1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9–10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Helicobacter pylori-induced gastritis is typically associated with a strong infiltration of the infected stomach mucosa by neutrophils and mononuclear inflammatory cells (Warren and Marshall, 1983; Marshall et al., 1985; Bayerdorffer et al., 1992; Fiocca et al., 1992). There is a good correlation between mucosal damage and neutrophil infiltration (Warren and Marshall, 1983; Davies et al., 1992; Fiocca et al., 1994). Several studies have shown the presence of protein component(s) in H. pylori water extracts that attract and activate neutrophils and other inflammatory cells (Karttunen et al., 1990; Craig et al., 1992; Mai et al., 1992; Nielsen and Andersen, 1992a; b; Kozolet al., 1993; Reymunde et al., 1993; Evans et al., 1995a; Marchetti et al., 1995). Indeed, H. pylori strains capable of neutrophil activation were found more frequently in patients affected by peptic ulcer disease than in those with active chronic gastritis only (Rautelin et al., 1993), although recent data contradict these findings (Hansen et al., 1999).

Yoshida et al. (1993) identified a protein in water extracts of H. pylori capable of promoting neutrophil adhesion to endothelial cells. This protein was purified as a 150 kDa decamer composed of identical 15 kDa subunits. It was termed HP-NAP because it induces adhesion of neutrophils to endothelial cells and production of reactive oxygen radicals, detected by nitro-benzo-tetrazolium reduction (Evans et al., 1995a) or with the quantitative and sensitive assay based on the peroxidase-mediated oxidation of homovanillic acid (Menegazzi et al., 1991; B. Satin, unpublished).

Evans et al. (1995b) reported a considerable variation in the level of neutrophil adhesion promoting activity among different H. pylori strains that suggests a variable level of expression of the protein, similar to that found for VacA (Cover et al., 1997). HP-NAP binds to mucin, a highly glycosylated protein that is the main component of the mucous layer (Namavar et al., 1998) and to neutrophil glycosphingolipids (Teneberg et al., 1997). Very recently, we found that HP-NAP is a major H. pylori antigen in the human immune response to this bacterium (our unpublished observations), thus making HP-NAP a strong candidate for a component of an anti-H. pylori vaccine. Such findings prompted us to characterize the structural and functional properties of this poorly studied molecule.

Amino acid sequence comparison has indicated that HP-NAP belongs to the family of DNA-protecting proteins (Dps) (accession no. PDOC00645 of the PROSITE databank), which has significant structural similarities with the ferritin protein family (accession no. PDOC00181). The crystal structure of Escherichia coli Dps was recently determined (Grant et al., 1998). It consists of a four-helix bundle similar to that of ferritins. However, E. coli Dps possess a short helix in the middle of the BC loop and lack the E helix present at the C-terminus of the ferritin monomer. Moreover, ferritin forms a icositetramer (24-mer) with a 432 symmetry and two pores of different types: one at the threefold axis and one at the fourfold axis, connecting an 8 nm diameter cavity where iron is stored (Harrison and Arosio, 1996). The E helix plays a major role in mediating the fourfold interactions in ferritins. In contrast, Dps forms a dodecamer with 32 symmetry with a central hole of 4.5 nm in diameter connected to the surface via two pores around the threefold axis, one of which is similar to the threefold pore of ferritins whereas the other is different. In agreement with the Dps crystallographic structure, electron microscopy and molecular weight determinations reveal that proteins of the Dps family form multimers of a smaller size than ferritins (Almiron et al., 1992; Chen and Helmann, 1995; Bozzi et al., 1997). To date, three proteins of the Dps family have been shown to bind DNA with no apparent sequence specificity, a process that is believed to protect nucleotides from oxidative damage (Almiron et al., 1992; Chen and Helmann, 1995; Evans et al., 1995b; Martinez and Kolter, 1997).

On the basis of molecular sieving chromatography, it was suggested that HP-NAP forms decamers (Evans et al., 1995a), but no structural and biochemical characterization has been carried out to date. Likewise, no data on the DNA- or iron-binding characteristics of HP-NAP have been described. Here, we report on the biochemical and structural properties of this important protein and suggest a molecular model of HP-NAP as well as a possible role in H. pylori physiology.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

A structural model for HP-NAP

A comparison of the available amino acid sequences of HP-NAP from different strains clearly shows their high similarity (Fig. 1A), suggesting a defined and important role of this protein in H. pylori growth and/or survival. Figure 1A also highlights homologous sequences present in databases, suggesting, in agreement with a previous study (Evans et al., 1995b), that HP-NAP is similar to bacterioferritins (Bfr) and to Dps proteins. Secondary structure prediction methods indicate that HP-NAP and Dps possess α-helical segments of similar length (Fig. 1B), in contrast to the predicted secondary structure of bacterioferritin. Both Bfr and Dps have a high-order quaternary structure: their four-helix bundle monomers oligomerize to form 24-mer and dodecamers respectively (Harrison and Arosio, 1996; Grant et al., 1998). The computer-assisted molecular replacement of the amino acid sequence of HP-NAP into the three-dimensional (3D) co-ordinates of Bfr and Dps provided a better fit in the Dps crystallographic structure, and in particular revealed the presence of the BC helix (yellow in Fig. 2) connecting helix B with helix C. The presence of the BC helix is not predicted by secondary structure prediction methods, but becomes evident upon molecular fitting of the HP-NAP sequence into the Dps structure. This is a strong indication that the structure of HP-NAP is closely similar, if not identical, to the model depicted in Fig. 2. Figure 2A shows the HP-NAP monomer and Fig. 2B depicts the structure of the putative dodecamer with one monomer in colour; the figure also highlights those residues putatively involved in iron binding, with their lateral chains pointing into the internal cavity of the oligomer.


Figure 1. . Primary and secondary structure similarities of HP-NAP isoforms. A. A selection of proteins obtained by blast (basic local alignment search tool at NCBI) search, against HP-NAP, with a score > 48. The gene encoding for HP-NAP has been sequenced from four different strains and (1) (2) (3) and (4) refer to the sequences reported in the following studies respectively: the present study; Evans et al. (1995a); Tomb et al. (1997); and Alm et al. (1999). TYF1-TRPE is antigen TYF 1 of Treponema pallidum (Noordhoek et al., 1989); FRI-LISIN is a non-haem iron-binding ferritin in Listeria innocua (Bozzi et al., 1997); MRGA-BACS is a metalloregulation DNA-binding stress protein of Bacillus subtilis (Chen and Helmann, 1995); SP-UK is an unknown protein of Streptococcus pneumoniae, GenBank accession no. AAC33862; RP-UK is an unknown protein of Rickettsia prowazekii (Andersson et al., 1998); napA-BP is the neutrophil-activating protein of Borrelia burgdorferi (Fraser et al., 1997); LR-SLP is a surface protein of Lactobacillus rhamnosus, GenBank accession no. AAB88605; YLT2-ANAVA is a low-temperature-induced protein of Anabaema variabilis, SWISSPROT accession number P29712; Dps is the DNA-binding protein from starved cells of Escherichia coli (Almiron et al., 1992); DpsA-SYNP is the DpsA protein of Synechococcus (Pen~a and Bullerjahn, 1995). The consensus sequence is marked by dots representing similar amino acids and asterisks representing identical amino acids. B. Secondary structure comparison between E. coli Dps and HP-NAP. The 2D structure of Dps was obtained from PDB (Protein Data Bank: entry 1dps). The secondary structure of HP-NAP was predicted according to the method of Garnier et al. (1978).

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Figure 2. . Three-dimensional model of the HP-NAP monomer and oligomer. A. Model of HP-NAP monomer obtained by substitution of the amino acid sequence of HP-NAP into the 3D co-ordinates of E. coli Dps (PDB entry 1dps) performed with the SWISS-model server (Peitsch et al., 1995). The carboxylate lateral chains of residues Glu-42, Glu-46, Asp-49, Asp-53 and Glu-56 are highlighted as they are putatively involved in iron binding. B. Quaternary structure of Dps showing the position of one monomer in colour within the dodecamer with the lateral chains of iron-binding residues pointing into the central cavity. Figures were generated using the program insight ii.

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Circular dichroism and fluorescence spectra of HP-NAP

To further test the structural model described above, sizeable amounts of pure proteins, which cannot be obtained directly from cultures of H. pylori, were required. Hence, HP-NAP was produced in recombinant form from Bacillus subtilis. The protein was obtained free from any other protein contaminants and was subjected to circular dichroic (CD) and fluorescence spectra determination. The heat and guanidinium-induced denaturation curves were determined, and electron microscopy was performed after negative staining.

Figure 3A shows the far UV CD spectrum of HP-NAP, which is typical of a protein with a very high α-helical content. Application of the method of Yang et al. (1986) led to an estimate of about 90%, a value that is in perfect agreement with the model of Fig. 2, which predicts an α-helical content of 88%. The near UV CD spectrum (Fig. 3B), considered to be a fingerprint of the 3D structure of the molecule, is very similar to the spectrum of Dps-related ferritin of Listeria innocua, which forms dodecamers in solution (Bozzi et al., 1997; Ilari et al., 1999). The large negative peak between 260 and 290 nm and its shoulders at 264 and 270 nm are attributable to the 10 phenylalanine residues present in the molecule. The shoulder at 290 nm and the negative peak at 292–308 nm can be ascribed to the 0–0 cm−1 1Lb and 1La transitions of tryptophan residues, whereas the shoulder at 275 nm is attributable to tyrosines. The fluorescence spectrum (Fig. 3C) shows an emission maximum at 333 nm, indicating that the tryptophan residues are exposed to the solvent.


Figure 3. . Far UV and near UV circular dichroism spectra and fluorescence emission spectrum of HP-NAP. A. Far ultraviolet spectrum of 0.1 mg ml−1 HP-NAP in 20 mM tris-HCl, pH 7.4, 50 mM NaCl. B. Near ultraviolet spectrum of 0.5 mg ml−1 HP-NAP in 20 mM tris-HCl, pH 7.4, 50 mM NaCl. C. Fluorescence emission spectrum of 0.02 mg ml−1 HP-NAP in 20 mM tris-HCl, pH 7.4, 50 mM NaCl.

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Denaturation curves of HP-NAP

Four-helix bundle folds are characterized by very high stability to both thermal denaturation and denaturing agents. This is also the case for HP-NAP, whose ellipticity content does not vary significantly at temperatures up to 80°C (not shown). Moreover, as much as 4 M guanidinium chloride is necessary to decrease the α-helical content of the molecule (Fig. 4). These properties are in agreement with HP-NAP being a four-helix bundle protein forming large oligomers. These properties are particularly relevant with respect to the design of simple procedures of purification of large amounts of HP-NAP.


Figure 4. . Guanidinium chloride-induced denaturation of HP-NAP. The process of protein unfolding was followed by monitoring the decrease of fluorescent emission intensity at 330 nm (excitation at 280 nm) as a function of increasing guanidinium concentration. Fluorescence data are reported as the ratio F/F0, where F0 is the intensity of fluorescence at 0 M guanidinium chloride.

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Electron microscopy of HP-NAP and ferritins

The dimensions of the oligomers formed by ferritins and protein of the Dps family are such that they can be studied at low resolution by electron microscopy. Table 1 summarizes the dimensions of these proteins as obtained by electron microscopy. Electron micrographs of negatively stained samples of both HP-NAP and horse ferritin, used here for direct comparison (Fig. 5), clearly show that HP-NAP forms dodecamers and not 24-mers. The average diameter of the HP-NAP rings was estimated to be 9.5 nm. Again, this provides direct evidence in favour of the model of Fig. 2A and suggests that HP-NAP is similar to Dps. From these data, one could further extrapolate that HP-NAP may have a biological activity similar to that of Dps.

Table 1. . Dimensional characteristics of ferritins and Dps-like proteins. a. Hudson et al. (1993).b.Bozzi et al. (1997).c.Chen and Helmann (1995).d.Almiron et al. (1992).Thumbnail image of

Figure 5. . Electron microscopy analysis of negatively stained HP-NAP. (insert) For multimer dimension comparison, HP-NAP was mixed with horse spleen ferritin (indicated by the arrow).

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HP-NAP binds iron but not DNA

The DNA-binding activity of HP-NAP was assayed according to Almiron et al. (1992) using both uncut and HindIII-digested H. pylori plasmid DNA followed by electrophoresis through a 1% Tris-acetate agarose gel. No change in DNA migration was observed in the presence of HP-NAP, indicating a lack of interaction between the protein and the DNA (data not shown).

The iron content of the recombinant protein obtained by expression in B. subtilis was estimated to be 0.2–0.3 atoms per monomer of HP-NAP by atomic adsorption. In contrast, the iron uptake of apo-HP-NAP in vitro, shown in Fig. 6, was very efficient, similar to that of other ferritins (Frolow et al., 1994; Harrison and Arosio, 1996; Bozzi et al., 1997). Iron uptake can be followed by UV absorption at 310 nm. The progression curve is hyperbolic at low iron to protein molar ratios and changes to a sigmoidal shape when larger amounts of iron are added to the apoprotein. The maximum number of iron atoms bound by apo-HP-NAP was estimated to be up to 40 per monomer, corresponding to about 500 atoms per dodecamer. When higher iron quantities were added, a brown precipitate formed, indicating saturation of the iron-binding capacity of the protein. This saturation point was similar to that of the ferritin from L. innocua (see Table 1).


Figure 6. . Progress curve of iron uptake by apo-HP-NAP. Ferrous ammonium sulphate 10 mM (a, 20 μl; b, 5 μl) was added to 1 ml of apo-HP-NAP 0.1 mg ml−1 in 20 mM MOPS, pH 7.0, and to a protein-free control (dashed lines). The formation of an iron core was monitored spectroscopically at 310 nm.

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Isoelectrofocusing of both the apo-HP-NAP and the iron-loaded HP-NAP gave the same single band at pI 6.75. The band of Fe-HP-NAP was visible without staining of the gel as a brown line. The neutrophil-activating activity of apo-HP-NAP and of the iron-saturated HP-NAP are identical (data not shown), suggesting that iron binding does not induce such structural changes resulting in different interactions with the neutrophil cell surface.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In this study, we have combined theoretical and experimental approaches to obtain novel information on the biochemical and structural properties of the H. pylori neutrophil-activating protein, which appears to play an important role in the pathogenesis of H. pylori-associated gastritis. This molecule is a major antigen in the human immune response to H. pylori infection and a strong candidate as one of the components of a multicomponent recombinant anti-H. pylori vaccine. Such findings clearly called for a definition of the properties of this molecule, which after the cloning of its gene (Evans et al., 1995a) was only studied with respect to its binding to mucin and to lipids (Teneberg et al., 1997; Namavar et al., 1998). Here, we provide evidence that the secondary, tertiary and quaternary structure of HP-NAP is closely similar to that of Dps. However, HP-NAP clearly is functionally different from Dps because it does not appear to interact with DNA as Dps does. At present, it is not known whether Dps binds iron or whether iron binding modulates its DNA interaction. HP-NAP binds up to 500 atoms of iron, but the presence of iron does not affect its neutrophil-activating activity.

HP-NAP and the ferritin of L. innocua would appear to belong to a new class of ferritins composed of 12, rather than 24, monomers capable of accumulating 500 atoms of iron per oligomer. The existence of this new protein class of small ferritins could be evidence for a common evolutionary origin between DNA-binding proteins and iron-accumulating protein (Pen~a and Bullerjahn, 1995).

Both HP-NAP and the L. innocua ferritin lack the ferroxidase centre conserved in eukaryotic H-chain ferritins and bacterial 24-mer ferritins. Ferroxidase activity of L. innocua ferritin was theoretically attributed to a cluster of carboxylate residues that in mammalian ferritin L chains are conserved and located in the B helix of the four-helix bundle protruding into the internal cavity of the assembled molecule (Bozzi et al., 1997; Stefanini et al., 1999). Interestingly, five of the six carboxylate residues, proposed as belonging to the carboxylate-based catalytic site in L. innocua ferritin, are conserved in HP-NAP (i.e. E42, E46, D49, D53, E56) and their position is indicated in Fig. 2A. In the 3D model of HP-NAP, these residues are located in the B helix and, as in the L chain multimers of mammalian ferritins, are pointing into the internal cavity of the HP-NAP oligomer.

It is now generally agreed that iron penetrates 24-mer ferritins through the pore along the threefold axes of symmetry (Harrison and Arosio, 1996; Harrison et al., 1998), the one which is conserved between ferritins and Dps. It could then be hypothesized that iron penetrates through this pore of HP-NAP to reach the central cavity.

Iron is an essential nutrient for bacterial growth and its acquisition is considered to be an important virulence factor for many bacteria. To date, a number of groups have identified and characterized mechanisms by which H. pylori acquires and then utilizes iron (Frazier et al., 1993; Housson et al., 1993; Dhaenens et al., 1997; Bereswill et al., 1998; Worst et al., 1999). What role HP-NAP plays in iron metabolism in H. pylori remains to be elucidated. We have not been able to detect changes in the level of expression of HP-NAP in different strains of H. pylori grown in iron-enriched or iron-depleted media (not shown). Recent evidence indicates that human neutrophils possess a specific receptor for HP-NAP, which is responsible for the transmembrane cellular activation of these cells (our unpublished observations). This is in keeping with the hypothesis of Blaser (1993) that H. pylori induces a moderate inflammatory reaction leading to alteration of the epithelial tight junctions and basal membranes so as to promote the release of nutrients from the mucosa to support the growth of H. pylori residing within the mucous layer.

Not only will the present structural data contribute to the design of novel protocols for the purification of HP-NAP more suitable to vaccine development but they will also contribute to the characterization of its interaction with neutrophils. In fact, the present model will allow the design of mutants in the exposed regions of the HP-NAP oligomer, which are likely to be directly responsible for the binding to the neutrophil receptor.

As shown in Fig. 1, HP-NAP sequence comparisons indicate that HP-NAP is one of a large family of proteins produced by a variety of pathogenic bacteria. The present approach of structure modelling followed by spectroscopic and biochemical investigations can be easily extended to those HP-NAP-like proteins that can be purified from the producing bacteria or from suitable bacteria in sufficient amounts.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

HP-NAP expression and purification

HP-NAP cloning in Bacillus subtilis and purification will be described elsewhere (G. Grandi, unpublished). The protein was pure as judged from overloaded gels composed of different percentages of polyacrylamide, run according to Laemmli (1970). Mass spectrometry analysis, performed with a Maldi Reflex (Brucker Analytik), confirmed that the protein consisted of a single molecule of 16 875 ± 20 Da. Protein concentration was determined by absorbance at 280 nm using a Perkin-Elmer lambda-20 spectrophotometer. The absorption coefficient at 280 nm and 0.1% (by mass) protein concentration was taken as 0.9 mg−1 cm2, determined according to the method of Gill and von Hippel (1989).

Structure predictive methods

Secondary structure of HP-NAP was predicted using the program gor (Garnier et al., 1978). Molecular modelling of the NAP 3D structure was obtained by submitting the HP-NAP sequence to the swiss-model protein server (Peitsch et al., 1995) ( html), using the first mode of approach among the four distinct modes of function. The Dps co-ordinate file in the Protein Data Bank (PDB) was automatically selected by the server as the template.

Spectroscopic characterization of HP-NAP

CD spectra were recorded on a Jasco-710 spectropolarimeter fitted with a thermostatted cell holder and interfaced with a Neslab water bath RTE-110. The molar ellipticity (deg cm2 dmol−1) in the far UV region ([Θ]MRW) was calculated on the basis of a mean residue molecular weight of 117 and in the near UV region ([Θ]M) on the basis of the molecular mass (16 864 Da). The percentage of secondary structure in the protein was estimated from the far UV CD spectrum with a computer program provided by Jasco and based on the method of Yang et al. (1986). Thermal denaturation of HP-NAP, 0.1 mg ml−1 in PBS (8 mM Na2HPO4, 1.5 mM KHPO4, 135 mM NaCl, 3 mM KCl), was monitored by recording the decrease in the CD signal at 222 nm as a function of sample temperature.

Fluorescence spectroscopy measurements were performed on a Perkin Elmer LS-50B spectrofluorimeter by excitation of the aromatic amino acid residues at 280 nm, 25°C in 20 mM tris-HCl, pH 7.4, 50 mM NaCl. The guanidinium-induced denaturation of HP-NAP was followed by monitoring the light emission at 330 nm. Samples were prepared by diluting a stock solution (1 mg ml−1) of HP-NAP to a final concentration of 20 μg ml−1 in phosphate buffer, pH 7.4, containing different percentages of an 8 M guanidine hydrochloride solution (Fluka).

Electron microscopy

One drop (10 μl) of HP-NAP 50 μg ml−1 in phosphate buffer and one of Fe-HP-NAP, 50 μg ml−1, mixed with an equimolar solution of horse ferritin (purchased from Pharmacia Biotech), were placed on thin carbon films supported by a copper grid. Unbuffered 1% uranyl acetate was used as a negative stain. Micrographs were taken with a Hitachi H-600 electron microscope at a magnification of 40 000 and recorded on SO-163 Kodak film.

DNA-binding experiments

Plasmid DNA was purified from H. pylori strain CCUG using the alkaline lysis method according to Ausubel et al. (1987). Purified HP-NAP (1 μg) was added to 2 μg and 1.5 μg of uncut and HindIII-digested plasmid DNA, respectively, in TE (10 mM tris-HCl, 1 mM EDTA, pH 8.0). The DNA–protein mixture was incubated for 30 min at 37°C and then electrophoresed through a 1% Tris-acetate agarose gel stained with ethidium bromide.

Iron incorporation

The iron content of the protein, as purified from B. subtilis, was determined using a Perkin Elmer 4000 atomic absorption flame spectrophotometer. Iron was removed from HP-NAP by incubation in 0.3% sodium dithionite in 20 mM MOPS, pH 7.0, containing 1 mM 2-2′ bipyridil (Fluka) added to chelate spurious ferrous iron.

Iron was incorporated by adding 10 mM ferrous ammonium sulphate, kept under a nitrogen atmosphere and used within a few seconds of its preparation, to 1 ml of 0.1 mg ml−1 apo-HP-NAP in 20 mM MOPS, pH 7.0. The iron oxidation and incorporation kinetics were followed spectroscopically at 310 nm (Trefry and Harrison, 1978). As a control, the rate of Fe(II) auto-oxidation was measured by adding 10 mM ferrous ammonium sulphate to 1 ml of protein-free buffer. After iron incorporation, 1 μg of iron-saturated HP-NAP and 1 μg of the apo-protein were subjected to isoelectric focusing (IEF) on Phast-gel 3-9 using a Phast-System (Pharmacia-LKB). The gel was stained with Coomassie blue.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by grants from the EC (BMH4-CT97-2410), from the MURST-CNR Programma Biotechnologie Legge 95/95 and from the Armenise-Harvard Medical School Foundation.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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