Laboratory of Physiology, Katholieke Universiteit Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium
Submitted 21 February 2003 ; accepted in final form 3 June 2003
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ABSTRACT |
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Rho; actin; swelling
An emerging concept with respect to the activation of volume-regulatory anion channels (VRACs) is their modulation by cellular or membrane architectural elements such as the cytoskeleton and caveolae (for a review, see Ref. 6). For example, efficient activation of the VRAC requires the expression of caveolin-1 and can be repressed by dominant negative caveolin-1 or caveolae/raft-targeted c-Src mutants, indicating that VRAC and/or its activation pathway are compartmentalized to caveolae/lipid rafts (41-43). Similarly, VRAC activation depends on a functional Rho/Rho kinase/myosin phosphorylation pathway (7, 22, 24, 28, 40). However, cell swelling does not directly activate the Rho pathway, indicating a permissive, but not a triggering, role for Rho/Rho kinase/myosin phosphorylation in VRAC activation (3). More controversial is the contribution of the actin cytoskeleton to volume-regulatory responses and, in particular, to the activation of VRAC. Swelling-induced rearrangements of the actin cytoskeleton have indeed been observed in cultured astrocytes, C6 glioma cells, opossum kidney cells, Ehrlich ascites tumor cells, and B lymphocytes (16, 20, 21, 29, 40) but not in calf pulmonary endothelial cells (3). Similarly, actin-depolymerizing agents such as cytochalasin B facilitate VRAC activation in some cell types such as cervical cancer HT-3 cells (38) and B lymphocytes (16) but not in human endothelial cells (25). Given the disparity of these data, it is not clear at the moment whether changes in the actin cytoskeleton are functionally relevant for RVD and/or for VRAC activation or whether they are coinciding epiphenomena.
The actin cytoskeleton is a dynamic structure under the control of the small Rho GTPases RhoA, Rac, and Cdc42 (18, 33). RhoA bundles actin filaments into stress fibers that traverse the cell and connect to focal adhesion complexes in the plasma membrane (34). Rac induces a submembranous actin network, which forms peripheral extensions (lamellipodia) that can lift up and fold back (membrane ruffles) (35). Finally, Cdc42 promotes the formation of fingerlike protrusions (filopodia) containing short actin bundles in the direction of the protrusion (13). These effects were first documented in quiescent fibroblasts where they control cell migration, but there is now compelling evidence that Rho GTPases via their effect on the actin cytoskeleton guide a whole array of cellular functions, including neuronal growth cone modeling (14) and epithelial morphogenesis (46).
Rho GTPases function as molecular switches that shuttle between an active GTP-bound state and an inactive GDP-bound state, which can form a complex with GDI (guanosine nucleotide dissociation inhibitor) proteins (for a review, see Ref. 39). GTPase activation requires dissociation from GDIs and exchange of GDP for GTP and is catalyzed by guanosine nucleotide exchange factors (GEFs) that are activated by upstream signaling events (45). Once activated, Rho GTPases interact with several effector proteins, such as Rho kinase and p21-activated kinase (PAK), or scaffolding proteins, such as mDia or Wiskott-Aldrich syndrome protein (WASP), to exert their effect on downstream processes such as the actin cytoskeleton (2, 33). Rho GTPases are turned off by intrinsic GTP hydrolysis, which is enhanced by interaction with GTPase-activating proteins (GAPs) (45).
In this study, we focus on the link between, on the one hand, hypotonic cell swelling and, on the other hand, actin cytoskeleton and Rho GTPases. Using a combination of morphological and biochemical approaches in Rat-1 fibroblasts, we show that hypotonic stimulation rearranges the actin cytoskeleton and induces the formation of membrane protrusions via activation of Rac1 and/or Cdc42.
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MATERIALS AND METHODS |
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Cell culture and transfection. We used Rat-1 fibroblasts, a kind gift from Pierre Courtoy (Institute of Cellular Pathology, Louvain, Belgium). The cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, GIBCO) containing 10% fetal calf serum (FCS), 4 mM L-glutamine, 20 mM glucose, 10 mM NaHCO3, 10 mM HEPES, 10 µg/ml streptomycin, and 66 U/ml penicillin, maintained at 37 °C in a fully humified atmosphere of 10% CO2 in air. Passaging of the cells was performed by brief exposure to 0.5 g/l trypsin in a Ca2+- and Mg2+-free solution.
Twenty-four hours after seeding, cells were transiently transfected with the bicistronic pCINeo/IRES-GFP vector (44), containing the cDNA encoding the constitutively active form of RhoA or the dominant negative forms of Rac1 and Cdc42. Rat-1 fibroblasts were seeded at 20,000-35,000 cells per 18-mm gelatin-coated coverslip. Transfection was performed using 1 µg of DNA, 7 µl of "Plus" reagent (Life Technologies), and 2 µl of LipofectAMINE (Life Technologies) per coverslip. Cells were analyzed on day 1 after transfection.
Visualization of the F-actin cytoskeleton. Rat-1 fibroblasts were grown on gelatin-coated coverslips at a density of 30,000-50,000 cells per coverslip. After stimulation with an isotonic solution (in mM: 105 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, and 90 mannitol, adjusted to pH 7.4 with NaOH; 320 mosM) or a 25% hypotonic solution (in mM: 105 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 10 HEPES, adjusted to pH 7.4 with NaOH; 240 mosM) for the indicated time periods, cells were fixed in 3.7 % fresh formaldehyde for 15 min at room temperature, washed twice, and permeabilized in 0.2 % Triton X-100. After permeabilization, cells were incubated in 2 units of rhodamine-phalloidin for 40 min at room temperature. Stained samples were treated with Vectashield mounting medium for fluorescence to retard photobleaching. All incubation and washing steps were performed in TBS (in mM: 10 Tris · HCl, pH 7.3, 150 NaCl, 1 MgCl2, and 1 EGTA) at room temperature.
Immunofluorescence. Cells were grown and treated as indicated above. After permeabilization, nonspecific binding sites were blocked during 2 h in 3% BSA. Primary and secondary antibodies were diluted in 0.3% BSA. Primary antibodies were incubated at 4°C overnight. The secondary AlexaFluor 488-labeled goat anti-mouse antibody is used in a 1:1,000 dilution. All washing and incubation steps were performed in TBS at room temperature, unless otherwise indicated. Stained samples were treated with VectaShield anti-fade reagent to retard photobleaching and analyzed using a Leica DMRB microscope and Nikon ACT-1 on DXM1200 Software. Colocalization of two signals was performed with Lucia G on DXM1200 version 4.71 software (Analysis S. A.). Omission of the primary antibody resulted in a weak background staining.
Rac and Cdc42 activation assays. BL21 Escherichia coli
bacteria were transformed with the pGEX2TK vector containing the GST-PAK-CD
construct that encodes the Cdc42/Rac interactive binding domain PAK (a
Cdc42/Rac effector protein) fused to glutathione-S-transferase.
GST-PAK-CD protein synthesis was induced with 0.1 mM isopropyl
-D-thiogalactoside (IPTG) at 37°C. After 2 h, the culture
was centrifuged (3,900 g, 20 min, 4°C), and the pellet was
sonicated in a bacterial lysis buffer [20% sucrose, 10% glycerol, 50 mM Tris
· HCl, pH 8, 200 µM Na2S2O5, 2 mM
MgCl2, 2 mM 1,4-dithiothreitol (DTT), 1 mM PMSF, 0.4 mM
Na-pervanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin]. The lysate
was centrifuged for 13,800 rpm, 20 min, 4°C, and the supernatant
containing the GST-PAK-CD fusion protein was incubated with 50%
glutathione-Sepharose 4B beads (Amersham) for 60 min at 4°C. The beads
were washed three times with and resuspended in GST-fish buffer (50 mM Tris
· HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 1%
NP-40, 2 mM DTT, 1 mM PMSF, 0.4 mM Na-pervanadate, 10 µg/ml leupeptin, and
10 µg/ml aprotinin).
Rat-1 fibroblasts were grown in 10-cm petri dishes until they were 80-90% confluent. Serum was omitted from the medium for 1.5 h. Cells were washed twice in isotonic solution (in mM: 105 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 10 HEPES, and 90 mannitol, adjusted to pH 7.4 with NaOH; 320 mosM) and stimulated for the indicated time periods with either the isotonic solution or a 25% hypotonic solution (in mM: 105 NaCl, 6 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 10 HEPES, adjusted to pH 7.4 with NaOH; 240 mosM) at room temperature. Rac- and Cdc42-GTP formation was assessed with a pull-down assay (47). After lysis in ice-cold Triton X-100 buffer (25 mM Tris, 100 mM NaCl, 2 mM DTT, 1 mM EGTA, 1 mM PMSF, 1% Triton-X100, 0.4 mM Napervanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 90 mM mannitol for the isotonic buffer) and centrifugation at 10,000 g,4°C for 10 min, the supernatant was incubated with GST-PAK-CD beads (±50 µg) at 4°C during constant rotation. After 60 min, the beads were precipitated at 500 g for 5 min at 4°C and washed six times with GST-fish buffer. Beads were resuspended in GST-fish buffer and Laemmli loading dye and were further analyzed with SDS-PAGE and Western blotting.
RhoA activation assay. Rat-1 fibroblasts were grown in 10-cm petri dishes until they were 80-90% confluent. Serum was omitted from the medium for 1.5 h. Cells were washed twice in isotonic solution and stimulated for the indicated time periods with hypotonic solution.
For the analysis of the amount of RhoA-GTP during cell swelling, we used the RhoA activation assay kit according the manufacturer's instructions (Cytoskeleton, Denver, CO).
SDS-PAGE and Western blotting. Proteins captured by GST-Sepharose beads or aliquots from whole cell lysates were separated on 12.5% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. The transfers were quenched overnight in TBS (10 mM Tris · HCl, pH 7.5, 153 mM NaCl) supplemented with 0.05% Tween-20 and 0.5% nonfat dry milk and then incubated with the corresponding primary antibodies overnight at 4°C. After thorough washing, the membranes were incubated with alkaline phosphatase-conjugated anti-mouse antibodies for 2 h. The blots were repeatedly washed in TBS-Tween and finally in TBS. Immunoreactive bands were visualized and quantified using the Vistra enhanced chemifluorescence detection kit on a Storm 840 Imager (Molecular Dynamics).
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RESULTS |
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Hypotonicity-induced membrane protrusions are mediated via activation of Rac1 and Cdc42. The hypotonicity-induced membrane protrusions consist of an actin-dense submembranous zone, followed by a cytosolic region largely devoid of F-actin. As such, they resemble growth-factor induced lamellipodia and membrane ruffles in quiescent fibroblasts (35) and urokinase-type plasminogen activator receptor (uPAR)-mediated membrane protrusions in growing fibroblasts (12). Because both lamellipodia and the uPAR-mediated membrane protrusions require activation of the small GTPase Rac, we next focused on the role of RhoA, Rac, and Cdc42 in the hypotonicity-induced actin remodeling.
Four observations indicate that Rho GTPases, and in particular activation of Rac1 and Cdc42, are indeed involved in the hypotonicity-induced membrane protrusions in Rat-1 fibroblasts. First, immunofluorescence staining revealed a change in subcellular distribution of Rac1 during hypotonicity (Fig. 2B). In control Rat-1 fibroblasts (upper row in Fig. 2B), Rac1 was more or less homogenously distributed within the cell with no apparent colocalization with F-actin filaments. However, in hypotonicity-exposed cells (lower row in Fig. 2B), a fraction of the cellular Rac1 pool translocated to the membrane protrusions where it colocalized with the submembranous F-actin patches. Hypotonicity-induced translocation of Cdc42 was also observed in some cells, although the redistribution was less pronounced than that of Rac1 (Fig. 2C). Although the distribution of RhoA changed upon cell swelling, it did not colocalize with F-actin patches (Fig. 2A). Second, a pull-down assay measuring the ratio of active GTP-loaded GTPase vs. total GTPase indicated a threefold activation of Rac1 and Cdc42 during hypotonic exposure for 10 min (Fig. 3A). In contrast, incubation of Rat-1 fibroblasts with an isotonic extracellular solution for 10 min did not affect Rac nor Cdc42 activity (Fig. 3A). Moreover, a pull-down assay for RhoA-GTP failed to detect an activation of RhoA (data not shown). Third, preincubation of Rat-1 fibroblasts with C. difficile toxin B, which inhibits RhoA, Rac, and Cdc42 GTPases by monoglucosylation (11), diminished the amount of stress fibers under isotonic conditions. Importantly, toxin B prevented the appearance of membrane protrusions and the actin reorganization during hypotonicity (Fig. 3B). Selective inhibition of RhoA with C. limosum C3 exoenzyme (1) reduced stress fibers under isotonic conditions, but it did not prevent the hypotonicity-induced F-actin redistribution (Fig. 3B), indicating that RhoA activity is not required for actin remodeling. Taken together, these data strongly point to an essential role for activation of Rac1 and Cdc42 in the formation of membrane protrusions and actin remodeling during hypotonic exposure.
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cRGD-induced changes in cytoskeleton. A major determinant of RhoA GTPase activity in adherent cells such as Rat-1 fibroblasts is the interaction between fibronectin in the extracellular matrix and integrin receptors in the plasma membrane. Engagement of integrins by fibronectin activates RhoA, which then triggers the formation of stress fibers and assembles focal adhesion complexes (4). In contrast, plating of fibroblasts on a fibronectin matrix leads to a transient activation of Rac (and formation of membrane ruffles), followed by a prolonged suppression (31, 37). Intriguingly, the morphological changes observed during hypotonic exposure (decrease of stress fibers and formation of lamellipodia-like membrane protrusions) suggest a hypotonicity-induced reversal of the RhoA/Rac balance. To explore this possibility, we tested whether inhibition of integrins would induce similar changes as hypotonicity. To this end, we incubated adherent Rat-1 fibroblasts with cRGD, a peptide that competes with the RGD-dependent binding of fibronectin to the integrin binding site (36). As shown in Fig. 4A, Rat-1 fibroblasts treated with cRGD (15 µg/ml) for 20 min under isotonic conditions became rounded and developed membrane protrusions that contained dense F-actin patches at the tip. The cRGD effects were also fully reversible (data not shown). The cRGD-induced changes depended on Rac1 and Cdc42 activity. Indeed, overnight incubation with C. difficile toxin B prevented the cRGD-induced membrane protrusions and actin rearrangement (Fig. 4B), whereas C. limosum C3 exoenzyme was without effect (Fig. 4C). Moreover, Rac1 and, to a lesser extent, Cdc42 translocated toward the F-actin patches formed upon cRGD application (Fig. 5), which is consistent with their activation.
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Dominant negative Rac1 and Cdc42 prevent the hypotonicity-and cRGD-induced rearrangement of F-actin. Further proof of the involvement of both Rac1 and Cdc42 in hypotonicity- and cRGD-induced F-actin remodeling was obtained from experiments with Rat-1 fibroblasts, transiently transfected with a bicistronic green fluorescent protein (GFP) expression vector for either dominant negative Rac1 (Rac1 Thr17Asn) or dominant negative Cdc42 (Cdc42 Thr17Asn). Transfected cells were identified by GFP fluorescence. Importantly, Rat-1 fibroblasts transiently expressing dominant negative Rac1 or Cdc42 were no longer able to rearrange the F-actin cytoskeleton when exposed to 25% hypotonicity or to cRGD (Fig. 6, A and B, left and middle columns). Control experiments indicated that the expression of GFP alone did not disturb the hypotonicity-induced F-actin remodeling (Fig. 6C). Furthermore, transient expression of constitutively active RhoA (RhoA Gly14Val) resulted in prominent stress fibers under isotonic conditions (data not shown), which did not disappear upon hypotonic exposure or cRGD incubation (Fig. 6, A and B, left and middle columns). In addition, there was no formation of membrane protrusions during hypotonicity or cRGD incubation in RhoA Gly14Val-expressing Rat-1 fibroblasts. Control experiments on nontransfected cells on the same coverslip showed that nontransfected cells were able to reorganize the F-actin cytoskeleton upon stimulation with hypotonicity cRGD (Fig. 6, A and B, right column).
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DISCUSSION |
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When interpreting these observations, one must take into account that the Rat-1 fibroblasts were seeded on gelatin (i.e., heat-denatured collagen)-coated coverslips and grown in the presence of serum, which, among other factors, also contains fibronectin. Because fibronectin is a multivalent adhesion protein that binds to collagen in the extracellular matrix and via an RGD tripeptide to integrin receptors in the plasma membrane (9), Rat-1 fibroblasts adopt a typical adherent phenotype after being seeded. This phenotype is a direct consequence of the fibronectin-integrin interaction that leads to activation of RhoA, formation of stress fibers, and consolidation of focal adhesion complexes (32). Another consequence of the fibronectin-integrin-mediated adhesion is a time-dependent activation/inactivation pattern of Rac1. Within 5 min of being plated on a fibronectin matrix, Rac1 activity increases, but it returns to baseline values within 1 h (31). Recently, Sastry et al. (37) proposed that the Rac1 activity in adherent cells is repressed via the PTP-PEST tyrosine phosphatase. Extrapolating from these data, one can predict that in adherent cells that have been seeded out for more than 24 h, RhoA outweighs Rac1 and Cdc42 with respect to their effect on the F-actin cytoskeleton. The phenotype of Rat-1 fibroblasts under isotonic conditions (well spread, with prominent stress fibers running through the cytoplasm and contacting the plasma membrane) is indeed consistent with a dominant role for RhoA in organizing the actin cytoskeleton.
Our experimental data clearly indicate that Rac1 and Cdc42 are activated during hypotonicity: Rac-GTP and Cdc42-GTP levels increase, and both GTPases translocate to the actin-dense submembranous patches. Furthermore, the hypotonicity-induced membrane protrusions are reminiscent of Rac1-induced lamellipodia and membrane ruffles observed in other cell types (12, 35). On the other hand, we have no direct evidence for RhoA inhibition by hypotonicity, because there is no change in the RhoA-GTP level, and RhoA does not translocate to the actin-dense submembranous patches. Nevertheless, hyperactive RhoA (e.g., the constitutively active RhoA Gly14Val) prevents the Rac1- and Cdc42-mediated actin remodeling during hypotonicity. This suggests that it is not so much the absolute Rac1/Cdc42 activity but rather the balance between RhoA and Rac1/Cdc42 that drives the actin remodeling during hypotonicity. We therefore propose that hypotonicity shifts the Rho GTPase balance from RhoA dominance toward Rac1/Cdc42 dominance, which then results not only in the formation of submembranous actin patches and membrane protrusions but also in the disassembly of stress fibers. Indeed, an increase in Rac1 activity and subsequent activation of PAK kinases can be sufficient to break down stress fibers (48).
How does hypotonicity activate Rac1 and Cdc42? One aspect of this question is the nature of the cellular sensor that responds to the extracellular hypotonicity and initiates a signal transduction cascade to activate Rac1 and Cdc42. A possibility is that hypotonic cell swelling induces a mechanical stress (e.g., membrane deformation) that disrupts integrin binding and/or clustering, which would then distort integrin-controlled signaling pathways, such as PTP-PEST-mediated inhibition of Rac (37). Consistent with this proposed mechanism is the observation that a disturbance of integrin signaling by cRGD, a competitor peptide for fibronectin-integrin interaction, triggered identical morphological changes, which also depended on Rac1 and Cdc42 activation. However, other intracellular signals (e.g., a change in intracellular ionic strength, intracellular ion concentration, or macromolecular crowding) cannot be formally excluded at this time. A second unresolved issue is the molecular identity of the signal transduction cascade responsible for Rac1 and Cdc42 activation during hypotonicity. The control of Rho GTPases is a complex process involving activating GEFs, inhibitory GAPs, and inhibitory GDIs (39). In principle, an activation of GEFs or an inhibition of GAPs and/or GDIs could account for the hypotonicity-triggered activation of Rac1 and Cdc42. Interestingly, an unconventional myosin (MyoM) containing GEF activity mediates the formation of actin protrusions upon hyposmotic stress in Dictyostelium discoideum (8). Whether a related or other GEF proteins are involved in the hypotonicity-induced activation of Rac1 and Cdc42 remains to be elucidated. Finally, although hypotonicity has been shown to activate both Rac and Cdc42, it is not clear whether these GTPases are activated in parallel or consecutively. Moreover, the exact contribution of each GTPase to the actin remodeling remains unresolved.
How general is the actin remodeling we have observed in hypotonicity-stimulated Rat-1 fibroblasts? The membrane protrusions in Rat-1 fibroblasts bear morphological resemblance to the membrane ruffles that have been described in swollen rat C6 glioma cells (21). Furthermore, cytosolic stress fibers disappeared during swelling of C6 glioma cells. In contrast, Tilly et al. (40) described precisely the opposite changes in human Intestine 407 cells, which displayed a transient increase in stress fibers and a transient decrease of peripheral F-actin (membrane ruffles) upon cell swelling. In addition, a loss of peripheral F-actin during cell swelling has also been observed in Ehrlich ascites tumor cells (29) and in B lymphocytes (16). Finally, in some cell types, e.g., calf pulmonary artery endothelial cells, the actin cytoskeleton remains stable during cell swelling with no detectable changes in either F-actin subcellular distribution or F-actin content (3). Thus, there is an enormous cell type-dependent variation in whether and how the F-actin cytoskeleton changes during cell swelling. At this moment, we have no molecular explanation for the intercellular variability, but differences in adhesion mechanisms, in expression of GEFs and GAPs, or in RhoA/Rac1/Cdc42 downstream effectors can all contribute.
A final question relates to the physiological significance of the hypotonicity-induced reorganization of the cytoskeleton. In our opinion, it is unlikely that actin remodeling plays a role in the activation of ion transporters or channels that contribute to RVD. First, there is no correlation between actin remodeling and activation of VRAC. Indeed, as mentioned above, rat C6 glioma cells, human Intestine 407 cells, Ehrlich ascites tumor cells, and B lymphocytes differ with respect to swelling-induced cytoskeletal changes, yet they all activate VRAC upon cell swelling (10, 23, 30, 40). VRAC activation and F-actin reorganization, therefore, seem to be swelling-induced phenomena that are not causally or mechanistically coupled. Second, not only cell swelling but also cell shrinkage can activate Rac1 and Cdc42, as reported by Kapus and colleagues (5, 17). Yet only cell swelling, but not shrinkage, results in the opening of VRAC. Thus, activation of Rac1 and Cdc42 is unlikely to be the critical factor for VRAC activity and, hence, for RVD.
What then could be the role of Rac1/Cdc42 activation and actin remodeling during cell swelling? It is interesting to note that the shrinkage-induced activation of Rac1 and Cdc42 resulted in the de novo formation of actin patches at the cell periphery (5, 17), similar to the swelling-induced membrane protrusions in Rat-1 fibroblasts. The explanation to this paradox (opposite stimuli but identical response) may be that both swelling and shrinkage interfere with integrin-dependent cell adhesion and, therefore, generate similar (identical?) intracellular signals leading to the activation of Rac1 and/or Cdc42 and actin remodeling. However, the functional relevance of the actin remodeling during both hypotonicity and hypertonicity remains to be elucidated.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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