From the Department of Neurology, The Agnes Ginges
Center for Human Neurogenetics, Hadassah University Hospital, and
§ Department of Molecular Biology, Hebrew University Medical
School, Jerusalem 91120, Israel
Received for publication, August 28, 2000, and in revised form, January 2, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The scrapie isoform of the prion protein,
PrPSc, is the only identified component of the
infectious prion, an agent causing neurodegenerative diseases such as
Creutzfeldt-Jakob disease and bovine spongiform encephalopathy.
Following proteolysis, PrPSc is trimmed to a fragment
designated PrP 27-30. Both PrPSc and PrP 27-30 molecules
tend to aggregate and precipitate as amyloid rods when membranes from
prion-infected brain are extracted with detergents. Although prion rods
were also shown to contain lipids and sugar polymers, no
physiological role has yet been attributed to these molecules. In this
work, we show that prion infectivity can be reconstituted by combining
Me2SO-solubilized PrP 27-30, which at best
contained low prion infectivity, with nonprotein components of prion
rods (heavy fraction after deproteination, originating from a
scrapie-infected hamster brain), which did not present any
infectivity. Whereas heparanase digestion of the heavy fraction after
deproteination (originating from a scrapie-infected hamster brain),
before its combination with solubilized PrP 27-30, considerably
reduced the reconstitution of infectivity, preliminary results suggest
that infectivity can be greatly increased by combining nonaggregated
protease-resistant PrP with heparan sulfate, a known component of
amyloid plaques in the brain. We submit that whereas PrP 27-30 is
probably the obligatory template for the conversion of PrPC
to PrPSc, sulfated sugar polymers may play an important
role in the pathogenesis of prion diseases.
PrPSc, the abnormal isoform of
PrPC, is the only known component of the prion, an agent
causing fatal neurodegenerative disorders such as bovine spongiform
encephalopathy and Creutzfeldt-Jakob disease (1). It has been
postulated that prion diseases propagate by the conversion of
PrPC molecules into protease-resistant and insoluble
PrPSc molecules by a mechanism in which PrPSc
serves as a template (2). Whereas some PrPSc may be
insoluble in vivo (3), it is well documented that most PrPSc, as well as its protease-resistant core denominated
PrP 27-30, precipitate into insoluble aggregates (also known as prion
rods) when membranes from scrapie-infected brains are extracted with detergents such as sarkosyl (4). In addition to PrPSc,
prion aggregates were shown to contain nonprotein components, which
include sphingolipids as well as polysaccharides (5-7). The traces
of nucleic acids present in prion rods are believed to be too small to
function as coding tools (8). No physiological role has ever been
attributed to any nonprotein components of prion rods.
Disruption of prion rods into detergent protein lipid complexes
resulted in the retention of their protease resistance property concomitantly with an increase in their prion infectivity, suggesting that solubilized PrPSc is more infectious than the
aggregated prion protein (9). Contrarily, disruption of prion rods by
sonication and SDS resulted in a protease-sensitive PrP with
complete loss of infectivity (10).
As opposed to methods to disrupt prion aggregates, we have recently
introduced a new experimental procedure that results in the production
of nonaggregated PrPSc or PrP 27-30 molecules by
inhibition of the primary detergent-induced aggregation used for rod
formation (11). When membranes from brains of hamsters terminally ill
with scrapie were incubated in the presence of Me2SO in
addition to sarkosyl and subsequently applied to a sucrose density
gradient, the protease-resistant PrP molecules (PrP 27-30) were
divided between the light fractions, containing soluble or poorly
aggregated PrP 27-30 molecules, and the heaviest fractions, containing
insoluble and heavily aggregated PrP 27-30 molecules (11).
Interestingly, when light and heavy fractions of such gradients,
containing similar concentrations of protease-resistant PrP, were
inoculated into hamsters, the infectivity of the light fractions was
lower by more than 2 logs than the infectivity of the heavy
fractions. Light fractions produced in parallel in the absence of
Me2SO, which did not contain any detectable PrP, presented
the same low infectivity as the Me2SO light fractions,
suggesting that this residual infectivity was not due to the presence
of Me2SO-solubilized PrP 27-30 molecules. We attribute the
low infectivity present in both light fractions to the fact that the
brain extracts were applied to the sucrose gradient from the top, and
therefore some small prion aggregates, containing undetectable PrP
27-30, may not have sedimented.
In this work, we investigated whether molecules other than
protease-resistant PrP might have a physiological role in prion infectivity. To this effect, we combined the low infectious
protease-resistant Me2SO-solubilized PrP described
above with nonprotein components that remain in prion aggregates
subsequent to denaturation and harsh protease digestion
(NPHSc).1 In some
experiments we substituted the NPHSc fraction for heparan sulfate, a known component of prion rods. Our results show that the
addition of the deproteinized sedimented fraction (NPHSc) to low infectious solubilized protease-resistant PrP restores the prion
infectivity to its original values. We therefore propose that in
addition to PrPSc, prion infectivity may depend upon, or at
least be largely facilitated by, the presence of other components of
prion rods.
Sucrose Gradients--
Three hundred µl of 10, 15, 20, 25, 30, and 60% sucrose in phosphate-buffered saline were loaded into
centrifuge tubes adapted for the TLS-55 rotor of the TL 100 ultracentrifuge (Beckman Instruments) to form a zonal gradient. Normal
or scrapie brain membranes (25 µl containing 15 µg/ml protein) were
diluted with STE buffer (100 mM NaCl, 10 mM
Tris, pH 7.4, 1 mM EDTA) containing 2% sarkosyl to a final
volume of 240 µl. When appropriate, Me2SO (10%) was added to the brain extract and incubated for 16 h at 4 °C
before the extract was loaded on top of the gradient and
centrifuged in a TLS-55 tube ultracentrifuge (Beckman) at 55,000 rpm
(gav = 100,000) for 1 h at 20 °C.
Before immunoblotting with anti-PrP monoclonal antibody 3F4 (12),
gradient samples of equal volume were collected and digested with
proteinase K (PK, 40 µg/ml) for 60 min at 37 °C.
Sample Preparation--
The three top fractions of each gradient
were pooled and denominated fraction L (light). The three pooled bottom
fractions (H, heavy) were tested either directly or after a
deproteination treatment that included the following: 1) denaturation
with 4.5 M guanidium thiocyanate (GndSCN) (final
concentration) for 15 min, 2) precipitation with methanol to wash out
the GndSCN, 3) resuspension in 2% sarkosyl STE buffer before digestion
with PK (100 µg/ml for 60 min at 37 °C) to form NPH (non protein
heavy) fractions. To form the combined fractions, original samples (L, NPH, or HS (heparan sulfate)) were mixed at equal volumes and incubated
for 16 h at 4 °C. Original samples as well as combinations (detailed in Fig. 2) were assayed for the presence of PrP and infectivity. All volumes of original samples were adjusted before inoculation to contain the same concentration of L or NPH samples present in the mixtures.
Heparan Sulfate--
400 µl of the
L Heparanase Digestion--
400 µl of the NPHSc
sample were precipitated in methanol and resuspended in STE buffer
before the addition of 25 units/ml of heparinase III (Sigma) for
16 h at 37 °C. The resulting sample was denominated NPH*.
In Vivo Infectivity Experiments--
Five male Syrian hamsters,
4 weeks old, were inoculated intracerebrally with 50 µl of each of
the samples to be tested for prion infectivity. Animals were tested
daily. Prion titers were measured by monitoring the incubation period
until the appearance of symptoms (13). Before inoculation into
hamsters, samples containing only L or H fractions were supplemented
with 2% sarkosyl to retain similar concentrations of the components in
the infectivity assay.
Immunoblotting of Brain PrPSc--
10% (w/v) of
brain tissue from scrapie-infected hamsters (frozen at Statistical Analysis--
To compare the study groups, Anova and
the nonparametric Kruskal-Wallis test were applied. In addition,
multiple pairwise comparisons were performed using the Dunnett and
Scheffe methods. The tests were performed using the SPSS for
Windows computer program.
Brain membranes from scrapie-infected and uninfected hamsters were
extracted with sarkosyl in the presence and absence of Me2SO and, following an overnight incubation, applied to a
10-60% sucrose gradient as described (11). Gradient fractions were digested with PK and immunoblotted with
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80°c
following flash freezing in liquid nitrogen) was homogenized in cold
sucrose buffer (10 mM Tris, 0.3 M sucrose in
phosphate-buffered saline). 2% sarkosyl was added to 50-µl samples
before digestion with 40 µg/ml PK for 60 min at 37 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PrP monoclonal antibody 3F4
(Fig. 1a). As can be seen in
the figure, the light fraction prepared in the presence of
Me2SO contained a considerable fraction of the total PrP
27-30. To assure that the concentration of PrP 27-30 present in the
light fractions obtained with Me2SO was significantly higher than that present in the light fraction without
Me2SO, we immunoblotted with an anti-PrP antibody several
10-fold dilutions of the Me2SO scrapie light fraction. As
can be seen in Fig. 1b, the concentration of PrP 27-30 in
the Me2SO light fractions was at least 1000 times larger
than its concentration in the light fraction produced without
Me2SO.
View larger version (46K):
[in a new window]
Fig. 1.
Solubilization of PrP 27-30 by
Me2SO. a, membranes from scrapie-infected
brains extracted with 2% sarkosyl in the presence or absence of 10%
Me2SO or from normal brain extracted with sarkosyl only
were subjected to a sucrose density gradient (10-60%) as described
(11). Fractions collected from the gradients were digested in the
presence and absence of PK (40 µg/ml for 60 min at 37 °C) and
immunoblotted with monoclonal antibody 3F4. N,
normal; Sc, scrapie. b, light fractions produced in the
presence of Me2SO (lane 1) were diluted several
times (10-fold) with 2% sarkosyl. Lane 2, 10-fold dilution;
lane 3, 100-fold dilution; lane 4, 1000-fold
dilution; lane 5, light fraction produced in the absence of
Me2SO.
Fig. 2 presents the organization of the
reconstitution experiments and the samples used for infectivity assays.
The three first fractions (of 12), as well as the three last fractions
of each sucrose gradient, were pooled and denominated L and H
fractions, respectively. All gradient fractions were digested with 40 µg/ml PK at 37 °C for 60 min. Part of each H fraction was totally
denatured with 4.5 M GndSCN and, after methanol
precipitation, digested again with PK to produce the NPH fraction.
Subsequently, L and NPH fractions from different sources (normal and
scrapie-infected with and without Me2SO) were used to
create the combined samples specified in Fig. 2.
|
All samples to be evaluated for infectivity were precipitated by
methanol (to remove traces of Me2SO) and resuspended into inoculation buffer (1% bovine serum albumin in phosphate-buffered saline) to contain PrP 27-30 at comparable concentrations (Fig. 3a). No protease-resistant PrP
was detected in the NPHSc samples following the
denaturation/protease digestion treatment (Fig. 3a,
lane 2). PrP 27-30 was also absent from the light
samples produced in the absence of Me2SO, as well as in the
NPHSc/LSc sample (Fig. 3a,
lanes 4 and 8). When the bioassays were
completed, we also tested the brains of the animals inoculated with
each sample for the concentration and electrophoretic pattern of PrP after PK digestion (Fig. 3b). Although incubation times for
the different samples varied widely (see Table
I), the concentration of PrP 27-30, as
well as the banding pattern of the protein, was the same regardless of
the inocula administered to the hamsters. Histoblot analysis of all
brains were also identical (data not shown). This suggests that the
manipulations performed in this work did not produce a new prion
strain. Although it was repeatedly shown that different strains of
prions can be characterized by these parameters (14-18), end point
titration analyses are required to prove this point conclusively.
|
|
All samples described in Fig. 2 (original
and combined) were bioassayed for prion infectivity (Fig. 4 and Tables
I and II). Table I presents the
individual and accumulative results of three infectivity experiments
performed by intracerebellar inoculation of Syrian hamsters with the
samples described in Fig. 2. Table II presents the significance of the
variability among the treatment groups (p values). Although
p < 0.05 (*) is considered significant enough in this
kind of test, we also noted the extremely significant comparisons where
p was smaller than 0.001 (**). p values of 1 or
close to 1 suggest similarity between samples. The disease incubation
times for animals inoculated with similarly prepared samples in the
different experiments were pooled in the general calculations because
no statistically significant difference was found between them. We also
calculated the titers (log ID50) from the median of disease
incubation time as described (13). However, because the accuracy of
titers calculated from disease incubation times (in days) is a
debatable issue, we based all the statistical analyses directly on the
disease incubation times. A graphic representation of the results can
be seen in Fig. 4.
|
|
Whereas very high infectivity was present in the HSc fraction, no infectivity whatsoever was observed when this fraction was first denatured with 4.5 M GndSCN and then digested with PK, resulting in the NPHSc fraction. These results indicate that the NPHSc fraction cannot convert in vivo PrPC into PrPSc, because even after a long incubation time (more than 300 days), no animals inoculated with these samples present any disease symptoms. Moreover, no traces of PrP 27-30 were observed in their brains even after 300 days (Fig. 3a), suggesting that no subclinical infection was established in these animals. Preliminary experiments (data not shown) also suggest that NPHSc cannot convert PrPC to PrPSc in vitro, because the brain inoculation of combined fractions containing NPHSc and light fractions from normal hamsters without PK digestion (containing large quantities of PrPC) did not result in any disease symptoms or PrPSc accumulation after more than 300 days.
As shown here and in our previous work (11), whereas samples
HSc and
L
No increase in infectivity was detected when
L
One of the candidate molecules for a prion second component is HS. This sugar polymer was found in brain amyloid deposits of Alzheimer's disease as well as of prion diseases (19). In addition, sulfated sugars seem to have an important role in the metabolism of PrPSc (20). Molecules such as pentosan sulfate and low molecular weight heparin have been shown repeatedly to inhibit the production of PrPSc in scrapie-infected neuroblastoma cells (ScN2a cells). HS itself has been shown to either increase or reduce PrPSc accumulation in these cells, depending on the experimental setup (21-23). These molecules have also been shown to inhibit prion disease pathogenesis in vivo (24-26). This suggests that sulfated sugars of specific size and properties may either help form prions or disrupt prion formation, probably depending on some kind of competition mechanism.
To test whether one of the NPHSc components is an HS-like
molecule, we digested the NPHSc fraction with heparanase in
two of the reconstitution experiments, prior to its combination with the L
Extensive experiments are required to establish whether the effect of
HS is unique or whether other sugar polymers, sulfated or not, may
serve as the backbone of prion rods.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results presented here indicate that production of prion infectivity requires the presence both of protease-resistant PrP and of nonprotein components of prion rods and suggest that these components may well be sulfated sugar polymers. It remains to be established whether sulfated sugar polymers are indeed a fundamental component of prions or whether their function, although not essential, greatly facilitates prion propagation and the establishment of prion infection.
Unsuccessful attempts to dissociate and reconstitute prion infectivity were performed years ago, even before PrPSc was identified as a necessary components for infectivity (28). Prion infectivity could not yet be associated with protease-resistant PrP molecules produced by an array of in vitro conversion protocols (29-31) and has even been suggested to exist in the absence of detectable protease-resistant PrP in the inocula (32). In view of our results presented here, which suggest both prion components are required, we suggest that prion infectivity can be transmitted by a few (and therefore undetected) molecules of PrPSc, if associated with the appropriate nonprotein components. Our results also open the way to a new line of in vitro conversion experiments, which may hopefully result in full in vitro production of prion infectivity.
Whereas the function of PrPSc as a template in the PrPC to PrPSc conversion stands on solid grounds (33), the pathological role of a putative second component is unclear. A possible role for any functional molecule present in the NPHSc fractions may be to anchor the PrPSc molecules associated with it to the appropriate target in the host. Without a polymer such as HS, most PrPSc molecules may be cleared from the brain before the PrPC to PrPSc conversion reaction has been established in enough cells to establish a process of infection (34). The kind of sugar polymer used as rod backbone and anchor may play a role in modifying parameters of prion infectivity.
The fact that sulfated sugar polymers such as HS may have a crucial
function in prion structure and propagation suggests several plausible
explanations of the fact that small sulfated sugar polymers such as
pentosan sulfate were shown to inhibit the production of
PrPSc in cells (21, 22). These molecules may compete with
the sugar polymer functioning as prion component for the right cell
targets to which the template PrPSc molecules should be
docked. Otherwise, these molecules may compete with the prion sugar
component for the binding of newly formed PrPSc molecules.
Interestingly, polyamines were shown recently to inhibit PrPSc accumulation from ScN2a cells (35). We suggest that
these highly positively charged polymers may bind to the highly
negatively charged sulfated sugars and thereby facilitate the clearance
of newly formed and still nonaggregated protease-resistant PrP. The results presented here therefore provide an explanation of the fact
that such disparate molecules as small sulfated sugar polymers and
positively charged polymers may both prove effective in the treatment
of prion diseases.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Professor Haim Ovadia for very fruitful discussions.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a grant from the Israel Center for the Study of Emerging Diseases.
Supported by grants from the European Community and by
the Agnes Ginges Center for Human Neurogenetics. To whom
correspondence should be addressed: Dept. of Neurology, Hadassah
University Hospital, Jerusalem, Israel 91120. Fax: 972-2-6429441;
E-mail: gabizonr@hadassah.org.il.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M007815200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NPH, nonprotein
heavy;
NPHSc, NPH fraction originating from a
scrapie-infected hamster brain;
PK, proteinase K;
L, light;
H, heavy;
GndSCN, guanidium thiocyanate;
HSc, heavy fraction
originating from a scrapie-infected hamster brain;
NPHN, heavy fraction after deproteination, originating from a normal hamster
brain;
LSc, light fraction from a scrapie-infected hamster
brain;
L
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Prusiner, S. B. (1998) Brain Pathol. 8, 499-513[Medline] [Order article via Infotrieve] |
2. | Cohen, F. E., and Prusiner, S. B. (1998) Annu. Rev. Biochem. 67, 793-819[CrossRef][Medline] [Order article via Infotrieve] |
3. | Jeffrey, M., Goodsir, C. M., Bruce, M. E., McBride, P. A., Scott, J. R., and Halliday, W. G. (1992) Neurosci. Lett. 147, 106-109[CrossRef][Medline] [Order article via Infotrieve] |
4. | Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A., and Prusiner, S. B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2310-2314[Abstract] |
5. | Stahl, N., Baldwin, M. A., Teplow, D. B., Hood, L., Gibson, B. W., Burlingame, A. L., and Prusiner, S. B. (1993) Biochemistry 32, 1991-2002[Medline] [Order article via Infotrieve] |
6. | Appel, T. R., Dumpitak, C., Matthiesen, U., and Riesner, D. (1999) Biol. Chem. 380, 1295-1306[Medline] [Order article via Infotrieve] |
7. | Klein, T. R., Kirsch, D., Kaufmann, R., and Riesner, D. (1998) Biol. Chem. 379, 655-666[Medline] [Order article via Infotrieve] |
8. | Kellings, K., Meyer, N., Mirenda, C., Prusiner, S. B., and Riesner, D. (1992) J. Gen. Virol. 73, 1025-1029[Abstract] |
9. | Gabizon, R., McKinley, M. P., and Prusiner, S. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4017-4021[Abstract] |
10. | Riesner, D., Kellings, K., Post, K., Wille, H., Serban, H., Groth, D., Baldwin, M. A., and Prusiner, S. B. (1996) J. Virol. 70, 1714-1722[Abstract] |
11. |
Shaked, G. M.,
Fridlander, G.,
Meiner, Z.,
Taraboulos, A.,
and Gabizon, R.
(1999)
J. Biol. Chem.
274,
17981-17986 |
12. | Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna DeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M., and Diringer, H. (1987) J. Virol. 61, 3688-3693[Medline] [Order article via Infotrieve] |
13. | Prusiner, S. B., Groth, D. F., Cochran, S. P., Masiarz, F. R., McKinley, M. P., and Martinez, H. M. (1980) Biochemistry 19, 4883-4891[Medline] [Order article via Infotrieve] |
14. | Kascsak, R. J., Rubenstein, R., Merz, P. A., Carp, R. I., Wisniewski, H. M., and Diringer, H. (1985) J. Gen. Virol. 66, 1715-1722[Abstract] |
15. | Bessen, R. A., and Marsh, R. F. (1994) J. Virol. 68, 7859-7868[Abstract] |
16. | Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C., Gowland, I., Collinge, J., Doey, L. J., and Lantos, P. (1997) Nature 389, 448-450[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Telling, G. C.,
Parchi, P.,
DeArmond, S. J.,
Cortelli, P.,
Montaga, P.,
Gabizon, R.,
Mastrianni, J.,
Lugaresi, E.,
Gambetti, P.,
and Prusiner, S. B.
(1996)
Science
274,
2079-2082 |
18. | DeArmond, S. J., Yang, S. L., Lee, A., Bowler, R., Taraboulos, A., Groth, D., and Prusiner, S. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6449-6453[Abstract] |
19. | Snow, A. D., Mar, H., Nochlin, D., Kimata, K., Kato, M., Suzuki, S., Hassell, J., and Wight, T. N. (1988) Am. J. Pathol. 133, 456-463[Abstract] |
20. |
Shyng, S. L.,
Lehmann, S.,
Moulder, K. L.,
and Harris, D. A.
(1995)
J. Biol. Chem.
270,
30221-30229 |
21. | Caughey, B., and Raymond, G. J. (1993) J. Virol. 67, 643-650[Abstract] |
22. | Gabizon, R., Meiner, Z., Halimi, M., and Ben Sasson, S. A. (1993) J. Cell. Physiol. 157, 319-325[Medline] [Order article via Infotrieve] |
23. | Caughey, B., Brown, K., Raymond, G. J., Katzenstein, G. E., and Thresher, W. (1994) J. Virol. 68, 2135-2141[Abstract] |
24. | Ehlers, B., and Diringer, H. (1984) J. Gen. Virol. 65, 1325-1330[Abstract] |
25. | Farquhar, C. F., and Dickinson, A. G. (1986) J. Gen. Virol. 67, 463-473[Abstract] |
26. | Ladogana, A., Casaccia, P., Ingrosso, L., Cibati, M., Salvatore, M., Xi, Y. G., Masullo, C., and Pocchiari, M. (1992) J. Gen. Virol. 73, 661-665[Abstract] |
27. | Kimberlin, R. H., and Walker, C. A. (1986) J. Gen. Virol. 67, 255-263[Abstract] |
28. | Michel, B., Tamalet, J., Bongrand, P., Gambarelli, D., and Gastaut, J. L. (1987) Rev. Neurol. (Paris) 143, 526-531[Medline] [Order article via Infotrieve] |
29. | Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T., and Caughey, B. (1994) Nature 370, 471-474[CrossRef][Medline] [Order article via Infotrieve] |
30. | Hill, A. F., Antoniou, M., and Collinge, J. (1999) J. Gen. Virol. 80, 11-14[Abstract] |
31. | Kaneko, K., Peretz, D., Pan, K. M., Blochberger, T. C., Wille, H., Gabizon, R., Griffith, O. H., Cohen, F. E., Baldwin, M. A., and Prusiner, S. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11160-11164[Abstract] |
32. |
Lasmezas, C. I.,
Deslys, J. P.,
Robain, O.,
Jaegly, A.,
Beringue, V.,
Peyrin, J. M.,
Fournier, J. G.,
Hauw, J. J.,
Rossier, J.,
and Dormont, D.
(1997)
Science
275,
402-405 |
33. | Pan, K. M., Baldwin, M., Nguyen, J., Gasset, M., Serban, A., Groth, D., Mehlhorn, I., Huang, Z., Fletterick, R. J., Cohen, F. E., et al.. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10962-10966[Abstract] |
34. | Safar, J., Wille, H., Itri, V., Groth, D., Serban, H., Torchia, M., Cohen, F. E., and Prusiner, S. B. (1998) Nat. Med. 4, 1157-1165[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Supattapone, S.,
Nguyen, H. O.,
Cohen, F. E.,
Prusiner, S. B.,
and Scott, M. R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14529-14534 |