1 Developmental Biology, 2 Pediatric Surgery, and 3 Bone Marrow Transplant Programs, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027
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ABSTRACT |
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The survival of
type 2 alveolar epithelial cells (AEC2) in the lung after hyperoxic
injury is regulated by signals from the cellular environment.
Keratinocyte growth factor and Matrigel can ameliorate the hallmarks of
apoptosis seen in hyperoxic AEC2 after 24-h culture on plastic
[S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson,
and D. Warburton. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18):
L714-L720, 1998]. We used the same model of in
vivo short-term hyperoxia to characterize the protective effects of
substrate attachment. Culture of hyperoxic AEC2 on various biological
adhesion substrates showed reduced DNA end labeling in cells grown on
all biological substrates compared with growth on plastic. In contrast,
the synthetic substrate
poly-D-lysine conferred no
protection. Hyperoxic AEC2 cultured on laminin showed an increased
ratio of expression of Bcl-2 to interleukin-1-converting enzyme
compared with culture on plastic. Laminin also partially restored
hyperoxia-depleted glutathione levels and conferred improved optimal
mitochondrial viability as measured by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Conversely, attachment to the nonphysiological substrate
poly-D-lysine afforded no such
protection, suggesting that protection against hyperoxia-induced damage
may be associated with integrin signaling. Increased activation of
extracellular signal-regulated kinase (ERK), as detected by increased
ERK tyrosine phosphorylation, was seen in hyperoxic AEC2 as soon as the
cells started to attach to laminin and was sustained after 24 h of
culture in contrast to that in control AEC2. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK
activation, the cells were also plated in the presence of 50 µM PD-98059, an inhibitor of the ERK-activating
mitogen-activating kinase. Culture for 24 h with PD-98059 abolished the
protective effect of laminin. We speculate that after hyperoxic lung
injury, signals through the basement membrane confer specific
protection against oxygen-induced DNA strand breakage and apoptosis
through an ERK activation-dependent pathway.
extracellular signal-regulated kinase; type 2 alveolar epithelial cells; hyperoxia-induced deoxyribonucleic acid damage; tyrosine phosphorylation
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INTRODUCTION |
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TYPE 2 ALVEOLAR EPITHELIAL CELLS (AEC2), which
are normally quiescent in the adult lung, respond to lung injury by
proliferating and migrating to repair the alveolar epithelium (24). In
the rat model of short-term hyperoxic injury, the response of AEC2 to
damage is reproducible and transient. Induction of key cell cycle genes
and downregulation of autocrine transforming growth factor-
secretion, followed by proliferation, occur within 24 h postinjury,
with complete resolution occurring by 72 h (6, 7).
Apoptosis, a genetically controlled cellular response to developmental or environmental stimuli that culminates in cell death, is an important mechanism of negative selection that removes damaged cells that may be deleterious to the host (31). Apoptosis, as measured by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL), has been reported in sections of hyperoxic lungs (15). Apoptosis has also been reported in fibrotic human lungs (25, 26) and after acute lung injury (3), suggesting that apoptosis may play a key role in the resolution of lung injury. In our previous study using a rat model of in vivo short-term hyperoxia, we (5) showed that AEC2 isolated from hyperoxic lungs showed the hallmarks of apoptosis after 24 h of culture on plastic: significant TUNEL labeling and DNA fragmentation together with increased expression of p53, p21, and Bax proteins as well as DNA laddering. Interestingly, the DNA damage could be ameliorated by incubation with 20 ng/ml of keratinocyte growth factor (KGF) or by culture on Matrigel (5).
Although the protective effects of KGF in a variety of damage models
are well documented (8, 20, 22, 30, 35) and cell-matrix adhesion is a
recognized physiological determinant of cell growth and survival (1,
21, 23), little is known about the protective role of specific basement
membrane components during recovery from acute hyperoxic injury. In
this study, we again utilized the rat in vivo hyperoxic injury model to
generate hyperoxic AEC2 that undergo reproducible DNA damage and
apoptosis in culture. We demonstrated a significant inverse correlation between glutathione levels and DNA strand breakage in hyperoxic AEC2
after 24 h of culture on plastic, confirming that glutathione measurement would be useful when protective strategies in our model of
hyperoxia were assessed. We compared hyperoxic AEC2 cultured on
plastic, biological adhesion substrates, and
poly-D-lysine, measuring various
parameters associated with DNA damage and apoptosis, including
glutathione levels, mitochondrial viability, DNA end labeling, and
expression of interleukin-1-converting enzyme (ICE) and Bcl-2
proteins. We showed that the significant protection against
hyperoxia-induced DNA damage conferred by laminin was associated with
increased activation of extracellular signal-related kinase (ERK)
during the early period of attachment. Finally, we abolished the
protective effect of laminin by blockade of the mitogen-activated
protein (MAP) kinase cascade using PD-98059, an inhibitor of
ERK-activating MAP kinase, showing that ERK activation protects against
hyperoxia-induced DNA damage in AEC2 cultured on laminin. This leads us
to speculate that in vivo, laminin in the alveolar basement membrane
may contribute to the survival of hyperoxia-damaged AEC2.
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MATERIALS AND METHODS |
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Oxygen treatment and recovery. Adult male Sprague-Dawley rats were exposed to short-term hyperoxia exactly as previously described (7). Briefly, the rats were placed in a 90 × 42 × 38-cm Plexiglas chamber, exposed to humidified >90% oxygen for 48 h, a 48-h oxygen exposure time inducing lung damage with minimal mortality. Control rats were kept in room air. At the end of the exposure period, the rats were anesthetized by an intraperitoneal injection of pentobarbital sodium. After complete exsanguination by saline perfusion via the pulmonary artery, the lungs were lavaged to remove macrophages. The lavaged lungs were then used for AEC2 isolation and culture.
Isolation and culture of AEC2. AEC2 were isolated from lavaged lungs by elastase digestion followed by differential adherence on IgG plates as described by Dobbs et al. (9). The cells were either used immediately for glutathione measurement or plated at 2 ×105 cells/cm2 in DMEM with 10% FCS on plastic or on commercially prepared plates coated with biological substrates for 24 h of culture. Collagen 1-, collagen IV-, fibronectin-, laminin-, Matrigel-, and poly-D-lysine-coated plates were all from Becton Dickinson (Franklin Lakes, NJ). Freshly isolated AEC2 attached more efficiently to the biological substrates than to plastic and less efficiently to poly-D-lysine; therefore, for some experiments, parallel wells were set up for cell counts to correct for varying plating efficiencies. After 24 h of culture, the cells were lysed on ice to extract proteins for Western blotting (7) or trypsinized from the plate and fixed in ice-cold 1% paraformaldehyde for analysis of FITC-dUTP staining by fluoresence-activated cell sorting (FACS) analysis. In some experiments with laminin as the substrate, the cells were lysed early after attachment (2-5 h after being plated) or were treated with 50 µM PD-98059 (Calbiochem, La Jolla, CA) from the time of plating. Immunostaining of attached cells after 24 h of culture with a surfactant protein (SP) C antibody confirmed that >95% of the attached cells were SP-C positive. The antibody to SP-C was kindly provided by Jeffrey Whitsett (Children's Hospital Medical Center, Cincinnati, OH).
Flow cytometric method for measuring DNA end labeling in fixed cells. DNA strand breaks were measured in 1% paraformaldehyde-fixed AEC2 by labeling with fluorescent FITC-dUTP with an APO-DIRECT kit (PharMingen, San Diego, CA) according to the manufacturer's instructions. Fixed cells were incubated at 37°C for 1 h with TdT and FITC-labeled dUTP before being counterstained with propidium iodide. The stained cells were analyzed with a Becton Dickinson FACScan equipped with a 488-nm argon laser, with control positive and negative cells end labeled in parallel with the test samples supplied with the kit. The percentage of gated cells that were FITC positive was compared for each treatment group. No FITC fluorescence was seen in either control or oxygen-treated AEC2 that had been incubated in the labeling reaction mixture in the absence of TdT. Cellquest software (Becton Dickinson) was used for the analyses.
Western blotting of proteins. Western analysis was performed on cell lysates as described by Bui et al. (7), with 10-20 µg protein/lane depending on the sensitivity of the antibody used. Equal loading was confirmed by blotting with an antibody to actin. Proteins of interest were detected with horseradish peroxidase-linked secondary antibodies and the enhanced chemiluminescence system following the manufacturer's instructions (Amersham, Arlington Heights, IL). Antibodies to phosphotyrosine, ERK, p-ERK, c-Jun NH2-terminal kinase (JNK), p-JNK, Bcl-2, and ICE were from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody to actin was from ICN (Irvine, CA). The secondary antibodies were from Sigma (St. Louis, MO).
Glutathione measurement. Measurement of total glutathione (GSH plus GSSG) was performed with AEC2 immediately after isolation and after 24 h of culture. Freshly lysed hyperoxic and control AEC2 were assayed immediately with a modification of the recycling method of Owens and Belcher (19), which colorimetrically measures the formation of 2-nitro-5-thiobenzoic acid from 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of glutathione reductase and NADPH (19). Glutathione levels in the AEC2 were calculated relative to 1-4 nmol glutathione standards freshly prepared for each assay.
Assay of mitochondrial viability. Mitochondrial viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay exactly as described by Atabay et al. (2). Briefly, control and hyperoxic cells growing on plastic or on various adhesion substrates were incubated with buffered MTT for 30 min at 37°C, and the purple formazan color generated by viable mitochondria was measured colorimetrically. Because the measurements were performed on 24-h cultures, the cells in parallel wells were counted to correct for the differing attachment efficiencies on the various substrates. All chemicals used were from Sigma.
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RESULTS |
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Hyperoxia-induced DNA strand breakage in cultured AEC2
is significantly correlated with depletion of cellular
glutathione. AEC2 from hyperoxic and control rats were
lysed and assayed for total cellular glutathione (GSH plus GSSG)
immediately after isolation and after 24 h of culture, by which time
DNA strand breakage in the hyperoxic population was apparent. Total
glutathione levels were significantly depleted (~70%) in the fresh
isolates of hyperoxic AEC2 compared with those in control cells (Fig.
1A).
This depletion was sustained after 24 h of culture. There was a
significant inverse correlation between DNA damage, as measured by
FITC-dUTP DNA end labeling, and glutathione levels, as measured in
fresh isolates of AEC2 (Fig. 1B).
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Attachment to biological substrates reduces
hyperoxia-induced DNA strand breaks in 24-h cultured
AEC2. Because we (5) have previously shown Matrigel to
be protective against hyperoxia-induced DNA damage, some Matrigel
components and other adhesion substrates, including laminin,
fibronectin, collagen I, collagen IV, and the attachment factor
poly-D-lysine, were compared.
Hyperoxic AEC2 were plated on plastic or substrate for 24 h, then
recovered and fixed for FITC-dUTP DNA end labeling. DNA damage measured
in hyperoxic AEC2 cultured on the various substrates was compared with
damage on plastic. Figure 2 shows that
all biological substrates tested were significantly protective
against hyperoxia-induced DNA damage. In contrast,
poly-D-lysine provided no
protection at all. Control cells, which showed no significant DNA end
labeling on plastic (~10% of cells were end labeled) showed no
change on any substrate (data not shown).
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Attachment to biological adhesion substrates increases
mitochondrial viability in 24-h cultured hyperoxic
AEC2. Because mitochondrial damage is more extensive
and persists longer than nuclear damage in oxidative stress (33), we
compared the mitochondrial viability of hyperoxic AEC2 grown for 24 h
on plastic and other biological substrates with the MTT assay. Figure
3 shows that all biological substrates
tested significantly improved mitochondrial viability compared with
that on plastic, whereas
poly-D-lysine afforded no
protection, in agreement with the DNA end-labeling data.
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Laminin increases the expression ratio of Bcl-2 to
ICE. Expression of the apoptosis-protective protein
Bcl-2 and the apoptosis-associated caspase ICE in lysates of hyperoxic
AEC2 growing on the various substrates were compared by Western
analysis (Fig. 4). The ratio of Bcl-2 to
ICE expression was increased by growth on laminin compared with growth
on plastic.
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Laminin increases total cellular glutathione levels in
hyperoxic AEC2. To see whether laminin, which was
protective against hyperoxia-induced DNA damage to AEC2, could restore
hyperoxia-depleted glutathione levels, we cultured control and
hyperoxic AEC2 on laminin and plastic for 24 h and compared total
cellular glutathione levels (Fig. 5). A
24-h culture on laminin resulted in a 1.5-fold increase in glutathione
levels in hyperoxic AEC2 compared with culture on plastic.
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During early attachment to laminin, increased
activation (phosphorylation) of ERK is seen in hyperoxic AEC2 compared
with that in control AEC2. Hyperoxic and control AEC2
were plated on laminin and plastic and lysed after 2, 3, 4, 5, and 24 h
on laminin and 24 h only on plastic. The cells attached to plastic so
slowly that there was insufficient material for analysis at the earlier time points. Western blotting for tyrosine phosphorylation showed increased phosphorylation of a ~45-kDa protein during the early period of attachment in the hyperoxic AEC2 compared with that in
control AEC2 (Fig. 6,
top). There was also increased
expression of a higher-molecular-mass protein, ~120 kDa,
after 24 h of culture in both hyperoxic and control cells. The latter
~120-kDa protein was later confirmed by Western blotting to be focal
adhesion kinase (FAK; data not shown), but because there was no
difference between hyperoxic and control AEC2, we limited our
investigations to the lower-molecular-mass protein. Based on the
approximate molecular mass, we performed Western analysis with p-ERK
and p-JNK and showed that p-ERK had a similar pattern of expression to
that seen with the phosphotyrosine antibody (Fig. 6,
middle). Although the activity of
p-ERK was increased during attachment of the hyperoxic AEC2 to laminin,
the expression of ERK was not significantly altered and the expression
and activity of JNK did not change significantly (data not shown).
Blotting with an anti-actin antibody confirmed equal loading (Fig. 6,
bottom).
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MAP kinase pathway inhibitor PD-98059 blocks the
protective effect of laminin on hyperoxia-mediated DNA damage to
AEC2. To confirm the involvement of ERK activation with
protection against hyperoxia-induced DNA damage, hyperoxic AEC2 were
plated on plastic and laminin for 4 and 24 h in the presence and
absence of 50 µM PD-98059, a specific inhibitor of ERK-activating MAP
kinase. Four hours was selected as the optimum time to observe
differences in p-ERK, whereas all measurements of DNA damage were made
at 24 h, by which time DNA damage was apparent. Inhibition of ERK activity by PD-98059 abolished the protective effect of laminin, resulting in increased DNA strand breakage (Fig.
7A).
Western analysis of ERK activation as hyperoxic AEC2 attach to laminin confirms successful blockade by PD-98059 (Fig.
7B,
top row). Expression of ERK did not
change with PD-98059 treatment (Fig.
7B, second row). Bcl-2 levels were
slightly offset by 24 h of PD-98059 treatment, in agreement with the
end-labeling data (Fig. 7B, third
row). Actin served as a loading control (Fig.
7B,
bottom row). ERK activation in control
AEC2, much reduced in comparison with hyperoxic AEC2, was also
inhibited by PD-98059, with no change in Bcl-2 levels (data not shown).
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DISCUSSION |
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The alveolar epithelium of the lung is a major target for oxidant injury. The survival of AEC2 is critical to ensure timely and efficient repopulation of the damaged alveolar surface during the repair process after hyperoxic injury. Depending on the net balance of environmental cues, AEC2 can remain quiescent or proceed toward proliferation or apoptosis. In this paper, we used an in vivo model of hyperoxia to examine the balance between DNA damage, which accompanies a depletion of cellular glutathione, and protective signaling in hyperoxic AEC2 grown on laminin, a key component of the alveolar basement membrane.
Glutathione is an efficient antioxidant, maintaining a reduced intracellular state in the face of a highly oxidizing environment. Epithelial lining fluid of the lung contains GSH at an ~100-fold higher concentration than that found in the fluid of many other tissues (27). The observation that certain tumor cells have evolved resistance to oxidant injury suggests that manipulation of the cellular redox status may provide a role in future therapies, a compelling reason to correlate antioxidant levels, apoptosis, and putative protective strategies in normal, untransformed AEC2. AEC2 from rats subjected to short-term hyperoxia were found to have significantly lower total glutathione (GSH plus GSSG) levels than control AEC2 both immediately on isolation and after 24 h of culture. DNA damage as measured by FITC-dUTP DNA end labeling after 24 h of culture had a significant inverse correlation with initial glutathione levels, making glutathione a useful parameter in assessing hyperoxia-induced DNA damage. Low glutathione levels associated with DNA damage and apoptosis have also been reported in AEC2 derived from a more chronic model of hyperoxia involving exposure to 60 or 85% oxygen for 7 days (27).
In a previous study using the same model of hyperoxia, we (5) showed that Matrigel could significantly reduce hyperoxia-induced DNA damage at 24 h, indicating that the initial in vivo damage is not lethal and may be reversed by a favorable environment in vitro. Antiapoptotic signaling through cell-matrix interaction has been reported in a variety of epithelial cells, including bronchial epithelial cells, mammary epithelial cells, and retinal pigment epithelium (1, 18, 23). We therefore decided to focus on some key components of the alveolar basement membrane as a potential source of protective signaling. We plated hyperoxic AEC2 for 24 h on a selection of biological adhesion substrates using commercially prepared, thinly coated plates to ensure reproducibility and measured DNA damage by FACS analysis of FITC-dUTP end-labeled cells. Because end labeling may not always discriminate between apoptosis and necrosis (14), we also examined expression of proteins specifically related to apoptosis, the caspase ICE and the protective protein Bcl-2. Bcl-2 acts upstream of the execution caspases, preventing their proteolytic processing into active mediators of cell death. Bcl-2 also dramatically alters intracellular glutathione compartmentalization, promoting sequestration of glutathione into the nucleus to restore nuclear antioxidant status and block apoptosis (28). Mitochondrial viability was also assessed because the mitochondria have been described as "the central executioner of programmed cell death" and can contribute to apoptotic signaling via the production of reactive oxygen intermediates (28). FACS analysis of FITC-dUTP DNA end labeling showed that significant protection against hyperoxia-induced DNA damage was conferred by all biological substrates tested but not by the synthetic attachment factor poly-D-lysine. MTT data were in agreement with the FACS analysis, showing that mitochondrial viability was significantly increased when the hyperoxic cells were grown for 24 h on biological substrates compared with those cultured on plastic or poly-D-lysine. Laminin was effective in reducing DNA strand breakage while increasing the ratio of Bcl-2 to ICE expression, suggesting an amelioration of apoptosis. The protective effect of laminin was confirmed further by the observation that depleted glutathione levels seen in hyperoxic AEC2 after 24 h of culture were significantly increased by culture on laminin.
Although the protective effects of laminin against hyperoxia-induced DNA damage and apoptosis in cultured AEC2 were apparent, the mechanism of protection was less clear. The fact that the hyperoxic AEC2 survived relatively poorly on poly-D-lysine or plastic compared with natural biological adhesion substrates suggested the involvement of integrins in protective signaling. Interactions of integrins with the extracellular matrix activates FAK and suppresses apoptosis in diverse cell types (29). Cell interaction with adhesive proteins or growth factors in the extracellular matrix is reported to initiate MAP kinase signaling and cell migration (16), an important function of the AEC2 when repopulating the pulmonary epithelium after lung injury. The observation that laminin strongly promotes AEC2 migration (17) suggests a possible further role for MAP kinase signaling through laminin in the hyperoxic lung because protection against oxidant damage combined with increased capacity for migration would ensure a timely and efficient repopulation of the damaged epithelium.
Comparison of phosphotyrosine expression by Western analysis during
attachment of hyperoxic and control AEC2 to laminin showed increased
activation of ERK in the hyperoxic population during early attachment.
In contrast, p-JNK expression did not differ between the control and
hyperoxic populations. Tyrosine-phosphorylated FAK expression was
increased in both control and hyperoxic cells after 24 h of culture on
plastic or laminin, but there was no difference between the groups.
Therefore, we concluded that ERK activation played at least a
correlative role in protection and used an inhibitory strategy to test
whether the protective signaling in hyperoxic AEC2 cultured on laminin
was occurring through an MAP kinase pathway. Blockade of the MAP kinase
pathway by 50 µM PD-98059 abolished the protective effect of laminin
against DNA strand breakage, showing that activation of ERK confers a
protective effect against DNA damage. The observation that the cells
attach faster to laminin than to plastic may indicate that timely
reattachment confers or maintains viability of damaged cells.
Alternatively, laminin may specifically select for a population of
"survivors," a population in which protective signaling has
already been initiated. Thus an important caveat concerning the data
presented herein is that the specific protective effects associated
with ERK activation may be due to signaling originating from more than
one source. The laminin itself may contribute diverse signals because
the commercial plates are prepared by extracting laminin from
Engelbreth-Holm-Swarm tumors and therefore may contain other factors.
Signaling through ERK activation can be antiapoptotic or apoptotic
depending on the cell type and the nature of the apoptotic stimulus.
Survival through ERK activation against a diverse range of apoptotic
signals has been reported, including serum-induced apoptosis of PC12
cells (34), tumor necrosis factor--induced apoptosis of L929 cells (12), and ultraviolet-induced apoptosis of human primary neutrophils (10). In contrast, apoptotic signaling through ERK activation has also
been described: Fas-mediated apoptosis in a neuroblastoma cell line was
blocked by interference with either the ERK or JNK pathways (with
dominant-interfering mutant proteins), indicating that ERK and JNK
cooperate in the induction of apoptosis by Fas (13). AEC2 express Fas
receptors and undergo Fas-dependent apoptosis under the appropriate
stimuli (11). Therefore, the survival of AEC2 in vivo after hyperoxic
injury will be determined by integration of diverse signals, both
protective and destructive, from the cellular environment through ERK
activation. In our model of short-term hyperoxic injury, the net signal
transmitted to the hyperoxic AEC2 cultured on laminin is protective. We
therefore speculate that laminin in the alveolar basement membrane may
promote survival of AEC2 during lung repair after acute hyperoxic injury.
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FOOTNOTES |
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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: D. Warburton, Childrens Hospital Los Angeles Research Institute, 4650 Sunset Blvd. (MS 35), Los Angeles, CA 90027 (E-mail: dwarburton{at}chla.usc.edu).
Received 11 November 1998; accepted in final form 18 March 1999.
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