HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: ps.041000905v1 14/4/942    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tahiri-Alaoui, A. Articles by James, W. Search for Related Content PubMed PubMed Citation Articles by Tahiri-Alaoui, A. Articles by James, W. Social Bookmarking                   What's this?

Rapid formation of amyloid from -monomeric recombinant human PrP in vitro

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom

Reprint requests to: William James, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK; e-mail: william.james{at}path.ox.ac.uk; fax: (+44) 1865 285756.

(RECEIVED July 20, 2004; FINAL REVISION October 20, 2004; ACCEPTED December 3, 2004)

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
The infectious agent of prion diseases is identified with PrP^Sc, a -rich, amyloidogenic and partially protease resistant isoform of the cellular glycoprotein, PrP^C. To understand the process of prion formation in vivo, we and others have studied defined misfolding pathways of recombinant PrP in vitro. The low-level infectivity of the in vitro misfolded murine PrP amyloid has recently been reported. Here we analyze the in vitro kinetics of amyloid formation from recombinant human PrP^90–231 in vitro in the context of two common allelic forms of PrP found in human populations that are associated with differences in prion disease susceptibility and pathological phenotype. We show that human PrP amyloid forms readily from its PrP^C-like state in vitro, that the lag time of the reaction can be further shortened by the presence of a "seed" of pre-formed PrP amyloid, and that amyloid propagation is more complex than a simple crystallization process. We further show that the kinetics of amyloid formation do not differ between the Met129 and Val129 allelomorphs of human PrP, and that amyloid from each functions as an equally effective seed in heterologous, as in homologous amyloid reactions. The results could illuminate the process of amyloid formation in vivo as well as help understanding prion pathogenesis.

Keywords: amyloid; prion; human PrP; polymorphism 129; CJD; misfolding

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041000905.

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
At the heart of prion diseases lies a poorly understood conformational conversion of an abundant, GPI-anchored glycoprotein, PrP, into abnormal forms (Prusiner 1996). These include PrP^Sc, which is commonly found in amyloid plaques at the site of neurodegeneration, and which appears to comprise the infectious agent. PrP^Sc is characterized by its affinity for amyloid-staining dyes, insolubility, relative resistance to digestion by proteinase K, high -sheet content, and a fibrillar appearance when examined under electron microscopy (Prusiner et al. 1983). However, there is clearly a great variety of disease-associated conformations of PrP, even within forms operationally classified as PrP^Sc. Different combinations of infectious strain and host genotype produce biochemically distinguishable variants of PrP, with different kinetics and with different anatomical distribution (Wadsworth et al. 1999). No detailed structure of any of these abnormal forms is yet available. Recently, however, analysis of 2D crystals of PrP27–30 and PrP^Sc106 by electron crystallography led to low-resolution projection maps (Wille et al. 2002; Govaerts et al. 2004), providing evidence for assembly of prions with left-handed -helices into trimers. In order to gain greater mechanistic insight into this pathophysiological complexity, we have isolated conformationally selective aptamer ligands for PrP (Rhie et al. 2003) and examined conformational switching exhibited by the interaction of peptides derived from the PrP polypeptide (Tahiri-Alaoui et al. 2003). As a complementary approach, we have studied the in vitro refolding of recombinant human PrP^90–231 into abnormal forms (Tahiri-Alaoui et al. 2004). Previous studies of this sort had identified three major routes of folding denatured, disulphide-oxidized PrP in vitro (see Fig. 1; Baskakov et al. 2001, 2002).

View larger version (19K): [in this window] [in a new window]   Figure 1. In vitro folding pathways of PrP discussed in the text. Three distinct routes of folding of recombinant, disulphide oxidized PrP have been discussed in the literature (Baskakov et al. 2001, 2002): (pathway 1) from the denatured state to -monomeric (physiological) state; (pathway 2) from denatured to a PK-resistant oligomeric state (maturing to a -sheet form) (Tahiri-Alaoui et al. 2004); (pathway 3) from the denatured state to a multimeric aggregate, and hence to an amyloid form. The present work describes experiments that commence with PrP in the -monomeric state (pathway 4).

The common polymorphism at residue 129 of the human PRNP gene modulates disease susceptibility and the pathological phenotypes in human transmissible spongiform encephalopathies (Collinge et al. 1991; Brown et al. 1994). The molecular mechanism by which the effect of this mutation is mediated remains unclear, and so we have been investigating the dynamics of misfolding of the two allelomorphs under in vitro conditions. It has already been shown that the folding, dynamics, and stability of the physiological, -helix-rich form of recombinant PrP are not affected by this polymorphism nor by other substitutions related to inherited human prion disease (Liemann and Glockshuber 1999; Hosszu et al. 2004). In contrast, we have recently shown that the misfolding pathway leading to the formation of -sheet-rich soluble oligomers from the fully denatured, unfolded state ("pathway 2" in Fig. 1) was favored by the presence of methionine, compared with valine, at this site (Tahiri-Alaoui et al. 2004). Data from short model peptides that contained either methionine or valine at position 129, suggested that the predisposition of homozygote individuals to sporadic and iatrogenic Creutzfeldt-Jakob Disease (CJD) could be explained by a nucleation-dependent polymerization mechanism during amyloid formation (Come and Lansbury 1994). Here we have used the recombinant human PrP^90–231 to examine the fourth potential pathway, which leads to PrP amyloid from the physiological, -monomeric state ("pathway 4" in Fig. 1), and investigate the effect of polymorphism at residue 129 on its kinetics.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Characterization of -monomeric forms of recHuPrP allelomorphs
The recHuPrP Met129 and Val129 were submitted to rapid refolding through size-exclusion high-performance liquid chromatography (HPLC) to ensure that the starting protein was monomeric and in -helical conformation. As demonstrated from the elution profile, both allelomorphs eluted as monomers (Fig. 2) and had CD spectra characteristic of -helical conformation (inset in Fig. 2) as indicated by the two minima at 208 and 222 nm. An undetectable amount of -oligomer can concomitantly be formed during the refolding of recPrP into the -helical monomer (Baskakov et al. 2004). To ensure the maximum conformational homogeneity of the -monomeric form used in these experiments we took specific precautions to avoid undesirable accumulation of -oligomers, such as keeping the pH above 5.0 and using -monomeric that has been freshly purified through size-exclusion HPLC under low (1 M) urea concentration (Tahiri-Alaoui et al. 2004).

View larger version (16K): [in this window] [in a new window]   Figure 2. Characterization of monomeric -helical human PrP90–231 Met128 and Val129 variants. Recombinant human PrP90–231 was expressed with either methionine or valine at position 129 in E. coli. Protein was fully oxidized and folded into its physiological, -monomeric form by rapid refolding using size-exclusion HPLC. Met 129 and Val129 PrP allelomorphs were eluted as monomeric peak with a retention time of 9.0 min in 50 mM sodium acetate pH 5.5, 150 mM sodium chloride, 1 M urea, and 0.02% azide. The -helical conformation for both PrP variants was assessed by circular dichroism (inset), the CD spectra were recorded at a protein concentration of 50 µM and 56 µM for Met 129 and Val129, respectively.

Spontaneous amyloid formation of human PrP from its -monomeric state

View larger version (21K): [in this window] [in a new window]   Figure 3. Spontaneous amyloid formation of human PrP90–231 Met128 and Val129 variants from the -monomeric state. Amyloid formation was initiated by dilution to a final concentration of 35 µM protein in PBS pH7.2 containing 3M urea, 1 M guanidine hydrochloride, and 0.02% azide. Samples were continuously agitated at 37°C, and samples were periodically withdrawn for assay of amyloid by thioflavin T fluorescence. (A) Met129 and Val129 allelomorphs of PrP studied individually at 35 µM. (B) A 1:1 mixture of Met129 and Val129 allelomorphs of PrP at a final total concentration of 35 µM. In A and B, the solid lines were drawn by nonlinear, least-square fits of the data using equation 1.

View this table: [in this window] [in a new window]   Table 1. Calculated kinetic parameters for the amyloid formation from -monomeric state of human PrP90–231

View larger version (152K): [in this window] [in a new window]   Figure 4. Transmission electron microscopy of negatively stained fibrils derived from recombinant human PrP90–231 allelomorphs. (A,B) Representative micrographs of Met129 and Val129 variants, respectively. The negative staining was performed with 1% uranyl acetate solution on fibrils derived from unseeded spontaneous amyloid reactions.

Substantial efforts have been devoted to identifying intermediate species that might be involved as precursors in the generation of PrP^Sc using recPrPs (Hornemann and Glockshuber 1998; Wildegger et al. 1999; Baskakov et al. 2001; Apetri and Surewicz 2002). Recently, and in order to find out whether fibril formation occurs through unfolding of -monomeric recPrP, Baskakov et al. examined the effect of increasing urea concentration on the lag phase of amyloid formation (Baskakov et al. 2004). The authors observed that the unfolding transition occurred between 1.4 M and 3.4 M urea, where both the native and the denatured states were abundant, and that the midpoint of the unfolding transition was close to 2.4 M urea, in which the kinetics of amyloid formation displayed the shortest lag phase (Baskakov et al. 2004). This led the authors to suggest that the conversion is favored by the presence of low abundant, partially unfolded intermediates present under mild denaturing conditions. Our data support this suggestion in that less than complete unfolding of the -monomer is required for conversion to the primary amyloidogenic form. This is supported by the observation that an extended lag phase is seen when amyloid formation is initiated from mature oligomeric state (Tahiri-Alaoui et al. 2004; I. Baskakov, W. James, and A. Tahiri-Alaoui, in prep.), which is known to be thermodynamically more stable than -monomeric recPrP (Baskakov et al. 2001). This may be a reflection, again, of the relative ease with which -monomeric forms can transition to the amyloid state, making the process relatively insensitive to the local, native conformational subtleties that might be conferred by the polymorphism at position 129.

Seeded amyloid formation
The lag phase preceding amyloid formation is considered to reflect the low probability of multimolecular PrP interactions that result in the formation of a nucleation site suitable for the propagation of amyloid fibrils. Like crystallization, a hallmark of this process is the capacity of fiber formation to be bypassed by providing exogenous seed from a previously conducted reaction (Miranker 2004). Consequently, admixture of a small amount of pre-formed amyloid is expected to provide suitable nucleation sites and eliminate the lag phase. Accordingly, we added various amounts of unsonicated "seed" of amyloid, derived from the procedure described above, to a solution of pure, -monomeric PrP under otherwise identical conditions to the spontaneous reaction (see Fig. 5; Table 1). As anticipated, this resulted in a reduction in the lag time for amyloid formation (P < 5%). However, the lag phase was not entirely eliminated, suggesting either that pre-formed amyloid does not provide a perfect nucleation site or that the nucleation model does not perfectly represent the reaction.

View larger version (18K): [in this window] [in a new window]   Figure 5. Amyloid formation of human PrP90–231 Met128 and Val129 variants from the -monomeric state seeded with pre-formed amyloid. Recombinant, -monomeric human PrP was prepared as described, and diluted to a final concentration of 35 µM in PBS pH7.2 containing 0.01%, 0.1%, or 1% mature amyloid, 3 M urea, 1 M guanidine hydrochloride, and 0.02% azide. Samples were incubated at 37°C with agitation and studied for amyloid formation as before. (A) Methionine 129 variant homologous seeding, in which the amyloid seed was of the same allelomorph as the bulk -monomeric protein. (B) Valine 129 variant homologous seeding, in which the amyloid seed was of the same allelomorph as the bulk -monomeric protein. (C) Heterologous seeding, in which the amyloid seed was from the alternative allelomorph. (D) Plot of the lag time as a function of the amount of seeds. In A,B,C, the solid lines were drawn by nonlinear, least-square fits of the data using equation 1.

The effect of seeding on the kinetic parameters of amyloid formation was seen in both the Met129 and Val129 variants, and the heterologous amyloid was as good as the homologous amyloid in promoting amyloid formation. Our seeding results are consistent with the observations made by Come and Lansbury (1994), who used short peptide models and showed that the seeding was insensitive to the polymorphism at codon 129.

Significantly, it has been reported recently that amyloid generated from recombinant mouse PrP ("pathway 3") caused a transmissible form of prion disease when inoculated to transgenic mice (Baskakov et al. 2004). This result is a major milestone in that it seems to confirm the "protein-only" hypothesis in mammalian prion disease (Prusiner 1991). It is not known whether amyloid generated from the -monomeric form of human PrP, as studied here, is also infectious, but if it is, our findings reveal a more efficient, as well as physiologically more plausible, route of prion formation in vivo.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Protein expression and purification
Genomic DNA encoding methionine or valine at codon 129 of the PRNP gene was extracted from blood of a heterozygote individual using the standard phenol-chloroform method. The fragment of PRNP spanning codon 90 through 231 was amplified and cloned as described before (Tahiri-Alaoui et al. 2004). Escherichia coli expression and purification of recombinant human PrP was performed as previously described (Tahiri-Alaoui et al. 2004). Stocks of highly purified proteins were kept in 6 M guanidine hydrochloride, 50 mM Tris-HCl pH 7.2.

Folding of PrP into an -monomeric form in vitro
Rapid refolding of proteins into -monomeric form was carried out by size-exclusion HPLC as described before (Tahiri-Alaoui et al. 2004) with the following modifications. Proteins, in 100-µL volume at 5 mg/mL from an unfolded state in 6 M guanidine hydrochloride, 50 mM Tris-HCl pH 7.2, were injected onto TSK-Gel SWXL G3000 HPLC column, 7.8 x 300 mm (Phenomenex), equilibrated in 50 mM sodium acetate pH 5.5, 150 mM sodium chloride, 1 M urea, and 0.02% azide. The peak corresponding to monomeric proteins was manually collected and the folding of the proteins was assessed by circular dichroism. CD spectra were recorded with a Jasco-720 spectrometer scanning at 50 nm/sec, with a band width of 1.0 nm and a resolution of 1 nm using a 1-mm cuvette. Four individual scans were averaged, and the buffer spectra were subtracted.

Formation of amyloid fibrils in vitro
Time course study of amyloid formation of recombinant human PrP^90–231 was carried out as previously described (Baskakov et al. 2002) by monitoring Thioflavin T (ThT) fluorescence. Briefly, proteins in the -monomeric fold were diluted to a final concentration of 35 µM in phosphate-buffered saline pH 7.2 containing 3 M urea, 0.02% azide, and a final concentration of 1 M guanidine hydrochloride. Samples were incubated at 37°C under continuous agitation in 1.5-mL tubes. To mimic the situation in heterozygote individuals, methionine 129 and valine 129 variants were mixed at a 1:1 molar ratio. The final protein concentration was kept at 35 µM. In the homologous and heterologous seeding experiments, an aliquot of pre-formed fibrils was taken from the stationary phase and considered to contain 100% seeds. Various amounts of unsonicated seed (0.01%, 0.1%, and 1%) were used to inoculate a freshly prepared amyloid reaction.

Kinetic data analyses
The kinetics of amyloid formation were analyzed by fitting time-dependent changes in ThT fluorescence of samples to the following sigmoidal equation using nonlinear, least-squares analysis (GraphPad Prism version 4.01).

(1)

where F_ThT is the fluorescence intensity of ThT, A is the ThT fluorescence intensity in the posttransition plateau, t_i is the inflection point, i.e., the midpoint of the transition region, B (h^–1) is the amyloid growth rate constant, and t is the time in hours. The lag time (t_lag) of the amyloid formation was calculated by extrapolation of the linear region of the sigmoidal transition phase of ThT fluorescence to the abscissa intercept (Wall et al. 1999).

Negative staining and transmission electron microscopy
Aliquots of 3 µL were taken from samples containing amyloid fibrils as judged from ThT fluorescence, loaded onto carbon-coated, glow-discharged 400-mesh copper grids, blotted, negatively stained with 1% uranyl acetate, air dried, and then viewed in a Zeiss (formerly Leo) Omega 912 electron microscope equipped with an in-column charge-coupled device camera (2048 x 2048 pixels) from Proscan (Tahiri-Alaoui et al. 2003).

	Acknowledgments

 
We thank Petra Disterer for helping with the protein expression and purification and Michael Shaw for helping with the transmission electron microscopie. This work was supported by grant 43/ BSD17731from the Biotechnology and Biological Sciences Research Council (BBSRC) to W.J., with A.T.-A. as the recognized researcher.

	References

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Apetri, A.C. and Surewicz, W.K. 2002. Kinetic intermediate in the folding of human prion protein. J. Biol. Chem. 277: 44589–44592.[Abstract/Free Full Text]

Baskakov, I.V., Legname, G., Prusiner, S.B., and Cohen, F.E. 2001. Folding of prion protein to its native -helical conformation is under kinetic control. J. Biol. Chem. 276: 19687–19690.[Abstract/Free Full Text]

Baskakov, I.V., Legname, G., Baldwin, M.A., Prusiner, S.B., and Cohen, F.E. 2002. Pathway complexity of prion protein assembly into amyloid. J. Biol. Chem. 277: 21140–21148.[Abstract/Free Full Text]

Baskakov, I.V., Legname, G., Gryczynski, Z., and Prusiner, S.B. 2004. The peculiar nature of unfolding of the human prion protein. Protein Sci. 13: 586–595.[Abstract/Free Full Text]

Brown, P., Cervenakova, L., Goldfarb, L.G., McCombie, W.R., Rubenstein, R., Will, R.G., Pocchiari, M., Martinez-Lage, J.F., Scalici, C., Masullo, C., et al. 1994. Iatrogenic Creutzfeldt-Jakob disease: An example of the interplay between ancient genes and modern medicine. Neurology 44: 291–293.[Abstract/Free Full Text]

Collinge, J., Palmer, M.S., and Dryden, A.J. 1991. Genetic predisposition to iatrogenic Creutzfeldt-Jakob disease. Lancet 337: 1441–1442.[CrossRef][Medline]

Come, J.H. and Lansbury, J.P.T. 1994. Predisposition of prion protein homozygotes to Creutzfeld-Jacob disease can be explained by a nucleation-dependent polymerization mechanism. J. Am. Chem. Soc. 116: 4109–4110.[CrossRef]

Govaerts, C., Wille, H., Prusiner, S.B., and Cohen, F.E. 2004. Evidence for assembly of prions with left-handed -helices into trimers. Proc. Natl. Acad. Sci. 101: 8342–8347.[Abstract/Free Full Text]

Hornemann, S. and Glockshuber, R. 1998. A scrapie-like unfolding intermediate of the prion protein domain PrP(121–231) induced by acidic pH. Proc. Natl. Acad. Sci. 95: 6010–6014.[Abstract/Free Full Text]

Hosszu, L.L., Jackson, G.S., Trevitt, C.R., Jones, S., Batchelor, M., Bhelt, D., Prodromidou, K., Clarke, A.R., Waltho, J.P., and Collinge, J. 2004. The residue 129 polymorphism in human prion protein does not confer susceptibility to Creutzfeldt-Jakob Disease by altering the structure or global stability of PrPC. J. Biol. Chem. 279: 28515–28521.[Abstract/Free Full Text]

Larson, J.L. and Miranker, A.D. 2004. The mechanism of insulin action on islet amyloid polypeptide fiber formation. J. Mol. Biol. 335: 221–231.[CrossRef][Medline]

Liemann, S. and Glockshuber, R. 1999. Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein. Biochemistry 38: 3258–3267.[CrossRef][Medline]

Miranker, A.D. 2004. Unzipping the mysteries of amyloid fiber formation. Proc. Natl. Acad. Sci. 101: 4335–4336.[Free Full Text]

Padrick, S.B. and Miranker, A.D. 2002. Islet amyloid: Phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis. Biochemistry 41: 4694–4703.[CrossRef][Medline]

Prusiner, S.B. 1991. Molecular biology of prion diseases. Science 252: 1515–1522.[Abstract/Free Full Text]

———. 1996. Molecular biology and pathogenesis of prion diseases. Trends Biochem. Sci. 21: 482–487.[CrossRef][Medline]

Prusiner, S.B., McKinley, M.P., Bowman, K.A., Bolton, D.C., Bendheim, P.E., Groth, D.F., and Glenner, G.G. 1983. Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35: 349–358.[CrossRef][Medline]

Rhie, A., Kirby, L., Sayer, N., Wellesley, R., Disterer, P., Sylvester, I., Gill, A., Hope, J., James, W., and Tahiri-Alaoui, A. 2003. Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion. J. Biol. Chem. 278: 39697–39705.[Abstract/Free Full Text]

Tahiri-Alaoui, A., Bouchard, M., Zurdo, J., and James, W. 2003. Competing intrachain interactions regulate the formation of -sheet fibrils in bovine PrP peptides. Protein Sci. 12: 600–608.[Abstract/Free Full Text]

Tahiri-Alaoui, A., Gill, A.C., Disterer, P., and James, W. 2004. Methionine 129 variant of human prion protein oligomerizes more rapidly than the valine 129 variant: Implications for disease susceptibility to Creutzfeldt-Jakob disease. J. Biol. Chem. 279: 31390–31397.[Abstract/Free Full Text]

Wadsworth, J.D., Hill, A.F., Joiner, S., Jackson, G.S., Clarke, A.R., and Collinge, J. 1999. Strain-specific prion-protein conformation determined by metal ions. Nat. Cell Biol. 1: 55–59.[CrossRef][Medline]

Wall, J., Schell, M., Murphy, C., Hrncic, R., Stevens, F.J., and Solomon, A. 1999. Thermodynamic instability of human 6 light chains: Correlation with fibrillogenicity. Biochemistry 38: 14101–14108.[CrossRef][Medline]

Wildegger, G., Liemann, S., and Glockshuber, R. 1999. Extremely rapid folding of the C-terminal domain of the prion protein without kinetic intermediates. Nat. Struct. Biol. 6: 550–553.[CrossRef][Medline]

Wille, H., Michelitsch, M.D., Guenebaut, V., Supattapone, S., Serban, A., Cohen, F.E., Agard, D.A., and Prusiner, S.B. 2002. Structural studies of the scrapie prion protein by electron crystallography. Proc. Natl. Acad. Sci. 99: 3563–3568.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				R. Gerber, A. Tahiri-Alaoui, P.J. Hore, and W. James Conformational pH dependence of intermediate states during oligomerization of the human prion protein Protein Sci., March 1, 2008; 17(3): 537 - 544. [Abstract] [Full Text] [PDF]

				R. Gerber, A. Tahiri-Alaoui, P. J. Hore, and W. James Oligomerization of the Human Prion Protein Proceeds via a Molten Globule Intermediate J. Biol. Chem., March 2, 2007; 282(9): 6300 - 6307. [Abstract] [Full Text] [PDF]

				A. Tahiri-Alaoui, V. L. Sim, B. Caughey, and W. James Molecular Heterosis of Prion Protein beta-Oligomers: A POTENTIAL MECHANISM OF HUMAN RESISTANCE TO DISEASE J. Biol. Chem., November 10, 2006; 281(45): 34171 - 34178. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: ps.041000905v1 14/4/942    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tahiri-Alaoui, A. Articles by James, W. Search for Related Content PubMed PubMed Citation Articles by Tahiri-Alaoui, A. Articles by James, W. Social Bookmarking                   What's this?