Toyota Central R&D Laboratories Inc., Nagakute, Aichi 480-1192, 1 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192 and 2 Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyogo, Setagaya, Tokyo 158, Japan
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Abstract |
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Keywords: antibodies/error-prone PCR/phage-display antibody/steroid/structural models
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Introduction |
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Since the development of PCR technology and phage-display systems, several groups have reported the construction of libraries of phage antibodies (Griffiths et al., 1994; de Kruif et al., 1995
; Vaughan et al., 1996
). Although these groups succeeded in isolating antibodies with high specificity and high affinity for several antigens just by screening their libraries, such libraries should be considered to correspond to a naive repertoire in view of the absence of stimulation with antigens, with the exception of the antibodies for some certain specific antigens. Introduction of random mutations in vivo appeared to be an efficient method for increasing both specificity and affinity in the presence of strong selective pressure. In the present study, we used error-prone PCR followed by competition panning to mimic the mechanism in vivo and we attempted to introduce changes in the specificity for steroid antigens.
In a previous study (Iba et al., 1998), we attempted to change the specificity of antibodies by introducing mutations at restricted residues. A monoclonal Ab, 1E9, specific for 17
-hydroxyprogesterone (17-OHP), was used as the starting Ab. Using a model that had been generated by a computer-driven model-building system, we constructed a phage-display library of antibodies in which 16 residues were mutated in three CDRs of the heavy chain that appeared to form the steroid-binding pocket. Although the clones that we isolated by screening the library with cortisol (CS) as antigen exhibited newly developed CS-binding ability, they still retained strong 17-OHP-binding activity (Iba et al., 1998
). Therefore, in the present study, we examined the effectiveness of random mutation by error-prone PCR that was followed by competition panning (Hawkins et al., 1992
).
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Materials and methods |
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11-Deoxycortisol (11-DOC), cortisol 3-(O-carboxymethyl) oxime (3-CMO) and ovalbumin (OVA) were purchased from Sigma. 11-Deoxycortisol was reacted with an equimolar amount of carboxymethoxy-HCl (Wako, Japan) to yield 11-DOC 3-CMO. Cortisol 3-CMO and 11-DOC 3-CMO were conjugated to OVA, in 0.1 M potassium phosphate buffer (pH 7.5), with N-dicyclohexylcarbodiimide and N-hydroxysuccinimide. The average stoichiometry for 11-DOC and CS conjugated with OVA was estimated to be 51 molecules and 27 molecules/one molecule of OVA, respectively.
Oligonucleotide primers
The following oligonucleotides were synthesized chemically and used as primers for construction of plasmid DNAs.
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Construction of plasmid DNA encoding mAb SCET
Plasmid pAALFab (Iba et al., 1997) was modified as follows. The CAA codon, corresponding to the fifth residue of the VH domain, was changed from CTGCAA to CTGCAG with the generation of a PstI site. The VH and V
genes encoding mAb SCET were amplified from SCET-VH cDNA and SCET-V
cDNA (Sawada et al., 1991
) by PCR with two sets of primers, SHPst plus SHBst and SLSpe plus SLXho, respectively. The PstI- and BstPI-digested VH gene-containing fragment and also the SpeI- and XhoI-digested V
gene-containing fragment were inserted into the modified pAALFab vector. The resulting plasmid, designated pFCA-SCHL, was used in subsequent experiments. The amino acid sequences of VH and V
domains encoded by pFCA-SCHL were slightly different from those of the authentic SCET mAb as follows: the N-terminus of the VH domain, QIQLV
QVQLQ; the N-terminus of the V
domain, SIVMTQTPKFLHVSVGDRVTITCKA
DIELTQ-SPASLSASVGETVTITCRT; and amino acid residues 96 and 97 of the V
domain, YT
RA.
Construction of a large set of mutant antibodies
A large set of mutant antibodies, in which a variety of amino acid residues were introduced at 14 positions in the three CDRs of the VH domain, was constructed as follows. As indicated schematically in Figure 1, introduction of mutations at three residues in CDR1 was performed by PCR with pFCA-SCHL DNA as template and primers HISa and HISb. Diversification at six and five residues in CDR2 and CDR3, respectively, was achieved by a similar PCR-based strategy. In this case, however, since we planned to introduce codons for Trp and Tyr at position 50, as well as codons for Arg and Tyr at position 98, we synthesized two similar but different primers to avoid the presence of termination codons in the degenerate primer sequences. Thus, we performed four separate PCRs with four kinds of primer combination: HIISa-1 plus HIISb-1, HIISa-1 plus HIISb-2, HIISa-2 plus HIISb-1 and HIISa-2 plus HIISb-2. After PCR, the four products were mixed together. Finally, we connected the three CDR-containing fragments by PCR, using a mixture of the product of the first PCR and the pooled products of the second PCR as templates, with primers HIIISa and SHBst. In order to remove products whose CDR2 sequences had not been diversified, we digested the products with NdeI and PstI. The recognition sequence CATATG for NdeI was present in the original template DNA but not in the primer sequences used in the present study. Conversely, the recognition sequence CTGCAG for PstI was present in primer HISb but not in the template DNA. The final products were digested with SnaI and BstPI and they were used to replace the corresponding region in pFCA-SCHL, sandwiched between SnaI and BstPI sites. The ligated DNAs were used to transfect Escherichia coli DH12S cells by electroporation, and a library, designated pFCA-SCHL-Lib1, was generated. While the number of clones with different sequences should have been 5x107, we estimated the number of independent clones in this first library was 1x108.
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Clones with newly developed CS-binding activity were isolated from pFCA-SCHL-Lib1 by panning. The method used for panning was essentially the same as described previously (Iba et al., 1998). The surface of an immunotube was coated with 10 µg/ml CS-OVA in phosphate-buffered saline (PBS) for 2 h at room temperature. After panning processes had been repeated five times in all, phagemid DNAs were prepared, digested with SalI, self-ligated and transduced into E.coli DH12S. The characteristics of the FabPP forms of antibodies encoded by this plasmid DNA have been described in previous work (Ito and Kurosawa, 1993
; Iba et al., 1998
; Ib et al., 1998). In brief, FabPP is an abbreviation that refers to the artificial form of Ab composed of Fab fragment fused to two Fc-binding domains of protein A.
Enzyme-linked immunosorbent assay (ELISA)
Two kinds of antibodies were subjected to ELISA. The wells of immunoplates were coated with 100 µl aliquots of solutions of CS-OVA or 11-DOC-OVA at various concentrations. In the case of phage antibodies, wells were blocked with 2% skimmed milk in PBS. Then, 1x1010 colony forming units (c.f.u.) of phages in 2% skimmed milk in PBS that contained 0.1% Tween 20 were added to wells. Phages that bound to the wells were detected with rabbit antibodies against M13, alkaline phosphate-conjugated antibodies against rabbit IgG and p-nitrophenyl phosphate. In the case of FabPP antibodies, the wells were blocked with 0.5% OVA in PBS. FabPP antibodies were prepared from periplasmic fractions as previously described (Iba et al., 1997, 1998
). The periplasmic fractions, each of which had been diluted 10-fold with PBS that contained 0.1% CHAPS, were added to the wells. The bonding of FabPP to CS or 11-DOC was detected with rabbit antibodies against mouse F(ab')2 with the addition of alkaline phosphatase-conjugated Ab against rabbit IgG and p-nitrophenyl phosphate. In the case of the competition ELISA for which results are shown in Figure 6
, the 10 ng aliquots of purified FabPPs were first preincubated with various amounts of 11-DOC in 100 µl PBS that contained 0.1% Tween 20 at room temperature for 30 min and then the mixtures were added to the wells that had been coated with CS-OVA.
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The reagents for error-prone PCR were 10 mM TrisHCl (pH 9.0), 50 mM KCl, 7.5 mM MgCl2, 0.5 mM MnCl2, 0.1% Triton X-100, 0.2 mM dATP, 0.2 mM dGTP, 1.0 mM dCTP, 1.0 mM TTP, 0.3 µM primers SHPst and SHBst, 100 ng of plasmid DcC16 and 2.5 U Taq DNA polymerase (Promega Corp., Madison, WI) in 100 µl. The reaction was allowed to proceed for 30 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 4 min. Then reaction mixtures were treated with phenol and chloroform and DNA was precipitated with ethanol. After digestion with PstI and BstPI, products were inserted into the corresponding region of pFCA-DcC16.
Isolation of CS-specific clones by competition panning
The phage library obtained by error-prone PCR, designated pFCA-SCHL-Lib2, was dissolved in PBS. Then 7.2x1011 phages were mixed with various amounts of 11-DOC in 3.8 ml PBS containing 2% skimmed milk and 0.1% Tween 20 and the mixtures allowed to stand at room temperature for 30 min. Each mixture was transferred to an immunotube that had been coated with 5 µg/ml CS-OVA and incubated at room temperature for 2 h. Other conditions were the same as those for standard panning. This competition panning was repeated four times in all.
Kinetic analysis by measurements of surface plasmon resonance (SPR)
The FabPP forms of representative antibodies were prepared from 2 liter cultures. Proteins in supernatants of overnight culture were precipitated with 70% of ammonium sulfate. Pellets were suspended in 20 ml PBS and dialyzed against PBS. Soluble proteins were loaded onto a column of IgG-linked Sepharose 6 Fast Flow (Pharmacia), bound proteins were eluted with 0.1 M glycine, pH 3.0, and eluates were immediately neutralized. Then proteins were loaded onto a column of CS-OVA-linked Sepharose 4B that had been prepared by immobilizing CS-OVA on CNBr-activated Sepharose 4B (Pharmacia). In the case of SCET mAb, 11-DOC-OVA-linked Separose was used instead of CS-OVA-linked Separose 4B. The purified FabPPs were analyzed by SDSPAGE and no unanticipated bands were detected in all cases. Protein concentration was determined from the absorbance at 280 nm (for Fab': E280 = 1.48 g1·l·cm1). The purified FabPPs were used for subsequent kinetic analysis.
Association and dissociation rate constants of the purified FabPPs for CS-OVA and 11-DOC-OVA were measured with a BIAcore instrument (Johnson et al., 1991; Chaiken et al., 1992
). We coupled the ligands to a CM5 sensor chip using the amino coupling kit supplied by the manufacturer. First, OVA of 1 mg/ml was immobilized in 10 mM sodium acetate, pH 3.5. Next, cortisol 3-CMO or 11-DOC 3-CMO, which had been pretreated with N-hydroxysucciimide, was conjugated with OVA. Then purified FabPPs at 10, 20, 50, 100 and 200 nM were injected into cells. Measurements of surface plasmon resonance (SRP) were made at a flow rate of 5 µl/min in 10 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.05% BIAcore surfactant P20 at 25°C. Surfaces were regenerated by treatment with 10 mM HCl. We calculated the `on' and `off' rate constants using the BIA evaluation program 2.1 (Pharmacia Biosensor AB).
Structural modeling of steroid-binding pockets
We constructed models using the method of homology modeling using the LOOK & SegMod module of GeneMine (Levitte, 1992). On the basis of sequence alignment, we chose the Fv fragment of 1DBB, whose crystal structure had been obtained from the Brookhaven Protein DataBank (Arevalo et al., 1993a
,b
), as the backbone of our starting structure. Identity of the amino acid sequences of VH domain between SCET and DB3 was calculated to be 80.7%. The addition of the necessary hydrogen atoms to the antibody structure and the building of the antigen structure were performed by the biopolymer module of Insight II 97.0.
In the first attempt to simulate the complexes, cortisol and 11-DOC were placed at the same position as progesterone, which is bound to 1DBB in the crystal structure (Arevalo et al., 1993a). Simulations were performed by the Discover 3.0.0. program (Molecular Simulations Inc., San Diego, CA) with energy minimization and molecular dynamic calculations. Nonbonded parameters were obtained by Cell Multipole method (Greengard and Rokhlin, 1987
; Schmnidt and Lee 1991
; Ding et al., 1992
) under the CVFF force field (Dauber-Osguthorpe et al., 1988
). Molecular dynamic calculation were performed in NVT ensemble at 296 K.
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Results and discussion |
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The molecular structures of 11-DOC and CS are almost identical, with the absence and presence, respectively, of a hydroxy group at the eleventh carbon of the steroid ring being the only difference between them. The original SCET mAb was isolated after immunization with 11-DOC that had been conjugated with BSA and it exhibited weak cross-reactivity with CS (0.2%; Hosoda et al., 1987). Since the three-dimensional structure of a progesterone-specific mAb, DB3, had been reported (Arevalo et al., 1993a
,b
) and since the VH gene of the SCET mAb belongs to the same family as that of DB3, we were able to predict the structure of the steroid-binding pocket of SCET with some confidence. In the present study, we attempted to isolate clones that had gained CS-binding activity and had lost some or all of their 11-DOC-binding activity. In a previous study (Iba et al., 1998
), we attempted a similar feat. An mAb, designated 1E9, that was specific for 17-OHP was mutated and the resulting clones had newly developed CS-binding ability. However, they retained 17-OHP-binding activity. In our earlier attempt, we introduced mutations at 16 residues that seemed likely to form the steroid-binding pocket. In the present study, therefore, we adopted a two-step procedure in an attempt to change the Ag specificity entirely. During the first step, we introduced mutations at specific positions in three CDRs of the VH domain that supposedly formed the steroid-binding pocket, generating an initial mutated library. This step was the same as that in our previous study. During the second step, we introduced mutations randomly into the entire VH gene of an isolated clone that had already developed CS-binding ability. This step can be performed by error-prone PCR followed by a competition panning.
Isolation of a clone with newly developed CS-binding activity
We chose to mutate residues at 14 positions, at which from two to seven possible amino acids are located, as summarized in Table I. The principles that governed the choice of positions and amino acids were essentially the same as those described in a previous paper (Iba et al., 1998
). In brief, since we could predict that the steroid ring should be sandwiched between TrpH50 and TyrH100 (Arevalo et al., 1993a
,b
), we chose 14 positions that would be located in the region surrounding the steroid. Since the CDRs of the VL domain should form a binding pocket for the A ring of the steroid, we did not mutate this domain. The first library, pFCA-SCHL-Lib1, was panned for selection of CS-binding clones. Although neither unselected clones nor pools of eluted phages after one to four rounds of panning exhibited CS-binding activity, a pool of phages after the fifth panning appeared to show evidence of CS-binding activity in a phage ELISA with CS-OVA-coated wells (data not shown). Phagemid DNA was prepared from the final pool of eluted phages, digested with SalI, self-ligated and used to transfect E.coli DH12S. Twenty clones were picked up and examined for CS-binding activity. One of them, designated DcC16, had high CS-binding activity, as shown in Figure 2a
. As had been the case in our previous study (Iba et al., 1998
), this clone had newly developed CS-binding activity but it retained strong 11-DOC-binding activity (Figure 2b
). The nucleotide sequence of DcC16 revealed substitution of amino acids at five positions in CDR2, as indicated in Table I
.
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As noted above, the DcC16 clone had newly developed CS-binding activity but retained strong 11-DOC-binding activity. As a second step, therefore, we introduced random mutations into the VH gene of DcC16, by error-prone PCR, in which the presence of Mn2+ ions and limited amounts of dATP and dGTP enhanced the frequency of incorporation of mismatched nucleotides (Leung et al., 1989). The number of clones in the second library, constructed by error-prone PCR, was estimated to be 5x106. We selected 20 clones and determined their nucleotide sequences. We found that all the mutations were nucleotide substitutions and the frequency of such mutations was 3.1 bases per 312 nucleotides.
We used the competition panning method to select clones that had increased CS-binding activity and little or no 11-DOC-binding activity. The appropriate concentration of free 11-DOC for use as a competitor was determined as follows. After incubation of the mutated phage library with free 11-DOC at various concentrations, the mixtures were subjected to ELISA in immunoplates that had been coated with 5 µg/ml CS-OVA. As Figure 3 shows, 50, 70 and 90% inhibition was observed in the presence of 11-DOC at 0.16, 0.32 and 1 µg/ml, respectively. Using 11-DOC at these three concentrations as the competitor, we eluted phage particles that had bound to CS-OVA-coated tubes. The panning procedure, namely, the binding, elution and growth of phages, was repeated four times in all. After each cycle, we examined the ability of the eluted phages to bind to CS and 11-DOC. In the presence of 11-DOC at 1.0 µg/ml, the eluted phages did not bind to CS. By contrast, at the other concentrations of 11-DOC, after two rounds of panning, the ratio of CS-binding activity to 11-DOC-binding activity exceeded unity, as shown in Figure 4
. Judging from these data, 0.32 µg/ml appeared to be the best of the tested concentrations. The following experiments were performed using the sample that was eluted after four rounds of panning in the presence of 11-DOC at 0.32 µg/ml.
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In order to estimate the changes in the absolute values of the affinity for CS and 11-DOC, we prepared FabPP forms of representative clones and subjected them to kinetic analysis with a BIAcore instrument. The results are shown in Table III. Although the SCET mAb showed 0.2% cross-reactivity to CS in a previous study (Hosoda et al., 1987), it did not give any evidence of binding to CS in this experiment. This apparent discrepancy might have reflected a difference of valency. We used a monovalent form of the Ab in the present experiment and a divalent form in our previous experiment. As shown by the ELISA results, DcC16 had newly developed CS-binding activity but had not lost 11-DOC-binding activity. The other three clones isolated by competition panning exhibited 1.63.8-fold increases in CS-binding activity, with a loss of 1060% of the 11-DOC-binding activity of DcC16. While the ELISA results indicate only 1.31.5-fold improvements in the relative affinity for CS versus 11-DOC, the results obtained by BIAcore analysis indicated 4.14.5-fold improvements in the relative affinity for CS versus 11-DOC, as well as 1.63.8-fold improvements in the absolute affinity for CS. Since the results obtained by the ELISA might not have been strictly quantitative, the values from the BIAcore analysis should be considered to be more accurate.
We examined the improvements in the relative affinity for CS versus 11-DOC in further detail by competition ELISA, as shown in Figure 6. In this experiment, purified FabPPs were first mixed with different amounts of free 11-DOC in solution. Then the mixtures were added to wells that had been coated with CS-OVA. In the case of DcC16, 1 µg/ml 11-DOC gave 90% inhibition. By contrast, in the case of cc-53, even with 11-DOC at 10 µg/ml, only 80% inhibition was observed.
Theoretical considerations related to the competition panning method
Under the conditions adopted in the present study, the frequency of mutations introduced by error-prone PCR was estimated to be 3.1 per 312 base pairs. In general, more mutations might provide more opportunities to generate clones with the desired affinity, but they would simultaneously increase the likelihood of destruction of the authentic antigen-binding activity. The number of mutations introduced into each clone should correspond to Poisson's distribution. Poisson's law (applied to the present case) states that
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While the results of the competition ELISA shown in Figure 6 indicated that preincubation of the Fab-PP antibodies of cc53, cc118 and cc96 with 11-DOC at 1 µg/ml resulted in only 4060% inhibition of the binding of CS in the ELISA, the phage antibodies eluted from the immunotubes when this concentration of 11-DOC was used as competitor did not have any CS-binding activity. Inclusion of 11-DOC at 0.32 µg/ml resulted in efficient enrichment for clones with enhanced relative affinity for CS as compared with 11-DOC. This apparent discrepancy might have resulted from the difference in the form of the Ab, namely, the phage form versus the FabPP form, or from a difference in population, namely, a very heterogeneous versus a homogeneous population.
During competition panning and during the ELISA, phage antibodies first formed complexes with free 11-DOC. Since the concentration of antigens were much higher than those of antibodies, the concentration of antigens can be considered to have remained constant during formation of antigenantibody complexes. Thus, the following relationship might be valid.
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Comparison of models predicted by computer modeling
The three-dimensional structure of a progesterone-specific antibody, DB3, has been well characterized by X-ray crystallography (Arevalo et al., 1993a,b
). Eight amino acid residues in the VH domain and six residues in the VL domain are involved in direct contacts with progesterone. In particular, the indole side chains of TrpH50 and TrpH100 sandwich the steroid ring. Methyl groups at positions 18 and 19 of progesterone are directed toward TrpH50. Although Tyr is located at position 100 in the mAb SCET instead of Trp, the model constructed by computer simulation suggested that the overall arrangement of the binding pocket in SCET should be similar to that in DB3. This predicted similarity might have been achieved by the identity of the amino acids, such as AsnH35, TrpH47 and TrpH50, which are direct contact residues to steroid in CDR1 and CDR2 in addition to high identity of the overall amino acid sequences (80.7%). As shown in Figure 7a
, the steroid ring was stacked between the aromatic rings of TrpH50 and TyrH100. The 20-keto group of progesterone makes a hydrogen bond with AsnH35 in DB3, while not only did the 20-keto group of 11-DOC make a hydrogen bond with AsnH35 but also the 21-hydroxy group appeared to form a hydrogen bond with TrpH50 (Figure 7b
).
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Perspective
In order to change the antigen specificity of antibodies from 11-DOC to CS, we adopted two kinds of mutagenesis in the present study. During the first step, mutations were introduced at restricted positions in CDRs. Although, in the present study, we isolated only one clone from the resulting library that newly developed a CS-binding activity, we analyzed many such clones in the previous study (Iba et al., 1998). The respective characteristics of the clones analyzed previously were similar to each other. In all cases, binding to the original antigen was unaffected. During the second step, mutations were introduced randomly into the entire VH-coding region by error-prone PCR. The clones with enhanced binding to CS and decreased binding to 11-DOC had mutations at positions located outside of the steroid-binding pocket. It has been reported that a majority of clones in libraries with random mutations in the entire V-coding regions failed to express detectable Fab (Casson and Manser, 1995
). These observations suggest how variations in sequences might be achieved for construction of a large Ig repertoire. During V(D)J rearrangement in Ig loci, random sequences can be generated only in CDR3 of the VH domain. The sequences in the other regions are predetermined in the germline V and J genes. We have been making libraries using human Ig genes. More than 70% of clones in the libraries, each of which has a random combination of VH and VL genes, can be expressed as an Fab form in E.coli (to be published elsewhere). Thus, the majority of Ig genes encoded on the germline genome appeared to be faithfully expressed even in E.coli. In general, appropriate sets of germline V genes should have been selected during evolution. Introduction of random mutations both in vivo and in vitro into the entire regions might destroy the integrity of the Ig-fold structure of V domains at high frequency. Therefore, somatic mutations in vivo can contribute only to the affinity maturation of antibodies in the presence of strong selection systems. This principle could also be applicable to in vitro systems. While introduction of random mutations into the entire V-coding regions might generate a large set of mutants, only minor fractions can be expressed as forms with antigen-binding ability. However, fine tuning for accommodation by the antigen-binding pocket of the target antigen would require a random process. Moreover, similar to the selection system in vivo, in the present study competition panning adopted in the second screening process should have facilitated selection of clones that changed the specificity.
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Acknowledgments |
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Notes |
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References |
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Received November 16, 1998; revised December 25, 1998; accepted January 28, 1999.