Division of Nephrology, Hypertension and Transplantation, Department of Medicine, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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Cytochrome
c-mediated activation of caspase-3 is the final common
pathway for most signals that induce apoptosis. Before release of cytochrome c from mitochondria, K+ and
Cl efflux and intracellular acidification must occur. We
have utilized an in vitro assay to examine the role of pH, cations,
anions, and uncharged molecules on the process of cytochrome
c-mediated activation of procaspase-3. In this cell-free
system, a pH above 7.4 severely suppressed the activation of
procaspase-3 but not the activity of caspase-3. KCl, NaCl, and other
salts all inhibited caspase activation, but uncharged molecules did
not. Comparison of the inhibitory capacity of various salts suggests
that the crucial element in causing suppression is the cation. The
inhibition of alkaline pH could be overcome by increasing
concentrations of cytochrome c, whereas the inhibition of
ionic charge could not, suggesting that pH and salts affect the
activation of caspase-3 by different mechanisms.
apoptosis; cell death; potassium
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INTRODUCTION |
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THE PROCESS of preordained cell death is known as apoptosis or programmed cell death. This process, distinct from necrosis, is characterized by a series of cellular events, such as cell shrinkage, phosphatidylserine externalization (14), membrane blebbing (29), and DNA fragmentation (35). Some of these changes are a direct result of enzymes, collectively termed caspases, which are constitutively produced, highly conserved, aspartic acid-specific cysteine proteases (10). Caspases, normally present in cells in the form of inactive zymogens, are activated by autoproteolysis or cleavage by other caspases. A number of independent pathways have been implicated in the activation of different caspases with a variety of triggering mechanisms, involving an assortment of accessory proteins.
Of the many stimuli that induce apoptosis, the majority of them act by releasing cytochrome c from the inner mitochondrial matrix (6, 8, 16, 17, 20, 21, 33). Cytosolic cytochrome c is a critical factor contributing to the ultimate formation of a large, ~700-kDa, heterogeneous protein assembly termed the apoptosome, consisting of oligomers of Apaf-1, cytochrome c, and procaspase-9 (7, 9, 37). Formation of the apoptosome requires dATP/ATP (23) and is critical for the activation of caspase-9 (28).
Intracellular acidification (22) and ion efflux (2, 5, 25) must precede cytochrome c release from mitochondria. The requirement for K+ efflux in apoptosis was partly explained by the demonstration that KCl inhibited both the nuclease activity from apoptotic thymocytes and the activation of caspase-3 in vitro (18). However, the mechanism of ionic inhibition is unknown. The identification of whether the cation or anion is responsible for the inhibition of caspase-3 activation may provide insight into the mechanism of apoptosome formation.
To investigate the role that ionic charge and pH play in cytochrome c-mediated apoptosis, we used an in vitro assay in which cytosol was dependent on addition of cytochrome c for activation of caspase-3. With this assay, we demonstrate that cytochrome c-mediated activation of caspase-3 is inhibited by alkaline pH and that the inhibition by high pH can be overcome with an excess of cytochrome c. By comparing the ability of a variety of salts to inhibit this activity, we demonstrate that cations appear to exert a stronger influence than anions on the activation of caspase-3. Although both cations and alkalinity inhibit caspase activation, we demonstrate that they inhibit by a different mechanism.
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MATERIALS AND METHODS |
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Materials and supplies. All tissue culture reagents were obtained from Life Technologies (Rockville, MD); all other reagents are from Sigma (St. Louis, MO) unless otherwise indicated.
Cell culture. The human embryonic kidney fibroblast cell line 293T Fas (a kind gift of Dr. Jurg Tschopp, Institut de Biochimie, University of Lausanne, Switzerland) was cultured at 37°C in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin, in a humidified atmosphere with 5% (vol/vol) CO2.
Preparation of the cytosol.
Cytosol preparation was performed at 4°C. Cells scraped from eight
150-mm tissue culture plates were resuspended in phosphate-buffered saline (137 mM NaCl, 2.6 mM KCl, 10 mM Na2HPO4,
and 1.7 mM KH2PO4, pH 7.4), pelleted at 300 g, washed twice, and resuspended in 1 ml of extraction
buffer (50 mM HEPES, 50 mM NaCl, 2 mM MgCl2, and 5 mM
EGTA). After addition of the protease inhibitors leupeptin, pepstatin,
and phenylmethylsulfonyl fluoride at 1 µg/ml, the suspension was
homogenized by 10 passages through a 25-gauge needle. The homogenate
was centrifuged at 20,000 g for 5 min at 4°C, and the supernatant was collected and centrifuged at 100,000 g for
1 h in a 70.1 Ti rotor in a Beckman L8-70M ultracentrifuge
(Beckman Instruments, Fullerton, CA) before being filtered through a
0.45-µm filter (Fisher Scientific, Atlanta, GA). The protein
concentration of a 1:50 dilution of homogenate (to decrease HEPES
interference) was measured using the Lowry procedure (24).
The protein concentration of the cytosol was in the range of 2-3
mg/ml. Cytosol was stored in 100-µl aliquots at 80°C until use.
Assay of caspase activity.
Components of a routine assay, 2.5 µg of lysate protein, 0.25 µg of
rat heart cytochrome c, an ATP regeneration system (1 mM
ATP, 8 mM phosphocreatine, and 5 µg/ml creatine kinase), and Z-DEVD-rhodamine110 substrate (Z-DEVD-R110; Molecular Probes, Eugene,
OR), were combined on ice and brought up to a final volume of 12 µl
in a buffer consisting of 10 mM PIPES, 10 mM dithiothreitol (experiments involving ZnCl2 used 20 mM -mercaptoethanol
instead of dithiothreitol to avoid Zn2+ binding), and 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), pH
7.1. Kinetic measurements of caspase activity were recorded using a
Bio-Tek FL600 Microplate Fluorescent Reader (Bio-Tek Instruments,
Winooski, VT). The cleavage of the rhodamine substrate was determined
with the use of a 485-nm excitation filter and a 530-nm emission filter
at a temperature of 25°C. The pH of the reaction mixtures was
adjusted for individual assays within a range between 6.8 and 7.8 by
addition of NaOH and was measured with a needle pH electrode (MI408
Microelectrodes, Bedford, NH) attached to an AB150 Accumet pH meter
(Fisher Scientific). The pH of each reaction was measured both before
and after the incubation period. In the assays examining the inhibition
of caspase-3 activation by salts, PIPES was eliminated from the
reaction mixture to limit particle concentration. Caspase inhibitors
(Calbiochem; La Jolla, CA) were redissolved in dimethyl sulfoxide.
Measurement of inhibitory activity. Inhibition of caspase activity of different salts and sugars was expressed in terms of the molarity of the compound required to reduce the activity to 50% of the control. The RFU values of all reactions were compared at the last time point at which the positive control reaction was still increasing linearly. At this time point, the RFU values were plotted vs. molarity, and the Marquardt-Levenberg algorithm (26) was used to derive the best fit for each curve. This equation was then solved to calculate the necessary molarity required to inhibit the reaction to half the maximum RFU value. This molarity was used to assign a relative inhibitory potency for each reagent.
Measurement of Na+/K+ concentration and osmoles. The osmolality of reaction mixtures was measured on a 5500 Vapor Pressure Osmometer (Wescor, Logan, UT). Samples (10 µl) of reaction mixture were applied to filter disks and vaporized in the osmometer, and the osmolality in osmoles was recorded. The total free K+ and free Na+ concentration in the cytosol was determined with a K+ and Na+ microelectrode (Microelectrodes, Bedford NH) attached to an AB150 Accumet pH meter.
Immunodepletion of caspase-3. Cytosol was incubated at 4°C rotating with either 2 µl of mouse monoclonal antibody, at 1 mg/ml, to caspase-3 (Pharmingen, San Diego, CA) or nonspecific mouse monoclonal antibody (IgG2a) at 1 mg/ml (Sigma). Antigen antibody complexes were precipitated by addition of a 5% (vol/vol) slurry of protein A agarose beads (Repligen, Needham, MA) in PBS with 1 mg/ml bovine serum albumin. Depleted cytosol was separated from beads by centrifugation at 5,000 g for 5 min and used in the caspase activity assays.
Immunoblot analysis of cytosols. A fixed quantity of cytosol was activated as above in the presence of KCl or ZnCl2 or at a pH of 7.8. The activated cytosol was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane as previously described (12). The blot was then probed with an anti-caspase-9 antibody that recognizes the activated form with greater affinity than the pro form (Oncogene, San Diego, CA) or with an anti-caspase-3 antibody that recognizes procaspase-3 preferentially over active caspase-3 (Chemicon, Temecula, CA). After being washed in PBS/0.2% Tween 20, the blot was overlayed with horseradish peroxidase-conjugated secondary antibody, and the immunoreactive polypeptides were detected by enhanced chemiluminescence (Pierce, Rockford, IL) and imaged using the Fluorchem Imaging System and software (Alpha Innotech, San Leandro, CA).
Statistical analysis. Statistical significance for the effects of salts and pH on activation of caspase-3 activity was determined by analysis of variance and Student's t-test (34).
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RESULTS |
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Cytochrome c-induced Z-DEVD-R110 cleavage is concentration
dependent and specifically due to caspase-3 activity.
Cytosol used for in vitro assays was shown previously to provide most
of the components required for caspase-3 activation (13).
The requirement for addition of cytochrome c to the cytosol varies with the method of cell homogenization. When cells were lysed by
shear force, as described in MATERIALS AND METHODS,
caspase-3 activation was measurable only after addition of cytochrome
c (Fig.
1A). This
finding is in accordance with previously published observations
(18, 23), although other protocols of cytosol preparation
have reported sufficient residual cytochrome c in lysate to
obviate the need for supplementation (9). Increasing concentrations of cytochrome c led to an increase in the
rate of substrate cleavage to a maximum value of 8 nmoles · min1 · mg cytosolic
protein
1 (Fig. 1B), although this maximum rate
varied slightly with each cytosol preparation. The addition of a
caspase-3 inhibitor, DEVD-fmk, or a caspase-9 inhibitor, Z-LEHD-fmk,
prevented the cytochrome c-induced caspase activity (Table
1). The fluorescence was specifically due
to caspase-3 activity, since cytosol immunodepleted of caspase-3 had
levels of substrate cleavage no greater than cytosol with no cytochrome
c added (Fig. 1C).
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Increasing pH diminishes cytochrome c-mediated activation of
caspase-3.
Intracellular acidification is associated with apoptosis
(15) and is necessary for cytochrome c release
(22). To test the influence of pH on cytochrome
c-mediated activation of caspase-3 within cytosol, we
performed the in vitro apoptosis assay at different physiological pH values. From pH 7.0 to 7.6, the activity of caspase-3 was inversely related to pH (Fig.
3A). At a
pH of 7.6, the caspase-3 activity was below that seen in the no
cytochrome c control. At the nonphysiological pH of 8.2, cleavage of substrate was not detected.
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Cations inhibit cytochrome c activation of caspase-3.
As reviewed in the Introduction, recent studies have demonstrated
that a lowering of cytosolic K+ activity is required for
apoptosis, but the ion specificity and target sites for the
effect of KCl have not been identified. We therefore tested the effect
of KCl on caspase-3 activation in vitro. The 293T Fas cell cytosol,
before addition of any inhibitory salts, had a measured osmolality of
225 mosmol, with a free K+ concentration of 13 mM
and a free Na+ concentration of 45 mM (as determined by
K+- or Na+-selective microelectrodes). Because
2.5 µl of cytosol were used in a final volume of 12 µl, the cytosol
contributed a cation concentration of 12 mM to the assay. As shown in
Fig. 4A, KCl inhibited
cytochrome c-mediated activation of caspase-3 in a
concentration-dependent manner, with 50% inhibition relative to the
cytochrome c-activated control at a KCl concentration of 20 mM (Table 2). As shown in Fig. 2,
lane 4, KCl addition to cytosol completely inhibited
cleavage (activation) of procaspase-9 and procaspase-3.
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Majority of cations do not inhibit active caspase-3.
To determine whether the cations have any effect on activated
caspase-3, salts were added to the reaction mixtures after the caspase-3 was activated by the addition of cytochrome c and
were allowed to reach maximum measurable activity. With the notable exception of Zn2+, the addition of salts at concentrations
that clearly inhibited the activation of caspase-3 had limited or no
effect on the cleavage rate of preactivated caspase-3 (Fig.
5). ZnCl2 at a concentration of 5 mM markedly inhibited active caspase-3.
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Inhibitory effect of KCl on activation of caspase-3 cannot be
overcome by the addition of cytochrome c.
As shown above, excess cytochrome c suppressed pH
inhibition. To determine whether additional cytochrome c
could overcome the inhibitory effect of salt on activation of
caspase-3, we added up to four times the normal cytochrome c
concentration to the reaction assay. In marked contrast to findings
with inhibition by high pH, increasing the cytochrome c
concentration did not overcome the suppression of caspase-3 activity by
KCl (Fig. 6). This finding indicates that
ionic charge is inhibiting caspase-3 activation by a mechanism
different from that of pH.
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Increasing osmolality does not inhibit caspase-3 activation. Cellular osmolality is determined by and equal to the extracellular environment of the cell. In the majority of circumstances, it is ~295 mosmol with ~150 mosmol contributed by monovalent cations. To determine whether overall osmolality affected cytochrome c-mediated caspase-3 activation, we added uncharged sugars to the assay. Increasing the final osmolality to >300 mosmol, by adding sucrose or sorbitol, did not inhibit caspase-3 activity (Table 2). These results suggest that charged molecules are required to inhibit cytochrome c-mediated caspase-3 activity.
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DISCUSSION |
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Caspase-3 involvement in the process of apoptosis has received considerable attention in recent years because it is the downstream effector caspase responsible for activating dismantling enzymes and cleaving structural and cell cycle proteins (10). Caspase-3 activity is detectable after the cell passes through a crucial commitment phase of apoptosis, during which time the apoptosome, composed of oligomerized Apaf-1/cytochrome c/procaspase-9/(dATP/ATP), is assembled, providing the necessary components to advance a self-destructive cascade and ensuring cell death. The assembly of the apoptosome apparently serves as one of the throttles controlling the decision for apoptosis to advance. Therefore, identifying what impels a cell toward that irrevocable decision is crucial. This study provides information concerning the influence of the pH, ionic charge, and osmolality of the cytosol on cytochrome c-mediated activation of caspase-3.
We used an in vitro system in which the addition of cytochrome
c to cytosol produced caspase-3 activation. This assay is
specific for activation of caspase-3 as demonstrated by inhibition of
the activity with DEVD-fmk (a caspase-3 inhibitor) and by elimination of the activity by immunodepletion of caspase-3 before the assay. The
rate of caspase-3 cleavage increased with increasing concentrations of
cytochrome c to a maximum of ~8 nmol
substrate · min1 · mg cytosolic
protein
1.
Previously, it had been demonstrated that purified, active caspase-3 has only slight changes in activity between the pH of 6.7 and 7.8. This finding is in contrast to our results demonstrating that the activation of caspase-3 is markedly affected in the pH range of 6.8-7.8. At a pH >7.6, no caspase-3 activity was detected above the negative control value. Our results for the effect of pH on caspase-3 activity correlate closely with those published recently (27). To exclude the possibility that the presence of other cytosolic proteins led to the inhibition of caspase-3 enzymatic activity at alkaline pH values, we activated caspase-3 before raising the pH and determining the enzymatic rate. The enzymatic rate of previously activated caspase-3 was not significantly affected, even if the pH value was increased to 7.8. The distinction between the alkaline effect on the activation and not the activity of caspase-3 suggested that alkaline pH had an effect on a step preceding activation of the enzyme, possibly the assembly of the apoptosome, or activation of caspase-9 activity. However, the enzymatic rate of recombinant active caspase-9 was not affected by pH between a pH of 7.0 and 7.8 (data not shown), and cleavage of both procaspase-9 and procaspase-3 was inhibited by alkaline conditions (Fig. 2). Additional studies are needed to determine whether pH is affecting apoptosome formation.
Our studies, consistent with previous reports, demonstrate that normal intracellular concentrations of KCl inhibit caspase-3 activation (Fig. 4) (19). All salts, monovalent as well as divalent, also inhibited caspase-3 activation (Table 2).
Maeno et al. (25) reported that blockade of
Cl or K+ channels results in inhibition of
cell shrinkage in apoptosis. We designed assays to determine
which charged particle is inhibiting the cytochrome c-mediated activation of caspase-3. Our studies suggest a
50% inhibition in caspase activity at a concentration of 20 mM for KCl
and 10.6 mM for NaCl (Table 2). If the anion were involved in the
inhibition of caspase-3 activation, both NaCl and KCl would be equally
effective in bringing about this inhibition because they dissociate to
the same extent (Table 3). In addition, if the anion were involved,
divalent cations associated with two Cl
atoms per
molecule should inhibit at the same free anion concentration as
monovalent cations, such as Na+ and K+. This
was not found to be the case. The concentration of free anions required
to obtain 50% relative inhibitory potential for KCl and NaCl was
clearly less than that required for the divalent cation salts
CaCl2 and MgCl2 and was clearly greater than
that required for ZnCl2 (Table 2). Thus our studies
indicate that cations, rather than anions, inhibit cytochrome
c-mediated activation of caspase-3. Efflux of
Cl
ions may be necessary only to allow the efflux of
K+ ions and not because the Cl
ions are
inhibitory themselves. If Cl
channels did not open,
cation transfer to the extracellular environment would be limited by
the electronegativity of the cell. It has previously been reported that
the intracellular KCl decreases to between 35 and 56 mM during
apoptosis (4, 5, 19). Our prepared cytosol had a
free K+ concentration of 13 mM and a free Na+
concentration of 45 mM, contributing a 12 mM cation concentration in
the final assay reaction. The addition of more than 20 mM
K+ (a total cation concentration of 32 mM) or 11 mM
Na+ (a total cation concentration of 23 mM) resulted in a
50% inhibition of caspase-3 activity.
Osmolality itself appears to play no role in the inhibition of
caspase-3 because uncharged osmoles added at a concentration as high as
300 mosmol had no effect on caspase-3 activation. Our results suggest
that divalent as well as monovalent cations can inhibit caspase-3
activation. Thus the overall cationic charge appears to suppress the
activation of caspase-3, rather than the concentration of any
individual cation. If ionic charge can inhibit the apoptotic
pathway at the release of cytochrome c from mitochondria (25), at cytochrome c-mediated activation of
caspase-3 (Fig. 4) (18), and at the effector enzyme
required for DNA degradation (18), then it appears that
ionic charge must be maintained at a permissive level for the time
required for the entire apoptosis process to occur. However,
K+ is the major intracellular osmole, and efflux of
K+ and Cl is followed by water efflux and
cell shrinkage (5, 25). If the driving force for water
efflux were the hyposmolality caused by the efflux of ions, water
efflux would continue until the intracellular osmolality was identical
to its environment (~290 mosmol). At that point, the intracellular
concentration would once again be 140 mosmol, a concentration that
would inhibit apoptosis. This paradox could be resolved in one
of two ways: either the apoptotic cells are hypotonic, which would
require impermeability to water, or there is production or accumulation
of other osmotic solutes. If the latter were the case, our data suggest
that these osmoles would need to be uncharged. This is known to occur
in cells when they are exposed to a hypertonic environment (3,
31, 32). Further investigations are needed to distinguish these possibilities.
Previously it was reported that KCl inhibits the nuclease activity required for the DNA fragmentation observed during apoptosis (18). In a series of experiments in which salts were added to preactivated caspase-3 within the cytosol, 140 mM NaCl or KCl had little effect on caspase-3 enzymatic activity. This finding demonstrates that ionic charge is not inhibitory at all steps along the apoptotic pathway.
We found that the inhibition of alkalinity could be overcome with
excess cytochrome c. In the standard assay, 2.1 × 10 11 moles of cytochrome c were added to 2.5 µg of cytosolic protein in 12 µl, yielding a final concentration of
20 µg/ml. This concentration is 100 times the concentration of
cytochrome c per milligram of cytosol found in rat
cardiomyocytes (1). This finding implies that only a
fraction of the cytochrome c added to the cytosol in our
assay is functional, that a component needed to accelerate caspase
activation at lower cytochrome c concentrations may be missing from the in vitro assay, or that in vivo caspase-3 activation occurs at a much slower rate than in our in vitro assay. Our
data suggest that the lower the concentration of cytochrome
c, the greater the effect of pH change.
The ability of alkalinity and cations to inhibit cytochrome c-mediated activation of caspase-3 is not specific for human embryonic kidney cell cytosol. When the assay is performed with cytosol derived from human umbilical vein endothelial cells, Jurkat cells, and insect cell cytosol derived from SF9 cells, similar results were obtained (data not shown). Thus the effect of pH and cations appears to be a general phenomenon of cytochrome c activation of caspase-3.
Although our studies found that both alkaline pH and elevated KCl inhibit caspase-3 activation, these conditions appear to act in different ways. An increase in the concentration of cytochrome c was able to overcome the inhibition by alkaline pH, whereas excess cytochrome c was unable to overcome the inhibition by KCl. Thus, unlike the modulatory effect of pH, if the cell is above a threshold KCl concentration, cytochrome c will not be able to mediate caspase-3 activation (Fig. 4A), and apoptosis will not proceed. This was confirmed by immunoblot analysis. While KCl completely eliminated cleavage of procaspase-9 and procaspase-3 (Fig. 2, lane 4), a pH of 7.8 decreased but did not eliminate cleavage of procaspase-9 while abrogating the cleavage of procaspase-3 (Fig. 2, lane 3). A reasonable hypothesis is that pH affects the cytochrome c/Apaf-1 oligomerization step. Cationic strength may inhibit either the interaction of caspase-9 with the apoptosome or the activation of caspase-9. The caspase recruitment domain of Apaf-1 consists of an acidic patch (D27, E39, E40, and E41) that interacts with a basic patch on caspase-9 (11, 36). Cations may interact with the acidic patch of Apaf-1 and prevent caspase-9 recruitment into the apoptosome. This hypothesis would predict that by adding additional procaspase-9 to cytosol, the inhibitory effects of ionic charge might be overcome as well. Our results provide a means to dissect out distinct steps in caspase-3 activation that will allow these possibilities to be examined in future studies.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Gluck for many helpful discussions and critical reading of the manuscript.
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FOOTNOTES |
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This work was financially supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-02537 and Gatorade Research funds.
Address for reprint requests and other correspondence: M. S. Segal, PO Box 100224, Gainesville, FL 32610 (E-mail: segalms{at}medicine.ufl.edu).
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.
Received 9 January 2001; accepted in final form 11 June 2001.
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