The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity

Giuliano Degrassi1, Milan Kojic1, Goran Ljubijankic2 and Vittorio Venturi1

Bacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano 99, I-34012 Trieste, Italy1
Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11000 Belgrade, Yugoslavia2

Author for correspondence: Vittorio Venturi. Tel: +39 040 3757317. Fax: +39 040 226555. e-mail: venturi{at}icgeb.trieste.it


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The Bacillus pumilus gene encoding acetyl xylan esterase (axe) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) purified from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar size and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C.

Keywords: Bacillus pumilus, acetyl xylan esterase, cephalosporin C deacetylase

Abbreviations: AXE, acetyl xylan esterase; rAXE, recombinant xylan esterase

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AJ249957.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Biodegradation of hemicellulose is a process that requires the cooperation of several enzymes. Xylan is the major constituent of hemicellulose and after cellulose it is the most abundant renewable polysaccharide in plants; several bacteria and fungi grow on xylan as a carbon source by using an array of enzymes, such as endoxylanases and ß-xylosidases. Xylan of hemicellulosic polysaccharide plant cell walls is predominantly a 1,4-ß-D-xylose polymer and is commonly substituted to various degrees with acetyl, arabinosyl and glucuronyl residues (Whistler & Richards, 1970 ). This structural complexity thus requires the cooperation of xylanases and ß-xylosidases with several accessory enzymes for its biodegradation. About 60–70% of xylose residues are esterified at the hydroxyl group with acetic acid; these acetylated xylans are abundant in hardwood (Lindberg et al., 1973 ). The existence of acetyl xylan esterases (AXEs) was first reported in fungal cultures (Biely, 1985 ; Biely et al., 1986 ), and also the synergistic activity of partially purified AXEs of Schizophyllum commune with xylanases, with production of xylo-oligomers, xylose and acetic acid. Following these reports, a number of enzymes with the same activity have been described from both fungi and bacteria, and have been reviewed by Christov & Prior (1993) . AXEs (EC 3 . 1 . 1 . 72) are enzymes that hydrolyse the ester linkages of the acetyl groups in position 2 and/or 3 of the xylose moieties of natural acetylated xylan from hardwood. These enzymes are one of the accessory enzymes which are part of the xylanolytic system, together with xylanases, ß-xylosidases, {alpha}-arabinofuranosidases and methylglucuronidases; these are all required for the complete hydrolysis of xylan (Biely, 1985 ).

The xylanolytic system of Bacillus pumilus has been investigated in part and genes encoding xylanase and ß-xylosidase enzymes have been identified and cloned (Moriyama et al., 1987 ; Panbangred et al., 1984 ). We have recently reported for the first time the purification and characterization of an inducible and secreted AXE from B. pumilus PS213 (Degrassi et al., 1998 ). We report here the cloning of the acetyl xylan esterase gene (axe) of B. pumilus, and its expression and purification in Escherichia coli. The recombinant protein had similar properties to the purified AXE of B. pumilus and belongs to the esterase family 7 (Coutinho & Henrissat, 1999 ). Interestingly, the highest identity (76%) of the B. pumilus AXE was observed with the cephalosporin C deacetylase of B. subtilis. The expression of the B. pumilus axe gene in E. coli resulted in high-level production of the enzyme. Finally, the recombinant B. pumilus AXE (rAXE) had activity on several acetylated substrates including cephalosporin C; esterase enzymes of this family possess broad substrate specificity.


   METHODS
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INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains, plasmids and culture conditions.
E. coli DH5{alpha} (Hanahan, 1983 ) and XL-1 Blue (Bullock et al., 1987 ) were used (Table 1). Growth was at 37 °C in Luria–Bertani (LB) broth supplemented when necessary with 100 µg ampicillin ml-1, 0·2 mM IPTG and 20 µg X-Gal ml-1. B. pumilus PS213 is an aerobic soil bacterium, initially isolated from rumen fluid; it was grown at 30 °C in LB medium. Plasmids used are listed in Table 1.


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Table 1. Strains and plasmids used

 
Isolation of B. pumilus axe gene and recombinant DNA techniques.
Two degenerate oligonucleotide probes were synthesized and used for the PCR amplification of a region of the axe gene of B. pumilus. The B. pumilus genomic DNA was prepared according to Ausubel et al. (1988) and used as a template for PCR. The oligonucleotide primers were an 18-mer called BPEN [5'-ATGCA(AG)(CT)T(ACGT)TT(CT)GA(CT)(CT)T(ACGT)-3'] and a 21-mer designated BPEI [5'-(CT)TC(AG)TC(ACGT)AC(CT)TC(ACGT)GG(AG)AA(ACGT) (GC) (AT)-3']. Digestion with restriction enzymes, agarose gel electrophoresis, purification of DNA fragments, ligation with T4 DNA ligase, end-filling with Klenow fragment of DNA polymerase, hybridizations, radioactive labelling by random priming and transformation of E. coli were performed as described by Maniatis et al. (1982) . Southern hybridizations were performed using Hybond-N+ membrane (Amersham Phamacia Biotech). Plasmids were purified as described by Birnboim (1983) and with Qiagen columns.

DNA sequence determination and analysis.
DNA fragments from plasmid pBPEB2 harbouring the 2·3 kb fragment containing the axe gene were prepared by digestion with HindIII and XbaI and cloned directly in pBluescript II SK(+) and pBluescript II KS(+). The constructs were either encapsidated as single-stranded DNA upon infection with helper phage VCSM13 (Stratagene) or used directly for DNA sequencing. Fifteen oligonucleotides (17-mers) were synthesized and used as primers in the sequencing reactions. DNA sequences were determined by the dideoxy chain-termination method of Sanger et al. (1977) , using [35S]dATP{alpha}S for labelling.

Enzyme assay and purification.
AXE was routinely assayed by measuring the conversion of 2 mM {alpha}-naphthyl acetate to {alpha}-naphthol at 37 °C for 10 min according to Poutanen & Sundberg (1988) , except that the pH of the reaction mixture was 7·0. One unit of activity was defined as the amount of enzyme required to produce 1 µmol product min-1 under the assay conditions. To determine the substrate specificity of rAXE, the deacetylation of p-nitrophenyl acetate, 4-methylumbelliferyl acetate, xylose tetraacetate and glucose pentaacetate (Sigma and see Table 3) was also determined, following the procedures previously described (Degrassi et al., 1998 ).


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Table 3. Substrate specificity and specific activity of rAXE

 
Acetyl esterase activity was also determined in situ after IEF, by spreading 2 mM {alpha}-naphthyl acetate onto the gel, incubating for 10 min at 37 °C and then adding 5% Fast Garnet GBC in 10% SDS. Optimal pH and temperature for the rAXE were determined in the range pH 3–9·5 (50 mM sodium acetate, pH 3·0–5·5; sodium phosphate, pH 6·0–7·0; Tris/HCl, pH 7·5–9·5) and 4–80 °C, respectively. In both cases determination was made using 2 mM {alpha}-naphthyl acetate in 10 min assays at 37 °C. For optimal temperature determination, 50 mM phosphate buffer, pH 7·0, was used.

rAXE was purified to homogeneity from the supernatant of 1 l recombinant E. coli culture, grown on LB medium overnight at 37 °C on a rotary shaker. Chromatography was performed at room temperature using a low-pressure liquid chromatography system (GradiFrac; Pharmacia Biotech), following the procedure adopted for the purification of the AXE from B. pumilus and previously described (Degrassi et al., 1998 ), except that the first step (Q Sepharose column) was omitted.

Protein analysis.
After gel filtration chromatography, the purified protein was analysed by SDS-PAGE (12% acrylamide). Molecular mass markers were from Amersham Life Science, as follows: myosin, 220 kDa; phosphorylase b, 97·4 kDa; albumin, 66 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20·1 kDa; {alpha}-lactalbumin, 14·4 kDa.

The pI of the rAXE was determined by using an Ampholine PAGplate precast polyacrylamide gel (Pharmacia Biotech), with pH values ranging from 3 to 10, and by using the broad-pI calibration kit (Pharmacia Biotech) as the pI marker, according to the instructions of the supplier. Protein bands in SDS-PAGE and IEF gels were stained with Coomassie blue R-250, according to Sambrook et al. (1989) .

Other enzyme assays.
Cephalosporin C deacetylase activity of the rAXE was assayed in 50 mM sodium phosphate buffer (pH 7·0) at 37 °C for 30 min and measured according to the procedure of Takimoto et al. (1994) . The reaction mixture was analysed by reverse-phase HPLC using a RP-C18 column (5 mm, 15x0·4 cm) and 90% 43 mM potassium phosphate buffer, pH 5·2/10% methanol as eluent. Flow rate was 0·8 ml min-1 and peaks were monitored at 254 nm. To determine the Michaelis constants, the reaction was performed as described above with concentration of cephalosporin C (Sigma) or 7-aminocephalosporanic acid (7-ACA; Sigma) ranging from 0·05 to 5 mM.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning of the acetyl xylan esterase gene (axe) of B. pumilus
The AXE enzyme of B. pumilus was previously purified to homogeneity and was subjected to N-terminal amino acid sequencing; in addition, the amino acid sequence of an internal fragment was also determined following trypsin digestion (Degrassi et al., 1998 ). On the basis of these two amino acid sequences, two degenerate oligonucleotides were synthesized and used in a PCR reaction using B. pumilus genomic DNA as template (see Methods); the BPEN oligonucletide was designed based on the N-terminal sequence (MQLFDLFLEELG; Degrassi et al., 1998 ), whereas BPEI was designed from an internal amino acid sequence (ALEVIQSFPEVDEHR, Degrassi et al., 1998 ). The product of the reaction was a DNA fragment of approximately 516 bp. This fragment was cloned in pUC18, giving rise to pMPAX. The sequence of this fragment confirmed the presence of both oligonucleotides used in the PCR reaction, and furthermore displayed significant high homology with the axe gene of Thermoanaerobacterium and of the cephalosporin C deacetylase of B. subtilis. It was concluded that the amplified DNA fragment was part of the axe gene of B. pumilus. To isolate the axe gene of B. pumilus, genomic DNA was digested with several restriction enzymes and after Southern transfer was hybridized with the PCR fragment as probe and under conditions of high stringency. A 2·3 kb EcoRI single discrete band of hybridization was seen (data not shown). The 2·3 kb EcoRI hybridizing fragment was then cloned into the EcoRI restriction site of pBluescript II SK(+), creating pBPEB2. It was assumed that the clone pBPEB2 contained the gene(s) which were previously amplified by PCR.

Nucleotide sequence of the B. pumilus axe gene
To determine the nucleotide sequence of the B. pumilus axe gene, different restriction fragments from plasmid pBPEB2 were cloned in pBluescript II SK(+) and KS(+) and sequenced. The 2·26 kb EcoRI fragment was sequenced in both directions and the sequence deposited in the GenBank/EMBL/DDBJ database under accession number AJ249957. Nucleotide sequencing of this fragment revealed the presence of an open reading frame starting at nucleotide 841 until 1800 encoding a protein of 320 amino acids with a predicted molecular mass of 35989 Da. This ORF, representing a gene designated axe, was preceded by a potential Shine–Dalgarno sequence (AAAGGGGAA) and a putative transcription termination signal was noted 57 bp after the stop codon (GCTAAAATGA–3bp–TCATTTTAGC). Analysis of the deduced amino acid of the AXE protein revealed striking identity (76%) with the cephalosporin C deacetylase protein isolated from B. subtilis (Mitsushima et al., 1995 ). This B. pumilus AXE also displayed high identity (42%) with the putative AXE of Thermotoga maritima and 38% identity with the AXE of Thermoanaerobacterium; in Fig. 1 an alignment of these four very related proteins is shown. All four AXE proteins have an esterase/lipase/thioester common sequence (protein prosite signature PS50187; Fig. 1). In the B. pumilus AXE it is located between positions 179 and 184 and is GGSQGG; the motif of this common sequence is GXSXG (Brenner, 1988 ). The AXE protein contained both the amino acid sequences previously determined (Fig. 1 and Degrassi et al., 1998 ) and, together with the protein similarities with other AXEs, it was therefore concluded that the gene encoding the B. pumilus AXE protein was isolated.



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Fig. 1. Protein sequence alignment using the one letter code of B. pumilus AXE, B. subtilis cephalosporin C deacetylase (P94388, Q59233), AXE of Thermotoga maritima (AAD35171) and the AXE of Thermoanaerobacterium sp. JW/SL (O30361). * indicates conserved amino acids; : indicates conserved substitutions; . indicates semi-conserved substitutions. The amino acids which are in the boxed region represent the signature of the esterase common sequence (see text for details).

 
Expression, purification and characterization of recombinant AXE
Acetyl esterase activity against {alpha}-naphthyl acetate was detected in the supernatant of the E. coli culture harbouring pBPEB2. It was observed that this enzyme present in supernatants was due to cell lysis and not as a result of protein secretion. This was verified by the observation via SDS-PAGE that other proteins are also released in the supernatant (data not shown). The specific activity of the supernatant was 5·5 U mg-1, eightfold higher than the 0·66 U mg-1 found in the supernatant of B. pumilus cultures (Degrassi et al., 1998 ). The enzyme was expressed and released due to cell lysis into LB medium and was purified from 1 l culture supernatant as shown in Table 2. SDS-PAGE analysis of the purified rAXE showed a single band of approximately 40 kDa (Fig. 2), whilst the molecular mass determined by gel filtration was 190 kDa, suggesting a homotetrameric or homopentameric structure of the enzyme, in accordance with the molecular mass of the native AXE from B. pumilus. The pI of the rAXE was determined by analytical IEF and found to be approximately 4·8. Activity staining confirmed the acetyl esterase activity of the band, but showed also another band of activity at pH 6·0, not corresponding with any sharp band focused in the Coomassie blue-stained gel (Fig. 2). The pH optimum of the purified rAXE was 8·5 and the temperature optimum was 45 °C. However, the range of pH and temperature where the activity was optimal was 7·5–9·0 and 42–65 °C, respectively, in accordance with the previously reported data for the AXE from B. pumilus (Degrassi et al., 1998 ). The kinetic parameters were determined using the purified rAXE on several acetylated substrates and compared with the same parameters previously reported (Degrassi et al., 1998 ), showing that the recombinant enzyme is at least as effective as the original one (Table 3). The best substrates for the rAXE were {alpha}-naphthyl acetate and p-nitrophenyl acetate, according to the Vmax/Km values, whilst the highest specific activity was on xylose tetraacetate (Table 3). It was also found that the specific activity of the recombinant AXE on {alpha}-naphthyl acetate (Table 3) was not lower than that of native AXE (Degrassi et al., 1998 ).


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Table 2. Purification of rAXE from E. coli supernatant

 


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Fig. 2. Analytical IEF (a) and SDS-PAGE (b) of the purified rAXE. (a) Lanes: 1 and 2, activity staining of 10 µg (1) and 2 µg (2) of purified rAXE; 3, Coomassie blue staining of 10 µg purified rAXE; 4, pI markers, consisting of myoglobin (7·35), myoglobin (6·85), human carbonic anhydrase B (6·55), bovine carbonic anhydrase (5·85), ß-lactoglobulin A (5·2), soybean trypsin inhibitor (4·55) and amyloglucosidase (3·5). (b) Lane 1, molecular mass standard, consisting of myosin (220 kDa), phosphorylase b (97 kDa), bovine serum albumin (66), ovalbumin (46) and carbonic anhydrase (30); lane 2, 20 µg rAXE; lane 3, 5 µg rAXE.

 
B. pumilus AXE has cephalosporin deacetylase activity
Due to the high level of identity between the B. pumilus AXE and the cephalosporin C deacetylase from B. subtilis, we tested the ability of rAXE to catalyse the deacetylation of cephalosporin C. We found that the recombinant enzyme can catalyse the reaction and that the specific activity is similar to the one reported for another AXE, from Thermoanaerobacterium sp., which shows amino acid sequence similarity to cephalosporin C deacetylase (Table 3). However, this value is lower than the one reported for the cephalosporin C deacetylase from B. subtilis (Mitsushima et al., 1995 ). The enzyme seems to be catalytically more specific for 7-aminocephalosporanic acid than for cephalosporin C, as suggested by the higher Vmax/Km, with also a higher specific activity (Table 3).


   DISCUSSION
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INTRODUCTION
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DISCUSSION
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AXE is an accessory enzyme absolutely required for the complete mineralization of many xylans. In contrast to the considerable data available on xylanases and xylosidases, little information is available on AXEs; this report being to our knowledge only the second one describing a gene encoding a secreted bacterial AXE protein. In this study, we have continued our previous studies and report here the cloning and characterization of the B. pumilus acetyl xylan esterase gene (axe). In addition, we also report the heterologous expression, purification and characterization of the rAXE protein from E. coli.

The gene was isolated using oligonucleotides designed on the basis of the amino acid sequences obtained from the purified AXE from B. pumilus (Degrassi et al., 1998 ). The axe gene was identified in a 2·2 kb EcoRI fragment; it encodes a protein of 320 amino acids with a calculated molecular mass of 36 kDa. Interestingly, the protein showed no significant similarity with most identified AXEs; however, it displayed high identity, approximately 40%, with two recently identified AXEs of Thermoanaerobacterium and Thermotoga maritima (Fig. 1). The highest identity (76%) was observed with the cephalosporin C deacetylase of B. subtilis. This identity was not only in the primary structure; the rAXE had enzyme activity towards cephalosporin C and 7-aminocephalosporanic acid. Thus, the two proteins also have functional similarity. This enzyme activity was also observed for the rAXE of Thermoanaerobacterium (Lorenz & Wiegel, 1997 ). This family of AXEs have an esterase domain located at a similar position (Fig. 1). It was also observed that all four proteins are of similar size and display regions of identity throughout the whole protein. Interestingly, there is a stretch of 10 amino acids, PPSTVFAAYN, located very near the C terminus, which is identical in all four proteins (Fig. 1).

The AXE of B. pumilus was purified and characterized from supernatant (Degrassi et al., 1998 ); thus, the enzyme in its parent strain is secreted, as expected due to the function on acetylated xylan. Exported proteins are synthesized initially as preproteins with an amino-terminal extension; this signal peptide, which distinguishes the secreted proteins from cytoplasmic ones, is needed for targeting to the export pathway (Simonen & Palva, 1993 ). It is surprising that the B. pumilus AXE does not have an apparent signal sequence as we have found that the parent strain secretes this enzyme. The AXE protein might possess an internal signal sequence. However, in prokaryotes, to our knowledge an internal signal sequence has not been reported; thus, further studies are needed to understand how this protein is secreted in Bacillus species. Interestingly, the Thermoanaerobacterium AXE protein and the cephalosporin C deacetylase of B. subtilis are reported not to be secreted (Lorenz & Wiegel, 1997 ), whereas the putative AXE of Tht. maritima has not been characterized.

The axe gene was successfully expressed in E. coli when cloned in pBluescript II SK(+); the expression was efficient, however, it led to a certain amount of cell lysis. The rAXE was not secreted in E. coli since the protein is of Gram-positive origin and thus cannot be secreted through the two membranes of Gram-negative bacteria. It was observed that the expression of the axe gene in pBluescript was not due to the lac promoter as the axe gene was cloned in the opposite orientation to this promoter; the expression was therefore due to its own promoter.

The characterization of the purified recombinant enzyme showed that it was highly similar to the AXE purified from Bacillus pumilus in terms of pH and temperature optima, and stability, molecular mass and pI. The heterologous rAXE protein was also assembled as a homotetramer or homopentamer of approximately 190 kDa, with a pI identical to the B. pumilus AXE. The N-terminal amino acid sequence of the rAXE was also found to be 100% identical with the deduced sequence from the gene. However, the IEF of the rAXE showed another band of activity at pI higher than expected not corresponding to any focused protein (Fig. 2), suggesting that the process of assembly and folding of the homopolymer in the heterologous system might be incomplete, perhaps due also to the high protein expression level.

Substrate specificity confirmed what was previously reported by Lorenz & Wiegel (1997) , with the highest specific activity for rAXE found on xylose tetraacetate. We could detect activity on acetylated xylan but much lower than in B. pumilus. As observed in Thermoanaerobacterium, the expression of AXE in E. coli leads to the reduction or loss of activity on acetylated xylan. This might be explained by problems in folding of the homopolymeric protein in the heterologous system, as suggested also by the result of IEF, with a consequent reduced affinity or binding capacity for the complex substrate.

Further investigation therefore needs to be carried out to understand the specificity of this class of enzymes and their mechanism of regulation, in relation to the real metabolic function. In fact, the gene encoding the acetyl esterase in B. pumilus, which showed activity on acetylated xylan and was also found to be induced by this substrate, is more similar to the B. subtilis cephalosporin C deacetylase than to the Thermoanaerobacterium or Thermotoga AXEs. Furthermore, this gene has esterase activity on several substrates. These observations raise the question whether the primary function of the AXE of B. pumilus reported here is deacetylation of acetyl xylan. On the basis that the enzyme is secreted and that its production is induced by xylan and corn cob (Degrassi et al., 1998 ), we postulate that deacetylation of xylan is a primary function for this enzyme. This AXE belongs to esterase family 7 (Coutinho & Henrissat, 1999 ). The other three enzymes that belong to this class have similar characteristics; the AXE1 of Thermoanaerobacterium has broad substrate specificity, it is not clear whether this enzyme is secreted, and indications are that it is an internal enzyme having its primary activity on acetylated xylo-oligomers (Lorenz & Wiegel, 1997 ). The other characterized enzyme of this family is the cephalosporin C deacetylase of B. subtilis, which has been identified for its activity on cephalosporins; it is possible that its primary biological function is not known. In general, it appears that this class of enzymes has a broad substrate specificity and that possibly the enzymes have divergently evolved from a common ancestral gene. Further work is required to understand further the mode of action of this new family of esterases.


   ACKNOWLEDGEMENTS
 
M.K. is a scientist on leave from the Institute of Molecular Genetics and Genetic Engineering, Belgrade, and is benefiting from an ICGEB fellowship.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 27 January 2000; revised 3 April 2000; accepted 12 April 2000.