PGE2-mediated inhibition of T cell p59fyn is independent of cAMP

Mashkoor A. Choudhry, Zulfiqar Ahmed, and Mohammed M. Sayeed

Trauma and Critical Care Research Labs, Departments of Surgery and Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We recently observed that prostaglandin E2 (PGE2)-mediated suppression of T cell functions could result from an attenuation of p59fyn protein tyrosine kinase activity. The present study evaluated the effects of an adenylate cyclase agonist (forskolin) and antagonist (SQ-22536), as well as those of cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cell p59fyn kinase activity. The study allowed us to assess whether PGE2-mediated activation of adenylate cyclase by itself or the elevation in intracellular cAMP levels is an integral event in the modulation of anti-CD3-linked p59fyn activation in T cells. The experiments were carried out with splenic T cells from male Sprague-Dawley rats. A 30-50% suppression in the autophosphorylation and the kinase activity of p59fyn in T cells incubated with PGE2 or forskolin was observed. Pretreatment of T cells with SQ-22536 prevented significant PGE2-mediated inhibition of T cell p59fyn kinase activity. In contrast, no change in p59fyn autophosphorylation and kinase activity in T cells treated with cAMP analogues was observed. These data suggest that PGE2-mediated suppression of p59fyn autophosphorylation and kinase activity in T cells is dependent on the activation of adenylate cyclase and independent of the elevation in cAMP levels.

protein tyrosine kinase; src kinase; adenylate cyclase; phosphodiesterase; rat; prostaglandin E2


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PROSTAGLANDIN E2 (PGE2) has been shown to suppress T cell interleukin-2 (IL-2) production and proliferation in a number of inflammatory conditions including burn, trauma, and sepsis (2, 9, 15, 20, 21, 33, 39, 40). In addition to inhibiting T cell proliferation, PGE2 has been shown to suppress macrophage antigen-presenting ability (4, 22). A decrease in T cell activation, either as a result of direct PGE2 effect or due to a defect in antigen presentation, could contribute to an overall decrease in host resistance and an increased susceptibility to both opportunistic and nonopportunistic pathogens.

The activation of T cells is primarily induced via stimulation of the T cell receptor (TCR)-CD3 complex; it could be initiated in vitro via TCR ligation with lectins or with the antibody against CD3 (1, 8, 12, 13). The earliest biochemical events after TCR ligation are increases in the activation of a number of protein tyrosine kinases including p59fyn, p56lck, and Zap-70 (19, 23, 28). The activation of these kinases leads to phosphorylation of phospholipase C-gamma (PLC-gamma ), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (8, 39). Whereas IP3 stimulates Ca2+ release from the intracellular stores (7), DAG activates the protein tyrosine kinase C activity (35). An increase in intracellular Ca2+ concentration ([Ca2+]i) sustained for several hours precedes T cell activation and its subsequent proliferation activity (42).

PGE2, acting on its receptors on T cells, stimulates adenylate cyclase to enhance the formation of cAMP (10, 11, 31, 38-40). The elevation of cAMP levels in T cells has been implicated in the inhibition of T cell IL-2 production, IL-2 receptor (IL-2R) expression, and proliferation (2, 5, 9, 20, 34, 38, 40). Our previous studies suggested that PGE2-mediated suppression of T cell proliferation could result from a disturbance in Ca2+ signaling (16, 17). Further, we have shown an inhibitory effect of PGE2 on p59fyn kinase activity, a component upstream to Ca2+ signaling (18). To investigate the mechanism of PGE2 modulation of p59fyn, the present study evaluated the effects of the incubation of T cells with cAMP analogues and with adenylate cyclase agonist forskolin on p59fyn activation. Also, this study ascertained the effects of adenylate cyclase inhibitor SQ-22536 on p59fyn kinase modulation by PGE2 to determine whether adenylate cyclase activation is vital to PGE2-mediated modulation of p59fyn in T cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. PGE2, forskolin, and enolase were purchased from Sigma (St. Louis, MO). Dibutyryl-cAMP (DBcAMP), 8-bromo-cAMP (8-BrcAMP), IBMX, and SQ-22536 were purchased from Calbiochem-Novabiochem (La Jolla, CA). Monoclonal antibodies to p59fyn (residues between amino acids 85 and 206 of human Fyn protein) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rat CD3 antibodies were purchased from Pharmingen. [gamma -32P]ATP was obtained from DuPont NEN (Boston, MA). The specific activity of [gamma -32P]ATP is 3,000 Ci/mmol. Reagents for the SDS-PAGE were obtained from Bio-Rad (Richmond, CA). An Immobilon-P membrane (polyvinylidene difluoride) for the transfer of proteins was obtained from Millipore (Bedford, MA). Protein molecular weight markers and other reagents needed for the preparation of lysis buffer, wash buffer, and kinase buffer were obtained from Sigma. Nylon wool was obtained from Polysciences (Warrington, PA). Ficoll-Paque was purchased from Pharmacia (Uppsala, Sweden).

T cell preparation. Adults rats (~250 g) were anesthetized (pentobarbital sodium; 65 mg/kg body wt) to remove the spleen and then were killed with a pentobarbital sodium overdose. Splenic T lymphocytes were isolated by previously described methods (16-18). Briefly, spleens were gently ground to prepare a single cell suspension. Red blood cells and the dead cells from the suspension were removed by density gradient centrifugation with Ficoll-Paque. Splenocytes appearing at the interface of Ficoll-Paque and the medium were collected and added to nylon wool-packed columns. These columns were preequilibrated with Hanks' balanced salt solution (HBSS) supplemented with 10 mM HEPES, 5% FCS, and 50 mg/ml gentamicin. The columns containing cells were incubated at 37°C for 50-60 min. T cells were obtained by eluting the columns with 30-40 ml of HBSS at a flow rate of 1 drop/s. Flow cytometric analysis was carried out to assess the purity of the CD3-positive cells by using anti-CD3 antibodies. It was found that 90-95% of the cells were CD3 positive (data not shown).

Stimulation of T cells and lysate preparation. Rat splenic T cells were stimulated with soluble anti-CD3 antibodies (1 µg/ml) for 180 s at 37°C. The stimulation was stopped by cell solubilization in a phosphorylation lysis buffer (PLB). PLB was prepared by mixing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 100 mM NaF, 1 mM MgCl2, 10 mM Na4P2O7, 200 µM Na3VO4, 0.5% Triton X-100, and 10% glycerol on ice for 45-50 min. Lysates were centrifuged at 10,000 rpm for 5 min at 4°C.

In some experiments, T cells were incubated with PGE2 (10 µM), forskolin (10 µM), or cAMP analogues (doses in the figure legends) for 2 h before stimulation with anti-CD3. These experiments allowed us to assess the effects of PGE2, forskolin, or cAMP analogues on anti-CD3-linked p59fyn autophosphorylation and kinase activity in T cells. For the measurements of SQ-22536 or IBMX effects, T cells were incubated with SQ-22536 or IBMX for 30 min and then with PGE2 or cAMP analogues, respectively, for an additional 2 h.

Immunoprecipitation. Lysates were incubated with monoclonal antibodies to p59fyn protein for 1 h, and then the mixture was incubated with protein G-Sepharose beads for another 2 h (18). These incubations were carried out at 4°C. The precipitates were washed three times in PLB without added glycerol.

In vitro kinase assay. This assay was performed by a previously described method (18). After the final wash, immune complexes were collected and washed two times with in vitro kinase buffer (50 mM Tris · HCl, pH 7.4, 10 mM MnCl2, 0.1% Triton X-100). After these washes, kinase assays were performed by incubating immune complexes first with 5 µg/ml acid-treated enolase and then for 30 min with 10 µCi [gamma -32P]ATP. These incubations were carried out at room temperature (28°C). Samples were analyzed by SDS-PAGE, and the proteins were transferred to the Immobilon-P membrane. Phosphoproteins were analyzed by autoradiography, and the intensities of the bands were assessed by densitometry.

Reprobing the membranes. Membranes were reprobed for equal protein loading after stripping the antibodies. For stripping, membranes were incubated with stripping buffer (65 mM Tris · HCl, pH 6.8, 100 mM 2-mercaptoethanol, 2% SDS). The membranes were saturated with blocking buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween 20 supplemented with 10% BSA) for 1 h at room temperature or for 16-20 h at 4°C; this was followed by an incubation with anti-p59fyn antibody (1:200 dilution) at 4°C. The membranes were washed three times with wash buffer (10 mM Tris, 150 mM NaCl, 0.05% Tween 20) and were incubated with a secondary antibody conjugated with horseradish peroxidase (1:3,000 dilution) and then washed. After the final wash, membranes were probed with enhanced chemiluminescence dye and proteins were autoradiographed.

Assessment of T cell cAMP levels. Splenic T cells were incubated with various reagents for 2 h, and the intracellular accumulation of cAMP was measured with an enzyme immunoassay kit from Cayman Chemical (Ann Arbor, MI).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of stimulation or blockade of adenylate cyclase on p59fyn. The effects of PGE2 on p59fyn autophosphorylation and its phosphorylation of enolase are shown in Fig. 1A. There was no detectable phosphorylation of p59fyn or enolase by p59fyn in T cells without their stimulation with anti-CD3 antibodies. Stimulation of T cells with anti-CD3 resulted in increased autophosphorylation of p59fyn as well as increases in its kinase activity. The autophosphorylation of p59fyn induced by anti-CD3 was significantly suppressed in T cells incubated with PGE2 compared with T cells incubated without PGE2. Likewise, the phosphorylation of enolase by p59fyn in PGE2-treated T cells was significantly lower than that observed in untreated T cells. Densitometric analyses of 10 or more similar experiments suggested a 30-50% inhibition of p59fyn autophosphorylation and its kinase activity in T cells treated with PGE2 compared with the untreated T cells (Fig. 1, C and D). Figure 1B shows the equal-protein-loading controls for blots shown in Fig. 1A.





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Fig. 1.   Effect of prostaglandin E2 (PGE2) on anti-CD3-stimulated p59fyn autophosphorylation and kinase activity (enolase phosphorylation) in T cells. A: lane 1, control cells without PGE2 treatment and without anti-CD3 stimulation; lane 2, cells without PGE2 treatment but stimulated with anti-CD3; lane 3, cells stimulated with anti-CD3 in presence of 10 µM PGE2. B: p59fyn protein loading control blot. Lanes 1-3 are as defined for A. C: densitometric analyses of blots showing p59fyn autophosphorylation in presence and absence of anti-CD3 and PGE2. * P < 0.025 for T cells treated with PGE2 vs. control T cells (ANOVA). D: phosphorylation of enolase by p59fyn in presence and absence of anti-CD3 and PGE2. * P < 0.05 for enolase phosphorylation by p59fyn in PGE2-treated T cells vs. control T cells (ANOVA). Densitometric analyses represent values (mean ± SE) from unstimulated control (n = 6), anti-CD3-stimulated control (n = 10), and PGE2-treated T cells (n = 12). Densitometric values from anti-CD3-stimulated p59fyn autophosphorylation and enolase phosphorylation of control T cells were normalized to maximum value, taken as 1.

As shown in Fig. 2, A and B, treatment with SQ-22536 prevented PGE2-caused inhibition of p59fyn autophosphorylation as well as kinase activity in T cells stimulated with anti-CD3. This SQ-22536 effect was found to be dose dependent. Blots from three or more experiments performed with various doses of SQ-22536 were analyzed by densitometry, and the pooled data from these experiments are shown in Fig. 2, C and D. SQ-22536 alone had no effect on the anti-CD3-linked autophosphorylation and kinase activity of p59fyn. The doses of SQ-22536 used in the present study had no effects on cell viability, as tested by the trypan exclusion test. To further ascertain the role of adenylate cyclase on p59fyn, we assessed the anti-CD3-linked autophosphorylation and kinase activity of p59fyn in T cells incubated with or without forskolin. Forskolin has been used widely to directly upregulate adenylate cyclase activity . As shown in Fig. 3, the stimulation of adenylate cyclase with forskolin significantly inhibited the autophosphorylation and kinase activity of p59fyn. These data suggested that the activation of adenylate cyclase was critical for PGE2-mediated inhibition of p59fyn in T cells.





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Fig. 2.   Effects of various doses of SQ-22536 on PGE2-mediated suppression of anti-CD3-linked p59fyn autophosphorylation and kinase activity in T cells. T cells were treated with various doses of SQ-22536 for 30 min before their incubation with PGE2. A: lane 1, T cells without PGE2 treatment; lane 2, T cells + 10 µM PGE2; lane 3, T cells + 10 µM PGE2 + 1 µM SQ-22536; lane 4, T cells + 10 µM PGE2 + 10 µM SQ-22536; lane 5, T cells + 10 µM PGE2 + 100 µM SQ-22536; lane 6, T cells + 100 µM SQ-22536. B: p59fyn protein loading control blots. Lanes 1-6 are as defined for A. Values (means ± SE from 3 animals) obtained from densitometric analyses were normalized as described in legend to Fig. 1 and plotted for p59fyn autophosphorylation (C) and phosphorylation of enolase by p59fyn (D). * P > 0.05 for T cells treated with PGE2 + SQ-22536 vs. control T cells (ANOVA).






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Fig. 3.   Effect of forskolin on p59fyn autophosphorylation and kinase activity in T cells. A: lane 1, control cells without forskolin treatment and without anti-CD3 stimulation; lane 2, cells without forskolin treatment but stimulated with anti-CD3; lane 3, cells stimulated with anti-CD3 in presence of 10 µM forskolin. B: p59fyn protein loading control blot. Lanes 1-3 are as defined for A. C: densitometric analysis of blots showing p59fyn autophosphorylation. * P < 0.025 for T cells treated with forskolin (n = 8) vs. control T cells (n = 8; ANOVA). D: densitometric analysis of blots showing p59fyn phosphorylation of enolase. * P < 0.05 for enolase phosphorylation by p59fyn in forskolin-treated T cells (n = 8) vs. control T cells (n = 8; ANOVA). Values (means ± SE) obtained from densitometric analyses were normalized as described in legend for Fig. 1.

Effect of cAMP analogues on p59fyn. T cells were incubated with various concentrations of DBcAMP before their stimulation with anti-CD3, and then p59fyn autophosphorylation and kinase activity were assessed. Surprisingly, there was no difference in p59fyn autophosphorylation and its ability to phosphorylate enolase whether or not T cells were treated with DBcAMP (Fig. 4A). We also assessed p59fyn autophosphorylation and kinase activity in T cells after their incubation with 8-BrcAMP. 8-BrcAMP also failed to affect autophosphorylation and kinase activity (Fig. 5A). The lack of effect of cAMP analogues on p59fyn could be due to an insufficient increase in intracellular cAMP levels resulting from the treatment of cells with these analogues. Intracellular levels of cAMP in T cells were measured after their incubation with PGE2, forskolin, or a cAMP analogue (DBcAMP or 8-BrcAMP). The data from these experiments (Table 1) show that all three agents, PGE2, forskolin, and the cAMP analogues, increased cellular cAMP. Prior treatments of T cells with SQ-22536 significantly inhibited PGE2-induced elevation of intracellular levels of cAMP. The potential role of intracellular cAMP on p59fyn was further examined by inhibiting T cell phosphodiesterases with IBMX. In these experiments, cAMP analogues (DBcAMP and 8-BrcAMP) were added to T cells in the presence of phosphodiesterase inhibitor IBMX. The results from these experiments are shown in Fig. 5, A and C. In the cAMP analogue-treated T cells, the presumed further increase in cAMP after phosphodiesterase inhibition did not have any effect on the activation of p59fyn. The above results suggested that although there was an increase in intracellular levels of cAMP after the treatment of T cells with PGE2, forskolin, or cAMP analogues (DBcAMP or 8-BrcAMP), such increases in cAMP levels did not contribute to the PGE2-related inhibition of p59fyn phosphorylation and its kinase activity.



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Fig. 4.   Effects of various concentrations of dibutyryl cAMP (DBcAMP) on p59fyn autophosphorylation and kinase activity in T cells. A: lane 1, control cells without DBcAMP treatment and anti-CD3 stimulation; lane 2, cells without DBcAMP treatment but stimulated with anti-CD3; lane 3, T cells stimulated with anti-CD3 in presence of 1 µM DBcAMP; lane 4, T cells stimulated with anti-CD3 in presence of 10 µM DBcAMP; lane 5, T cells stimulated with anti-CD3 in presence of 100 µM DBcAMP; lane 6, T cells stimulated with anti-CD3 in presence of 1 mM DBcAMP. B: p59fyn protein loading control blot. Lanes 1-6 are as defined for A. Blots are representative of determinations from 4 animals.






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Fig. 5.   Effects of cAMP analogues [DBcAMP or 8-bromo-cAMP (8-BrcAMP)] on p59fyn autophosphorylation and kinase activity (enolase phosphorylation) in T cells in absence or presence of IBMX. A: lane 1, cells without 8-BrcAMP treatment but stimulated with anti-CD3; lane 2, T cells stimulated with anti-CD3 in presence of 1 mM 8-BrcAMP; lane 3, T cells stimulated with anti-CD3 in presence of 1 mM 8-BrcAMP and 100 µM IBMX; lane 4, T cells stimulated with anti-CD3 in presence of 100 µM IBMX. B: p59fyn protein loading control blot. Lanes 1-4 are as defined for A. C: lane 1, cells without DBcAMP treatment but stimulated with anti-CD3; lane 2, T cells stimulated with anti-CD3 in presence of 1 mM DBcAMP; lane 3, T cells stimulated with anti-CD3 in presence of 1 mM DBcAMP and 100 µM IBMX. D: p59fyn protein loading control blot. Lanes 1-3 are as defined for C. Blots are representative of 4 animals.


                              
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Table 1.   Effects of stimulants on intracellular levels of cAMP in T cells


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Whereas some studies have suggested that PGE2-mediated alteration in the transcriptional regulation of IL-2 and IL-2R results in attenuated T cell proliferation (2, 14, 33, 36), others have shown an impairment in the TCR-related early signaling events contributing to disturbed T cell functions (11, 15, 25, 30-33, 39). In our previous studies (16-18), we found that PGE2-mediated inhibition of p59fyn kinase activity could be a component of the early signaling events that impaired the downstream signaling, including the elevation in [Ca2+]i and the transcriptional regulation of IL-2. The present study evaluated a cross talk between the anti-CD3- and PGE2-mediated signaling pathways. Our data demonstrated that PGE2-mediated suppression of p59fyn is dependent on the activation of adenylate cyclase. However, the elevation of the intracellular cAMP level appeared to have no effect on the anti-CD3-mediated activation of p59fyn, suggesting that the block in p59fyn activation was dependent on the activation of adenylate cyclase but independent of cAMP.

The accumulation of intracellular cAMP after T cell stimulation with PGE2 is a common finding in a number of previous studies (10, 24, 25, 29, 38, 40). The correlation between elevated levels of cAMP and suppression of Ca2+ signaling and IL-2 transcription in T cells has been generally taken to indicate cAMP mediation of T cell dysfunction (2, 6, 15, 32, 34, 39, 41, 44). Our study has shown that although cAMP modulation did occur because of PGE2 action, cAMP itself was not required for the PGE2-mediated suppression of p59fyn. The inability of cAMP to modify p59fyn signaling in T cells is also supported by experiments in which intracellular cAMP hydrolysis was prevented by means of phosphodiesterase inhibition.

That cAMP might be involved in the suppression of TCR-mediated signaling events was demonstrated primarily by studies using cAMP-elevating agents, cholera toxin, and forskolin (3, 5, 10, 31, 39, 40). Both cholera toxin and forskolin act on Gs protein to stimulate adenylate cyclase, leading to the accumulation of cAMP (3, 10, 25, 27). cAMP is known to stimulate protein kinase A (PKA) in a number of cell types including T cells (2, 5, 6, 26, 32, 43). Furthermore, PKA has been shown to inhibit the PLC-gamma -mediated hydrolysis of PIP2 to IP3 and DAG (6, 24, 31, 32, 37, 38). An inhibition of PLC-gamma , PIP2 hydrolysis, and the IP3-related elevation in [Ca2+]i has been observed after adenylate cyclase activation of T cells with forskolin (24, 30-32, 44). Thus a direct activation of adenylate cyclase via forskolin and cholera toxin leading to cAMP accumulation could inhibit Ca2+ signaling in T cells and thereby prevent their activation and the expression of T cell functions. However, other studies have shown that although cholera toxin-mediated activation of adenylate cyclase, subsequent to Gsalpha activation, could effect both an increase in cAMP and a decrease in the Ca2+ signal in T cells, the decrease in Ca2+ signaling with increased accumulation of cellular cAMP could not be shown (3, 24, 25, 27, 29, 31). The intracellular Ca2+ signaling decrease could result not only from Gsalpha activation but also from an interference with PLC-gamma activation caused by p59fyn inhibition. The latter possibility is indicated by the dependence of PLC-gamma activation on p59fyn activity (8, 13, 28). Our previous studies had shown that PGE2-mediated inhibition of p59fyn is correlated with downregulation of the Ca2+ signal (18). However, we do not know whether the downregulation of the Ca2+ signal was due exclusively to p59fyn inhibition or due additionally to an inhibitory effect of PGE2-mediated Gsalpha activation and cAMP accumulation on the Ca2+ signal. Because we have found that PGE2 downregulates p59fyn in a cAMP-independent manner, we postulate that the Ca2+ signal downregulation by PGE2 is partly through p59fyn inhibition and partly due to accumulation of cAMP.

The precise mechanism for p59fyn suppression has remained unclear. A speculative mechanism for p59fyn inhibition could be that PGE2-mediated activation of the G protein and adenylate cyclase couples to some adapter protein, which could lead to a downregulation of p59fyn tyrosine kinase and/or an upregulation of protein tyrosine phosphatases. An alternative possibility could be that, as for cholera toxin, PGE2 activation of the G protein and adenylate cyclase modifies the TCR-zeta homodimer (zeta zeta ) chain (1, 8) located upstream of p59fyn, as has been shown by Haack et al. (25). A modulation in the zeta zeta -chain could alter p59fyn autophosphorylation and kinase activity.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of L. Amato.


    FOOTNOTES

This study was supported by National Institute of General Medical Sciences Grants RO1-GM-53235 and RO1-GM-56865.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. M. Sayeed, Trauma and Critical Care Research Labs, Dept. of Surgery, Stritch School of Medicine, Loyola Univ. Chicago, Maywood, IL 60153 (E-mail: msayeed{at}luc.edu).

Received 31 December 1998; accepted in final form 22 April 1999.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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