PEDS Advance Access originally published online on January 3, 2006
Protein Engineering Design and Selection 2006 19(3):91-97; doi:10.1093/protein/gzj006
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High-yield expression in Escherichia coli of soluble human 
-hemoglobin complexed with its molecular chaperone
INSERM U473, 78 rue du Général Leclerc, F-94275 Le Kremlin-Bicêtre Cedex, France
1 To whom correspondence should be addressed. E-mail: baudin{at}kb.inserm.fr
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Abstract |
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The
-subunits of human hemoglobin (Hb) have been more difficult to express than ß-chains owing to the high instability of -chains. Here, we describe the production in Escherichia coli of a soluble recombinant -Hb with human -hemoglobin-stabilizing protein (AHSP), its molecular chaperone. To succeed in this expression, we have constructed a vector pGEX--AHSP which contains two cassettes arranged in tandem in the same orientation permitting to express -hemoglobin and human AHSP. While the GST--Hb alone was expressed in E.coli as insoluble protein, even after adding lysate containing recombinant AHSP, the expression vector pGEX--AHSP permits the co-expression of soluble GST--Hb and GST-AHSP. The -Hb, produced at a high yield of 12 to 20 mg per liter of culture, was then purified as a complex with its chaperone. Biochemical and biophysical properties of recombinant AHSP/recombinant -Hb complex were similar to those of recombinant AHSP/native -Hb complex as assessed by UV/visible and CO or O2 binding properties. This co-expression technique can be use to study the interaction between a molecular chaperone and its target protein and, more generally, this system would be particularly interesting for the study of partner proteins when one or both proteins are individually unstable.
Keywords: -hemoglobin-stabilizing protein (AHSP)/Escherichia coli AHSP and -hemoglobin expression system/glutathione S-transferase protein/human -hemoglobin/human hemoglobin/molecular chaperone AHSP
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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Molecular chaperones are involved in the acquisition and maintenance of the native conformation of their target proteins and thereby help prevent aggregation of these proteins. Chaperones comprise several highly conserved families of related proteins, many of which are heat shock proteins. Chaperones selectively recognize and bind to the exposed hydrophobic surfaces of the target protein in a non-covalent interaction in order to inhibit irreversible aggregation.
-hemoglobin-stabilizing protein (AHSP), a recently discovered molecular chaperone, specifically binds to -hemoglobin (-Hb) to form a stable soluble heterodimer protecting freshly synthesized -Hb chains (Gell et al., 2002
; Kihm et al., 2002). Unlike the ß-hemoglobin (ß-Hb) chains, which are soluble and form homologous tetramers, the free -chains are highly unstable and act as active oxidants, causing apoptosis and inefficient erythropoiesis (Weatherall and Clegg, 2001). AHSP, a small protein of 102 amino acids, is present at a high level (0.1 mM) in human red blood cell precursors. The role of AHSP would be the prevention of free -Hb from forming inclusion bodies, which might damage membrane structures and trigger cell apoptosis.
Different expression systems in bacteria have been developed to produce -chains but their instability resulted in low yields in Escherichia coli compared with the expression of ß-chains. A fusion protein system was used to express -globin alone (Jessen et al., 1994), as previously done for ß-globin using N-terminal fusion peptides cleaved by factor Xa to yield native N-terminal subunit (Nagai and Thogersen, 1984). This fusion -globin protein was insoluble and accumulated as inclusion bodies in the bacterial cytoplasm and the purification steps are long and arduous. Monomeric -globin did not accumulate in the cytoplasm even when using the high-expression T7 promoter system, apparently owing to rapid proteolysis of unstable apo--globin (Weickert and Curry, 1997). Expression of -globin as soluble fusion protein with exogenous heme has been attempted, but expression and isolation from bacteria have been more difficult than for ß-globin, which is apparently stable enough to accumulate to levels as high as 10% of total cell protein (Hernan et al., 1992). The heme molecule also plays an important role in the stability of -chains. This interaction promotes the formation of the tertiary structure of the growing -chain on polyribosomes (Komar et al., 1993, 1997); it has been shown that the nascent -chain possesses a spatial structure that will permit binding of the heme molecule as soon as the first 86 amino acids (of the total of 141) are formed. The study of assembly of Hb A, by expression in an in vitro cell-free system, also suggests that ß-chains associate with -chains during or soon after translation, preventing the formation of unstable -chain monomers (Adachi et al., 2002). Others methods such as co-expression with methionine aminopeptidase (MAP) in the presence of exogenous heme permit a production of soluble -Hb (Adachi et al., 2000), but this system requires two chromatography steps for recombinant -Hb purification with moderate yield. Hence protein engineering using chaperones is an interesting strategy to produce a high yield of proteins, in particular for insoluble proteins when the assembly partner(s) are absent. In this paper, we describe an over-expression system for human -Hb in E.coli achieving high production of the soluble -Hb by co-expression with its chaperone under the control of the two tac promoters. The two recombinant proteins were produced as independent fusion proteins with glutathione S-transferase (GST) and after bacterial lysis, the -Hb was obtained as a complex with AHSP. This purified recombinant AHSP/recombinant -Hb complex (recAHSP/rec-Hb complex) has all the characteristics of functional recombinant AHSP/native -Hb complex (recAHSP/native -Hb complex). Our engineering of a system to produce both the -Hb with its chaperone demonstrates a new strategy for obtaining soluble -Hb in a high yield.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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Plasmids and strains
The plasmid pHE7 containing human -globin cDNA was provided by Dr Chien Ho of Carnegie Mellon University (Pittsburgh, PA, USA). The pGEX4T-1 vector and E.coli BL21 and BL21(DE3) were purchased from Amersham Bioscience (Uppsala, Sweden). The construction of pGEX-AHSP containing the human AHSP cDNA (Figure 1a) was described previously (Baudin-Creuza et al., 2004).
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Construction of plasmid pGEX-
expressing GST--Hb
The coding sequence of human -globin cDNA was obtained by polymerase chain reaction (PCR) with pHE7 (Shen et al., 1997) as template and two synthesized primers, 5'-GGGGGATCCGTGCTGTCTCCTGCCGACAAG-3' and 5'-CCGCTCGAGTTAACGGTATTTGGAGGTCAGCACGGT-3', which are complementary to the 5'- and 3'- end sequence of the human -globin cDNA and contain a BamHI site and a XhoI site, respectively. After digestion with BamHI and XhoI and desalted on a microspin HR400 column (Amersham Biosciences), this fragment was ligated in frame with GST into pGEX-4T-1 plasmid (Figure 1b).
Construction of pGEX--AHSP co-expressing GST--Hb and GST-AHSP
The expression cassette containing the ‘tac promoter-GST-AHSP’ gene coding sequence was obtained by PCR with pGEX-AHSP as template and two synthesized primers, 5'-ATAAGAATGCGGCCGCCCGACATCATAACGGTTC-3' and 5'-TATTGACGTCCTCGAGCTAGGAGGAGGG-3', which contain a NotI site and an AatII site, respectively. After digestion with NotI and AatII, the PCR product (1.12 kb) was inserted between the NotI and AatII sites of pGEX-. The resulting pGEX--AHSP vector contains two cassettes in the same orientation: (1) tac promoter-GST-human -globin cDNA coding sequence and (2) tac promoter-GST-human AHSP cDNA coding sequence (Figure 1c). The three plasmid constructs have a pGEX-4T-1 background with the pBR322 origin of DNA replication, the ampicillin resistance gene and a thrombin cleavage recognition site between GST gene and the gene of interest. These three constructs were checked by DNA sequencing.
Expression of different fusion proteins
The pGEX-AHSP, pGEX- and pGEX--AHSP constructs were expressed in E.coli BL21 cells according to the manufacturer's instructions with minor modifications. Expression of pGEX-AHSP construct was induced by adding isopropyl ß-thiogalactopyranoside (IPTG) at 0.1 mM at 37°C. Expression of pGEX- construct was induced by 0.1 mM IPTG and then supplemented with hemin (30 µg/ml) and the expression of pGEX--AHSP construct was induced by 0.2 mM IPTG and supplemented with hemin (30 µg/ml). In the three cases, the growth was continued for 4 h at 37°C. An aliquot of these cultures was withdrawn and prepared by boiling in SDS loading buffer. The bacterial cells were harvested by centrifugation.
Purification of different fusion proteins
The three different cell pellets were resuspended in PBS (150 mM NaCl, 10 mM Na2HPO4, pH 7.4) and stored frozen at –20°C until needed for purification. The different GST fusion proteins were solubilized as described previously (Baudin-Creuza et al., 2004). The sample containing GST--Hb was saturated with CO gas. The soluble fractions containing the GST fusion protein(s) were mixed with glutathione Sepharose 4B beads (Amersham Bioscience) for 1 h with a volume ratio of 4:1 (supernatant:bead). The beads were then washed with 10 (bead) volumes of PBS. In the first procedure, the GST fusion proteins were eluted by the addition of two bead volumes of elution buffer (50 mM Tris–HCl pH 8.0) containing 10 or 20 mM reduced glutathione. In the second experiment, direct thrombin cleavage of GST proteins bound to glutathione beads was achieved by the addition of the thrombin (100 or 200 units/ml bead) and the incubation was then continued overnight at room temperature under gentle agitation. The released recombinant proteins were recovered in the supernatant after centrifugation, while GST remained bound to the matrix. Then, the recombinant proteins were concentrated by ultracentrifugation (Centriprep 10, Millipore, Billerica, MA, USA). In all constructs, an additional Gly–Ser dipeptide remains at the N-terminus of the protein. The recAHSP/native -Hb was prepared as described previously (Baudin-Creuza et al., 2004).
Characterization of recombinant proteins
Different aliquots were withdrawn during expression, solubilization and purification of recombinant proteins. These samples were analyzed by SDS–PAGE. To determine the molecular mass, size-exclusion chromatography was carried out on a Superose 12 10/300 GL column (Amersham Biosciences) equilibrated with a 150 mM Tris–acetate buffer pH 7.5 at room temperature with a flow rate of 0.4 ml/min (Manning et al., 1996). Protein concentrations for AHSP and for complexes were determined by UV–visible spectrophotometry using extinction coefficients at 280 nm (calculated from the primary amino acid sequence with software from the Infobiogen site, http://www.infobiogen.fr) and at 414 nm (specific for the heme molecule), respectively.
Spectra and ligand binding kinetics
UV and visible spectral measurements were carried out with an HP 8453 or Varian Cary 50 spectrophotometer. The spectra of the oxidized (ferric state) and deoxy (ferrous state) forms of the free -Hb and -Hb bound to AHSP (7 µM on a heme basis) at 25°C were performed in 100 mM potassium phosphate buffer at pH 6.0, 7.0 or 8.0. We used 100 mM Tris–HCl buffer for pH 9.0. The deoxy sample was obtained by equilibration under nitrogen and adding an excess of sodium dithionite. The pure oxy form was obtained after adding the deoxy protein to a cuvette with the appropriate buffer equilibrated under 1 atm O2.
In order to assess the ligand binding parameters, laser flash photolysis was used to determine the bimolecular CO and O2 association rate constants (kon), as described previously (Marden et al., 1988). The kinetics were initiated with 10 ns YAG laser pulses (Quantel, France) at 532 nm. Detection of the sample absorption was in the Soret band, typically at 436 nm, using a 50 W quartz halogen lamp and interference filters. Samples from 1 to 10 µM were in 4x 10 mm quartz cuvettes as for the absorption spectra.
The CO dissociation rate (koff) was measured using a replacement-type reaction. A carbonylated sample was mixed with a buffer containing 1 mM ferricyanide at pH 7.0 and 25°C. After CO dissociation, the ferricyanide may induce the oxidation of the heme iron.
Measurement of auto-oxidation rates
The protein was prepared by incubation on ice in 100 mM potassium phosphate buffer at pH 7.0 with 1 mM EDTA, adding catalase and superoxide dismutase (Sigma Aldrich, St. Louis, MO, USA) to prevent the degradation of heme by superoxide anion O2–, H2O2, etc., as described previously (Brantley et al., 1993). After flushing under nitrogen, an excess of dithionite was added to obtain the ferrous protein; the reduced solution of protein was immediately loaded on to a small G-25-medium gel filtration column under air and eluted quickly with 100 mM potassium phosphate pH 7.0, adding 1 mM EDTA, in a cold room (
3°C) to eliminate the dithionite. The oxygenated protein was thus obtained. The solution was then added to the sample cuvette and the kinetics started.
The auto-oxidation kinetics were followed by measuring the entire absorption spectra of the proteins versus time. We analyzed the auto-oxidation reaction at 412, 540 and 576 nm for the -Hb and the AHSP/-Hb complexes over time. All spectra and kinetics were measured using a thermostated diode-array spectrophotometer (HP 8453); samples were in 4x 10 mm quartz cuvettes at 25°C; the temperature was further checked with a classical thermometer.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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Solubilization of the
-Hb by co-expression with its chaperone
In a first experiment, the -Hb was expressed as fusion protein with GST under the control of a tac promoter (Figure 1b). The construct pGEX- was derived from the pGEX expression system that used to obtain a high-level expression of recombinant (rec) proteins in E.coli, enhancing the solubility and permitting the purification of proteins in a single step. The SDS–PAGE analysis shows that both the GST--Hb (Figure 2a, lane 4) and GST-AHSP (Figure 2a, lane 2) fusion proteins were expressed at high level with the pGEX construct. Whereas the GST-AHSP was obtained in the soluble fraction (Figure 2a, lane 3), the GST--Hb was not solubilized (Figure 2a, lane 5). This result indicates the instability of -Hb even as a GST fusion protein. In an attempt to produce soluble -Hb protein, the E.coli cells expressing GST--Hb were disrupted in the presence of bacterial lysate containing the GST-AHSP protein (Figure 2a, lane 6), but in this case the fusion GST--Hb protein was not solubilized in the presence of its molecular chaperone. This result shows that the AHSP could play a crucial role early in the synthesis of -chain in the cell. This is in agreement with the recent study of dos Santos et al. (2004), which showed a progressive increase in AHSP gene expression following the expression of -Hb gene.
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In the second experiment, we developed a new expression pGEX-
-AHSP vector (Figure 1c), derived from the two original plasmids, pGEX-AHSP and pGEX-. The pGEX--AHSP contains two expression cassettes arranged in tandem in the same orientation, the -globin cDNA fused to GST gene under the control of first tac promoter and the AHSP cDNA fused to GST gene under the control of second tac promoter. With this pGEX--AHSP co-expression vector, SDS–PAGE of whole cell lysate after 4 h induction expression displays two intense polypeptide bands corresponding to GST--Hb and GST-AHSP, respectively (Figure 2a, lane 7). The GST--Hb and GST-AHSP proteins were both present in the soluble fraction (Figure 2a, lane 8). After purification by affinity chromatography on glutathione Sepharose 4B, the SDS–PAGE analysis revealed the presence of the two bands corresponding to GST--Hb and GST-AHSP (Figure 2b, lane 2). Gel permeation chromatography on Superose HR 10/300 GL revealed the presence of two peaks (Figure 3a) with an absorbance at 415 nm characteristic of the heme found in the -Hb. From the calibration curve, the elution volumes of the two peaks correspond to 241 kDa (Ve = 10.68 ml) and 91 kDa (Ve = 12.09 ml); these values would correspond to 3.0 and 1.1 subunits of [GST-AHSP/-Hb-GST], respectively, considering the theoretical masses of GST--Hb (42.1 kDa) and GST-AHSP (38.1 kDa). Note that calibrated on this scale, GST alone, which migrates as a dimer (Walker et al., 1993), elutes at 1.2 times the value expected. It is therefore not clear if the higher molecular weight species corresponds to a dimer or trimer (via GST interactions) of [GST-AHSP/-Hb-GST].
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After direct thrombin cleavage of the complex bound to glutathione beads, the SDS–PAGE analysis of the released proteins revealed the presence of the two bands corresponding to
-Hb (15.9 kDa) and AHSP (12 kDa) with a purity of not less than 98% (Figure 2b, lane 3). The analysis of these purified proteins on Superpose 12 10/300 GL (Figure 3b) showed the presence of one pure peak of the order of 99%. Based on the same calibration curve as above, the elution volume of this recombinant protein corresponded to 30.7 kDa (Ve = 13.66 ml). This value was similar to that obtained with the recAHSP/native -Hb complex (Ve = 13.71 ml) and is consistent with a complex formed from one molecule of AHSP and one molecule of -Hb (1.1 subunits).
The yield of purified recAHSP/rec-Hb complex was 20–35 mg/l culture, corresponding to 12–20 mg of -Hb/l culture (Table I). This was higher than those obtained when -Hb was co-expressed with methionine aminopeptidase (Adachi et al., 2000) or with ß-chains (Shen et al., 1997). The co-expression of -Hb with its chaperone was also achieved without supplementation of hemin during induction. In this case, the same quantity of soluble -chains was obtained but only 15% contained the heme molecule. Our experimental results clearly show that AHSP permits the solubilization of -Hb as the complex form and confirm the stabilizing role of AHSP towards the -chains. Furthermore, our procedure is rapid and requires only one chromatographic purification step (Table I).
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Spectral characteristics of recAHSP/rec
-Hb
The recAHSP/rec-Hb complex was characterized by spectroscopic analysis and compared with that observed with the recAHSP/native -Hb complex. The absorbance spectra of the recAHSP/rec-Hb complex with different ligands and iron state measured between 350 and 700 nm (Figure 4) were similar to those observed previously for the recAHSP/native -Hb complex (Baudin-Creuza et al., 2004). All the spectra were independent of pH. The spectra of the ferrous complexes were similar to those of the free -chains, and the spectra of ferric forms were similar to those observed for hexacoordinated globins such as neuroglobin (Dewilde et al., 2001) and cytoglobin (Burmester et al., 2002) with a Soret band at 413 nm and two bands at 532 and 560 nm. Our results are in agreement with those obtained from the crystal structure of oxidized -Hb bound to AHSP (Feng et al., 2005). Indeed, the oxidized -Hb bound to AHSP reveals a His–Fe3+–His heme conformation as the most likely binding scheme of the hexacoordinated protein.
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Functionality of recAHSP/rec
-Hb
The ligand binding kinetics of CO and O2 followed by means of the flash photolysis experiments were explored (Table II). The rate parameters describing the ligand binding dynamics (kon and koff) to the recAHSP/native -Hb and recAHSP/rec-Hb complexes were similar whatever the ligand. The laser photolysis time course of CO and O2 recombination exhibits a monophasic shape which follows the behavior expected for a bimolecular process (data not shown). The rate for the CO rebinding of recAHSP/rec-Hb complex was 2 µM–1 s–1, as observed previously for the recAHSP/native -Hb complex. The association rate of CO was three times lower than that observed for free native -Hb (Table II). The dissociation rate of CO increases by a factor of nearly three once AHSP is bound to the -Hb chains. A smaller change was observed in the association rate of O2, 35 µM–1 s–1 for the complexes and 50 µM–1 s–1 for the -Hb control (Table II). The major effect for oxygen was observed in the dissociation rate; the association of AHSP to -Hb leads to a factor of five decrease in the O2 dissociation rate, suggesting a weakening of the hydrogen bonds that normally stabilize the O2 bound to the heme. Based on the kinetic experiments, the affinity for the CO and O2 of the complexes shows a decrease by a factor of eight and five, respectively, relative to free -chains (Table II). These results show that AHSP affects the ligand binding dynamics of the -Hb, essentially in the dissociation process for oxygen and both association and dissociation processes for CO.
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Addition of 1 equiv. of ß-Hb to the recAHSP/rec
-Hb complex leads to the formation of tetrameric Hb (2ß2), as described previously for the recAHSP/native -Hb complex (Baudin-Creuza et al., 2004). The release of the -Hb chains from the complex and Hb reconstitution could be followed by the CO recombination kinetics, based on the specific biphasic shape of the CO recombination to cooperative Hb tetramers. After addition of ß-Hb, CO association displayed a fast phase with a rate near 8 µM–1 s–1 characteristic of the (high-affinity) R-state and a slow phase of rate 0.2 µM–1 s–1 corresponding to the (low-affinity) T-state conformation of Hb.
We next investigated the auto-oxidation kinetics of recAHSP/rec-Hb complex in the oxygenated form compared with those obtained for the recAHSP/native -Hb complex, -Hb chains and tetrameric Hb. Figure 5 illustrates the time courses of auto-oxidation. The transition from the oxy to the met spectrum of recAHSP/rec-Hb complex shows well-defined isosbestic points at 593 and 577 nm (inset in Figure 5). The standard auto-oxidation mechanism for hemoproteins (Weiss, 1964) involves the dissociation of the superoxide anion O2– from the heme. All auto-oxidation curves could be simulated as a monoexponential process (Figure 5). The recAHSP/rec-Hb and recAHSP/native -Hb complexes have the same kox = 2.8 h–1, which is 70 times greater than the value for isolated -Hb chains at 25°C, pH 7.0 (Table II). The oxidation of the AHSP/-Hb complex, as for many hexacoordinated globins, shows a very high rate in comparison with Mb or Hb. This indicates that the AHSP facilitates the oxidation of -Hb as reported by Feng et al. (2004).
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) compared with those of recAHSP/native Conclusion
We have described an especially interesting method, requiring only one purification step, which produces a high yield of -Hb stabilized by its AHSP chaperone. The GST--Hb fusion protein could be expressed alone in bacteria, but is not soluble in native conditions. This confirms the crucial role of AHSP toward the -Hb stability, especially in inhibiting the aggregation of -Hb. AHSP plays the same role in bacteria and in the red blood cells in protecting the -Hb from precipitation and thereby maintaining a soluble form of -Hb in the cell. The co-expression of -Hb with AHSP thus overcomes the instability of isolated -Hb.
The overall results of the functional characteristics of recAHSP/rec-Hb complex show that the recombinant complex exhibits similar properties to those of the recAHSP/native -Hb. The rec-Hb is capable of binding ß-Hb to form ß dimers and subsequently 2ß2 tetramers, suggesting that rec-Hb is correctly folded as for native -Hb.
The expression yields indicate that the chaperone protein is sufficient for the -Hb stability, even in the absence of heme. This is an additional advantage of this technique, since there would be a choice of the stage at which to add heme to form the holoprotein. With this new method, the host system does not necessarily have to supply the heme.
This method could be especially interesting for studying some -Hb variants described as highly unstable. Indeed, the purification of -Hb from hemolyzate is long and arduous and, in the case of unstable variants -Hb, the yield of preparation was very low. This expression vector could be an interesting tool to study the interaction between -Hb and its chaperone, especially for certain disorders involving the -globin gene.
Furthermore, the expression system described here may be generally useful for producing proteins that are individually insoluble and for studying interactions with their partner proteins.
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We thank Dr H.Wajcman for helpful advice and discussion and G. Caron for skilful technical assistance. This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Université Paris XI and the Délégation Générale de l'Armement (France).
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Received October 20, 2005; revised December 6, 2005; accepted December 6, 2005.
Edited by Hans Thogerson
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