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Nutrient-Gene Interactions

RNA Polymerase II Association with the Phosphoenolpyruvate Carboxykinase (PEPCK) Promoter Is Reduced in Vitamin A–Deficient Mice¹

Departments of Nutritional Sciences and Molecular and Cellular Biology, The University of Connecticut, Storrs, CT 06269

²To whom correspondence should be addressed. E-mail: mmcgrane{at}canr.uconn.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Phosphoenolpyruvate carboxykinase (PEPCK) gene expression is decreased in vitamin A–deficient (VAD) mice. However, the underlying molecular mechanism at the PEPCK promoter that contributes to this alteration in gene expression remains unexplained and thus serves as the basis for our investigation in this report. Using liver from vitamin A–sufficient (VAS) and VAD mice in the chromatin immunoprecipitation (ChIP) assay, we determined that histones H3 and H4 were in the acetylated or active state in VAS mice at each of the three retinoic acid response elements (RARE1, RARE2 and RARE3) of the PEPCK promoter. The same acetylation pattern was seen in VAD mice, but with relatively lower levels of acetylated H3 and H4 bound at the region encompassing PEPCK RARE1/RARE2. In ChIP assays conducted with an antibody to RNA polymerase II (RNA Pol II), the association of RNA Pol II with PEPCK RARE1/RARE2 was significantly decreased in vitamin A deficiency. The reduction in RNA Pol II association is indicative of an interruption in the direct interactions of RNA Pol II with the PEPCK promoter, with general transcription factors and/or with coregulator molecules that contribute to the activation of the PEPCK gene. These results increase our understanding of the molecular basis for decreased PEPCK gene expression in VAD mice in vivo and offer additional insight into the regulation of other retinoid-responsive genes.

KEY WORDS: • phosphoenolpyruvate carboxykinase • retinoic acid response element • mice • retinoic acid • RNA polymerase II

Phosphoenolpyruvate carboxykinase [PEPCK² (EC 4.1.1.32)] is the enzyme that catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, the first committed step in hepatic gluconeogenesis. In vitamin A–sufficient (VAS) mice, PEPCK gene expression is highly induced in the food-deprived state, when blood glucose levels are reduced (1,2). In their vitamin A–deficient (VAD) counterparts, however, there is little to no induction of PEPCK gene expression (2). When food-deprived VAD mice are provided with supplemental retinoids, PEPCK gene expression is readily restored, indicating the indispensable role of vitamin A in the regulation of this gene (2).

Two hormone response elements associated with retinoid receptor binding have been identified in the rat PEPCK promoter and were classified as retinoic acid response elements (RARE) (3–5). In more recent years, a third, less characterized RARE that lies further upstream was studied in conjunction with regulation of the PEPCK gene in rat adipose tissue (6). The corresponding PEPCK RARE in mice are RARE1 (-451/-439), RARE2 (-337/-321) and RARE3 (-1018/-1006). RARE1 and RARE3 are both direct repeat (DR) elements arranged with one intervening nucleotide (DR-1), whereas RARE2 is a DR-5 element. Retinoid receptors and other nuclear receptors participate in the retinoid-mediated regulation of gene transcription by binding at RARE, with varying dimeric orientations depending on the configuration of the DNA and the ligand bound (7–9).

Molecular interactions among nuclear receptors and their coregulatory proteins occur within the context of the nucleosomal core structure of chromatin. In most cases, nuclear receptor binding serves to increase gene transcription by contributing to the weakening of histone/DNA interactions, making the DNA more accessible to basal transcription factors associated with RNA polymerase II (RNA Pol II) in the preinitiation complex (PIC) at the transcription start site (7,10,11). Nuclear receptors enhance gene transcription directly by interacting with components of the PIC and indirectly through interactions with coregulator molecules that associate with the PIC (7,11–15).

To more fully delineate the molecular complexes that form at PEPCK RARE in vivo, we began to explore the retinoid-mediated regulation of the PEPCK gene using the chromatin immunoprecipitation (ChIP) assay. Although this assay has become an increasingly popular method for identifying protein/DNA and protein/protein interactions in various cell systems, we are conducting the ChIP assay using chromatin isolated from mouse liver. Overall chromatin structure is an important parameter in the formation of molecular complexes that participate in coordinated regulation of the PEPCK gene. For example, histone acetylation is generally associated with the activation of gene transcription, whereas histone deacetylation is associated with the inhibition of gene transcription. We hypothesize that with alterations in vitamin A status, there are corresponding changes in the chromatin structure at the RARE and/or changes in coregulator binding and activity. In addition, interactions between the molecular complexes at the PEPCK RARE and the PIC including RNA Pol II at the transcription start site might also be subject to change under different nutritional conditions.

In this report, we present data generated using the ChIP assay that shows an alteration in RNA Pol II-association with hepatic PEPCK RARE1/RARE2 in vitamin A-deficiency. These data correspond to previous work conducted in our laboratory that showed a decrease in PEPCK gene expression under conditions of vitamin A-deficiency, thereby demonstrating a molecular basis for decreased PEPCK gene expression in VAD mice.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Mice.

For the generation of VAD mice, pregnant C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were fed the AIN76A diet without vitamin A (Dyets, Bethlehem, PA) (16) from d 10 of gestation until the pups were weaned. Pups were fed the VAD diet until they were killed at 9–10 wk of age. VAS mice were fed the same diet with 3600 retinol equivalents of retinyl esters/kg diet (Dyets). In earlier studies conducted in our laboratory, plasma retinol levels in VAD-deficient mice were 20% of those measured in VAS mice (2). In the current study, mean liver retinol levels in VAS mice were 2300 nmol/g, whereas mean liver retinol levels in VAD mice were decreased to 5.5 nmol/g liver, as measured by HPLC (18–20). At the time of killing, VAD mice did not differ from VAS mice in physical activity, body weight or food consumption.

Three hours before chromatin isolation, VAD mice undergoing retinoid treatments were supplemented with 10 mg/kg body weight of all-trans retinoic acid (RA) (Sigma-Aldrich, St. Louis, MO), 9-cis RA (Toronto Research Chemicals, North York, Canada) or both, delivered in peanut oil by gavage, with control counterparts administered only peanut oil.

All mice (VAS, VAD and treated) were food deprived for 15 h before they were killed for chromatin isolation. The above protocols were approved by the Institutional Animal Care and Use Committee at the University of Connecticut (Animal Care Protocol #E1200501).

Chromatin immunoprecipitation (ChIP) assay.

Chromatin isolation was carried out according to the methods of Farnham and colleagues (20) and Bennett and Osborne (21) with the following modifications. Dissected livers were minced and crosslinked with 1% formaldehyde for 30 min at room temperature. Phenylmethylsulfonyl fluoride and a protease inhibitor cocktail (both from Sigma-Aldrich) were used as protease inhibitors. The chromatin was sonicated on ice in 0.5-mL aliquots and pulsed 4 times for 5-s intervals on setting #5 of a Fisher Model 60 sonicator (Fisher Scientific, Pittsburgh, PA). Samples were frozen at -80°C until the immunoprecipitation (IP) step.

Before IP, chromatin was precleared (to reduce nonspecific binding) twice for 1 h at 4°C with rotation using either 70 µL protein A or G Sepharose (1.5 g/L; Zymed Laboratories, S. San Francisco, CA) that had been previously treated with 0.2 g/L salmon sperm DNA and 0.5 g/L bovine serum albumin.

The equivalent of 0.03 g of original liver was used in each ChIP assay. Either 25 µL of an antibody (Ab) to RNA Pol II [sc-899 (Santa Cruz Biotechnologies, Santa Cruz, CA)] or 1 µL of an Ab to acetylated or nonacetylated H3 or H4 [07–108, 06–866, 06–942, or 06–911 (Upstate Biotechnology, Lake Placid, NY)] was added to the chromatin sample (for IP) and an equal amount of TE (100 mmol/L Tris-Cl, pH 8.0/10 mmol/L EDTA, pH 8.0) to the corresponding chromatin sample being used as a negative control (No Ab). These samples were incubated with rotation overnight (14–16 h) at 4°C.

Equal amounts of IP and No Ab samples were added to 35 µL pretreated protein A or G Sepharose beads in a microspin chromatography column (BioRad Laboratories, Hercules, CA) for the IP step and incubated 1–2 h at 4°C with rotation. The columns were centrifuged at 1000 x g for 2 min at 4°C, and the beads in the columns were washed twice with 2 volumes of ice-cold wash buffer (1 mL/L Triton X-100/20 mmol/L Tris-Cl, pH 8.0/150 mmol/L NaCl/2 mmol/L EDTA), high salt wash buffer (1 g/L SDS/10 mL/L Triton X-100/2 mmol EDTA/20 mmol Tris-Cl, pH 8.0/500 mmol NaCl), LiCl wash buffer (0.25 mmol/L LiCl/10 mL/L Igepal/10 g/L deoxycholate/1 mmol/L EDTA/10 mmol/L Tris-Cl, pH 8.1) and once with TE, followed by 2-min of centrifugation at 1000 x g.

The immune complexes were eluted by two successive 3-min incubations with 150 µL elution buffer [10 g/L SDS/50 mmol/L NaHCO₃] at 65°C. After each elution, the samples were centrifuged at 1000 x g for 2 min and the eluates pooled for the respective conditions.

To reverse the formaldehyde crosslinks, the NaCl concentration was adjusted to 0.3 mol/L, and 1 µL RNase A (10 g/L) was added per 200 µL of original diluted chromatin. The samples were mixed briefly on a vortex mixer and incubated at 65°C for 3–4 h. DNA was purified using the Qiaquick Purification Kit (Qiagen, Valencia, CA) and subjected to PCR.

PCR.

PCR reactions (50-µL) contained 4 µL DNA from IP or No Ab samples, along with 0.6 µmol/L of each primer, 200 µmol/L each dATP, dCTP, dGTP and dTTP, 1X PCR buffer containing 1.5 mmol/L MgCl₂ (Qiagen) and 1.25 U HotStarTaq DNA Polymerase (Qiagen). After 29 cycles of PCR amplification with an annealing temperature of 57°C, PCR products were run on a 1.5% agarose gel, visualized and quantified by ethidium bromide staining using Quantity One v.4.1 software (BioRad Laboratories). Primers used for the region encompassing mouse PEPCK RARE1/RARE2 were 5'-AGGTAACACACCCCAGCTAAC-3' and 5'-GGCTCTTGCCTTAATTGTCAG-3' and for mouse PEPCK RARE3, 5'-GGCATGAAGGTCTGTGGCTAC-3' and 5'-TAGACACCATCACCCTTGGAG-3' (see Fig. 1A).

View larger version (36K): [in this window] [in a new window]   FIGURE 1 Acetylated histones H3 and H4 are bound at phosphoenolpyruvate carboxykinase (PEPCK) retinoic acid response element (RARE)1/RARE2 and RARE3 of the PEPCK promoter in vitamin A–deficient (VAD) or –sufficient (VAS) mice. Chromatin immunoprecipitation (ChIP) assays were conducted using antibodies specific to nonacetylated and acetylated (Ac) histones H3 and H4 and primers specific to the regions encompassing RARE1/RARE2 or RARE3 of the PEPCK regulatory domain (A). Negative controls (No Ab) are ChIP assays performed without the use of antibodies, and the relative intensity of the positive signal is given as a fold-increase over that of the No Ab signal, which is set to 1 for each experiment. H3 and H4 at PEPCK RARE1/RARE2 in VAS (B) and VAD (D) mice. H3 and H4 at PEPCK RARE3 in VAS (C) and VAD (E) mice. The results shown are representative of a minimum of three ChIP assays that were conducted using chromatin isolated from at least three individual mice; the fold differences are given as mean ± SEM.

Results of ChIP assays were reported as mean image densities ± SEM, with the negative control (No Ab) for each ChIP assay set to a value of 1. Differences between group means were analyzed by Student’s t tests and were considered significant at P < 0.05. All data were evaluated using MS Excel 97 (Microsoft, Redmond, WA).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Acetylation of histones H3 and H4 at PEPCK RARE.

PEPCK gene transcription is responsive to vitamin A and regulated at least in part by multiple RARE in the PEPCK promoter. To directly examine RARE involvement in transcription complex formation, we investigated the acetylation states for histones H3 and H4 at PEPCK RARE1/RARE2 and at RARE3 because H3 and H4 are frequent targets for many known histone acetyltransferases (HAT) [for reviews, see (22,23)]. Both H3 and H4 were predominantly acetylated in VAS mice at each RARE (Figs. 1B and 1C); using chromatin isolated from VAD mouse liver, the same pattern of histone acetylation was evident (Figures 1D and 1E). In VAS mice, the relative amount of acetylated H3 and H4 bound at RARE1/RARE2 was greater than that in VAD mice (Figures 1B and 1D).

Bridging between PEPCK RARE1/RARE2 and RNA Pol II.

To investigate a potential bridging effect between molecular complexes at the PEPCK RARE and the PIC at the transcription start site, we conducted ChIP assays using chromatin isolated from food-deprived VAS mice, an antibody to the large subunit of RNA Pol II and primers specific to PEPCK RARE1/RARE2 or RARE3. There was an increase in RNA Pol II association with the PEPCK promoter as measured by its linkage to the region encompassing PEPCK RARE1/RARE2 (Fig. 2A). RNA Pol II was not associated with PEPCK RARE3, however, which is centered at –1012 bp from the transcription start site (Fig. 2A). The interaction of RNA Pol II with the PEPCK RARE, therefore, is limited to the RARE that are relatively close to the start site of transcription and are likely linked through some intermediary regulatory protein.

View larger version (16K): [in this window] [in a new window]   FIGURE 2 RNA Pol II association at the PEPCK promoter in vitamin A–deficient (VAD) or –sufficient (VAS) mice. ChIP assays were conducted using an antibody to RNA polymerase II (Pol II) and primers specific to the regions encompassing RARE1/RARE2 or RARE3 (panel 1A). (A) RNA Pol II is associated with PEPCK RARE1/RARE2 (-451/-321) but not with RARE3 (-1018/-1006) in VAS mice. ChIP assays were conducted comparing RNA Pol II association with PEPCK RARE1/RARE2 in food-deprived and fed VAS mice (B) and in food-deprived VAS and food-deprived VAD mice (C). Negative controls (No Ab) are ChIP assays performed without the use of antibodies, and the relative intensity of the positive signal is given as a fold-increase over that of the No Ab signal, which is set to 1 for each experiment. The results shown are representative of a minimum of four ChIP assays that were conducted using chromatin isolated from at least two individual mice; the fold differences are given as means ± SEM. See legend to Figure 1 for other abbreviations.

Because PEPCK gene expression is upregulated when blood glucose levels are low, we would expect to see a difference in the relative association of RNA Pol II with the PEPCK promoter in the fed and food-deprived states. When fed and food-deprived VAS mice were compared, the association of RNA Pol II with the PEPCK promoter, as measured by its linkage to RARE1/RARE2, was almost 2 times greater in food-deprived mice than in their fed counterparts (P = 0.0004) (Fig. 2B). These results indicate that RARE1/RARE2 bridging or association with RNA Pol II occurs under the same conditions as does increased transcription of the PEPCK gene, demonstrating that an interruption in that linkage is at least partly responsible for decreased PEPCK gene expression.

Effect of vitamin A deficiency on RNA Pol II association with the PEPCK promoter.

Past work in our laboratory showed that in VAD mice, PEPCK gene expression is significantly decreased even after prolonged food deprivation (2). There was a nearly 60% decrease in the association of RNA Pol II with the PEPCK promoter in VAD compared with VAS mice (Fig. 2C). These data are consistent with our previous work that showed decreased PEPCK gene expression under the same nutritional conditions (2). These results also provide evidence that an interruption in RNA Pol II association contributes to decreased PEPCK gene expression.

In combination, all-trans and 9-cis RA increase the association of RNA Pol II with the PEPCK promoter.

All-trans and 9-cis RA are the identified ligands for the nuclear receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), respectively (24,25). PEPCK gene expression is enhanced by treatment with all-trans RA and 9-cis RA in vitro in H4IIE rat hepatoma cells (3–5,26,27). In addition, we showed that supplementation of VAD mice with all-trans RA restores PEPCK gene expression (2). The results of supplementation of VAD mice with all-trans RA, 9-cis RA or both, as outlined in Materials and Methods are shown in Figure 3. There was no difference in RNA Pol II association at RARE1/RARE2 when mice were given individual retinoids, but when all-trans and 9-cis RA were given in combination, there was an increase in RNA Pol II association (P = 0.001) (Fig. 3). These data suggest that the presence of both retinoids is necessary to restore the RNA Pol II linkage to PEPCK RARE1/RARE2 that is lost in vitamin A deficiency.

View larger version (43K): [in this window] [in a new window]   FIGURE 3 Supplementation of vitamin A–deficient (VAD) mice with all-trans retinoic acid (RA) and 9-cis RA leads to the recruitment of RNA polymerase (Pol) II to mouse PEPCK RARE1/RARE2. ChIP assays were conducted as described in Figure 2 using chromatin from food-deprived VAD mice that were untreated, or treated with vehicle alone (peanut oil), all-trans RA, 9-cis RA, or both all-trans and 9-cis RA. Primers used were specific to the region encompassing RARE1/RARE2 (as shown in Fig. 1A). Negative controls (No Ab) are ChIP assays performed without the use of antibodies, and the relative intensity of the positive signal is given as a fold-increase over that of the No Ab signal, which is set to 1 for each experiment. The results shown are representative of a minimum of three ChIP assays that were conducted using chromatin isolated from at least two individual mice; the fold differences are given as means ± SEM. See legend to Figure 1 for other abbreviations.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The potential decrease in coregulator activity does provide some explanation for the decreased PEPCK gene expression we measured with vitamin A deficiency, but it is likely that decreased histone acetylation is merely one contributing factor. Because coactivators can act as bridging molecules between the PEPCK RAREs and the PIC, and because there was a reduction in the amount of RNA Pol II that was bound at the promoter, it may be that one result of vitamin A deficiency is a decrease in HAT activity and another is a decrease in the ability of a coactivator(s) to act as an intermediary factor(s) between the PEPCK RARE and the transcription start site.

In the generalized activation of gene transcription, the following events occur at the promoter: 1) nuclear receptors bind at DNA hormone response elements; 2) regulatory proteins, some with inherent HAT activity, are recruited to nuclear receptors either as preformed complexes or in succession; and 3) general transcription factors and RNA Pol II assemble at the transcription start site. The purpose of nuclear receptor and cofactor assemblages, individually and collectively, is the same, i.e., to loosen chromatin for increased access of the basal transcription machinery and RNA Pol II to the DNA for activation of gene transcription. The sequence in which these related events takes place remains unclear, but from the work presented here, it can be confirmed that vitamin A induction of PEPCK gene transcription corresponds to a relative increase in histone acetylation and an increase in RNA Pol II association with the PEPCK promoter.

In an ongoing examination of the molecular mechanism that drives retinoid regulation of the PEPCK gene, we identified the primary nuclear receptors that bind to the three RARE in the PEPCK promoter using nuclear extracts from mouse liver and the electrophoretic mobility shift assay (Fig. 4) (28). Of the nuclear receptors tested, hepatic nuclear factor-4 binds in the greatest abundance, and only to RARE1 (28). RXR binds to RARE1, 2 and 3 with an intermediate affinity, whereas RAR and chicken ovalbumin upstream promoter transcription factor-2 bind both RARE1 and RARE2, although in much lower amounts (28). Knowledge of the predominant nuclear receptors that bind the PEPCK RARE is valuable information for identifying accessory molecules, such as coactivators, that might be involved with regulation of the PEPCK gene.

View larger version (24K): [in this window] [in a new window]   FIGURE 4 Nuclear receptors bound at RARE in the mouse PEPCK promoter. This schematic shows the positioning of the RARE in the mouse PEPCK promoter in relation to the transcription start site. The primary nuclear receptors that bind to the RARE are depicted with hypothetical dimeric orientations based on the configurations of the DNA response elements. The PIC is comprised of general transcription factors (TFIID/TBP, TFIIB, TFIIF, TFIIE and TFIIH) and RNA polymerase II. Abbreviations: COUP, chicken ovalbumin upstream-transcription factor-2; DR-1, direct-repeat element with one intervening nucleotide; DR-5, direct-repeat element with five intervening nucleotides; HNF, hepatic nuclear factor-4; RAR, retinoic acid receptor-; RARE, retinoic acid response element; RXR: retinoid X receptor-.

All-trans RA and 9-cis RA are ligands for the nuclear receptors, RAR and RXR (24–26), respectively, both of which bind mouse PEPCK RARE1 and RARE2 (28). 9-cis RA can also bind the - and ß-isoforms of RAR with an affinity that is comparable to that of all-trans RA (24). In this study, supplementation of VAD mice with all-trans RA and 9-cis RA increased the association of RNA Pol II with the region encompassing PEPCK RARE1/RARE2, suggesting that increased transcription is mediated by retinoid receptors binding at these sites. In VAD mice expressing a transgene driven by either a -355 bp (containing RARE2) or a -460 bp (RARE1 and RARE2) PEPCK promoter sequence, there were differences in the response to retinoid supplementation. All-trans RA stimulated PEPCK gene expression in both transgenic lines, whereas 9-cis RA enhanced gene transcription only at the PEPCK(-460) transgene and to an even greater extent than does all-trans RA (28). These data indicate that although RARE2 is sufficient for the response to all-trans RA, RARE1 is essential for the PEPCK gene’s response to 9-cis RA. Our latest data show that in the context of the native promoter, both retinoids were necessary to induce a response in VAD mice. All-trans and 9-cis RA might be working in a synergistic fashion, a possibility that is consistent with the concept of a nonpermissive RXR/RAR heterodimer bound at RARE2 (7,47). On the basis of results from in vitro studies, a consensus DR-1 site such as PEPCK RARE1 is potentially bound by an RXR/RXR homodimer, and a consensus DR-5 such as RARE2 by an RXR/RAR heterodimer (48,49). For the PEPCK gene prototype, complexes at both RARE contribute to an overall increase in gene transcription in VAD mice supplemented with all-trans and 9-cis RA.

The RARE comprise only one of many recognized hormone response units that reside in the PEPCK promoter, all of which contribute to the multifaceted regulation of the PEPCK gene. Despite existing data that has focused on other regulators such as glucocorticoids, cAMP and thyroid hormone, there remain a number of unanswered questions specific to the regulation of the PEPCK gene by retinoids. It is also relevant that much of the research on the PEPCK gene was conducted using cell systems, whereas in this report, we show data from studies conducted in the whole animal. This is the first report that presents data on retinoid-mediated PEPCK regulation using the ChIP assay and chromatin isolated directly from mouse liver. Further study is warranted to elucidate which coregulator molecules are involved with the coordinated regulation of the PEPCK gene and, specifically, by retinoids. In addition, it will be interesting to determine which components of the molecular complexes at the RARE are associated with general transcription factors and which interrupted associations are the cause of the decreased association of RNA Pol II with the PEPCK promoter in vitamin A deficiency. Resolving these questions will further our current understanding of retinoid-mediated PEPCK gene regulation and contribute to a more comprehensive understanding of other genes that are regulated by retinoids.

	ACKNOWLEDGMENTS

 
We thank Mary K. Bennett in the laboratory of Timothy F. Osborne at the University of California at Irvine for technical assistance with the ChIP assay.

	FOOTNOTES

 
¹ Supported by U.S. Department of Agriculture Grant #2003–00883 (to M.M.M.).

³ Abbreviations used: Ab, antibody; CBP (cAMP response element binding protein)-binding protein; ChIP, chromatin immunoprecipitation; DR, direct repeat; HAT, histone acetyltransferase; PEPCK, phosphoenolpyruvate carboxykinase; PIC, preinitiation complex; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element; RNA Pol II, RNA polymerase II; RXR, retinoid X receptor; SRC-1, steroid receptor coactivator-1; VAD, vitamin A–deficient; VAS, vitamin A–sufficient.

Manuscript received 31 July 2003. Initial review completed 20 August 2003. Revision accepted 19 September 2003.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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