Molecular Human Reproduction, Vol. 9, No. 12, pp. 793-798, 2003
© European Society of Human Reproduction and Embryology 2003; all rights reserved
Characterization of human trophoblast as a mineralocorticoid target tissue
Division of Medical Sciences and Fetal Medicine, University of Birmingham, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, UK
1 To whom correspondence should be addressed. e-mail: m.d.kilby{at}bham.ac.uk
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Abstract |
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In mineralocorticoid target tissues, 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) confers mineralocorticoid receptor selectivity by metabolizing hormonally active cortisol to inactive cortisone, allowing aldosterone access to the receptor. This enzyme is also expressed in high abundance in fetal tissues, particularly in placental trophoblast, where a role has been proposed in regulating fetal growth and development by protecting the fetus from maternal hypercortisolaemia and modulating local glucocorticoid receptor (GR), rather than mineralocorticoid receptor-mediated responses. As such the placenta has not been considered a mineralocorticoid target tissue. We have used conventional RT–PCR and real-time quantitative RT–PCR to demonstrate that primary cultures of term human cytotrophoblast express the mineralocorticoid-responsive genes Na/K-ATPase (
1 and ß1 subunits), epithelial sodium channel (ENaC, and 
subunits) and the serum and glucocorticoid-inducible kinase (SGK). SGK expression was found to be rapidly and strongly induced by corticosteroids (24- and 38-fold by 10–7 mol/l aldosterone and 10–7 mol/l dexamethasone respectively after 1 h). Dexamethasone-, but not aldosterone-stimulated SGK induction was inhibited by GR antagonist (RU38486), confirming the presence of a functional mineralocorticoid receptor and suggesting that placental trophoblast expresses a functional mineralocorticoid receptor, which is in part responsible for the corticosteroid regulation of SGK expression. Placental 11ß-HSD2 may protect the MR in a fashion analogous to classical mineralocorticoid tissues to modulate trophoblast sodium transport. Key words: cytotrophoblasts/mineralocorticoid receptor/serum and glucocorticoid inducible kinase/trophoblast
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Introduction |
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The classical action of aldosterone is to stimulate sodium transport across epithelial cells in the distal nephron, distal colon and salivary gland (Verrey et al., 1987). At a cellular level, this is now known to involve the induction of the basolateral Na/K-ATPase (Verrey et al., 1987; Escoubet et al., 1997) and apical epithelial sodium channel (ENaC) (Baxendale-Cox et al., 1997; Garty, 2000), the latter being dependent upon de-novo transcription of serum and glucocorticoid-inducible kinase (SGK) (Chen et al., 1999). The mineralocorticoid receptor (MR) is non-selective in vitro, demonstrating the same inherent affinity for both aldosterone and cortisol (Arriza et al., 1988). Aldosterone (Aldo) gains access to the MR in vivo due to ‘pre-receptor metabolism’ of cortisol (F) to inactive cortisone (E) by the type 2 isozyme of 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) (Edwards et al., 1988; Funder et al., 1988). If expression of this enzyme is compromised, as seen in the inherited syndrome of apparent mineralocorticoid excess (AME) (Stewart et al., 1987) or following liquorice ingestion (Stewart et al., 1996), cortisol interacts with the MR and severe mineralocorticoid hypertension ensues. In the ‘classical’ mineralocorticoid target tissues, distal nephron, colon and salivary gland, 11ß-HSD2 is co-expressed with the MR. This serves to protect the receptor from glucocorticoid excess in an autocrine fashion.
11ß-HSD2 is also expressed in high abundance in specific fetal tissues, particularly trophoblast, where its role remains uncertain (Brown et al., 1993; Stewart et al., 1994, 1995; Sun et al., 1997; Pepe et al., 1999; Driver et al., 2001). Glucocorticoid excess in fetal life impairs fetal growth and the suggestion has arisen that placental 11ß-HSD2 acts as a ‘barrier’, protecting the fetus from high maternal cortisol concentrations, thereby facilitating normal growth and development (Edwards et al., 1993). Enzyme expression is attenuated in pregnancies complicated by severe intrauterine growth restriction (IUGR), in keeping with this suggestion (Shams et al., 1998; McTernan et al., 2001). The association of low birthweight with adult diseases including hypertension (Barker et al., 1989a), diabetes mellitus (Hales et al., 1991) and coronary artery disease (Barker et al., 1989b) has added a new impetus to these studies and 11ß-HSD2 has emerged as a putative factor in explaining Barker’s epidemiological data. In specific fetal tissues, notably kidney and colon, 11ß-HSD2 is co-expressed with the glucocorticoid receptor (GR) rather than MR (Condon et al., 1998). This suggests that any modulatory effects of 11ß-HSD2 upon fetal growth may impact through the GR rather than the MR. However, data analysing the corticosteroid regulation of 15-hydroxyprostaglandin dehydrogenase support the expression of a functional MR within human placenta (Patel et al., 1999). Recent studies have also noted MR immunoreactivity (Hirasawa et al., 2000) and MR mRNA together with specific binding of aldosterone to MR (Driver et al., 2001) in human syncytiotrophoblast and cytotrophoblast cells. Furthermore, trophoblast itself is inherently an ‘epithelial tissue’ involved in the transport of numerous ions (including sodium) between mother and fetus (Clarson et al., 1996; Stulc, 1997). The possibility that mineralocorticoids may regulate placental sodium transport throughout gestation should be considered, particularly in view of the current paucity of data on the pathogenesis of pre-eclampsia and factors controlling fetal growth.
In this paper we have identified the expression of mineralocorticoid-responsive genes required for epithelial sodium transport (Na/K-ATPase, ENaC, SGK) in human primary cultures of cytotrophoblast and have documented their regulation by corticosteroids.
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Materials and methods |
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Subjects
Placentae were obtained from women (23–33 years of age) undergoing elective Caesarean section at term (38–41 weeks) at the Birmingham Women’s Hospital, according to local ethical committee approval. All participants in the study gave written informed consent. All were uncomplicated pregnancies with appropriately grown fetuses. None of the mothers had any medical conditions.
Primary cultures of human cytotrophoblast cells
Term placental cytotrophoblast (CT) cells were from isolated term human placentae using previously described methods (Driver et al., 2001). In brief, CT cells were plated at a density of 4x106 cells/well in 6-well plates and cultured at 37°C and 5% CO2 in medium consisting of Dulbecco’s modified Eagle’s medium with 25 mmol/l HEPES (Life Technologies Ltd, UK), 4.4% F12–Ham’s nutrient mix (Life Technologies), 10% fetal bovine serum (Life Technologies), 1% PSG [0.6 g benzyl penicillin, 1 g streptomycin sulphate, 2.92 g L-glutamine (Sigma) in 100 ml ultra-pure water] 0.1% gentamicin (50 mg/ml; Sigma, UK). CT cells were cultured for 72 h and media changed to charcoal-stripped fetal bovine serum (FBS) for 24 h prior to experimentation.
Conventional RT–PCR analysis of 1-Na-K-ATPase, ß1-Na-K-ATPase, -ENaC, -ENaC, and SGK expression
Total RNA was extracted from 72 h primary cytotrophoblast cultures, adult human liver and kidney tissue biopsies (11ß-HSD type 1 and 2 positive tissues respectively) using classic methods based on that of Chomczynski et al. (1987) (RNAzol B; ams Biotechnology, UK). cDNA was synthesized from 1 µg total RNA, using avian myeloblastoma viral (AMV) reverse transcriptase (RT)-driven primer extension from random hexamer oligonucleotides (Promega, UK). 10% of each RT reaction was used as a template for PCR amplification using oligonucleotide primers designed specifically for 1-Na-K-ATPase (5'-GATGCTGCTCACCATCAGTG-3' and 5'-TATTTGGGCTGCAGGAGTTT-3') and amplified a region of 594 bp, primers for ß1-Na-K-ATPase (5'-CCAGCATGTTCAGAAGCTCA-3' and 5'-GGAGCATCCACAGGA GAGAG-3') amplified a region of 597 bp. Primers for -ENaC (5'-CCAGCTACCAGCTCTCTGCT-3' and 5'-TTCTCACACCAAGGCAGATG-3') and the -ENaC subunit (5'-GTGCCAATCAGGAACATCTACA-3' and 5'-CACTTTCAACTCTGCTTTGCAC-3') amplified regions of 601 and 696 bp respectively. Primers for SGK (5'-GCTTTGTCCTGTCCTTCTGC-3' and 5'-AGGGCAGTTTTGGAAAGGTT-3') amplified a region of 699 bp. For semi-quantitative PCR, primers and competitors (1:4) for 18S ribosomal RNA, which yield a product of 488 bp, were incorporated according to the manufacturer’s guidelines (Ambion, UK). For all primers, an initial denaturing step (95°C for 6 min) was followed by 32 cycles of annealing (55°C for 1 min), extension (72°C for 1 min) and denaturing (95°C for 1 min), and a single extension termination step (72°C for 10 min). Negative controls employed products from reverse transcriptase reactions without template AMV. Positive/negative controls of adult human liver and kidney RNA were also used where appropriate. PCR products were visualized using 2% agarose gel electrophoresis. Semi-quantitative analysis was performed by densitometry on the products of triplicate reactions each run twice on 2% agarose gels (UVP:Gelbase/Gelblot).
Regulation of SGK mRNA expression
Following 24 h incubation in media containing charcoal-stripped FBS, cells were incubated with aldosterone (10–7 to 10–10 mol/l) or dexamethasone (10–7 mol/l) (Sigma), with or without a 100-fold excess of the GR antagonist RU38486 (Roussel Uclaf, France). Total RNA was collected (as above) following 0, 0.5, 1 and 8 h for aldosterone and dexamethasone (DEX) time-course experiments, and 1 h for dose–response and RU38486 assays. RNA was then subject to RT–PCR for SGK (as above). Dilutions of aldosterone, dexamethasone and RU38486 were performed such that final ethanol concentrations were <0.01%.
Real-time quantitative PCR analysis of SGK expression
Real-time quantitative PCR was also employed as a method of monitoring changes in SGK expression levels. This technique was used on cDNA generated from total RNA extracts taken from cells treated as above.
Probes and primers specific for human SGK were designed from the published gene sequences (NM005627) using the Primer ExpressTM software (PE Applied Biosystems). Primers were synthesized by Alta Bioscience (UK). Probes were produced by Oswell DNA Services (UK). PE Applied Biosystems supplied 18S housekeeping primers and probe, which were multiplexed in reactions with the primers and probes for the mRNA of interest. SGK forward primer had the sequence 5'-CATGCAAACACCCTGAAGTTCA-3', the reverse primer was 5'-GAAGGGTTGGCATTCATAAGCT-3', and the hybridization probe was 5'-TCCATCTTGAAGATCTCCCAACAACCT CAGGA-3'. Reactions were performed in 96-well plates using an ABI 7700 sequence detection system (PE Applied Biosystems). 25 µl reactions incorporated TaqMan Universal PCR Master Mix (3 mmol/l Mn[Oac]2, 200 µmol/l dNTP, 1.25 IU AmpliTaq Gold polymerase, 1.25 IU AmpErase UNG), 900 nmol each primer, 100 nmol hybridization probe, and 50 ng cDNA. Once loaded, samples were heated at 50°C for 2 min and 95 °C for 10 min, followed by 44 cycles of 95°C for 15 s and 60°C for 1 min.
Statistical analysis
All statistical analysis was performed using Sigma Stat for windows 2.03 (SPSS Inc.). The analysis employed was one-way analysis of variance with all pair-wise multiple comparison procedures (Tukey–Kramer test). Results were expressed as the mean of three experiments ± SE. For real-time quantitative PCR, potential bias due to averaging data that had been passed through the equation 2–
Ct was avoided by analysing data at the Ct stage to give relative fold-changes in mRNA levels. With comparison between groups being achieved by an unpaired t-test.
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Results |
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RT–PCR analysis of
1-Na-K-ATPase, ß1-Na-K-ATPase, -ENaC, and -ENaCRT–PCR analyses of mRNA isolated from primary cultures of cytotrophoblasts, at 72 h, demonstrated the presence of mRNA for
1-Na-K-ATPase and ß1-Na-K-ATPase, with products corresponding to the predicted sizes of 594 and 597 kb respectively (Figure 1). Further analyses showed expression of both -ENaC and -ENaC, with RT–PCR products at 601 and 696 kb respectively (Figure 2). However, the ß-ENaC subunit was not detectable.
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RT–PCR and real-time quantitative PCR analysis of SGK expression
RT–PCR also showed the presence of mRNA encoding SGK. Using a semi-quantitative method of duplexing SGK primers with those specific for 18S ribosomal RNA, 10–7 mol/l aldosterone and 10–7 mol/l dexamethasone increased SGK expression 24.3 ± 1.0 (mean ± SEM) and 38.9 ± 0.7-fold respectively after 1 h (Figure 3A and Figure 4A). These data were supported by the findings of real-time quantitative PCR, where incubation with 10–7 mol/l aldosterone resulted in significant increases in SGK mRNA expression after 0.5, 1 and 8 h (6.55-, 17.34- and 19.74-fold respectively, compared to expression at time 0), as shown in Figure 3B (P < 0.01 in all cases). These data were calculated from mean ± SEM
Ct values of 15.6 ± 0.1, 12.9 ± 0.2, 11.4 ± 0.2 and 11.3 ± 0.4 (0, 0.5, 1 and 8 h respectively) and were normalized for 18S ribosomal RNA expression. Incubation with 10–7 mol/l dexamethasone resulted in 9.6-, 16.5- and 22.1-fold increases in SGK mRNA expression after 0.5, 1 and 8 h respectively, compared with expression at time 0 (P > 0.01 in all cases), as shown in Figure 4B. These data were calculated from mean Ct values of 15.7 ± 0.1, 12.4 ± 0.1, 11.4 ± 0.3 and 11.1 ± 0.6 (0, 0.5, 1 and 8 h respectively) and were normalized for 18S ribosomal RNA expression.
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Furthermore, the increase in expression of mRNA encoding SGK following incubation with aldosterone appeared to be dose dependent. Figure 5A shows semi-quantitative RT–PCR for SGK and 18S ribosomal RNA, following incubation with a range of aldosterone concentrations. Repetition of this experiment using the quantitative real-time PCR technique indicated that significant increases in SGK mRNA expression occurred with all concentrations of aldosterone used. Compared with untreated (control) cells, the fold increases in SGK mRNA expression after 1 h pre-treatment with 10–10, 10–9, 10–8, 10–7 and 10–6 mol/l aldosterone were 4.9, 8.3, 16.3, 20.7 and 21.4 respectively. These data were calculated from mean
Ct values of 15.7 ± 0.1, 13.7 ± 0.1, 12.3 ± 0.5, 11.8 ± 0.3, 11.3 ± 0.5, and 11.1 ± 0.1 (control, 10–10, 10–9, 10–8, 10–7 and 10–6 mol/l aldosterone respectively) where P < 0.05 with 10–10 mol/l aldosterone and P > 0.01 with 10–9 through to 10–6 mol/l aldosterone. All reactions were normalized for 18S housekeeping gene expression (Figure 5B).
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Receptor functionality
As can be seen in Figure 6A, SGK induction seen following incubation with 10–7 mol/l dexamethasone was reduced by co-incubation with a 100-fold excess of the GR antagonist RU38486. By contrast RU38486 had a minimal effect on the aldosterone induction of SGK mRNA, suggesting that dexamethasone exerts its effect via the GR and aldosterone via the MR.
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Again these data were endorsed by findings from real-time quantitative PCR which showed that, compared with untreated (control) cells, the fold increase in SGK mRNA expression following 1 h incubation at 37°C with 10–7 mol/l aldosterone, 10–7 mol/l aldosterone and 10–5 mol/l RU38486, 10–7 mol/l dexamethasone and 10–7 mol/l dexamethasone + 10–5 mol/l RU38486, was 16.95, 8.27, 14.09, 18.29 and 4.08 respectively. These data were calculated from mean
Ct values of 15.1 ± 0.2, 11.0 ± 0.2, 11.2 ± 0.5, 10.9 ± 0.3, and 12.8 ± 0.3 (control, 10–7 mol/l aldosterone, 10–7 mol/l aldosterone + 10–5 mol/l RU38486, 10–7 mol/l dexamethasone or 10–7 mol/l dexamethasone + 10–5 mol/l RU38486 respectively). The change in expression between treatment with 10–7 mol/l aldosterone and 10–7 mol/l aldosterone + 10–5 mol/l RU38486 was not significant. However, the loss of induction between cells treated with 10–7 mol/l dexamethasone + 10–5 mol/l RU38486 was significant with P < 0.01 (Figure 6B). |
Discussion |
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The exact role of 11ß-HSD2 within human trophoblast is unknown. In human adult tissues, this isozyme is closely associated with mineralocorticoid-responsive epithelia—kidney collecting ducts, colonic mucosal epithelial cells and salivary gland—where it co-localizes with the MR to protect the receptor from illicit occupancy by cortisol. Congenital deficiency of the enzyme (AME syndrome) or inhibition of the enzyme with liquorice are rare causes of hypertension in which cortisol acts as a potent mineralocorticoid (Stewart et al., 1987, 1996).
Since the original description of 11ß-HSD activity in human placenta in 1960 (Osinski, 1960), a series of publications has highlighted the abundant expression of 11ß-HSD2 in human trophoblast (Brown et al., 1993; Stewart et al., 1994, 1995; Sun et al., 1997; Pepe et al., 1999; Driver et al., 2001). Correlations between enzyme activity and birthweight in some studies (Stewart et al., 1995), but not all (Rogerson et al., 1997), support a role for the enzyme in regulating fetal growth. Placental 11ß-HSD2 efficiently inactivates the relatively high maternal cortisol concentrations protecting the developing fetus from the potentially harmful effects of this steroid. Further support for this hypothesis comes from studies that have documented reduced expression of 11ß-HSD2 in pregnancies complicated by severe intrauterine growth restriction (Shams et al., 1998; McTernan et al., 2001). An alternative endocrine role for placental 11ß-HSD2 is that by maintaining relatively low fetal cortisol concentrations, 11ß-HSD2 facilitates the ontogeny of the fetal HPA axis (Pepe and Albrecht, 1990; Stewart et al., 1995).
One possibility, hitherto largely overlooked in placenta, was that 11ß-HSD2 might serve to protect the MR in an analogous fashion to ‘classical’ mineralocorticoid target tissues such as kidney and colon. In earlier studies, we had identified MR mRNA in human trophoblast tissue (Driver et al., 2001). Recent studies have also immunolocalized MR protein to human syncytiotrophoblast and cytotrophoblast cells (Hirasawa et al., 2000). In addition, data from Patel et al. (1999) indirectly suggested the presence of a functional MR within placenta. These experiments analysed the expression of placental 15-hydroxyprostaglandin dehydrogenase, and demonstrated inhibition of this enzyme following addition of cortisol with an IC50 of 0.1 nmol/l, results that are more in keeping with a MR-mediated rather than GR-mediated event.
In our studies in primary cultures of human trophoblast, we have demonstrated the binding of aldosterone to high affinity sites (Kd 
1 nmol/l) in the presence of the GR antagonist RU38486, highly suggestive of expression of a functional MR (Driver et al., 2001).
In contrast to GR-mediated gene transcription, very few MR target genes have been characterized. In sodium-transporting epithelia, mineralocorticoids increase sodium transport by increasing the expression of subunits of the basolateral Na-K-ATPase (Mujais et al., 1984; Verrey et al., 1987; Whorwood et al., 1995) and apical sodium channel (Denault et al., 1996; Baxendale-Cox et al., 1997; Escoubet et al., 1997; Garty, 2000). However, such changes are probably not mediated by a direct transcriptional effect but by secondary changes to intracellular sodium or by early-inducible intermediate factors. A mineralocorticoid target gene that undergoes rapid induction is SGK, which then plays a crucial role in activating the apical sodium channel through a process involving phosphorylation of Nedd4 and the ubiquitination of ENaC (Chen et al., 1999; Staub et al., 2000). In addition to defining the expression of Na-K-ATPase subunits and ENaC subunits, we have further characterized the corticosteroid regulation of SGK within placental trophoblast. In keeping with observations in mineralocorticoid-responsive tissues such as kidney and colon, both glucocorticoids (dexamethasone) and aldosterone induced a rapid, dose-dependent increase in SGK mRNA levels. The GR antagonist RU38486 blocked the induction seen following incubation with dexamethasone, but RU38486 had no significant effect upon the aldosterone induction of SGK, confirming the presence of a functionally active MR within human trophoblast.
Sodium transport across the placenta has been studied in some detail across many species. Many sodium transporters including extrusion of sodium by placental Na-K-ATPase have previously been documented (Stulc et al., 1993; Clarson et al., 1996), but this is the first description of expression of the epithelial sodium channel subunits and SGK within trophoblast. Radioactive tracer studies indicate that placental transport of sodium does indeed occur and that this greatly exceeds the fetal uptake of sodium, suggesting a bi-directional process. Whilst most transfer is thought to be passive, active transport, particularly by Na-K-ATPase as gestation advances, has been documented (Stulc, 1997). Our data suggest that corticosteroids may regulate this process. In other sodium-transporting epithelia, this is controlled by aldosterone or glucocorticoid (cortisol in man) depending upon the activity of 11ß-HSD2. As 11ß-HSD2 is expressed in abundance in human trophoblast and increases further with advancing gestation, inactivating F to E, the placental MR is likely to be highly selective for aldosterone in vivo.
It is of interest that Pepe et al. (2001) have recently described differential localization of 11ß-HSD1 (in the microvillous membranes) and 11ß-HSD2 (associated with the basal membrane) within the baboon and human syncytiotrophoblast. The expression of 11ß-HSD2 in and around the basal membrane may be important in its role in controlling glucocorticoids delivery to specific areas within the trophoblast cell (Pepe et al., 2001).
It remains to be seen whether aberrant mineralocorticoid regulation of placental sodium transport is of pathological significance. Fetuses affected with AME grow poorly and have abnormal placental morphology (Kitanaka et al., 1996; Geller et al., 2000) but whether this can be attributed to increased mineralocorticoid activity within the placenta is uncertain. Mutations within the ligand-binding domain of the MR have recently been described that result in progesterone (normally an MR antagonist) acting as a potent agonist. Hypertension across pregnancy was the resulting phenotype, but the underlying mechanism was thought to represent renal mineralocorticoid activation (Milford et al., 1995). The possibility that mineralocorticoid excess within the placenta might represent a novel autocrine system in the aetiology of pregnancy-induced hypertension and/or pre-eclampsia is a logical extension of these observations.
In conclusion, human trophoblast expresses a functional mineralocorticoid receptor; the aldosterone regulation of SGK indicates that mineralocorticoids may play a role in placental sodium transport. As with ‘classical’ mineralocorticoid tissues such as kidney and colon, the MR is protected from illicit occupancy from F by 11ß-HSD2. Further studies are required to define the physiological and pathophysiological consequences of mineralocorticoid-dependent processes within trophoblast.
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Acknowledgements |
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We wish to thank Colin Sibley and Susan Greenwood for advice and technical assistance regarding the primary cell culture technique. This work was supported by the MRC, Action Research, and ESAC. P.M.S. is an MRC Senior Clinical Fellow.
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Submitted on February 6, 2003; resubmitted on July 23, 2003. accepted on July 30, 2003
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