In vivo copper- and cadmium-binding ability of mammalian metallothionein ß domain

Neus Cols*, Núria Romero-Isart1,*, Roger Bofill1, Mercè Capdevila1, Pilar Gonzàlez-Duarte1, Roser Gonzàlez-Duarte and Sílvia Atrian2

Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, 08071-Barcelona, Spain and 1 Departament de Química, Universitat Autònoma de Barcelona, 08193-Bellaterra, Barcelona, Spain


    Abstract
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 Abstract
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 Materials and methods
 Results
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 References
 
The ß domain of mouse metallothionein 1 (ßMT) was synthesized in Escherichia coli cells grown in the presence of copper or cadmium. Homogenous preparations of Cu–ßMT and Cd–ßMT were used to characterize the corresponding in vivo-conformed metal-clusters, and to compare them with the species obtained in vitro by metal replacement to a canonical Zn3–ßMT structure. The copper-containing ßMT clusters formed inside the cells were very stable. In contrast, the nascent ß peptide, although it showed cadmium binding ability, produced a highly unstable species, whose stoichiometry depended upon culture conditions. The absence of ßMT protein in E.coli protease-proficient hosts grown in cadmium-supplemented medium pointed to drastic proteolysis of a poorly folded ß peptide, somehow enhanced by the presence of cadmium. Possible functional and evolutionary implications of the bioactivity of mammalian ßMT in the presence of monovalent and divalent metal ions are discussed.

Keywords: ß domain/in vivo copper binding/in vivo cadmium binding/metallothionein/recombinant expression


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Non-enzymatic metalloproteins are both storage/transport agents for essential metals inside organisms and defence mechanisms against xenobiotic elements. Among them, metallothioneins (MT) show the highest and most general metal coordinating capacity. They are small (60–65 residues), cysteine-rich (up to 30% in mammals) proteins notably abundant in animals, although also reported in other phyla. The observation that in response to metal exposure, the physiological levels of MT increase dramatically has led to the assignment of a protective and detoxifying function for MT. Besides, a house-keeping role on metal homeostasis has also recently been claimed (Kägi, 1993Go).

Mammalian MT, the most evolved form, folds over metal atoms constituting two domains: ß, the N-terminal half, and {alpha}, the C-terminal half. The ß domain includes nine Cys residues, mostly in an alternate pattern, -NCNCNor -NCNNCN-, and binds three divalent ions (Zn or Cd), giving rise to a M3(SCys)9 aggregate where each metal ion is tetrahedrally coordinated. The {alpha} domain comprises 11 Cys, in an -NCNCN- or -NCCNCC- array, and it is able to bind four divalent ions tetrahedrally, affording a M4(SCys)11 aggregate (Otvos and Armitage, 1980Go; Furey et al., 1986Go). These features are generally used to classify non-mammalian MT as {alpha} domain-like or ß domain-like peptides (Nemer et al., 1985Go). According to this criterion, yeast and fungal MT, the most primitive eukaryotic forms, are constituted by single ß domains (Peterson et al., 1996Go), crustacean MT has two (ß–ß) domains (Narula et al., 1995Go), eqinodermal MT is made of two ({alpha}–ß) domains (Wang et al., 1995Go) and vertebrate MT shows two (ß–{alpha}) domains. From these data, it seems plausible that duplication and divergence of an ancestral ß unit may have led to the present situation in crustacean, eqinodermal and vertebrates. If we consider their metal-chelating properties, fungal MT appears optimized for Cu-binding (Winge et al., 1985Go), whereas divalent ions, especially cadmium, are preferentially bound by the mammalian forms. Not only has the high stability of the Cd4{alpha}MT mammalian cluster been assessed in vitro (Bernhard et al., 1986Go; Capdevila et al., 1997Go), but dramatic decreases in cadmium resistance in transformed cells has also been reported for mutants where the {alpha} domain is nonfunctional (Chernaik and Huang, 1991Go). A housekeeping metal-regulatory role for MT in lower organisms is supported by the affinity of native ßMT forms for physiological metals (Cu, Zn). This function may have been extended to a detoxifying capacity, mainly acquired by the new {alpha} domain. Coordination studies have shown the differential chelating behaviour of both MT portions towards these metals in vitro (Nielson and Winge, 1984Go; Nielson et al., 1985Go; Okada et al., 1985Go, 1986Go; Li and Otvos, 1996Go), but genetic engineering strategies allow characterization of the metal binding ability in vivo. We have previously shown that both mammalian recombinant domains and the entire MT are able to coordinate Zn, leading to Zn3–ßMT, Zn4{alpha}MT and Zn7–MT respectively (Capdevila et al., 1997Go; Cols et al., 1997Go), and time-coincident studies have also been reported for the same species (Xiong and Ru, 1997Go). The corresponding MT aggregates were recovered from a GST–MT fusion construct rendered by a bacterial heterologous expression system, previously described for plant and cyanobacterial MT (Tommey et al., 1991Go; Shi et al., 1992Go). Although the recombinant Cd4{alpha}MT complex has been described, attempts to synthesize heterologous ßMT in Cd-supplemented media were reported unsuccessful (Sewell et al., 1995Go; Kurasaki et al., 1996Go; Kurasaki et al., 1997Go). Furthermore, no data about in vivo mammalian {alpha} or ßMT copper binding have been reported up to now. Here we describe the synthesis of mouse ßMT in E.coli cells grown in the presence of Cu and Cd, and provide data on the corresponding metal clusters. The factors inhibiting expression when cadmium is present in the culture medium have also been addressed.


    Materials and methods
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 Materials and methods
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Recombinant expression and purification of MT ß domain as different metal-complexes

In order to express the ßMT domain as a Cu- and Cd-complex, E.coli strains JM105 (protease proficient) and BL21 (lon and ompT protease deficient) were transformed with the plasmid pGEX-4T-ßMT (Capdevila et al., 1997Go). To synthesize Cu–ßMT or Cd–ßMT, E.coli cultures were supplemented with CuSO4 or CdCl2, to final concentrations of 300 and 500 µM Cu2+, or 100 and 300 µM Cd2+, respectively. To verify whether ßMT production was possible in non-metalsupplemented media, cultures without added metals were performed. As a control, the {alpha}MT domain was also assayed for expression in cadmium-supplemented media. Basic procedures for the expression and purification of the ßMT–metal complexes were as reported (Capdevila et al., 1997Go). Aliquots of the protein-containing FPLC fractions were separated by 15% SDS–PAGE, which were stained with Coomassie blue. Positive samples were pooled, and aliquots were stored at –70°C for further analysis.

Analysis of the binding properties of in vivo synthesized Cu–ßMT and Cd–ßMT

Inductively coupled plasma-atomic emission spectrometry (ICP-AES), in a Thermo Jarrell Ash, Polyscan 61E (measuring S at 182.0 nm, Zn at 231.8 nm, Cd at 228.8 nm and Cu at 324.7 nm) was used to assess S, Zn, Cd and Cu contents, to calculate the amount of protein present in the preparation and the metal-to-protein ratios. Additionally, the reaction with DTNB [5,5'-dithiobis(nitrobenzoic acid)], as described elsewhere (Birchmeier and Christen, 1971Go), allowed us to determine the percentage of thiol groups over total sulfur in our samples except for those containing Cu, as it is widely accepted that the presence of this metal in MT prevents determination of SH content by Ellman's method (Winge, 1991Go).

Electrospray ionization mass spectrometry (ESI-MS) was performed on a Platform II (Micromass) equipped with Max Lynx software, calibrated using horse heart myoglobin (0.1 mg/ml). Different sets of assay conditions were required depending on the sample. For apo-ßMT: source temperature, 60°C; capillary-counterelectrode voltage, 3.5 kV; lenscounterelectrode voltage, 0.5 kV; cone potential, 40 V; m/z range, 600 to 1700; scan rate, 4 s/scan; interscan delay, 0.2 s; and the carrier, a 1:1 mixture of acetonitrile and a 0.05% trifluoroacetic acid solution, at pH 2.5. For Cu–ßMT: source temperature, 120°C; capillary-counterelectrode voltage, 4.5 kV; lens-counterelectrode voltage, 1 kV; cone potential, 35 V; m/z range, 700 to 2000; scan rate, 4 s/scan; interscan delay, 0.2 s; and the carrier, a 5:95 solution of methanol and 3 mM ammonium formate/ammonia, at pH 7.0. To avoid the masking effect of Tris over Cu–ßMT, samples were dialyzed for 90 min against 2.5 mM Tris–HCl, pH 7.0, using Pierce Slide-A-Lyzer membranes (cut-off: 2000) prior to injection. For Cd–ßMT: source temperature, 120°C; capillary-counterelectrode voltage, 4.5 kV; lens-counterelectrode voltage, 1 kV; cone potential, 35 V; m/z range, 1000 to 1800; scan rate, 5 s/scan; interscan delay, 0.5 s; and the carrier, a 5:95 solution of methanol and 3 mM ammonium formate/ammonia at pH 7.5.

Electronic absorption measurements were performed on a HP-8452A Diode array UV-visible spectrophotometer. A Jasco Spectropolarimeter (J-715) interfaced to a computer (GRAMS/32 Software) was used for circular dichroism determinations.


    Results
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 Materials and methods
 Results
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 References
 
Expression of the ßMT domain in the presence of different metals

We had previously shown that successful biosynthesis of the ßMT domain, as well as that of {alpha}MT, was achieved in Zn-supplemented media using both JM105 and BL21 E.coli strains, and as expected the latter always rendered higher protein yield (Capdevila et al., 1997Go). In the present study, however, markedly different results were obtained for the synthesis of ßMT in the presence of other metals. When the pGEX-4T-ßMT construct was expressed in E.coli JM105, the GST–ßMT fusion protein was recovered at comparable yields in both non-metal and Zn2+-supplemented media (Figure 1Go, lanes 2 and 3); showed significant levels of synthesis when the medium was Cu2+-supplemented (lane 5); and was almost negligible in the assays of Cd2+-supplemented media (lane 4). Unlike Zn–MT and Cd–MT, the yield of Cu(I)–MT synthesis is affected by the required reduction of the Cu(II) available to the E.coli cell prior to the formation of the corresponding clusters. To further test the behaviour of the recombinant ßMT in the presence of cadmium, the same construct was transformed and tested for expression in BL21 cells. Figure 2Go shows the PAGE results for GST–ßMT, and GST–{alpha}MT as a control, for the two E.coli strains in Cd-supplemented media. It is well reported that pure, homogenous, metal-loaded MT migrates heterogeneously on SDS–PAGE, often rendering diffuse bands, and shows an electrophoretic mobility much lower (apparent 14 kDa) than that corresponding to the molecular mass of the apo-form (3.5 kDa), due to the fact that the SDS sample and gel conditions are not able to cause full denaturation of the metal–protein clusters, and thus the protein migrates as a metal aggregate along the gel (McCormick et al., 1991). The behaviour of the {alpha}MT fusion protein was similar to that in Zn medium, with significant production from both strains (lanes 1–4), but higher amounts in BL21 (lanes 3 and 4), and a slight reduction at higher cadmium concentrations. However, while the ßMT fusion protein was again practically absent from the homogenates of JM105 cells grown in cadmium (lanes 5 and 6), amounts comparable to those obtained for GST–{alpha}MT were recovered from the BL21 host (lanes 7 and 8). These results clearly indicate that recombinant synthesis of the ßMT domain in cadmium-supplemented media was essentially dependent upon the E.coli host strain.



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Fig. 1. SDS–PAGE, in 12.5% gels stained with Coomassie blue, of purified fusion GST–ßMT domain synthesized by E.coli JM105 cells grown in lane 2, non-metal supplemented medium; lane 3, 300 µM Zn2+ supplemented medium; lane 4, 300 µM Cd2+ supplemented medium; lane 5, 300 µM Cu2+ supplemented medium. Lane 1, molecular weight markers of 97.4, 66.2, 55.0, 42.7, 40.0, 31.0 and 21.5 kDa.

 


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Fig. 2. (A) SDS–PAGE, in 12.5% gels stained with Coomassie blue, of purified fusion GST–MT proteins, synthesized by different E.coli strains, grown in cadmium media: lane 1, GST–{alpha}MT, E.coli JM105, 100 µM Cd2+; lane 2, GST–{alpha}MT, E.coli JM105, 300 µM Cd2+; lane 3, GST–{alpha}MT, E.coli BL21, 100 µM Cd2+; lane 4, GST–{alpha}MT, E.coli BL21, 300 µM Cd2+; lane 5, GST–ßMT, E.coli JM105, 100 µM Cd2+; lane 6, GST–ßMT, E.coli JM105, 300 µM Cd2+; lane 7, GST–ßMT, E.coli BL21, 100 µM Cd2+; lane 8, GST–ßMT, E.coli BL21, 300 µM Cd2+. (B) SDS–PAGE, in 15% gels stained with Coomassie blue, of Cd–ßMT recovered after thrombin cleavage and FPLC chromatography from the fusion GST–ßMT of two independent preparations (lanes 2 and 3). Molecular weight markers correspond to 97.4, 66.2, 55.0, 42.7, 40.0, 31.0, 21.5 and 14.4 kDa.

 
Characterization of the binding abilities of the ßMT domain synthesized in the presence of Cu

Cu–ßMT was obtained by thrombin digestion of the GST–ßMT protein recovered from E.coli JM105 cells grown in the presence of 500 µM Cu2+. The metal content of the samples, determined by ICP-AES, was 6.3 to 6.8 Cu atoms per total sulfur content, which is consistent with the stoichiometry reported for the native mammalian ßMT (Nielson and Winge, 1985Go). Thus, our results showed that when the ßMT domain was recombinantly synthesized in the presence of copper, it was recovered as a fully loaded Cu–ßMT species. Comparison of the CD spectrum with those of the Cu–ßMT species generated in vitro by Zn/Cu replacement (Figure 3Go) (Bofill et al., 1999Go) showed that the sample recovered from biosynthesis of the ß domain in Cu-supplemented media contained Cu6–ßMT and Cu7–ßMT. Specifically, the CD spectral trace of the in vivo Cu–ßMT species precisely matched the computer-generated spectrum corresponding to 75% of Cu7–ßMT plus 25% of Cu6–ßMT. This explains the Cu/protein ratios of 6.3 and 6.8 found by ICP-AES. The molecular mass of the single peak detected by ESI-MS of the Cu–ßMT sample was 3599.27 (data not shown), which is consistent with the expected value of 3596.36 for the Cu7–ßMT species (apo–ßMT + 7 Cu–7H). The peak corresponding to Cu6–ßMT probably remains undetectable due to the low contribution of this species (25%) and to the excessive background produced by the Tris buffer in the sample, which cannot be exhaustively dialyzed if integrity of the Cu(I) aggregates is to be preserved. Thus, our data pointed towards a major formation of a Cu7–ßMT cluster inside the cell.



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Fig. 3. Comparison of the circular dichroism spectra of the in vivo-synthesized Cu–ßMT species (— —) with those corresponding to the Cu6–ßMT (— - —) and Cu7-ßMT (- - -) species generated in vitro by Zn/Cu replacement (Bofill et al. 1999Go). A computer generated spectrum corresponding to the sum of the CD spectral traces of Cu6–ßMT (25%) and Cu7–ßMT (75%) has also been included (—— ).

 
Characterization of the binding abilities of the ßMT domain synthesized in the presence of Cd

In order to analyze the features of the in vivo-structured Cd-cluster, and compare it with the Cd3–ßMT generated in vitro from Zn3–ßMT by Zn/Cd replacement, fusion GST–ßMT was purified from total protein extracts of BL21 cells grown in 300 µM cadmium medium, and used to recover the ßMT portion. Two types of culture were set up: small-scale 0.5 l cultures in Erlenmeyer flasks, grown in a New Brunswick Orbital Incubator, and large-scale 30 l cultures grown in a Biostat U (Braun Biotech) Fermentor. In both cases approximately 0.5–0.9 mg of the ß peptide were recovered per litre of culture after thrombin digestion and FPLC purification. ICP-AES measurements of the cadmium versus total sulfur content invariably yielded a ratio of 1.35–1.40, in contrast to the stoichiometric relationship of 3 reported for the Cd3–ßMT species generated in vitro. Neither Zn nor Cu was detected in the sample. Cadmium content was consistent with a 60–63% oxidation rate indicated by the DTNB reaction for samples purified from both types of culture. So far, the results point to the presence of undermetaled cadmium species, due to partial oxidation of the thiol groups. Surprisingly, further characterization revealed a different composition of the ßMT preparations obtained from the different culture conditions.

The CD spectrum of the Cd–ßMT species obtained from small-scale cultures paralleled that of the Cd3–ßMT species generated in vitro from Zn3–ßMT (Figure 4Go), which indicated the presence of some well-structured Cd3–ßMT clusters. The mass of these molecules was 3489.74 at pH 7.0, which coincides with the expected value of 3489.74 for Cd3–ßMT (apo-ßMT + 3 Cd – 6 H) (Figure 5AGo). Acidification of this sample yielded an apo-form with a molecular mass of 3159.69, which agrees well with the calculated value of 3158.54 (data not shown).



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Fig. 4. Circular dichroism spectra of the in vivo-synthesized Cd–ßMT species obtained from a 0.5 l, small-scale (— —) or a 30 l, large-scale culture (— - —). The Cd3–ßMT species generated in vitro by Zn/Cd replacement (Capdevila et al., 1997Go) has also been included for comparison (——).

 


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Fig. 5. ESI-MS mass spectra of the in vivo-synthesized Cd–ßMT species. (A) The spectrum at pH 7 of the sample obtained from a small-scale culture (0.5 l) indicates the presence of Cd3–ßMT. (B) The spectrum at pH 3 of the sample obtained from a large-scale culture (30 l) shows the presence of apo-ßMT. The molecular mass found in each case is given in the upper right-hand corner.

 
In contrast, the CD features observed for the Cd–ßMT species obtained from the large-scale cultures closely resembled those shown by aged Cd3–ßMT samples obtained in vitro and kept for several days at 4°C, indicating the absence of any Cd3–ßMT cluster in the preparation (Figure 4Go). Mass spectrometry at pH 7.0 did not detect either Cd3–ßMT or any definite Cd species, but acidification of the sample yielded an apo-form of molecular mass 3159.45, in agreement with the presence of whole ßMT molecules in the preparation (Figure 5BGo), and thus indicating that the lack of well-defined metal species could not be attributed to the degradation of the ß peptide.


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
To date, the widely accepted preference of the ßMT domain for Cu(I) over divalent ions had been inferred from in vitro data, and direct evidence of the ability of this fragment to form Cu or Cd aggregates in vivo has not been reported. Here, we describe the successful synthesis of recombinant mouse Cu–ßMT and Cd–ßMT, the latter only attainable in a protease-deficient E.coli strain, and the study of their metal-binding capacity.

In vivo, recombinant ßMT binds copper to form aggregates comparable with those obtained in vitro by Zn/Cu replacement from Zn3–ßMT (Bofill,R., Capdevila,M., Gonzàlez-Duarte,P., Palacios,O., Cols,N., Gonzàlez-Duarte,R. and Atrian,S., manuscript submitted). The coexistence of Cu7–ßMT (75%) and Cu6–ßMT (25%) species in the samples recovered from the transformed E.coli cells could be due to several factors: (i) equilibrium between the two species inside the cell; (ii) a partial loss of the seventh copper ion during sample manipulation; or (iii) the restrictive source of Cu(I) ions. The formation of the Cu7–ßMT cluster in vivo, as revealed by ESI-MS and CD spectroscopy, is consistent with previous in vitro copper-coordination data (Nielson and Winge, 1985Go), and reinforces the hypothesis of the primeval copper affinity of the ß domain.

In contrast, Cd–ßMT aggregates were recovered only in special conditions: in a protease-deficient E.coli strain, BL21, and expressed as a fusion protein, GST-tailed. The almost complete absence of Cd–ßMT–GST fusion protein in JM105 cells (Figures 1 and 2GoGo) fully agrees with reported results (Sewell et al., 1995Go; Kurasaki et al., 1996Go, 1997Go). In addition, the stability of the Cd–ßMT aggregates was found to be dependent upon culture conditions. On the one hand, preparations obtained from small-scale cultures were a mixture of approximately one third Cd3–ßMT, with identical structure to that generated in vitro through Zn/Cd replacement (Capdevila et al., 1997Go), and two thirds of fully oxidized protein, devoid of Cd(II) ions. On the other, large-scale cultures did not produce the Cd3–ßMT species, but probably less structured and partially oxidized Cd–ßMT aggregates. Our data are also in accordance with several in vitro ßMT cadmium-binding studies, showing that the apo-ßMT peptide is not prone to form de novo stable Cd3 clusters (Nielson and Winge, 1985Go; Kull et al., 1985; Stillman et al., 1987Go).

It is tempting to speculate on the lack of ßMT recovery from cadmium cultures. The finding that a protease-deficient host could yield high amounts of the ß domain argues in favour of a post-translational proteolytic degradation of ßMT. Indeed, the evidence that ßMT mRNA is present in transformed E.coli JM105 cells, which never accumulates the recombinant peptide, was reported by Kurasaki et al. (1996). Furthermore, the low affinity of the ß peptide towards cadmium would impair correct folding or lead to conformational changes and so favour its vulnerability to proteolysis. Finally, it had been shown that hydrolysis of thioamide peptides by carboxypeptidase A increases 240–970% in the cadmium-substituted enzyme (Bond et al., 1986Go). This is consistent with the hypothesis that cadmium, a thiophilic metal, increases the positive character of the carbonyl carbon and facilitates the nucleophilic attack of substrates by the proteolytic metalloenzymes.

From the present data, functional and evolutionary implications could also be drawn. If primeval ßMT forms were selected on the basis of their contribution to homeostasis of physiological metals, mainly Cu and Zn, this particular feature would have been retained by the present mammalian ß domain, although probably blurred by its interaction with the {alpha} counterpart. This has previously been shown for Zn (Capdevila et al., 1997Go), and is now confirmed for Cu. As a further evolutionary step, the duplication of the ßMT domain may have led to a derived {alpha}MT peptide, which conferred upon the organisms the capacity to respond to newly encountered toxic metals, such as cadmium. This would account for the poor in vivo reactivity of ßMT towards cadmium, as also described in this work, mainly if compared with its affinity for metals associated with life processes, such as Cu or Zn. Further in-depth investigation of the interaction mechanisms between different metal ions and the ßMT thiol groups should be now facilitated by the availability of functional recombinant ßMT peptide.


    Acknowledgments
 
This work was supported by the Spanish Ministerio de Educación y Ciencia (grants PB94-0695 and PB96-0225) and the Departament de Medi Ambient de la Generalitat de Catalunya. We thank the Serveis Científico-Tècnics, Universitat de Barcelona, for allocating instrument time to this research. M.C. is indebted to the Fundación Caja de Madrid for a post-doctoral scholarship and R.B. to the Comissionat per a Universitats i Recerca de la Generalitat de Catalunya (CIRIT) for pre-doctoral scholarships. We also thank R.Rycroft for revising the English version of this manuscript.


    Notes
 
2 To whom correspondence should be addressed Back

* Made equal contributions to this study Back


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 Introduction
 Materials and methods
 Results
 Discussion
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Received April 28, 1998; revised November 23, 1998; accepted December 11, 1998.