bisected. (The authors were too exhausted by the experimental work and its reporting to make this not-quite-proven observation.) This perfected entity would have 25 monosaccharides and a molecular weight of 4,500 at each position, so about 1/4 of the total molecular weight. This is a far better situation than wholly different designs for each rodent species or differen designs at the two positions (or within one position). This could change outside of rodents however.
Modified amino acids are common in proteins (eg, the arginines in prion protein) and the glycans could be viewed as much-modified asparagines, veritable tails 40x the size wagging the dog. The nmr structures have been done on a non-glycosylated domain; the structural effect is hoped small. The glycans are highly polar and may extend harmlessly out in the aqueous mileau, despite comprising so much ot total molecular weight. There is no way to structurally model these compounds; we have 25 monosaccharides at each site, each about the size of a histidine.
For the same reason, it is unlikely that glycan subtleties really influence the structure of prion fibrils all that much. That structure is a universal cross-beta design that applies across the board to include-non glycoproteins. Here too the glycans may extend harmlessly out in the aqueous mileau, though steric hindrance may drive helix parameters of the fibril to some extent and thus govern recruitment of incremental monomers, screening them for structural compatibility with the initial seed. Keep in mind that the CJD mutant Y145stop has no carbohydrate to begin with.
Now the glycans on Collinge strain types are very poorly characterized. The focus has been on all-or-none attachment, not on degree of completion. However, no free asparagines have been demonstrated, these could equally have core glycans attached. Saying that one aspargine is occupied does not distinguish between 180 occupied, 196 vacant or vice versa. Further, sporadic CJD, say of type 1, is likely a mix of easily differentiated classes of glycan types. This would be important to pursue because cwdCJD would apparently lie within the type 1 'classical' CJDs.
The authors are moving forward to examine the glycans in different strains of scrapie in mice. Let us hope they can determine the glycan structure in other species such as deer, mink, cow, sheep, and human. It would be instructive to characterize individual cases of sporadic CJD as well as nvCJD. This is one glycoprotein where all this detailed work is really worthwhile.
Science May 21 1999: 1372-1376. Oliver T. Keppler, Stephan Hinderlich, Josmar Langner, .. and Michael PawlitaThis paper, while not directed specifically at sialylation of Lewis-x tetraantennary prion protein, though probably applicable to it, shows how complex the overall regulation of glycan formation must be at the level of cell type and what a great over-simplification current strain typing must represent. Figure 1A of the paper locates this obscure but important enzyme relative to the CMP-NeuAc sialyltransferase itself.
No wonder CJD phenotypes are so variable: figure in the catabolism side as well, for both donor and host, as well as alleles of these enzymes; the combinatorics of recruiting seed and accretable monomer are quite large yet evidently satisfied at variable locations.
|"Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction. Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway. UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules. "
"Glycoproteins expressed at the cell surface can be modified to varying degrees by the addition of sialic acids [N-acetylneuraminic acid (NeuAc)]. Because of their widespread distribution, structural versatility, and peripheral position on oligosaccharide chains of glycoconjugates, sialic acid residues are well suited as molecular determinants of specific biological processes. For example, they are involved in cell-cell interactions, T and B cell activation, and hematopoietic cell differentiation. Differential sialylation, that is, quantitative or linkage-specific differences in sialylation, of cell surface molecules is also implicated in the tumorigenicity and metastatic behavior of malignant cells. "
"Sialyltransferases, which reside in the Golgi apparatus, add cytidine monophosphate (CMP)-activated sialic acid residues to specific terminal nonreducing positions on oligosaccharide chains of proteins and lipids. The differential expression of sialyltransferases explains some but not all examples of differential sialylation. ...We investigated whether differential sialylation could be a result of limitations in available intracellular CMP-NeuAc, a substrate of all sialyltransferases."
"Sialyl-Lewisx (sLex, CD15s) is an important sialylated component of carbohydrate ligands that bind to E- and P-selectin molecules. These ligands are expressed by leukocytes and are involved in leukocyte recruitment of selectin-expressing endothelial cells in response to injury or inflammation . "
"... Our data show that UDP-GlcNAc 2-epimerase, which catalyzes a rate-limiting step in the biosynthesis of sialic acids, is an important regulator of cell surface glycoconjugate sialylation in hematopoietic cell lines. Activity of the enzyme can be controlled at transcription and can affect the sialylation and function of specific cell surface molecules expressed on B cells and myeloid cells. Further insight into the regulation of sialylation by UDP-GlcNAc 2-epimerase in normal human cells and primary tumors may contribute to the understanding of physiological as well as pathological sialic acid-dependent processes in adhesion, signaling, differentiation, and metastasis."
Arch Virol 1999;144(3):431-45 Elsen JM, Amigues Y, ... Poivey JP, Lantier F, Laplanche JL INRA, SAGA, Auzeville, France[The article itself is off-line unless you are a paid subscriber; full text is on order. It is not possible to purchase access to a single article.]
Information from a scrapie epidemic in a closed INRA Romanov flock is presented. Performances, pedigree, histopathological diagnoses and PrP genotypes were recorded from the beginning of the outbreak (in 1993).
Between April, 1993 and May, 1997, 1015 animals were exposed to scrapie, and 304 died from this disease. A major influence of the polymorphisms at codons 136, 154 and 171 is shown, A136, H154, Q171 allele carriers proving to be nearly as resistant as A136, R154, R171 carriers.
A possible relationship between gastrointestinal parasitism and scrapie is discussed. There is evidence of maternal transmission, with a risk ratio for artificially fed lambs of 67 percent of the risk of lambs fed by their mother. Our results strongly suggest that resistant animals were not healthy carriers or at least were less infectious when comparing risk for lambs born to healthy dams either of resistant (risk = 0.431) or of susceptible (risk = 1.000) genotype.
Comment (webmaster): Let us hope for claims on carrier animals that they relied on the most sensitive possible immunohistochemistry rather than bulk statistics for determining what a non-carrier animal was. But it doesn't sound like it. The high penetrance, 30%, might have been even higher with IHC or if they had monitored the situation longer than 4 years.
Comment (Prof Jeanne Brugere-Picoux on 12 May 1999): "The first description of genetic susceptibility in natural scrapie observed in an INRA flock Romanov (and Ile-de-France) was presented by Laplanche and Coll in September 1991 at a London meeting. This work was made with the collaboration of the national veterinary school of Alfort without financial help from INRA (Institut National de la Recherche Agronomique) except the sheep with scrapie.These results were at the origin of the work (many years later) about the problem of genetic susceptibility in natural scrapie by researchers of INRA.
Citation: J.L. LAPLANCHE, J. CHATELAIN, S. THOMAS, M. DUSSAUCY, J. BRUGERE-PICOUX et J.M. LAUNAY : - Prp gene allelic variants and natural scrapie in French Ile-de-France and Romanov sheep.In "Prions diseases in humans and animals", Ed. S. PRUSINER et coll, Ed. Ell's Horwood, New-York 1992, p 329-33
Conference web site as of 24 Apr 99Here's what they have posted for the program:
Thursday, September 23rd, 1999 Registration and mounting of posters Introductory lectures: - The pivotal role of prion protein - Update on the BSE epidemic - Update on nvCJD cases Oral presentations on the following topic: - PrP conversion Friday, September 24th, 1999 Oral presentations on the following topics: - Prion pathogenensis - Clinical and preclinical in-vivo tests for prion diseases - Poster session - Post-mortem diagnosis in animals and man - Conventional and transgenic models for the detection of BSE and scrapie infectivity - Round-table discussion on the 'Chances and limitations of diagnostic techniques currently available' Saturday, September 25th, 1999 Oral presentations on the following topics: - Characterization of the infectious agent - Factors modulating susceptibility to disease
Scientific Board: A. Aguzzi, Z¸rich J. Collinge, London D. Dormont, Paris M. Groschup, T¸bingen H. Kretzschmar, G–ttingen C. Weissmann, LondonWhat characterizes the pathogenesis of prion diseases in animals and man?
Eur J Neurol 1998 Jul;5(4):375-379 Chatelain J, Delasnerie-Laupretre N, Lemaire MH, Cathala F, Launay JM, Laplanche JLBetween 1992 and 1995, the annual incidence of CJD in one of the 96 French departements (administrative districts) was found to be about six times higher than the CJD national incidence. Among the 12 definite or probable CJD patients referred during this period within this departement, nine originated from a small confined area (30 x 30 km) and seven patients carried the E200K mutation in their prion protein gene (PRNP).
Genealogical data showed that these seven cases, together with three other ones previously referred during the 1970-82 period, probably belonged to different branches of the same family which could be traced to the beginning of the eighteenth century.
Interestingly enough, all but two patients presented as sporadic cases before the genealogic and genetic studies. To our knowledge, this study is the first describing in France a focal accumulation of CJD associated with the PRNP E200K mutation.
Note the German archives of old messages are being moved to a new location. The old link is not working. BSE-L has been moved to a new server, but the new web archive interface has not yet been installed.
To search the archives of list BSE-L, you must currently use e-mail commands, an old-fashioned method. For example, send the command
search pseudogene in BSE-L
in the body of an e-mail message to
and after a few minutes, LISTSERV will send a list of articles about pseudogenez to you. You will then be able to retrieve these articles by the 'getpost' command, for example:
GETPOST BSE-L 229
This worked fine for me and is useful to know as a search method. Many articles on this web site appeared first on the BSE listserve.
ESB-liste is a new list for French speaking people. This list announced last week the 58 th case of BSE in France in Orne "departement" (western part of the country).
La liste ESB-liste a ÈtÈ lancÈe la semaine derniËre. Elle est associÈe ý la page "Vache Folle en ligne" de la Mission Environnement et SociÈtÈ de l'Institut national de la recherche Agronomique (INRA). Ouverte ý tous ceux - scientifiques ou non- que prÈoccupent les problÈmes posÈs par l'encÈphalopathie spongiforme bovine et les questions qui lui sont liÈesPour plus de dÈtails ou pour s'inscrire...
Comment (webmaster): This signs you up at a very generic Internet service that potentially sells your email and personal data to various unsolicited junk mail services in the language of your choice. However, here is the impressive policy of One list about privacy and spam:
On Restricted lists, the list owner must approve all subscriptions to the list, allowing for elimination of unwanted/problem members. Despite the significant resources we have allocated to prevent abuse of our system, spammers will eventually find ways to use the site to further their efforts. OneList will continue to modify our site to prevent spam, as these abuses become apparent. However, it is important to note that despite our best efforts, at a cost of providing subscriber and list owner services, some abuse will occur. While we can't prevent all spam, we can promise to do all we can to reach this end."
Archives of Virology 144 Issue 4 (1999) pp 829-834 Offline unless you are a paid subscriber. It is not possible to purchase access to this single article. A. Bossers, F. L. Harders, M. A. Smits DLO-Institute for Animal Science and Health, Lelystad, The Netherlands Received July 20, 1998, accepted October 1, 1998Polymorphisms within the prion protein (PrP) gene are associated with scrapie susceptibility. We analysed the PrP genes of 140 Romney Marsh sheep, the dominant breed in New Zealand, a country free from scrapie. We found PrP alleles that are associated with a high susceptibility to scrapie. Sheep with these PrP genotypes would probably succumb to scrapie when born and raised in a scrapie endemic environment. These findings correspond to those obtained in minor breeds from New Zealand. We conclude that scrapie development not only depends on host genetic factors but also requires exogenous factors. Our findings demonstrate the effectiveness of the measures taken by New Zealand to maintain free from scrapie.
In sheep, codon A136V, Q171R, and to a minor extent 154H contribute to the susceptibility of sheep for scrapie . The polymorphisms at codons 112, 137, 141, 171 (Q to H), and 211 are rare and have not been associated yet with a scrapie phenotype. In sheep breeds that carry PrP VRQ at codons 136, 154, and 171, there is a high susceptibility to scrapie. The PrP ARR allelic variant is associated with resistance in all breeds investigated so far. In breeds in which the PrP VRQ allele is rare or absent, for instance the Suffolk breed, the wildtype PrP ARQ allele is associated with susceptibility to scrapie. In breeds that contain the PrP VRQ allele, the PrP VRQ /PrP VRQ sheep are at greatest risk from scrapie followed by the PrP VRQ /PrP ARQ sheep, at least in Britain and Europe. In breeds in which the PrP VRQ allele is rare or absent, the PrP ARQ /PrP ARQ sheep are at greatest risk. In sheep with the latter homozygous PrP genotypes survival times of less than 14 months have been recorded (L. van Keulen, pers. comm.).
Is scrapie is a genetic disease arising spontaneously from sheep with certain PrP alleles in the absence of any infectious agent? Observations on the transmission of scrapie in embryo transfer experiments however indicated that scrapie is not purely a genetic disease. In addition, sheep with PrP genotypes at greatest risk from scrapie have been found in Cheviot and Suffolk sheep from Australia and/or New Zealand. Since these countries are free from scrapie for decades and scrapie has become a notifiable disease in New Zealand since 1955, it is likely that scrapie is not a spontaneous genetic disease linked to the PrP gene alone, although development of the disease is strongly influenced by PrP. [Surely the authors are speaking solely to the 3 alleles and bulk scrapie. Other mutations, such as 8 extra repeats, are very likely to cause scrapie in and of themselves. How else could scrapie have originated? -- webmaster]
Here we determined the PrP allele- and genotype frequencies of the most dominant sheep breed in NewZealand since 1900, the Romney Marsh. This breed was first imported from Britain into New Zealand in 1852. It was used to develop two new breeds, the Perendale and the Coopworth. The Romney Marsh and these two new breeds form the bases of the entire New Zealand sheep flock, which consists of about 46 million sheep, 58% Romney Marsh, 17% Perendale and Coopworth, and 25% other breeds. No new Romney Marsh sheep have been imported into New Zealand since the beginning of this century.
The authors collected blood samples of 140 random animals less than two years of age from 14 different flocks, scattered all over the country. These farms normally breed their own replacement animals and only purchase rams. The sampled flocks are likely representative for the entire Romney Marsh flock.
PCR amplified products were analysed by DGGE) which not only identifies known PrP alleles but also new ones. However no new ones were found, though AF141RQ was found in 7 sheep. The analysis revealed the presence of five different PrP alleles in the New Zealand Romney Marsh breed, including alleles linked to the highest scrapie susceptibilities (PrP VRQ allele, linked to the highest susceptibility, was present at a frequency of 2.5%, but never in homozygotes). Flock VIII had an unusually high PrP VRQ frequency of 15%, so VRQ / VRQ of2.25%, one per 44 animals expected; flock X had a relatively high frequency of PrP AHQ, and flocks XII and XIV were high in PrP AF141RQ .
PrP genotype frequencies: homozygotes in blue ARQ/ARQ 21 wildtype ARR/ARR 32 resistance AHQ/AHQ 1 weak susceptibility VRQ/ARQ 4 first allele is high susceptibility VRQ/ARR 3 double difference ARQ/ARR 54 ARQ/AF141RQ 5 ARQ/AHQ 9 ARR/AHQ 9 ARR/AF141RQ 2 Total 140No VRQ homozygotes were found in 140 sheep; however they should be present in the population at a frequency of about one per 1666 animals. Three of the 140 tested animals (2,9%) contain the PrP VRQ /PrP ARQ genotype. In an environment where scrapie is endemic, such animals would also succumb to scrapie.
Similar data were reported for Merino sheep of Australia and Poll Dorsetts of New Zealand [Hunter N, Cairns D (1998) J Gen Virol 79: 2 079‚2 082]. See also: Hunter N, Cairns D, Foster JD, Smith G, Goldmann W, Donnelly K (1997) Is scrapie solely a genetic disease? Nature 386: 137.
Opinion (webmaster): Is scrapie a genetic disease? Now no one doubts that 8 extra prion repeats in a genetically engineered sheep would cause scrapie in and of itself. How else could scrapie have originated in the first place?
However, such mutations have not been seen in actual scrapie sheep so far. Instead, various alternatives alleles to wildtype are found, with A136V homozygotes being the most suspicious of these. Because of pre-existing infection and facility contamination issues, it is not an easy matter to simply to raise a flock of these and see if they contract scrapie.
If one takes New Zealand's claims to be scrapie-free at face value (no outside group has ever validated this), and if a flock of VRQ/VRQ sheep could be identified and raised in isolation to advanced age and compared to an ARQ/ARQ control group, the question of A136V causality might be settled. (Alternatively, a super-sensitive test for scrapie might allow young sheep to be screened for incipient scrapie.)
The first issue has been settled by the paper above. That is, the highest risk allele does exist in New Zealand at about 1 in 1,666 sheep, or 27,611 sheep in the population of 43 million at any given time. However, tens of thousands of sheep need to be genotyped in order to collect an adequate study population of VRQ/VRQ. While this seems like an extremely excessive effort for a simple test of a point mutation, there are significant implications to breeding out scrapie. No sheep were actually tested for scrapie here or in the papers from Hunter's group.
I would like to see New Zealand adopt an explicit policy of allowing foreign researchers to do what they need to do, ie, IHC on a sample of these 27,611 sheep. Otherwise, spare us the scrapie-free anecdotes. If you truly are scrapie-free, what is there to be afraid of? The fact is, you really don't know.
June 2, 1999, 8:30 am to 5:30 pm and June 3, 1999, 8:30 am to 4:00 pm. Holiday Inn-Gaithersburg, Ballroom, 2 Montgomery Village Avenue, Gaithersburg, MD 20879. Contact Person: William Freas or Sheila D. Langford, (301) 827-0314, or FDA Advisory Committee Information Line, 1-800-741-8138 (301-443-0572 in the Washington, DC area) code 12392.Agenda: On June 2, 1999, the committee will continue the discussion from its December 18, 1998, meeting on the possible deferral of blood or blood product donors based on geographical criteria linked to possible food-borne exposure to the agent of Bovine Spongiform Encephalopathy as a measure to reduce the potential for transmission of new variant Creutzfeldt-Jakob Disease (nvCJD).
The transcripts [really a summary] of this December meeting are available on the FDA Home Page
The potential effects of such deferrals on the supply of blood and blood products will be considered as part of the committee's deliberations. The results of a survey of blood donors for duration and time periods of their visits to U.K. countries are expected to be presented. On June 3, 1999, the committee will receive an update on dura mater allograft materials. The committee will then discuss precautions needed to assure safe sources of sheep-derived and goat-derived materials contained in or used to manufacture injectable or implantable FDA-regulated products.
Oral Presentations: Between approximately 1:45 to 2:45pm on June 2, 1999, and 1:00 to 1:30pm on June 3, 1999 oral presentations from the public will be scheduled. Those desiring to make formal oral presentations should notify the contact person before May 26, 1999.
Closed Committee Deliberations: On June 3, 1999, from 3:45 to 4:00pm the meeting will be closed to permit discussion and review of trade secret and/or confidential information (5 U.S.C. 552b(c)(4)). CBER Advisory Committee Meetings - June 1999
Comment (webmaster): Why doesn't FDA simply post these announcements on the professional listserve? Answer: they don't really want anyone at their meeting yet are required under the law to announce it, the more obscurely the better.
In my opinion there will be no trade secrets or confidential information discussed during the secret session. Perhaps someone who attends will blow the whistle on this; hopefully 5 U.S.C. 552b(c)(4) provides for severe penalties.
Yeah, right:the FDA sends its regrets on military nvCJD blood deferrals:
"FDA regrets that it was unable to publish this notice 15 days prior to the June 2, 1999, Transmissible Spongiform Encephalopathies Advisory Committee meeting. Because the agency believes there is some urgency to bring these issues to public discussion and qualified members of the Transmissible Spongiform Encephalopathies Advisory Committee were available at this time, the Commissioner concluded that it was in the public interest to hold this meeting even if there was not sufficient time for the customary 15-day public notice. Notice of this meeting is given under the Federal Advisory Committee Act (5 U.S.C. app. 2).
Dated: May 11, 1999. Michael A. Friedman, Deputy Commissioner for Operations. [FR Doc. 99-12653 Filed 5-19-99; 8:45 am] BILLING CODE 4160-01-F"This meeting was actually announced at the Vancouver BC meeting on May 5-6 and probably was announced to insiders at the last meeting in November 1998. There was also a belated notice posted to Blood-CJD, another listserve. Let's do a FOIA to determine how many months back they reserved the room at the hotel.
Meanwhile, what have they done to look at this case of potential US nvCJD?:
Female, 65, died November 7, 1998, 3 weeks after diagnosis. She was a homemaker, mother and grandma. She was from the UK and went back periodically after migrating to the States forty years ago. She went back for several months four years ago to care for her mother-in-law. The hospice nurse stated that she was the third case he had treated for CJD.
6 May 1999 webmaster opinionNothing makes the webmaster more uncomfortable than a GenBank entry where the authors do not get their own names spelled right. On AF139166, the first author is actually PH Frederikse at the NIH National Eye Institute, who has worked somewhat on presenilin, beta-amyloid, and oxidative stress affects on amyloid.
Sure enough, they report a most improbable deletion of G Q G S P G G N R Y P (which follows SYRP in the entry). This has been a strongly invariant region of the prion protein in some 85 other species. This is then followed by a first repeat SHSGGTWGQ that is by itself a radical change without precedent in the lack of initial proline. So we are looking again at PCR error in this region, which has known stem-loop structure and a long history of sequencing problems. The authors have not responded to a polite request for clarification by 27 May 99.
The guinea pig has been used in expoerimental prion research for close to 3 decades, the earliest references on Medline being Science 1973 Oct 5;182(107):67-8 (Gibbs and Gajdusek) and PNAS 1976 Jan;73(1):223-7 (Manuelidis EE, Kim, Angelo, Manuelidis L). This has mostly been transmission and immunological studies. We know today that serial transmission cannot be interpreted without knowing the prion sequence of each intermediate species.
Laura Manuelidis notes that guinea pigs were used for the first successful serial transmission of human CJD to non primates (E.E. Manuelidis in Science 190:571-2, 1975); the lab made several other important observations (relevent today to transfusion and transplantation risks) on CJD in guinea pigs such as:
1) the reproducible demonstration of agent in white blood cells (Science 200:1069-71, 1978),
2) transmission of CJD via the eye with infected cornea (New Engl. J. Med. 296: 1134-6, 1977),
3) experiments on maternal transmission (Proc. Soc. Exp. Biol. Med. 160: 233-6, 1979)
Guinea pigs occupy an interesting and ambiguous spot in the evolutionary tree closely related to the rodent-primate divergence. As such the prion sequence is of exceptional interest despite being a very long branch. Here, only a partial sequence is reported, starting 14 residues before the end of the signal peptide and ending very regretably shortly after 'codon 129." Immunological work had previously established in DWEDR that the guinea pig had ancestral tyrosine like primates instead of tryptophan in rodents.
Looking at the better parts of the sequence, which make considerably more sense, we see that most of the guinea pig substitutions come down on the side of rabbit and primate, rather than rodent. This gives a measure of support to those that have declared that 'the guinea pig is not a rodent.' It would be very helpful if the Eye Institute group would complete the sequence and validate their result.
1 ctggttcttt ttgtggccac atggagtgac actggcctct gcaagaagcg accaaaacca 61 ggaggagggt ggaacaccgg gggcagccgt tacccatccc acagcggggg gacctggggt 121 cagccccatg gaggtagctg gggccagcct catggtggtg gctggggcca gccccatggt 181 ggtagctggg gacagcctca tggtggtggc tggggccatg gaggtggctc gtacaatcag 241 tggaacaaac ccagtaagcc caaaaccaac atgaagcaca tggcgggggc tgcggcggcc 301 ggggcagtgg tggggggcct tggcggctac atgctgggaa gcgccatgag cag
Transfus Med 1999 Mar;9(1):3-14 Foster PR SNBTS Protein Fractionation Centre, Edinburgh, UK.Although there is no evidence that classical CJD (cCJD) can be transmitted by human blood or blood products in clinical practice, uncertainties surrounding new variant CJD (nvCJD) have led to the safety of plasma products derived from UK donors being questioned.
To better define whether or not there is a risk of nvCJD being transmitted it is necessary to determine how the causative agent would partition across the separations processes used in the preparation of plasma products. The abnormal prion protein which is associated with transmissible spongiform encephalopathies (TSEs), such as CJD, has a low solubility, a high tendency to form aggregates and adheres to surfaces readily. If the physicochemical properties of the agent of nvCJD are similar to those of abnormal prion protein then nvCJD may be removed by precipitation and adsorption technologies used in plasma fractionation.
Available data on the removal of TSE agents by such bioprocess technologies have been used to estimate the potential degree of reduction expected from each step in the plasma fractionation processes used by the SNBTS. The overall process reduction factors estimated are: 10(13) (albumin). 10(9) (immunoglobulins), 10(7) (factor IX, thrombin), 10(5) (fibrinogen), 10(4) (factor VIII) and 10(3) (factor II, IX and X); however, it will be necessary to establish the accuracy of these estimates by practical validation studies.