University of Witten/Herdecke, Institute of Microbiology and Virology, Stockumer Str. 10, D-58448 Witten, Germany
Correspondence
Manfred H. Wolff
mhwolff{at}uni-wh.de
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ABSTRACT |
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INTRODUCTION |
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The cell's ability to sense external stimuli and to react by initiating a programme of gene expression often involves propagation of a cell-surface-initiated signal along specific pathways of protein kinases whose targets are nuclear-acting transcription factors. Factors such as the type of stimulus, its duration and the cell type can influence whether the transduced signals are interpreted as growth stimulatory, growth inhibitory or apoptotic events causing cell stress (Ip & Davis, 1998; Kyriakis et al., 1995
; Robinson & Cobb, 1997
; Whitmarsh & Davis, 1996
). Many of these stimuli are mediated through the activation of distinct mitogen-activated protein kinase (MAPK) cascades. The best characterized of these are the extracellular-signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38/MAPK pathways (Kyriakis & Avruch, 1996
; Seger & Krebs, 1995
). While the ERK pathway is activated by proliferative stimuli, UV irradiation, environmental stress and proinflammatory cytokines stimulate the JNK/SAPK and p38/MAPK cascades. Although there is coordinated regulation of JNK/SAPK and p38/MAPK, they have distinct upstream activators. p38/MAPK is activated by MAPK kinases (MKKs) 3 and 6, whereas MKK 4 and MKK 7 activate both JNK/SAPK and p38/MAPK (Derijard et al., 1995
; Lawler et al., 1997
).
JNK/SAPK and p38/MAPK phosphorylate a number of transcription factors, including c-Jun, which is specifically phosphorylated by JNK/SAPK, and ATF-2, which is phosphorylated by both JNK/SAPK and p38/MAPK (Derijard et al., 1994; Gupta et al., 1995
; Kyriakis et al., 1994
). c-Jun binds the 12-O-tetradecanoate-13-acetate response element (AP-1/TRE), and the so-called cAMP response element (ATF/CRE) is recognized by ATF-2 (Angel et al., 1987
; Castellazzi et al., 1991
; Montminy, 1997
; Northrop et al., 1993
). Recently, we identified a large number of AP-1/TRE and ATF/CRE sites inside VZV promoters (Rahaus & Wolff, 2003
).
Viruses are ultimately dependent upon the host cell for their replication. Because they reorganize or utilize various cellular functions, it seems likely that viruses would take advantage of pre-existing signalling pathways to induce cellular and/or viral gene expression to promote virus replication. Recent reports have shown that several viruses can induce the activation of MAPK pathways in infected cells, such as human immunodeficiency virus type 1 (Kumar et al., 1998; Li et al., 1997
), human cytomegalovirus (Rodems & Spector, 1998
), echovirus (Huttunen et al., 1998
), Sindbis virus (Nakatsue et al., 1998
) and herpes simplex virus type 1 (HSV-1) (McLean & Bachenheimer, 1999
; Zachos et al., 1999
). However, the precise role of MAPK in the replication of viruses remains unclear. No data concerning the activation of MAPK pathways during the replication cycle of VZV are available.
This investigation was initiated to determine whether MAPK plays a role in the replication of VZV. We report that VZV induced the activation of MAPKs such as JNK/SAPK and p38/MAPK in the early phase of infection. The use of specific inhibitors or activators of MAPKs resulted in severe variations in VZV replication. We also showed that the JNK/SAPK and p38/MAPK downstream targets were activated, but subsequent cascades leading to JNK/SAPK-mediated apoptotic events failed to be notably stimulated, whereas a strong increase in IL6 secretion was observed as a consequence of p38/MAPK activation. Furthermore, we demonstrated that the activation of stress pathways depended on VZV gene expression.
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METHODS |
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Reagents and cell treatment.
SB202190 (an inhibitor of p38/MAPK), anisomycin (an activator of JNK/SAPK) and the JNK inhibitor I (L-form; cell-permeable, biologically active peptide consisting of HIV-TAT4857-PP-JBD20) were purchased from Calbiochem. Cells were treated with SB202190 or JNK inhibitor at varying concentrations for 30 min prior to infection; treatment with anisomycin (activator of JNK/SAPK) was carried out for 45 min before infection. Actinomycin D (ActD) and cycloheximide (CHX) were purchased from Sigma-Aldrich; cell treatment was performed 1 h prior to infection using 0·5 µg ActD ml1 and 10 µg CHX ml1.
Whole-cell and nuclear-extract preparation and immunoblotting.
Nuclear protein extracts for the transcription factor assay were prepared using the Nuclear Extract kit (Active Motif) according to the manufacturer's instructions.
To prepare whole-cell extracts at the indicated time points, cells were harvested in PBS supplemented with 10 µM PMSF and protein concentrations were determined by the Bradford method. Samples were then dissolved in SDS sample buffer (40 mM Tris/HCl, pH 6·8, 2 % SDS, 280 mM -mercaptoethanol, 10 % glycerol, 0·01 % bromophenol blue).
Preparation of nuclear extracts for immunoblotting was done as described by Andrews & Faller (1991). Immunoblotting of 25 µg of the respective protein extract (whole-cell or nuclear extract) was done as described previously (Rahaus & Wolff, 2000
). Protein detection was done using anti-JNK/SAPK, anti-p38/MAPK, anti-PARP (all from New England Biolabs), anti-VZV pIE4, anti-VZV pE29 and anti-VZV pIE63 (kindly provided by P. Kinchington, R. Cohrs and B. Rentier, respectively), anti-gE (Biodesign International) and anti-USF2 (Santa Cruz) polyclonal antibodies. All antibodies were diluted 1 : 1000 in 5 % BSA, 1x TBS, 0·1 % Tween 20 and detected using an alkaline phosphatase-conjugated secondary antibody (Santa Cruz; diluted 1 : 1000) and NBT/BCIP (ICN).
Fast activated cell-based ELISA (FACE).
FACE kits to monitor the levels of JNK/SAPK and p38 MAPK activation were obtained from Active Motif. Procedures were performed strictly according to the manufacturer's instructions. Briefly, cells were seeded in 96-well plates 1 day prior to manipulation. After treatment and/or infection, cells were fixed with 4 % formaldehyde in PBS. After washing and blocking, cells were reacted overnight with an anti-JNK, anti-phospho-JNK, anti-p38 or anti-phospho-p38 antibody. Following incubation with an HRP-conjugated secondary antibody, colorimetric analysis was performed. A450 was determined using a plate spectrophotometer. Cell quantification was then carried out using crystal violet staining and determination of the A595.
To calculate the amount of total and phosphorylated protein, background results (samples without primary antibody) were subtracted from the values for each sample. Data were normalized according to the cell number by calculation of A450/A595.
Transcription factor assays.
The transcription factor assay systems (TransAM) to monitor activation of c-Jun and ATF-2 were obtained from Active Motif. Procedures were performed according to the manufacturer's instructions. Briefly, 15 or 12 µg nuclear proteins was used per binding reaction to detect c-Jun or ATF-2 phosphorylation, respectively, which was carried out in oligonucleotide-coated 96-well plates. The oligonucleotides contained an AP-1/TRE element [TGA(C/G)TCA; c-Jun assay] or an ATF/CRE element [TGA(C/G)GTCA; ATF-2 assay]. After pre-incubation in binding buffer (10 mM HEPES/NaOH, pH 7·5, 8 mM NaCl, 12 % glycerol, 0·2 mM EDTA, 0·1 BSA, 1 mM DTT, 50 ng poly[d(I-C)]) for 1 h at room temperature, phospho-specific antibodies directed against c-Jun (diluted 1 : 500) or ATF-2 (diluted 1 : 1000) were added for 1 h at room temperature. Following incubation with an HRP-conjugated secondary antibody (diluted 1 : 1000) for 1 h at room temperature, colorimetric analysis was performed and the A450 read on a plate spectrophotometer. To calculate levels of activation, background results (samples without protein lysate) were subtracted from the values for each sample. Positive controls of the respective kits were performed using double-stranded oligonucleotides with either wild-type or mutated Jun or ATF-2 binding sites according to the manufacturer's instructions.
Haematoxylin-stain based VZV plaque assay.
At 24 h post-treatment/-infection, cells were fixed in 4 % paraformaldehyde for 30 min and stained with Mayer's haemalum solution (Merck) and eosin Y (Merck) as described previously (Rahaus & Wolff, 2003). Evaluation was done by counting clearly visible viral plaques in an area of 2 cm2 located precisely in the centre of each well. Absolute plaque numbers were later converted to relative numbers as a percentage. Control experiments showed at least 150 plaques per evaluated area. Experiments were done in triplicate.
IL6 ELISA.
The high-sensitivity human IL6 ELISA kit (Diaclone Research) was used according to the manufacturer's instructions. Briefly, 100 µl cell culture supernatant from VZV-infected cells was taken at various time points post-infection (p.i.) and introduced into the assay. Supernatants were then reacted with a biotinylated anti-IL6 antibody for 3 h. After washing, streptavidinHRP was added, followed by a colorimetric reaction. A450 was determined using a plate spectrophotometer and IL6 concentrations were calculated based on a standard curve.
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RESULTS |
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MEWO cells were treated with medium containing 5 or 20 µM JNK inhibitor I for 30 min before infection. At a concentration of 5 µM, we found an increase in VZV replication up to 149·1 %, while using 20 µM inhibitor increased the replication efficiency up to 193·9 % compared with virus progeny in untreated cells (taken as 100 %) (Fig. 1c). Contrasting results were obtained after inhibition of p38/MAPK by SB202190, with a reduction in the replicative activity to 55·78 % (using 10 µM inhibitor) and 34·68 % (using 30 µM inhibitor) compared with untreated cells (Fig. 1d
). Inhibition of the ERK1/2 pathway did not affect VZV replication (data not shown).
Activation of JNK/SAPK and p38 MAPK during the replicative cycle
We next focused on the possible activation of JNK/SAPK and p38/MAPK during the replicative cycle. VZV-infected MEWO cells at early and late times p.i. were used for FACE assays to monitor both the early phase of infection and long-term events. The FACE system allowed the quantification of activated JNK/SAPK and p38/MAPK in a high throughput series; results from three independent infection series are shown in Fig. 2. A 2-fold increase in activation of phosphorylated JNK/SAPK was observed at 1 h p.i., followed by a peak 34-fold increase in activation at 6 h p.i. The level of JNK/SAPK phosphorylation subsequently fell to its basal level as determined at 0 h p.i. and remained constant (Fig. 2a
). In contrast, phosphorylation of p38/MAPK decreased between 1 and 6 h p.i. from its basal level as determined at 0 h p.i. and showed only a 1·8-fold peak increase in activation at 12 h p.i. (Fig. 2a
). A corresponding experiment was performed to determine the total amounts of JNK/SAPK and p38/MAPK protein. In both cases, we observed a decrease in protein accumulation in the early phase of infection followed by an increase, peaking at 6 h p.i. JNK/SAPK total protein increased up to 380 %, whereas accumulation of p38/MAPK protein only reached 125 %. Following these peaks, the accumulation levels of both proteins decreased and remained constant at 6075 % from 12 to 48 h p.i. compared with the protein level at 0 h p.i., which was taken as 100 % (Fig. 2b
).
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As a control for infection, the presence of VZV IE63 was monitored by immunoblotting of whole-cell lysates derived at the respective time points (Fig. 2d). To ensure that the observed effects were specific and did not reflect an overall increase in protein concentration, the cellular protein USF2 was also analysed. It is known that expression of USF2 is not influenced by VZV infection (Rahaus et al., 2003
); expression levels of USF2 remained unchanged (Fig. 2d
).
Propagation of JNK/SAPK and p38/MAPK signals for activation of downstream cascades during the replicative cycle
To confirm that activation of JNK/SAPK and p38/MAPK following VZV infection truly resulted in activation of their respective downstream targets c-Jun and ATF-2, respectively, the phosphorylation of these two transcription factors was measured in infected cells by transcription factor assays. In triplicate experiments, 15 or 12 µg nuclear extract prepared from VZV-infected cells and harvested at different time points was used for a binding reaction to immobilized oligonucleotides containing an AP-1/TRE element (c-Jun binding) or an ATF/CRE element (ATF-2 binding), respectively. Using a phospho-specific anti-c-Jun antibody, we obtained a steady increase in c-Jun phosphorylation starting at 1 h p.i. and reaching a plateau of elevated activation between 5 and 6 h p.i. In the subsequent phase of the replicative cycle, the level of c-Jun phosphorylation dropped off, returning to the basal level measured at 0 h p.i. by 36 h p.i. (Fig. 3a).
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In order to determine whether phosphorylation of c-Jun following VZV infection led to stimulation of subsequent cellular signal cascades, especially in the activation of apoptotic pathways, we analysed the cleavage of poly(ADP-ribose) polymerase (PARP) as an indicator of apoptosis. Whole-cell extracts prepared from VZV-infected MEWO cells at 0, 2, 4, 6, 8, 10, 12, 24 and 48 h p.i. were immunoblotted and reacted with an anti-PARP antibody. At all time points, the 116 kDa band of the full-length protein was detected (Fig. 4a). Between 2 and 24 h p.i. an additional but very weak band of 89 kDa was found, representing the activated PARP protein. At 48 h p.i., a clear increase in cleaved PARP was found, indicating an increase in apoptotic events. Densitometric evaluation of detected bands and comparison of the calculated protein amounts demonstrated that only 57·5 % of the total PARP protein was cleaved into its active form, representing an insignificant apoptotic activity as a consequence of VZV infection and following activation of the JNK/SAPK pathways (data not shown).
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These data indicated activation of c-Jun and ATF-2 in the early phase of VZV infection. The time course of phosphorylation corresponded to that described above for JNK/SAPK phosphorylation, affecting both c-Jun and ATF-2. p38/MAPK affected ATF-2 activity in the later phase of the infection. Moreover, these data suggested that activation of these MAPKs as a consequence of infection led to a propagation of the signal to downstream targets. No significant activation of JNK/SAPK-mediated apoptosis was detected, but there was a remarkable increase in IL6 secretion.
Variations in virus replication after activation of JNK/SAPK
We compared syncytium formation as a marker of virus replication with the levels of phosphorylation of JNK/SAPK and p38/MAPK after treatment of cells with the corresponding inhibitor or anisomycin. At subinhibitory concentrations, anisomycin strongly activates JNK/SAPK and p38/MAPK in mammalian cells. If used at inhibitory concentrations (10 µg ml1), anisomycin is known to act as an inhibitor of protein synthesis at the translational step (Cano et al., 1994; Hazzalin et al., 1998
). Plaque formation and phosphorylation of JNK/SAPK or p38/MAPK in untreated, VZV-infected cells were taken as 100 %; all experiments were done in triplicate. As shown above, after treatment of cells with JNK inhibitor, the number of VZV plaques increased 2-fold and no phosphorylation of JNK/SAPK was detected (Fig. 5
a). Using anisomycin at the subinhibitory concentration of 50 ng ml1, we detected activation of JNK/SAPK up to 230·4 %, whereas the number of viral plaques decreased to 44·83 %. This effect was intensified when 500 ng anisomycin ml1 was used: JNK phosphorylation increased to 747·6 %, while the plaque number decreased to 7·37 % (Fig. 5a
). These results revealed that activation of JNK/SAPK caused strong inhibition of the replication of VZV. Different data were obtained when the number of VZV plaques was compared with the level of p38/MAPK phosphorylation. Again, inhibition of p38 using SB202190 (30 µM) led to a loss of p38/MAPK phosphorylation, as well as a reduction in plaque number to 35 %. Surprisingly, neither 50 nor 500 ng anisomycin ml1 resulted in any increase in p38/MAPK phosphorylation in MEWO cells (Fig. 5b
). The number of viral plaques matched that found when virus replication versus JNK/SAPK phosphorylation was investigated.
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Importance of viral gene expression for the activation of JNK/SAPK and p38/MAPK
The absence of significant JNK/SAPK and p38/MAPK activities at a very early time after infection led us to assume that neither VZV-caused induction of a receptor-mediated signal cascade nor the presence of VZV tegument proteins was sufficient to activate these two MAPKs, but that the expression of viral genes or virus-mediated cellular gene expression was necessary. To confirm this hypothesis, we performed cell-based ELISAs to measure JNK/SAPK and p38/MAPK phosphorylation in cells that had been treated with CHX (10 µg ml1) prior to infection to block gene expression. In order to determine whether accumulation of transcribed but not translated viral and cellular mRNAs caused a stress situation that triggered the activation of JNK/SAPK or p38/MAPK, cells were also treated with ActD (0·5 µg ml1) to inhibit gene expression at the transcriptional step. Fig. 6(a) shows that, as a consequence of the cell treatment, expression of VZV E and L genes, represented here by the products of ORF29 (single-stranded DNA-binding protein) and ORF68 (gE), respectively, was in fact inhibited, whereas components of the viral tegument of input virions, represented by the product of the IE gene ORF4, were still detectable.
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Based on these findings, we concluded that virus entry and release of the tegument proteins is not sufficient to activate stress-related MAPKs. Expression of viral genes or VZV-mediated expression of cellular genes seems to be required.
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DISCUSSION |
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Signals leading to MAPK activation are often generated at the cell surface when receptors bind to their appropriate ligand. Some reports have shown that binding of a virus to a cell-surface receptor mimicking normal ligand-receptor events activates downstream pathways. Simian immunodeficiency virus activates ERK, JNK/SAPK and p38, depending on cell type and receptor (Popik & Pitha, 1998). Upon binding to the surface of permissive cells, cytomegalovirus generates intracellular signals that induce the translocation and activation of NF-
B (Sambucetti et al., 1989
; Yurochko et al., 1997
). In this study, we showed that VZV infection also resulted in short-term activation of upstream MAPK pathways. In the early phase of infection, a 34-fold and a 2-fold elevation of JNK/SAPK and p38/MAPK phosphorylation, respectively, were detected. Our data also indicated a block in both pathways after their peaks of phosphorylation and, in consequence, activity of the downstream targets c-Jun and ATF-2 also decreased. The fact that two peaks of ATF-2 activation were found can be explained by the postponed activation of JNK/SAPK and p38/MAPK. However, the mechanisms leading to the repression of MAPK activities remain undefined. We also observed weak induction of JNK/SAPK within the first hour of infection, corresponding to interactions of viral glycoproteins located on both the virus envelope and the infected-cell surface with receptors of as yet uninfected cells. These data contrast with the findings of McLean & Bachenheimer (1999)
, who did not detect any activation within this early attachment and penetration phase of HSV infection. However, following VZV penetration, strong activation began no earlier than 4 h p.i. for JNK/SAPK and 5 h p.i. for p38/MAPK, which is compatible with data from HSV during the corresponding phase of infection (McLean & Bachenheimer, 1999
).
Activation of JNK/SAPK and p38/MAPK could be a non-specific response by the cell, but results from experiments using CHX- or ActD-treated cells argue against this interpretation. The data showed that expression of viral genes or, alternatively, virus-mediated expression of cellular genes is necessary to induce JNK/SAPK and p38/MAPK phosphorylation. Due to the onset of JNK/SAPK and p38/MAPK activation that occurs at 4 and 5 h p.i., respectively, it is likely that expression of VZV-encoded genes is the necessary event for initiation of MAPK activation. By this time, expression of IE and E genes has started. IE proteins have already reached high levels of accumulation and have ample time to perform their functions. The presence of viral tegument proteins or the capsid seemed not to be sufficient to achieve this activation.
Experiments using highly specific inhibitors provided evidence that both JNK/SAPK and p38/MAPK have substantial but opposing effects on VZV replication. Blocking the ability of JNK/SAPK to act on its nuclear substrates resulted in increased VZV replication, whereas inhibition of p38/MAPK led to a reduction in virus replication. It is known that promoters of all kinetic classes of VZV genes contain cognate binding sites for a variety of cellular transcription factors, e.g. Oct-1, USF, Sp1, NF-B and AP-1 (Ito et al., 2003
; Kinchington et al., 1994
; Meier et al., 1994
; Rahaus et al., 2003
; Rahaus & Wolff, 2000
), which are all targets of upstream signalling pathways. Downregulation of the expression of AP-1 affects the efficiency of transactivation and effectiveness of replication (Rahaus & Wolff, 2003
). Similar reports underlining the importance of specific cellular components for herpesviral gene regulation have described the physical interaction of VZV IE62 with Sp1 or USF (Peng et al., 2003
; Rahaus et al., 2003
) and Patel et al. (1998)
showed that blocking HSV-mediated translocation of NF-
B into the nucleus reduced virus yields by 90 %.
JNK/SAPK is known to be responsible for complex and cell-type-specific effects, since its activation in different conditions and cell types correlates with proliferation, oncogenic transformation and apoptosis (Ip & Davis, 1998; Whitmarsh & Davis, 1996
). No significant activation of PARP cleavage as a marker of apoptotic response after activation of the upstream cascade JNK/SAPK
c-Jun
(Bax/Bak/Bid)
(cytochrome c/caspase 9/Apaf-1)
caspase 3 (Li et al., 2004
, and references therein) was detected in MEWO cells, leading to different assumptions: firstly, that the host defence induction triggered by VZV is not strong enough to activate apoptotic pathways and, secondly, that VZV is able to modulate the activation of apoptotic events in order to enhance its survival and spread. The second hypothesis is substantiated by the report of Hood et al. (2003)
showing differences in the apoptotic responses to VZV depending on the cell type. We propose that JNK/SAPK, once activated, could phosphorylate cellular or viral transcription factors to enhance the expression of VZV-encoded genes but is shut down afterwards by an as yet unknown mechanism to suppress the activation of cellular defence systems and the apoptotic response. The activation of ATF-2 downstream of p38/MAPK activation seems to be necessary for the expression of VZV genes. This is substantiated by the fact that inhibition of p38/MAPK and knockout of ATF-2 transcription (Rahaus & Wolff, 2003
) led to a severe decrease in VZV replicative activity. In contrast to JNK/SAPK and p38/MAPK, ERK1/2 seems not to play an important role in the VZV replicative cycle since its inhibition did not affect the production of virus progeny. HSV also fails to activate ERK (McLean & Bachenheimer, 1999
). Moreover, several lines of evidence exist to support a role for MEKs and ERKs in the inhibition of IL6 activity. However, results shown in this report demonstrated that infection of cells with VZV led to an increase in IL6 secretion, indicating that VZV does not seem to cause activation of the MEK/ERK pathway leading to inhibition of IL6.
IL6 production was blocked after inhibition of the p38/MAPK pathway. The role of the MKK3p38/MAPK
MAPKAP kinase 2 signalling cascade in the production of IL6 is well established (Miyazawa et al., 1998
). Iordanov et al. (2000)
reported a similar activation of IL6 secretion after infection of cells with encephalomyocarditis virus and postulated a possible role for proinflammatory cytokines in combating viruses. Our results demonstrated some similarities in MAPK activation patterns, and previous data from our laboratory showing that neither the protein kinase R nor RNase L pathways for host defence are activated after VZV infection (Desloges et al., 2004
) support the hypothesis that activation of p38/MAPK and JNK/SAPK after viral infection may represent a new host-defence mechanism. Moreover, activation of cellular signalling pathways is mainly achieved after expression of VZV components and triggers the production of cellular transcription factors, which have important functions in transactivating VZV genes. The cellular defence systems and apoptotic responses that are activated by the same pathways may be repressed by as yet unknown mechanisms to ensure virus replication. Further studies are necessary to investigate this virushost interaction more precisely.
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ACKNOWLEDGEMENTS |
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Received 7 June 2004;
accepted 21 August 2004.