SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Temporal/spatial expression of retinoid binding proteins and RAR isoforms in the postnatal lung

Matthew Hind, Jonathan Corcoran, and Malcolm Maden

Medical Research Council Centre for Developmental Neurobiology, King's College London, London SE1 9RT, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endogenous retinoids have been implicated in alveologenesis in both the rat and the mouse, and exogenous retinoic acid (RA) can reverse or partially reverse experimental emphysema in adult rat and mouse models by an unknown mechanism. In this study, we examine the cellular and molecular biology of retinoid signaling during alveologenesis in the mouse. We describe the temporal and spatial expression of the retinoid binding proteins CRBP-I, CRBP-II, and CRABP-I using RT-PCR and immunohistochemistry. We identify the retinoic acid receptor isoforms RAR-alpha 1, RAR-beta 2, RAR-beta 4, and RAR-gamma 2 and describe their temporal and spatial expression using RT-PCR and in situ hybridization. We demonstrate that both retinoid binding proteins and RAR isoforms are temporally regulated and found within the alveolar septal regions during alveologenesis. These data support a role of dynamic endogenous RA signaling during alveolar formation.

lung development; alveolar regeneration; bronchopulmonary dysplasia; emphysema


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALVEOLI ARE FORMED as a developmentally regulated, largely postnatal event in rats, mice, and humans (1, 4, 41). Retinoids are a family of molecules derived from vitamin A (retinol) and include the biologically active metabolite retinoic acid (RA). Alveologenesis is associated with dramatic changes in the metabolism of endogenous retinoids in both rats and mice from storage forms such as retinyl esters to metabolites such as retinol and RA (11, 14, 27). Both retinol and RA are bound in the cytoplasm by binding proteins, cellular retinol binding protein (CRBP-I and CRBP-II), and cellular retinoic acid binding protein (CRABP-I and CRABP-II). These families of binding proteins regulate the biological action of retinol and RA (31). Previous Northern blot analysis of CRBP-I and CRABP-I mRNA has demonstrated upregulation of both genes during alveologenesis in both whole rat lung tissue and isolated lipid-laden fibroblasts (27). Dexamethasone administered to postnatal rat pups between postnatal day 4 (P4) and P14 disrupts alveolar septation (21) and results in downregulation of CRBP-I and CRABP-I (37). The spatial distribution of CRBP-I and CRABP-I within lung tissue during alveologenesis has not been described.

The cellular effects of RA are mediated through the action of two classes of nuclear receptors, the retinoic acid receptors (RARs), which are activated by all-trans-RA and 9-cis-RA, and the retinoid X receptors (RXRs), which are activated by 9-cis-RA only (16). RARs are of three major subtypes, alpha , beta , and gamma , of which there are numerous isoforms created by alternative splicing and differential promoter usage (17). RARs form heterodimers with RXRs and act as ligand-activated transcription factors to regulate downstream gene expression. RXRs can act as homodimers or heterodimers with a variety of orphan receptors such as peroxisome proliferator-activated receptor (16). Elastin is a major structural component of the alveolus. Both the expression of its precursor gene, tropoelastin, and elastin deposition are regulated during alveologenesis (9, 32, 35). RA has been shown to regulate transcription of the tropoelastin gene in vitro (26), suggesting that tropoelastin may be a target gene of RA during alveologenesis.

It has been suggested, on the basis of differential expression patterns in the embryo, that each of the RAR isoforms has specific roles in development (5). Alveologenesis in the rat is associated with transcriptional upregulation of the RAR genes (27), and recent analysis of alveologenesis in RAR null mutants has demonstrated altered patterns of alveolar formation, with RAR-beta a negative (24) and RAR-gamma a positive (25) factor in the regulation of alveologenesis. Exogenous RA has been shown to increase the number of alveoli in the neonatal rat (22) and can reverse features of pulmonary emphysema in the adult rat (3, 23, 36). From these observations, it is clear that RARs may provide a unique therapeutic target in the development of highly specific agents to manipulate the formation of alveoli.

In this study, we sought to identify elements of the retinoid-signaling pathway during postnatal alveologenesis in the mouse. Using RT-PCR together with primers specific to the gene of interest, we have searched for 25 known retinoid-signaling genes. Of these genes, we identify and characterize temporal expression patterns of the retinoid binding proteins CRBP-I, CRBP-II, and CRABP-I, the specific isoforms of the retinoid receptors RAR-alpha 1, RAR-beta 2, RAR-beta 4, and RAR-gamma 2, through alveolar formation. We use immunohistochemistry to localize CRBP-I and CRABP-I proteins and in situ hybridization with specific RNA riboprobes to describe the spatial expression of the RAR genes in the postnatal mouse lung. We suggest a molecular model of RA signaling involving retinoid binding proteins and RARs that regulate the effects of endogenous RA during alveologenesis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care. All experiments were conducted in accordance with local ethics committee guidelines using outbred TO mice (Harlan, UK), both male and female neonatal mice at postnatal P1, P4, P9, P15, and adult female (over 8 wk of age). All animals were given access to water and laboratory chow ad libitum.

Tissue preparation. P1, P4, P9, P15, and adult animals were culled by neck dislocation, and their lungs were dissected free. Lung tissue for RT-PCR analysis was removed, washed in phosphate-buffered saline (PBS), and immediately stored at -70°C. Lungs for in situ hybridization studies and immunocytochemistry were dissected out and removed. The trachea was cannulated, tied firmly in place, and infused with either 4% paraformaldehyde (PFA) or perfix (4% PFA, 20% isopropryl alcohol, and 2% trichloroacetic acid) at a pressure of 20 cmH2O for 24 h. The lungs were then dehydrated through a graded series of alcohol solutions and xylene and embedded in paraffin wax. The lungs were sectioned at 5 µm, and the sections were mounted on polylysine-coated glass slides (BDH, Dorset, UK).

RT-PCR analysis. To identify which retinoid binding proteins and RAR isoforms were present during alveologenesis, semiquantitative RT-PCR was used. RNA was extracted using a Qiagen RNAeasy kit, and cDNA was prepared with the use of an Amersham first-strand cDNA synthesis kit, as described in the manufacturer's instructions. RNA extractions were performed from at least four lungs from each age group and analyzed separately. The primers used (see Table 1) were from mouse CRBP-I, CRBP-II, CRABP-I, CRABP-II, RARs (RAR-alpha 1-7, RAR-beta 1-4, RAR-gamma 1-7), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (15, 18, 29, 40). Amplification was carried out in the linear range for each primer, and their levels of expression were compared with GAPDH. Amplification was carried out as follows: denaturation for 30 s at 95°C, annealing for 30 s at 55°C, and extension for 30 s at 72°C. One-fifth of the resultant product was then run on a 1% agarose gel. Gels were scanned and analyzed by gel analyst software from Scion (available free of charge at http://www.scioncorp.com). The gels were normalized for GAPDH expression. Each experiment was repeated at least three times with similar results.

                              
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Table 1.   Specific mouse primers used in RT-PCR

In situ hybridization. Digoxygenin-labeled riboprobes were synthesized from the appropriate cDNA. Slides were rehydrated, washed once with PBS, and fixed in 4% PFA for 30 min. They were then washed twice for 5 min in PBS-0.05% Tween (PBT) and dehydrated through graded ethanol solutions. Hybridization was carried out at 55°C overnight with a 1:100 dilution of RNA probe. The buffer consisted of 50% formamide, 5× SSC, 0.05% heparin, 0.5% Tween 20, and 1% yeast tRNA. Slides were washed sequentially for 15 min at 55°C in 50% hybridization buffer, 50% 2× SSC, 2× SSC, and finally in 0.2% SSC. They were then washed at RT for 5 min each in 75% 0.2× SSC, 25% PBT, 50% 0.2× SSC, 50% PBT, 25% 0.2× SSC, 75% PBT, and PBT. Slides were blocked in 2% sheep serum in PBT for 1 h and incubated with anti-digoxygenin antibody overnight at 4°C. Slides were then washed eight times in PBT for 2 h and were developed using nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt according to instructions (Boehringer Mannheim). Control experiments using sense probes and absent probe revealed no signal.

Immunohistochemistry. Slides were dewaxed to PBS through graded alcohol solutions and blocked with goat serum for 1 h. They were then incubated in primary antibody at 4°C overnight and washed three times in PBS. Subsequent steps using secondary antibody, avidin-biotin complex reagent, and diaminobenzidine were performed according to a Vector ABC Elite kit (Vector Labs, Peterborough, UK). The monoclonal CRABP-I antibody was obtained from Affinity Bioreagents, and the affinity-purified CRBP-I and CRABP-I antibodies were gifts from Dr. U. Eriksson (Stockholm, Sweden). Appropriate dilutions of antibody were previously established. Controls for immunoreactivity were absent primary antibody, nonimmune serum, and other IgG antibodies. Slides were lightly counterstained with hematoxylin after immunohistochemistry.

Statistical analysis. A time course analysis of the RT-PCR data was performed using one-way analysis of variance statistics with Bonferroni's adjustment for multiple comparisons. Results were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The temporal expression of the retinoid binding proteins during alveolar septation. Previous studies have indicated that, in the mouse, alveolar septation occurs between P4 and P14 (1). To identify mRNA for retinol and RA binding proteins during alveologenesis, we used RT-PCR with RNA isolated from P1, P4, P9, P15, and adult mouse lung tissue and primers specific to CRBP-I, CRBP-II, CRABP-I, and CRABP-II. We positively identified CRBP-I, CRBP-II, and CRABP-I in all stages of postnatal lung; we could not identify CRABP-II in any of the tissues.

The temporal expression of CRBP-I, CRBP-II, and CRABP-I mRNA can be seen in the RT-PCR studies (Fig. 1, A-C, respectively). CRBP-I, CRBP-II, and CRABP-I mRNAs are all significantly upregulated in the early postnatal mouse lung during alveolar septation. CRBP-I peaks at P9 (P < 0.05 compared with adult), CRBP-II peaks at P4 (P < 0.05 compared with adult), and CRABP-I peaks in the P9 lung (P < 0.05 compared with adult). These data are in general agreement with the previous Northern blot analysis of CRBP-I and CRABP-I mRNA with both binding proteins significantly upregulated during the period of alveologenesis in the postnatal rat lung (27, 34). CRBP-II mRNA has been identified previously in postnatal lung tissue (33) but was not studied during the period of alveologenesis.


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Fig. 1.   Semiquantitative RT-PCR data demonstrating temporal expression of retinoid binding protein mRNA in the postnatal mouse lung. PCR products were visualized using ethidium bromide, and a representative gel is displayed. The PCR products of the gene of interest were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using Scion image analysis software and are represented graphically. A: expression of cellular retinol binding protein (CRBP)-I mRNA in the postnatal lung demonstrating a peak at postnatal day 9 (P9; P < 0.05 compared with adult when time course data are compared with ANOVA). B: CRBP-II peaks at P4 (P < 0.05 compared with adult). C: cellular retinoic acid binding protein (CRABP)-I peaks at P9 (P < 0.05 compared with adult). Data are expressed as means ± SE from at least 4 separate experiments. *P < 0.05. M = marker, bluescript cut with HpaII; Ad, adult.

CRBP-I protein localization in the postnatal mouse lung. To verify our RT-PCR analysis and examine the spatial distribution of CRBP-I protein during alveolar septation, we used an affinity-purified CRBP-I-specific antibody on sections of P1, P4, P9, P15, and adult lung. The preparation and specificity of this antibody for mouse tissue have been reported previously (10). Specifically, it does not cross-react with CRBP-II or CRABP-I (13). CRBP-I protein was identified in the alveolar septal tissue but not in the bronchial epithelium (Fig. 2, A and B). The locations of cells expressing signal changes through the stages examined are shown in Fig. 3, A-D. In the P1 lung, only occasional weakly stained cells are identified in the thick walls of the alveolar saccules (Fig. 3A). The pattern of staining is increased in the P4 lung with CRBP-I protein identified in the erupting primary septa and alveolar septal tissue (Fig. 3B). There is widespread, but not ubiquitous, CRBP-I protein labeled in the P9 lung, with strongly labeled alveolar septal cells adjacent to unlabeled alveolar septal cells (Fig. 3C). The P15 lung has a much more restricted pattern of CRBP-I protein expression, with only scattered cells labeled. The pattern of intracellular labeling is different in the P15 lung compared with the P9 lung, in which the labeling is more discrete and punctate (Fig. 3D).


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Fig. 2.   Immunohistochemical localization of CRBP-I demonstrating the spatial distribution of CRBP-I protein in sections of P9 mouse lung counterstained with hematoxylin. A: CRBP-I protein is not detected in the bronchial epithelium (Br). B: in contrast, CRBP-I is abundant in alveolar septal regions (Al). Arrows indicate CRBP-I-positive cells (labeled brown). Note some cells in the alveolar septa remain unlabeled. Control experiments using nonimmune serum showed no labeling (data not shown). Scale bar = 15 µm.



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Fig. 3.   Immunohistochemical localization of CRBP-I protein demonstrating both the temporal and spatial restriction of CRBP-I during postnatal alveologenesis on sections of P1, P4, P9, and P15 mouse lung. A: there are a few scattered cells staining for CRBP-I in the thick walls of the alveolar saccules (arrow). B: in the P4 lung, CRBP-I labeling is more intense with a greater number of cells labeled. There is CRBP-I protein identified in the erupting alveolar septa (arrows). C: the widespread spatial pattern of distribution of CRBP-I is maintained in the P9 lung, with the most intense labeling. D: in the P15 lung, signal is less intense and has a restricted pattern of distribution with fewer cells labeled and a different pattern of intracellular labeling. The signal appears punctate and restricted within the cells to small inclusions. Arrows indicate labeled cells (identified as brown). All immunohistochemistry was carried out under identical experimental parameters. Control experiments revealed no signal (data not shown). Scale bar = 15 µm.

CRABP-I localization in the postnatal mouse lung. We used both an affinity-purified polyclonal antibody and a commercially available monoclonal antibody to CRABP-I to observe the spatial expression of this protein during the period of alveologenesis. The preparation and specificity of the affinity-purified antibody for mouse has been previously reported (20). Specifically, it does not cross-react with CRABP-II (10). CRABP-I protein is identified in the alveolar septal regions in a similar distribution to the CRBP-I protein (Fig. 4). Consistent with the RT-PCR data of CRABP-I mRNA, we identify CRABP-I protein at all stages examined. Like the distribution of the CRBP-I protein, CRABP-I protein is identified strongly in the alveolar septal regions and pleural mesothelial cells but not in bronchial epithelium. Within the alveolar septal region, there are strongly labeled cells adjacent to unlabeled alveolar septal cells. The precise identity of CRABP-I-labeled cells is unknown.


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Fig. 4.   Immunohistochemical localization of CRABP-I demonstrating the spatial distribution of CRABP-I protein in the P9 mouse lung. CRABP-I protein has a similar, though more restricted, pattern of distribution to CRBP-I protein. A: CRABP-I is not detected in the bronchial epithelium. B: CRABP-I is identified in the alveolar septal regions. Using a commercially available monoclonal CRABP-I antibody, we detected CRABP-I protein in all stages examined, consistent with the RT-PCR data for mRNA. Arrows indicate labeled CRABP-I cells (identified as brown). Scale bar = 15 µm.

RAR isoform identification and temporal expression in the postnatal mouse lung. Previous Northern blot and RT-PCR studies have revealed changes in RAR gene expression during alveolar formation in the postnatal rat lung (27). The expression of the various RAR isoforms has not been reported during alveologenesis in mouse. In this study, we used RT-PCR with primers specific to each of the seven RAR-alpha , four RAR-beta , and seven RAR-gamma isoforms, to identify the major isoforms of the three RAR genes through alveolar septation. Of the 18 isoforms analyzed, only RAR-alpha 1, -beta 2, -beta 4, and -gamma 2 mRNA were identified in the postnatal mouse lung, and all showed dramatic changes in temporal expression during alveologenesis. All identified isoforms are significantly upregulated at P4 (P < 0.05) compared with adult (Fig. 5, A-D).


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Fig. 5.   Semiquantitative RT-PCR analysis of the retinoic acid receptor (RAR) isoforms during alveologenesis in the mouse. The RAR isoforms alpha 1, beta 2, beta 4, and gamma 2 were identified using specific primers and cDNA isolated from P1, P4, P9, P15, and adult lung. PCR products were visualized with ethidium bromide. A representative gel is shown in each case. To normalize the data, gels were scanned with Scion image analysis software, and the gene of interest was normalized to the housekeeping gene, GAPDH. The graphs represent data from a minimum of 4 separate experiments for each stage and are represented as means ± SE. A: RAR-alpha 1 mRNA expression in the postnatal lung. B: RAR-beta 2 mRNA expression. C: RAR-beta 4 mRNA expression. D: RAR-gamma 2 mRNA expression. The temporal expression patterns reveal significant regulation of all identified isoforms in the neonatal lung. All isoforms are significantly upregulated at P4 compared with adult (P < 0.05). *P < 0.05.

Spatial expression of RARs in the postnatal mouse lung. The in situ hybridization studies confirm the RT-PCR findings that RAR-alpha , -beta , and -gamma are present in postnatal mouse lung tissue (Fig. 6). RAR expression is localized to bronchial epithelium, bronchial and vascular smooth muscle, pleura, and scattered cells within the alveolar regions, some of which have the characteristic morphology of type II pneumocytes. More specific cell identification is beyond the resolution of this study. These results are in general agreement with previous studies identifying RARs in bronchial epithelium (8), lipid-containing fibroblasts (27), and type II cells (30). No obvious differences in the distribution of these receptors were noted over time.


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Fig. 6.   In situ hybridization studies using digoxigenin-labeled antisense riboprobes demonstrating the spatial expression of RARs in the adult lung. Signal can be identified as blue. RAR-alpha (A), RAR-beta (B), and RAR-gamma (C) mRNA expression can be identified in bronchial epithelium (BE), bronchial and vascular smooth muscle surrounding large blood vessels (BV), and in scattered cells in the alveolar septal regions (arrows). There were no obvious differences in the spatial distribution of the RARs. Control experiments using sense probes and absent probe were carried out under identical experimental conditions with no signal detected. Scale bar = 50 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have chosen to examine retinoid signaling during mouse alveologenesis to exploit both the powerful genetics and molecular biology available to this model. Alveolar formation in the rat is associated with transcriptional regulation of CRBP-I and CRABP-I (27) mRNA (37). Our RT-PCR data in the mouse support this association. In addition, we identify another retinoid binding protein gene, CRBP-II, that is also transcriptionally regulated during alveologenesis. The function of CRBP-II is not well understood. It has been largely studied in the intestinal epithelium, where it is thought to function in the uptake of dietary retinoids (33). Both the temporal and spatial restriction of the retinoid binding proteins suggest a role for these genes in modulating RA signaling during alveolar formation. Interestingly, neither CRBP-I nor CRABP-I protein is detected in the bronchial epithelium, a tissue known to be extremely retinoid sensitive. One of the earliest features of vitamin A deficiency is the metaplastic transformation of pseudostratified epithelium into squamous keratinizing epithelium (38, 39). Therefore, it would be interesting to examine the distribution of CRBP-II in the postnatal lung. We speculate that bronchial epithelium might express this protein.

Although retinoid binding proteins are temporally and spatially associated with alveologenesis, CRBP-I null mutants have no reported lung phenotype (12). However, more recent in vitro results demonstrate that CRBP-I null mutant lungs are more sensitive to the effects of a pan-RAR antagonist than wild-type controls (28), suggesting that the CRBP-I null mutant lungs do indeed have a subtle phenotype. To our knowledge, a detailed morphometric analysis of alveolar architecture in the CRBP-I null mutants has not been published.

Of the 18 reported isoforms of the RARs, we identify RARs -alpha 1, -beta 2, -beta 4, and -gamma 2, all of which are transcriptionally regulated in the postnatal mouse lung. All identified isoforms are significantly upregulated in the neonatal period compared with expression in the adult, suggesting a role in endogenous RA signaling. Data from RAR-beta null mutants, together with the use of RAR-beta -specific agonists, suggest that RAR-beta functions as an endogenous inhibitor of alveologenesis (24). Conversely, analysis of the RAR-gamma -/-/RXR-alpha +/- compound null mutants has revealed defects in alveologenesis, suggesting a requirement for RAR-gamma (25). In light of the identification of the RAR isoforms, this suggests that either RAR-gamma 2 may function as a positive regulator of alveologenesis or RAR-beta 2/RAR-beta 4 may function as negative regulators of alveologenesis, or both. We have previously reported the value of retinoid receptor-specific agents, both agonists and antagonists in nerve and limb regeneration studies (6, 7, 19). Further use of these agents may be important in deciphering the role of RAR regulation in both alveologenesis and alveolar regeneration.

The temporal and spatial distribution of the RARs suggests not only a role in alveologenesis but also a function in the adult lung. The effects of vitamin A deficiency on the bronchial epithelium are well known (38, 39). More recently, vitamin A depletion has been reported to result in defects in the alveolar epithelium (2) with areas of emphysema-like changes. It is tempting to speculate that, in the adult animal, endogenous RA acting via binding proteins and RARs has a role in mediating endogenous alveolar repair.

In summary, this study demonstrates, in a second species of altricial animals, the association of alveologenesis with transcriptional regulation of retinoid binding protein and RAR genes. We provide data on both the temporal and spatial distribution of CRBP-I and CRABP-I and identify the specific isoforms of the RARs associated with alveolar formation. This is further evidence to support a central role of retinoid signaling in postnatal alveologenesis. Understanding the molecular and cellular basis of alveolar formation will be fundamental to the development of novel therapies that promote alveolar regeneration or repair in diseases such as bronchopulmonary dysplasia or emphysema.


    ACKNOWLEDGEMENTS

We thank Dr. Ulf Eriksson, Ludwig Institute, Stockholm, Sweden, for the CRBP-I and CRABP-I antibody and Professor P. Chambon, IBCM, Strasbourg, France, for the RAR-alpha , RAR-beta , and RAR-gamma plasmids; and Katie Adams for technical assistance.


    FOOTNOTES

All work is funded by Wellcome Trust. M. Hind is a Wellcome Trust Research Training Fellow.

Address for reprint requests and other correspondence: M. Hind, Centre for Developmental Neurobiology, Fourth Floor New Hunts House, King's College London, Guy's Campus, London SE1 9RT, United Kingdom (E-mail: matthew.hind{at}kcl.ac.uk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00196.2001

Received 5 June 2001; accepted in final form 15 October 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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