Does protein aging apply to prions?
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Detection of scrapie at 4 months via tonsil biopsy
No altered aspartyl residues in scrapie
Alterations in Potassium Currents May Trigger Neurodegeneration
Number of extra repeats determines the type of cerebellar deposits
Microbiological hazards related to xenotransplantation of porcine organs into man.
CJD In Austria and Europe
Can loss-of-function prion-related diseases exist?
Extra repeat mutations in tau
New prion antibodies
E219K never occurs in sporadic CJD
Phosmet induces up-regulation of surface levels of the cellular prion protein

Maternal transmission of BSE: offspring of BSE-affected pedigree suckler cows.

Vet Rec 1998 May 23;142(21):579-580
Donnelly CA

CJD after blood product transfusion from a donor with CJD.

Neurology 1998 Jun;50(6):1872-1873
Patry D, Curry B, Easton D, Mastrianni JA, Hogan DB
We report a second case of an association between an albumin transfusion and Creutzfeldt-Jakob disease. On balance, we believe our case represents a chance and not a causal relation.

Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie.

Vet Rec 1998 May 23;142(21):564-568
Schreuder BE, van Keulen LJ, Vromans ME, Langeveld JP, Smits
Preliminary findings have indicated that in naturally infected sheep, fully susceptible to scrapie (VRQ-homozygous), PrPSc can be detected in the tonsils approximately one year before the expected onset of clinical disease, whereas no immunostaining can be detected in animals with a semi-resistant genotype. This paper describes the technique for taking tonsillar biopsies from sheep and gives the results of the completed experiment. In another experiment PrPSc was detected even earlier in comparable VRQ-homozygous sheep born and raised in different surroundings. At three-and-a-half months of age no PrPSc could be detected in three homozygous susceptible sheep (VRQ/VRQ), but PrPSc was detected at four months in one similar sheep.

At eight months of age all seven sampled VRQ/VRQ sheep showed positive immunostaining in the biopsies, but none of the biopsies from three VRQ/ARQ heterozygotes showed any immunostaining; they were positive when sampled at 14 to 15 months of age. Biopsies from VRQ/ARR sheep were negative throughout this period. On the basis of the established or expected incubation period, PrPSc could thus be detected in the tonsils of live susceptible animals at between one-third and a half of the incubation period, more than one-and-a-half years before clinical signs normally appear in both these genotypes Determination of the frequency and distribution of vascular and parenchymal amyloid with polyclonal and N-terminal-specific PrP antibodies in scrapie-affected sheep and mice.

Vet Rec 1998 May 16;142(20):534-537
 Jeffrey M, Goodsir CM, Holliman A, Higgins RJ, Bruce ME, McBride PA, Fraser JR
Brains from 17 histopathologically confirmed cases of scrapie, five of which had congophilic vascular amyloid, were stained immunohistochemically for prion protein (PrP) using a polyclonal antibody. Two clinically suspect but pathologically unconfirmed cases of natural sheep scrapie and the brains of four mice infected with the 111A murine scrapie strain were also examined. Selected sections containing amyloid were stained with each of two peptide antibodies which recognise the N-terminal amino acid residues which are lost following protease digestion of the disease-specific isoform of PrP.

The mice infected with the 111A murine scrapie strain had large numbers of hypermature plaques. All the amyloid plaques from both natural sheep scrapie brains and experimental murine brains were heavily immunostained by the polyclonal and both peptide antibodies. In addition, disease-specific accumulations of PrP were detected in endothelial cells or in the intima of blood vessels of the cerebral cortex of sheep scrapie brains. The affected blood vessels were located in areas which otherwise lacked typical scrapie pathology.

Vascular accumulations of PrP were also found in leptomeningeal and choroid plexus blood vessels. Vascular amyloid was found mainly in the neocortex. Vascular amyloid and disease-specific parenchymal accumulations of PrP were found in two sheep which showed clinical signs of scrapie but lacked its typical vacuolar pathology.

These results show that the mature amyloid of scrapie is composed of, or contains a substantial proportion of, whole length PrP protein. Thus truncation of PrP is not essential for the aggregation of PrP into amyloid. The vascular amyloid of natural sheep scrapie originates from the accumulation and release of PrP from endothelial cells presumably following systemic scrapie infection.

The topography of vascular amyloid distribution in Great Britain differs from that reported in the Netherlands. As amyloid deposition in mice is largely controlled by the strain of the infecting agent it is possible that the strain of the agent may influence vascular amyloid deposition.

The use of transgenic mice in the investigation of transmissible spongiform encephalopathies.

Rev Sci Tech 1998 Apr;17(1):278-290
Weissmann C, Fischer M, Raeber A, Bueler H, Sailer A, Shmerling D, Rulicke T, 
Brandner S, Aguzzi A
....The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.

Measurement of Altered Aspartyl Residues in the Scrapie Associated Form of Prion Protein.

Biochem Biophys Res Commun 1998 May 29;246(3):606-608
Weber DJ, McFadden PN, Caughey
In transmissible spongiform encephalopathies (TSE), the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to a pathologic form (PrP-res) that has greatly enhanced proteinase K resistance, insolubility, and beta sheet content. To investigate the possibility that alterations at aspartyl or asparaginyl residues in the form of d-aspartate and/or l-isoaspartate could play a role in either the formation or stabilization of PrP-res in TSE-infected animals, we assayed for the presence of these abnormal residues in PrP-res.

Protein d-aspartyl/l-isoaspartyl carboxyl methyltransferase (PIMT) was used to methylate and radiolabel altered aspartyl residues, which were detected in PrP-res, but at low levels (0.5 mole%). The scarcity of d-aspartyl and/or l-isoaspartyl groups in PrP-res suggests that this modification is unlikely to be primarily responsible for the differences between PrP-res and PrP-sen. However, it remains possible that such modifications in substoichiometric numbers of PrP molecules could help to initiate the PrP-res formation or stabilize PrP-res polymers in vivo.

Supplemental information -- webmaster: There are 5 aspartates in mature prion protein, only one, D178N, is involved in CJD:

Medline snippets: L-Asparaginyl and L-aspartyl residues in proteins are subject to spontaneous degradation reactions that generate isomerized and racemized aspartyl derivatives. Proteins containing L-isoaspartyl and D-aspartyl residues can have altered structures and diminished biological activity. These residues are recognized by a highly conserved cytosolic enzyme, the protein L-isoaspartate(D-aspartate) O-methyltransferase (EC The enzymatic methyl esterification of these abnormal residues in vitro can lead to their conversion (i.e., repair) to normal L-aspartyl residues and should therefore prevent the accumulation of potentially dysfunctional proteins in vivo as cells and tissues age. Particularly high levels of the repair methyltransferase are present in the brain, although enyzme activity is present in all vertebrate tissues.

Methyltransferase knockout mice revealed a striking accumulation of protein substrates for this enzyme in the cytosolic fraction of brain, heart, liver, and erythrocytes. The knockout mice showed significant growth retardation and succumbed to fatal seizures at an average of 42 days after birth. These results suggest that the ability of mice to repair L-isoaspartyl- and D-aspartyl-containing proteins is essential for normal growth and for normal central nervous system function.

The experiment here is thus somewhat unmotivated since there is efficient repair. One wonders, why not look at the other aging reactions such as N and Q deamination, methionine and cysteine sulphoxides, nitrotyrosine, and so on. Why not resolve the arginine issue, which are known to be modified?

Alterations in Potassium Currents May Trigger Neurodegeneration in Murine Scrapie.

Exp Neurol. 1998 Jun; 151(2): 326-333
Johnston AR, Fraser JR, Jeffrey M, MacLeod N
Conventional electrophysiological intracellular recording techniques were used to test the hypothesis that enhanced calcium entry via voltage-gated calcium channels or the N-methyl-d-aspartate (NMDA) subtype of glutamate receptor-channel complex may be a primary pathological mechanism triggering neurodegeneration in scrapie and related diseases. This study was carried out at a time when cell loss is known to occur and when hippocampal pyramidal cells in area CA1 are rendered hyperexcitable following scrapie infection.

There was no change to the NMDA receptor-mediated component of the Schaffer collateral evoked excitatory postsynaptic potential (EPSP) or the level of spontaneous firing activity of CA1 cells following addition of the specific NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV, 20 muM), to the perfusate in scrapie-infected mice, indicating that the NMDA receptor-channel complex is not compromised by scrapie. There was also no change seen in the non-NMDA mediated component of the EPSP. The calcium spike of CA1 pyramidal cells was not significantly altered by scrapie infection, indicating that high threshold voltage-gated Ca2+ channel function is not compromised by scrapie.

By contrast, cells from scrapie-infected mice fired calcium spikes repetitively and the long, slow AHP, which in control cells inhibited repetitive firing, was absent. Cells from scrapie-infected mice showed more depolarized membrane potentials than controls but this difference in potential was no longer observed after exposure to TEA. These data indicate a loss of TEA-insensitive and TEA-sensitive potassium conductances. We suggest that altered potassium currents rather than increased calcium entry via voltage-sensitive calcium channels or the NMDA receptor complex may be the primary pathological mechanism triggering neurodegeneration in scrapie and related diseases.

Prion encephalopathy with insertion of octapeptide repeats: the number of repeats determines the type of cerebellar deposits.

Neuropathol Appl Neurobiol 1998 Apr;24(2):125-130
Vital C, Gray F, Vital A, Parchi P,... Jarnier D, Julien J, Gambetti P
We studied modifications of the molecular layer of the cerebellum in three patients with octapeptide repeat insertion (OPRI). Two brothers carrying a six-OPRI showed only spongiosis in haematoxylin & eosin preparations (H&E), whereas immunocytochemical examination (ICC) with an antiprion protein (PrP) antibody revealed numerous elongated PrP deposits. The third patient from a family with an eight-OPRI had numerous plaques visible in H&E preparations and had been diagnosed as Gerstmann-Straussler-Scheinker syndrome.

So far, 15 other cases from seven families and three individual cases with OPRI have undergone neuropathological examination. Characteristic PrP deposits were seen in six other cases, two isolated cases with a four- and a seven-OPRI, whereas four cases with a six-OPRI came from three different families.

Such deposits have never been reported in other cases of prion encephalopathy, without OPRI.

Genuine plaques were observed in five out of the 15 other patients. Interestingly, four had an eight-OPRI and one a nine-OPRI. Cases with OPRI are prone to develop different PrP deposits: those only visible on ICC are not to be confused with genuine plaques visible in H&E preparations. Elongated PrP deposits are present in cases with a four- to seven-OPRI, whereas plaques are present when there is an eight- or a nine-OPRI. All these cases should be termed prion encephalopathy with OPRI.

Microbiological hazards related to xenotransplantation of porcine organs into man.

Infect Control Hosp Epidemiol 1998 May;19(5):355-365
Borie DC, Cramer DV, Phan-Thanh L, Vaillant JC, Bequet JL, Makowka L, Hannoun L
Pigs are emerging as the most likely providers of genetically engineered organs and cells for the purpose of clinical xenotransplantation. Introduction of clinical trials has been delayed primarily by uncertainties regarding the risk of swine pathogen transmission that could harm the recipient. The concern that xenotransplantation carries the potential for a new epidemic has been highlighted by recent experiences with both bovine spongiform encephalopathy and human immunodeficiency diseases. As clinical trials have been postponed ... Both current diagnostic tools and those under development are described, along with breeding strategies to provide donor animals that would not put the recipient or the general population at risk.

CJD In Austria

Wien Med Wochenschr 1998;148(4):101-106 .[Article in German] 
Radbauer C, Hainfellner JA, Jellinger K, Pilz P, Maier H,  Kleinert R, Budka H
]Between 1969 and 1996, transmissible spongiform encephalopathy was definitely diagnosed by autopsy and/or biopsy in 98 Austrian patients. The yearly incidence increased significantly in past years (1996: 1.41 cases per million inhabitants). This increase likely results from increased awareness in the medical community and effectuation of the diagnostic autopsy. The new variant of Creutzfeldt-Jakob disease (CJD), probably transmitted from bovine spongiform encephalopathy (BSE), has not occurred in Austria.

The percentage of patients older than 70 years increased until 1989 and declined slightly thereafter. One patient received a dura mater graft 11 years before death. Another patient had familial CJD with a glutamate-lysine mutation on codon 200 of the prion protein (PrP) gene PRNP. One more patient died from Gerstmann-Straussler-Scheinker disease (GSS), three patients from fatal familial insomnia (FFI). Another patient received intramuscular injections of a purified RNA preparation (Regeneresen) produced from various organs including brain.

The age at death symmetrically distributes around a median of 64 years. Two CJD patients were unusually young (27 and 30 years). Most patients (72.7%) died within 6 months of disease. Retrospectively, 81% of patients had clinical diagnostic criteria of probable or possible CJD (52% probably and 29% possible). In 19%, clinical criteria for CJD were not fully met. There is no case clustering with specific professional groups or geographic areas. However, residents of Vienna, or Vienna and Lower Austria, respectively, had CJD diagnosed twice or three times more frequently than the rest of the country, indicating regionally differing qualities of case retrieval.

Descriptive epidemiology of Creutzfeldt-Jakob disease in six European countries, 1993-1995

Ann Neurol 1998 Jun;43(6):763-767
Will RG, Alperovitch A, ... Zerr I, van Duijn C
After the occurrence of bovine spongiform encephalopathy (BSE), there has been concern that transmission of BSE to the human population might result in a change in the epidemiological characteristics of Creutzfeldt-Jakob disease (CJD). A collaborative study of CJD in the European Union was performed from 1993 to 1995, to compare data from national registries for CJD in France, Germany, Italy, The Netherlands, Slovakia, and the United Kingdom.

Five hundred seventy-five patients with definite or probable CJD died in the study period with an overall annual mortality rate of 0.71 cases per million. The incidence rates for CJD were similar in all participating countries despite variations in postmortem rates, and age-specific incidence rates were also relatively consistent, with the exception of an increased incidence of CJD in patients younger than 39 years of age in the United Kingdom.

In relation to etiological subtypes of CJD, 87% of cases were sporadic, 8% genetic, and 5% iatrogenic. Genetic forms of CJD comprised 80% of all cases in Slovakia, and iatrogenic forms of CJD occurred most frequently in France and the United Kingdom.

The statistical data reported here do not provide evidence of a causal link between BSE and CJD in Europe as a whole. However, the study has established baseline epidemiological parameters for CJD in participating European countries, which may be important in the assessment of any future change in the characteristics of CJD as a result of the epidemic of BSE.

Can loss-of-function prion-related diseases exist?

Molecular Psychiatry May 1998, Volume 3, Issue 3 196
HB Samaia and RR Brentani
A good question. However, a little tricky to prove if you don't know what normal function was to begin with. Here is a title and inadequate abstract. Recall also a recent article, Cell 1998 Apr 17;93(2):203-214 by Shmerling D et all suggesting del 32-121 or del 32-134 as candidates of this type. The above authors reported N171S as associated with schizophrenia; however, a second family with this allele was asymptomatic.

If the argument is absence of typical plaques, absence of immunoreactivity, absence of transmissibility, absence of this and that, then the case is as only as strong as the method is sensitive. For example, if wild type is dominant to mutant in heterozygotes, nothing is learned from absence of transmission to wild type background.

Abstract: Discovery of mutations of the PrnP gene without typical plaque formation and the characterization of two prion receptors led us to postulate a new class of prion-related disease: 'loss of function'.

Extra repeat mutations in tau

Lancet Volume 351, Number 911813 June 1998
Hannah Wunsch
In the Annals of Neurology, a US team report a point mutation of valine to methionine in the coding region of tau in a family with FTDP-17 (1998; 43: 815-25), whereas another paper to be published soon reports on a G to A transition in an intron of tau in another FTDP-17 family ( Proc Natl Acad Sci USA 1998; 95: 7737-41). In the study to be reported in Nature, a mixture of exon and intron tau mutations were found in ten different families (1998; 393: 702-05). None of these tau mutations were in any controls. A large proportion of the mutations seem to be at the exon-10 splice site. This mutation results in more frequent alternative splicing at exon 10 and a higher proportion of tau protein molecules that have four rather than three binding-domain repeats.

In many, but not all, FTDP-17 families, the neurofibrillary tangles consist of tau with four repeats, prompting speculation that a larger amount of protein with four repeats could be a cause of the disease. "Before this work, we couldn't tell if tangles were an end product or an active participant in the disease", explains Gerard Schellenberg (University of Washington, Seattle, WA, USA), an author of the Annals article. "We now know that if we alter the tau structure, we will get neurons to die." So, Tau mutations may also be involved in other dementia disorders. But the scientists are cautious of proposing any link between tau mutations and Alzheimer's disease because this disorder is characterised not only by tangles but also by brain amyloid plaques not seen in FTDP-17.

Various serine residues in the repeats may be phosphorylated by camp kinase.

FT   REPEAT      251    281       TAU/MAP MOTIF.
FT   REPEAT      282    312       TAU/MAP MOTIF.
FT   REPEAT      313    343       TAU/MAP MOTIF.
FT   REPEAT      344    375       TAU/MAP MOTIF.
FT   VARSPLIC    175    192       MISSING (IN FORM 2).
SQ   SEQUENCE   448 AA;  46332 MW;  078D38B2 CRC32;

Note moderate resemblance to mammalian prion repeat PHGGGWGQ PHGGGWGQ:

Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17

 Nature 393, 698 (1998)  M Hutton, et al.
Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), historically termed Pick's disease. Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics. Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q21.11; the tau gene also lies within this region. The authors have now sequenced tau in FTDP- 17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon 10.

The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10. This causes more frequent usage of the 5' splice site and an increased proportion of tau transcripts that include exon 10. The increase in exon 10+ messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17.

Monoclonal antibody F89/160.1.5 defines a conserved epitope on the ruminant prion protein.

J Clin Microbiol 1998 Jun;36(6):1750-1755 
O'Rourke KI, Baszler TV, Miller JM, Spraker TR, Sadler-Riggleman I, Knowles DP
The transmissible spongiform encephalopathies are a heterogeneous group of fatal neurodegenerative disorders occurring in humans, mink, cats, and ruminant herbivores. The occurrence of novel transmissible spongiform encephalopathies in cattle in the United Kingdom and Europe and in mule deer and elk in parts of the United States has emphasized the need for reliable diagnostic tests with standardized reagents.

Postmortem diagnosis is performed by histologic examination of brain sections from affected animals. The histopathological criteria for transmissible spongiform encephalopathies include gliosis, astrocytosis, neuronal degeneration, and spongiform change. These lesions vary in intensity and anatomic location depending on the host species and genetics, stage of disease, and infectious agent source. Diagnosis by histopathology alone may be ambiguous in hosts with early cases of disease and impossible if the tissue is autolyzed.

Deposition of the prion protein (an abnormal isoform of a native cellular sialoglycoprotein) in the central nervous system is a reliable marker for infection, and immunohistochemical detection of this marker is a useful adjunct to histopathology. In the present paper we describe monoclonal antibody (MAb) F89/160.1.5, which reacts with prion protein in tissues from sheep, cattle, mule deer, and elk with naturally occurring transmissible spongiform encephalopathies. This MAb recognizes a conserved epitope on the prion protein in formalin-fixed, paraffin-embedded sections after hydrated autoclaving. MAb F89/160.1.5 will be useful in diagnostic and pathogenesis studies of the transmissible spongiform encephalopathies in these ruminant species.

An antibody raised against a conserved sequence of the prion protein recognizes pathological isoforms in CJD and BSE.

Am J Pathol 1998 Jun;152(6):1415-1420 
Piccardo P, Langeveld JP, Hill AF, Dlouhy SR, Young K, Giaccone G, Rossi G, Bugiani M, Bugiani O, Meloen
RH, Collinge J, Tagliavini F, Ghetti B
Antibodies to the prion protein (PrP) have been critical to the neuropathological and biochemical characterization of PrP-related degenerative diseases in humans and animals. Although PrP is highly conserved evolutionarily, there is some sequence divergence among species; as a consequence, anti-PrP antibodies have a wide spectrum of reactivity (from strong immunopositivity to lack of reactivity) when challenged with PrP from diverse species.

We have produced an antibody (anti-PrP95-108) raised against a synthetic peptide corresponding to residues 95 to 108 of human PrP and have characterized it by epitope mapping, Western immunoblot analysis, and immunohistochemistry. The antibody recognizes not only human PrP isoforms but also pathological PrP from all species tested (ie, cattle, sheep, hamsters, and mice). This is probably due to the fac that the epitope recognized by this antibody includes residues 100 to 108 of human PrP, a sequence that is also present in PrP of several other species. Thus, this reagent is valuable not only for the study of human prion diseases but also for analysis of the possible relationship between human and animal disorders.

The distribution of scrapie-associated fibrils in neural and non-neural tissues of advanced clinical cases of natural scrapie in sheep.

Res Vet Sci 1998 Mar;64(2):141-146
 Stack MJ, Chaplin MJ, Aldrich  AM, Davis LA
The distribution of scrapie-associated fibrils (SAFs) throughout four brain regions, the pituitary gland, along the whole length of the spinal cord and in the sciatic nerve was assessed in 10 sheep terminally affected by scrapie and in four control sheep. Tonsils, retropharyngeal, broncho-mediastinal and mesenteric lymph nodes, the distal ileum, proximal colon and spleen were also examined for fibrils in all 14 sheep. Fibrils were detected in all four brain regions and throughout the length of the spinal cord in nine of the scrapie affected sheep. SAFs were not detectable in any of the sciatic nerve samples tested. In one of the 10 clinically affected sheep only minimal lesions were found by histopathology and fibrils were detected only from the cerebrum and one spinal cord region (taken at the C1 C2 vertebrae). Fibrils were not detected in the tonsils or retropharyngeal lymph nodes but were detected in other non-neural tissues of some of the scrapie-affected sheep. These tissues included pituitary gland, broncho-mediastinal and mesenteric portal lymph nodes, distal ileum, proximal colon and spleen. Fibrils could not be detected in any of the tissues taken from the four control sheep.

Codon 219 Lys allele of PRNP is not found in sporadic Creutzfeldt-Jakob disease

 Ann Neurol 1998 Jun;43(6):826-828
Shibuya S, Higuchi J, Shin 
RW, Tateishi J, Kitamoto T
The polymorphism at codon 219 of the prion protein gene (PRNP) was found in the general Japanese population with 6% allele frequency. Herein, we examined 85 cases of sporadic Creutzfeldt-Jakob disease (CJD) for the codon 219 polymorphism. The codon 219Glu/Lys heterozygous polymorphism was not found in these CJD cases. In addition, we examined 43 patients with dementia of non-CJD origin, and 4 were found to have the codon 219Glu/Lys heterozygous polymorphism with a similar allele frequency as in the general population. Thus, the codon 219Glu/Lys heterozygous polymorphism might be uniquely excluded from sporadic CJD.

Phosmet induces up-regulation of surface levels of the cellular prion protein.

Neuroreport 1998 May 11;9(7):1391-1395
Gordon I, Abdulla EM, Campbell IC, Whatley SA
Chronic (2 day) exposure of human neuroblastoma cells to the organophosphate pesticide phosmet induced a marked concentration-dependent increase in the levels of PrP present on the cell surface as assessed by biotin labelling and immunoprecipitation. Levels of both phospholipase C (PIPLC)-releasable and non-releasable forms of PrP were increased on the plasma membrane. These increases appear to be due to post-transcriptional mechanisms, since PrP mRNA levels as assessed by Northern blotting were unaffected by phosmet treatment. These data raise the possibility that phosmet exposure could increase the susceptibility to the prion agent by altering the levels of accessible PrP.

Comment (webmaster):

This is consistent with the idea that normal prion over-production, from whatever mechanism, is not a good idea expecially in a feedback setting. However, it is going to be very difficult to establish the historical in vivo relevence, if any, of individual boosters as there could be a considerable number.

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