Molecular Human Reproduction, Vol. 7, No. 9, 887-894,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Transcriptional regulation of human placental leucine aminopeptidase/oxytocinase gene
1 Department of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, 466-8550, Japan and 2 Laboratory of Cellular Biochemistry, Institute of Physical and Chemical Research (RIKEN), Saitama, 351-0148, Japan
Abstract
Human placental leucine aminopeptidase (P-LAP) plays a major role in the clearance of oxytocin, which is a key hormone in regulating labour pain. To explore the transcriptional regulation of P-LAP gene expression in placenta, we performed systematic studies using human choriocarcinoma cells, BeWo and JEG-3, as a model of placental trophoblastic cells. Transient transfection and luciferase assays using various 5'-deleted P-LAP-luciferase constructs showed that the region from –297 to +49 of the transcription start site was responsible for promoter activity in these cells. Footprinting analysis with nuclear extracts from both cell lines demonstrated at least four sites for nucleoprotein interactions in this region (FP1 to FP4). Site-directed deletion of FP1–4 in luciferase assays indicated the significance of the FP3 region (–214 to –183) for high promoter activity in the cells. Electrophoretic mobility shift assays to identify the proteins interacting with DNA at FP3 revealed three retarded bands, one of which was generated by activator protein-2 (AP-2) binding. Our findings suggest that AP-2 may be one of the important factors regulating P-LAP gene expression in human placenta.
aminopeptidase/AP-2/oxytocinase/placenta/promoter
Introduction
Levels of bioactive peptides depend on synthesis and degradation. Oxytocin is a key hormone in labour pain during pregnancy, and the enzyme responsible for hydrolysing oxytocin is termed oxytocinase. Previous studies demonstrated that oxytocinase is cystine aminopeptidase (EC 3.4.11.3), which is identical with human placental leucine aminopeptidase (P-LAP) (Tsujimoto et al., 1992
). Maternal serum P-LAP activity is increased with gestational age just before the onset of labour (Mizutani et al., 1976), suggesting that P-LAP may be involved in suppressing labour pain by hydrolysing oxytocin. Moreover, P-LAP plays an important role in regulating the functional levels of several other hormones, such as vasopressin and angiotensin III (Tsujimoto et al., 1992; Mizutani and Tomoda, 1996) which are involved in controlling feto-placental circulation by vasoconstriction. Thus P-LAP has been believed to be critical for maintaining the physiological condition of gestation.
Previously, we have isolated the P-LAP cDNA clone (Rogi et al., 1996) and revealed that this enzyme is a homologue of rat insulin-regulated membrane aminopeptidase (IRAP) (Keller et al., 1995) which is present in glucose transporter isotype GLUT4 vesicles of rat adipocytes (Kandror et al., 1994; Mastick et al., 1994). Since P-LAP is co-translocated from the cytosol to the cell membrane with GLUT4 by insulin stimulation in adipocytes and skeletal muscle cells, P-LAP may also be involved in glucose homeostasis by insulin-induced trafficking of GLUT4 vesicles.
Recent studies concerning P-LAP gene expression elicit two interesting points. One is the wide distribution of P-LAP in tissues besides placenta as demonstrated by Northern blot analysis (Rogi et al., 1996; Laustsen et al., 1997) and immunohistochemistry (Nagasaka et al., 1997). According to Northern blot analysis, P-LAP mRNA is expressed in placenta, brain, heart and skeletal muscle, while little or no P-LAP mRNA can be detected in lung, liver and kidney. Immunohistochemical analysis both in the adult and fetus has revealed a similar tissue distribution to that found by Northern blotting. Although these results indicate that P-LAP tissue distribution is regulated at transcriptional levels in a tissue-specific manner, the detailed mechanism of P-LAP gene regulation has not been clarified. It should be noted that the widespread distribution of P-LAP does not contradict the significance of serum P-LAP activities during pregnancy, because the soluble form P-LAP is thought to be present only in the serum of pregnant women (Nakanishi et al., 2000; Yamahara et al., 2000). There is also an exponential increase in P-LAP mRNA expression levels in human placenta throughout gestation (Yamahara et al., 2000). This is compatible with immunohistochemical analysis of P-LAP in human placenta showing that P-LAP staining is restricted to syncytiotrophoblast cells throughout gestation (Nagasaka et al., 1997; Yamahara et al., 2000). It is likely that P-LAP expression in placenta is associated with cell differentiation, as has been demonstrated in rat adipocytes (Ross et al., 1998). Gene regulation through cell differentiation is generally controlled by the interplay between transcription factors and the gene promoter/enhancer region. Investigation of the transcriptional regulation of the P-LAP gene, which has remained unclear, may provide clues to understanding the mechanism of trophoblast differentiation.
To obtain insights into the molecular mechanisms underlying the tissue-specific and gestation-induced regulation of human P-LAP gene expression, we first isolated genomic clones containing the 5'-upstream region of the human P-LAP gene (Horio et al., 1999). The P-LAP promoter contains GC-rich regions and the consensus sequences for the binding of several transcription factors, such as activator protein-2 (AP-2) and selective promoter factor 1 (Sp1); however, the interplay of these proteins on the P-LAP promoter gene has not been determined. We have also found that the human P-LAP gene is assigned to the chromosomal region 5q14.2–q15. Recently, the P-LAP gene was reported to span ~75 kb containing 18 exons and 17 introns (Rasmussen et al., 2000).
In order to begin a systematic study of the human P-LAP gene regulation in placenta, we performed transient transfection assays of the P-LAP gene promoter linked to the luciferase gene, DNase I footprinting analysis, and an electrophoretic mobility shift assay (EMSA) using BeWo and JEG-3 choriocarcinoma cells. These choriocarcinoma cells have retained several properties of the placenta (Ringler and Strauss, 1990) and express P-LAP mRNA (Horio et al., 1999), and are therefore considered to be a suitable model to investigate the molecular mechanism of P-LAP gene regulation in the placenta. We identified a functional region for high promoter activity located between –214 bp and –183 bp from the transcription initiation site. At least three nuclear factors bind to this region, one of which was identified as AP-2.
Materials and methods
Materials
The following items were purchased: Roswell Park Memorial Institute (RPMI) 1640 medium and fetal calf serum (FCS) from Sigma (St Louis, MO, USA); LipofectAMINE PLUSTM Reagent and custom-synthesized oligonucleotides from Life Technologies Inc. (Gaithersburg, MD, USA); the luciferase reporter vector pGL3-Enhancer, the control reporter vector pRL-TK, Dual-Luciferase reporter assay kit and RQ1 DNase from Promega Corp. (Madison, WI, USA); restriction enzymes and Klenow DNA polymerase from New England Biolabs Inc. (Beverly, MA, USA); Poly (dI-dC), [
-32P]dCTP, [-32P]dTTP, ProbeQuant G-50TM columns from Amersham Pharmacia Biotech Inc. (Piscataway, NJ, USA); the BCA Protein assay kit and bovine serum albumin (BSA) from Pierce (Rockford, IL, USA); polyclonal rabbit anti-NF-1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell cultures
BeWo (ATCC CCL-98) and JEG-3 (ATCC HTB-36) human choriocarcinoma cells were maintained in monolayer cultures in RPMI 1640 supplemented with 10% FCS.
Preparation of luciferase reporter constructs
A human P-LAP genomic clone containing the 5'-flanking region sequence was obtained and sequenced (Horio et al., 1999). The P-LAP promoter-luciferase constructs were prepared by cloning polymerase chain reaction (PCR)-derived fragments of the P-LAP 5'-flanking region into the vector pGL3-Enhancer. PCR was performed with 10 different sense oligonucleotides and one antisense oligonucleotide (Table I). A KpnI restriction site was added to the 5'-portion of all the primers. PCR fragments were digested with KpnI and subcloned into a similarly digested pGL3-Enhancer vector. Clones –752/+49, –396/+49, –297/+49, –265/+49, –242/+49, –216/+49, –172/+49, –144/+49, –94/+49 and –28/+49 (the transcription initiation site was numbered as +1) were all confirmed by restriction enzyme and sequencing analyses.
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Transient transfections of deletion constructs and luciferase assay
BeWo and JEG-3 cells were plated (0.8x106 cells/6-well plates) 24 h before transfection. Firefly luciferase reporter constructs (3 µg) and pRL-TK (0.3 µg) as an internal control to standardize transfection efficiency were transiently co-transfected into cultured cells at 50–60% confluence using LipofectAMINE PLUSTM Reagent according to the manufacturer's recommendations. Three hours after transfection, media were replaced by fresh normal growth media. Cells were harvested 36 h later, lysed and assayed for Firefly and Renilla luciferase activities using a dual-luciferase reporter assay system.
Preparation of nuclear extracts
Crude nuclear extracts were prepared from exponentially growing BeWo cells as described previously (Andrews and Faller, 1991). The concentration of the protein in the nuclear extracts was determined by the BCA protein assay. The nuclear extracts, which contained 2–6 mg/ml protein, were stored in aliquots at –70°C until use.
DNase I footprinting analysis
Probe preparation and DNase I footprinting analysis were performed as previously described (Galas and Schmitz, 1978; Nomura et al., 1997) with slight modification. Briefly, the luciferase vector containing the P-LAP –297/+49 fragment was digested with Bsu36I and the 3'-ends were labelled by Klenow fill-in reactions incorporating [-32P]dCTP and [-32P]dTTP. A second digestion by SacI generated the P-LAP insert labelled solely on the coding strand, which was then purified by agarose gel elecrophoresis. An aliquot of this probe (2–3x104 c.p.m.) was incubated with nuclear proteins or 10 µg BSA in the buffer composed of 20 mmol/l Tris–HCl (pH 7.9), 2 mmol/l MgCl2, 50 mmol/l NaCl, 1 mmol/l EDTA, 10% glycerol, 0.1% NP-40, 1 mmol/l dithiothreitol, followed by RQ1 DNase (0.25 unit) digestion. The final samples were run on an 8% sequencing gel with Maxam and Gilbert G tracks of the same DNA in an adjacent lane. The gels were exposed to X-ray film with intensifying screens at –70°C.
Electrophoretic mobility shift assay
Complementary single-stranded oligonucleotides designed with a 5'-overhang were annealed, labelled by end-filling using Klenow fragment and [-32P]dCTP, and purified by ProbeQuant G-50TM columns. 100 fmol of labelled probe (3x104 c.p.m./reaction) was incubated with nuclear extracts from BeWo cells in the binding buffer used for footprinting analysis. In the competition experiments, a 150-fold molar excess of the unlabelled competing DNA fragments was added before the addition of labelled probe. The oligonucleotides used as probes or competitors are indicated in Table II (with mutation sites underlined). For supershift experiments, 10 µg of rabbit polyclonal NF-1 antibodies which cross-react NF-1 isoforms were preincubated with the nuclear extracts for 20 min at room temperature. The protein–DNA complexes were separated on 5% non-denaturing polyacrylamide gels which were then dried and autoradiographed at –70°C.
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Statistical analysis
The transfection assays were performed three times in triplicate. All values (in the case of Figures 2 and 4
) were pooled for calculation of mean ± SD for paired t-test statistics using Statview 4.5 software (Abacus Concepts, Berkeley, CA, USA).
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Results
Analysis of the P-LAP 5'-flanking region for promoter activity
We have previously reported the sequences of P-LAP 5'-flanking region (Acc. No. AF145455) (Horio et al., 1999) and repeated analyses of the sequence revised the three nucleotides (Figure 1, shown in underlined bold type, `A' instead of `G' at –674; `A' removed from `TAG' at –529; `A' instead of `G' at –344). No typical mammalian TATA boxes are present but consensus sequences for several putative regulatory elements, Sp1, AP-2 and NF-1 (nuclear factor-1) are present. To define the DNA sequences of the 5'-flanking region responsible for promoter activity of the human P-LAP gene, we prepared chimera luciferase reporter constructs containing serially deleted fragments of the 5'-flanking sequence of P-LAP and transiently introduced these constructs into BeWo and JEG-3 cells for measuring promoter activity. Each plasmid construct and its corresponding relative luciferase activity, which was corrected for variations in transfection efficiency by reference to Renilla luciferase activity, is shown in Figure 2
. In both BeWo and JEG-3 cells, the elimination of sequences from position –752 to –396 or –297 had no significant effects on promoter activity; however, further deletion up to position –172 decreased promoter activity to 50% of the initial level. Subsequent deletions to position –94 reduced the activity to background levels and the shortest fragment, –28/+49, behaved similarly to fragment –94/+49. Since these results suggested that the region from –297 to –94 may contain positive control elements that are essential for efficient transcription of the human P-LAP gene, we focused on this region.
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DNase I footprinting analysis of the promoter region identifies four protected regions
To locate potential regulatory elements of the human P-LAP gene promoter, DNase I footprinting analysis was performed. As shown in Figure 3
, the addition of increasing amounts of crude nuclear extracts from BeWo cells resulted in at least four distinct regions protected from DNase I: FP1, –253 to –241; FP2, –233 to –219; FP3, –214 to –183; FP4, –170 to –150. A similar protection pattern was obtained with JEG-3 nuclear proteins (data not shown). DNase hypersensitive sites were clearly detected at positions –241, –233, –219, –214, –183, –170 and –150 (Figure 3, shown in asterisks).
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Functional analysis of protein-binding regions for P-LAP promoter activity
To determine the functional significance of the protein-binding regions detected by footprinting analysis, we constructed a series of serially deleted mutants extending from –297 to –94 and tested their ability to promote transcription in BeWo and JEG-3 cells (Figure 4
). Deletion of Sp1-binding sequence (in construct –265/+49), FP1 (in construct –242/+49) and FP2 (in construct –216/+49) had little effect on the relative promoter activity, while abolition of FP3 (in construct –172/+49) resulted in 62% suppression of luciferase activity in BeWo cells. In JEG-3 cells, similar results were obtained. These results indicate that the promoter region from –216 to +49 is sufficient to express efficient luciferase activity in these cells and suggest that functional elements for high level promoter activity reside within the boundaries of FP3.
Identification of proteins interacting with DNA at FP3 by EMSA
To study protein binding to FP3, we performed EMSA experiments using BeWo nuclear extracts. The labelled double-stranded oligomer which encompasses the FP3 footprint, wt3, (Table II) formed three major complexes and one minor complex (Figure 5A, lane 2 and 3). The major complexes C1–C3 are specific, since the formations were completely abolished by 150-fold molar excess of unlabelled wt3, whereas the fastest complex was hardly affected and therefore represented a non-specific interaction (Figure 5A, lane 4). Database analysis for transcriptional factors revealed that the FP3 region contains consensus binding sequences for NF-1, AP-2 and MZF-1 (myeloid zinc finger-1), which prompted us to test the binding ability of these factors to FP3. Synthetic oligonucleotides used in EMSA are shown in Table II. MZF-1 oligonucleotide was inefficient as a competitor for any of the retarded complexes (Figure 5A, lane 10). The NF-1 oligonucleotide appeared to compete weakly with C1 and C3 complexes, and more so with C2 (Figure 5A, lane 5), while mutated NF-1 oligonucleotide did not compete with any of the complexes (Figure 5A, lane 7). Therefore we tested the effect of anti-NF-1 antibodies on the mobility of the complexes (Figure 5A, lane 6), and revealed no marked shift of any complex. The AP-2 oligonucleotide competed weakly with C1 and C3 complexes and strongly with C2 (Figure 5A, lane 8), while the mutated AP-2 oligonucleotide was much less competitive (Figure 5A, lane 9). Simultaneous addition of oligonucleotide NF-1 and oligomer AP-2 completely out-competed only the C2 complex.
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In order to clarify the above findings, we mutated AP-2 and NF-1 sites in the wt3 oligonucleotide and tested the ability of these mutated sequences to interact with BeWo nuclear extracts. The labelled wt3 oligonucleotide with point mutations in the AP-2 binding site (mAP-2-wt3) only formed C1 and C3 complexes (Figure 5B
, lane 6), which were not competed by AP-2 and NF-1 oligonucleotides (Figure 5B, lanes 8 and 9). On the other hand, binding of nuclear proteins to the labelled wt3 oligonucleotide mutated in the NF-1 binding site (mNF-1-wt3) generated the same three complexes as those generated by wt3 (Figure 5B, lane 2). The AP-2 oligonucleotide efficiently out-competed the C2 complex (Figure 5B, lane 5), although addition of the competing NF-1 oligonucleotide did not modify any of the complexes (Figure 5B, lane 4). These results indicated that wt3 was able to produce an AP-2 complex, but not an NF-1 complex, with BeWo extracts. Discussion
The placenta is thought to be a main source of serum P-LAP during pregnancy (Mizutani et al., 1976; Yamahara et al., 2000) and play important roles in controlling labour pain through inactivating oxytocin (Mitchell and Wong, 1995; Mizutani and Tomoda, 1996). However, the basal transcriptional regulation of the P-LAP gene in any of the tissues, including placenta, has not been investigated. In the current study, we identified the sequence responsible for high promoter activity of the P-LAP gene and demonstrated the possible involvement of AP-2 transcription factor.
Our previous isolated clone containing 5'-flanking region of the human P-LAP gene (Horio et al., 1999) lacks obvious TATA boxes but possesses the initiator element (YY1; a consensus sequence; PyPyA+1NT/APyPy, where Py is a pyrimidine) and GC-rich region as seen in most TATA-less promoters (Smale, 1997). Although the human P-LAP promoter has highly conserved consensus binding sequences for Sp1, elimination of these sites did not significantly affect promoter activity in this study. A similar result was reported in the other gene promoters (Haun et al., 1993, Nishikawa et al., 2000). Sp1 is a minor protein in normal term human placenta (Hu et al., 1996), and this may account for the lower correlation between P-LAP promoter activity and Sp1 binding in choriocarcinoma cells. As another explanation, it is reported that Sp1 can bind to the GC-rich sequence within a few hundred base pairs upstream of the transcription start sites and activate transcription of certain genes, especially housekeeping genes (Bohm et al., 1995). Taking account of the positions of Sp1 sites in the P-LAP promoter region, the effects of Sp1 may be less potent on P-LAP promoter activity.
The 5'-deletion analysis of the P-LAP promoter region in BeWo and JEG-3 cells suggested that the sequence between –297 and –94 was important for driving high promoter activity in choriocarcinoma cells. P-LAP promoter analysis has also been performed with HEK293 cells (Rasmussen et al., 2000), demonstrating a functional region for high promoter activity upstream from our sites. Although the reasons for this difference are not clear, it is interesting that the sequence responsible for the high promoter activity in HEK 293 cells contains two Sp1 binding sites. The cell types used in the studies may contribute to this discrepancy, because HEK 293 cells are derived from the kidney and presumably possess different amounts of AP-2 and Sp1 proteins compared with choriocarcinoma cells. In fact, AP-2 is known to be abundant in placenta but in poor supply in kidney (Shi et al., 1997). Further, it is noteworthy that P-LAP gene expression in kidney is relatively low according to Northern blot analysis (Rogi et al., 1996).
Footprinting analyses on the region from –297 to –94 indicated that the sequence can be subdivided into at least four discrete protein binding regions surrounding the DNase I-hypersensitive sites, leading us to examine which protected region is essential for the high promoter activity. We therefore prepared a new series of P-LAP-luciferase constructs according to the results of the footprinting analyses and found that the FP3 region was important for the high level luciferase expression in choriocarcinoma cells. A computer search for potential binding sites of transcription factors in FP3 region showed the presence of a putative NF-1 half-site, as well as AP-2 and MZF-1 binding sites. In EMSA, binding of BeWo nuclear proteins to FP3 generated three retarded complexes C1, C2 and C3. Several oligomers were used as competitors in EMSA to test the ability of AP-2, MZF-1 and NF-1 factors to interact with FP3.
The three pieces of evidence from EMSA indicated that the second retarded band, C2, was generated by AP-2 binding to FP3: (i) C2 complex was competed out by the consensus sequence for AP-2; (ii) mutation in the AP-2 binding site in the competitor failed to compete with the C2 complex; (iii) the labelled FP3 oligomer with point mutations in the AP-2 binding site formed only C1 and C3 complexes. AP-2, originally described as the trophoblast-specific element-binding protein, has been identified as important for the placental expression of genes including those for human chorionic gonadotrophin (HCG) and ß subunit (Johnson et al., 1997), human growth hormone (HGH) (Courtois et al., 1990), human placental lactone (PL) (Richardson et al., 2000) as well as other genes (Yamada et al., 1995; Shi et al., 1997). It has been reported that AP-2 gene expression is induced during trophoblast differentiation (Johnson et al., 1997), and that differentiation-dependent transcription of the CG- gene in villous trophoblasts is mainly governed by increasing expression of AP-2 (Knofler et al., 2000). AP-2 has also been investigated for its role in mediating cAMP regulation of many genes including HCG (Johnson and Jameson 1999; LiCalsi et al., 2000). Although further experiments (such as investigating the effects of the mutation in AP-2 binding sites on P-LAP promoter activity) are required, the enhancement of human P-LAP gene expression during placental development, as demonstrated by Northern blot and immunohistochemical analyses may be regulated by AP-2. Interestingly, AP-2 binding sites are also present on the promoters of other genes that may link to the functions of P-LAP, i.e. the genes for oestrogen receptor (McPherson and Weigel, 1999), oxytocin receptor (Inoue et al., 1994) and vasopressin receptor (Rabadan-Diehl et al., 2000). Recently, AP-2 has also been reported to have implications for oestrogenic regulation (Perissi et al., 2000). Thus, AP-2 may also play an important role in controlling the activity of bioactive peptides such as oxytocin and vasopressin through the regulation of their receptor levels.
MZF-1 is preferentially expressed in haematopoietic cell lines, while recently it has been reported that MZF-1 may regulate LIMK2 gene expression in choriocarcinoma cell lines (Nomoto et al., 1999). Our competitive gel shift assays using a consensus binding sequence for MZF-1 (Kida et al, 1999) indicated little possibility of it binding to FP3. AP-2 protein may have a higher affinity with FP3 and interfere with MZF-1 binding at overlapping sites. Alternatively, a few nucleotides of MZF-1 binding site on the P-LAP gene are different from those on the LIMK2 gene, so MZF-1 may not bind to the P-LAP gene effectively.
The binding of AP-2 and NF-1 in a mutual way, which was reported on the HGH promoter (Courtois et al., 1990), was also not indicated on the P-LAP promoter, because the NF-1 consensus oligonucleotide did not behave as a competitor to any of the complexes. One possible explanation for our findings may relate to the different NF-1 isoforms. At least four proteins constitute a family of NF-1 transcription factors (Gronostajski, 2000) and the quantity of the different forms of NF-1 varies with the growth condition of the cells. To our knowledge, a detailed analysis concerning NF-1 in BeWo cells has not been performed. Another possibility is that the affinity of NF-1 to FP3 region may be rather weak, because the FP3 sequence contains just a half-site sequence for NF-1 binding. NF-1 binds strongly to the palindromic sequence, but weakly to a half-site sequence.
Until now we have been unable to identify the two other factors which form C1 and C3. AP-2 has been reported as an interactive partner of YY1 (Wu and Lee, 2000) and so in the human P-LAP promoter, YY1 and AP-2 might also be involved in regulatory network. We are currently performing a study to identify other proteins interacting with FP3.
In conclusion, our study is the first detailed analysis of the human P-LAP gene promoter and indicates that possible interactions between AP-2 and the FP3 region should modulate P-LAP gene expression. Elucidation of the underlying mechanisms for P-LAP gene regulation will pave the way to understanding the functional roles of P-LAP in vivo in labour pain. Further investigations of extracellular stimuli that could act as specific inducers or depressors of P-LAP gene expression may also facilitate the eventual development of therapeutic strategies for controlling oxytocin levels based on the modulation of P-LAP activities.
Acknowledgements
This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, and from the Ministry of Posts and Telecommunications of Japan for the specific medical research (collaboration with Nagoya Teishin Hospital).
Notes
3 To whom correspondence should be addressed. E-mail: snomura{at}med.nagoya-u.ac.jp 
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Submitted on January 24, 2001; accepted on July 9, 2001.
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