1Department of Physiology, Emory University School of Medicine; and 2Department of Medicine, Atlanta Veterans Affairs Medical Center and Emory University Medical Center, Atlanta, Georgia
Submitted 6 January 2005 ; accepted in final form 12 April 2005
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ABSTRACT |
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aldosterone; epithelial sodium channel; serum- and glycocorticoid-inducible kinase
NO is a highly diffusible, short-lived free radical that is synthesized from the amino acid L-arginine in a reaction catalyzed by NO synthase (NOS). In vivo NOS catalyzes the conversion of L-arginine, NADPH, and O2 to L-citrulline, NADP+, and NO. Two types of NOS have been identified: the constitutively active forms neuronal and endothelial NOS (nNOS and eNOS, respectively) are dependent on Ca2+ and calmodulin for activity; in contrast, inducible NOS (iNOS) is Ca2+ and calmodulin independent. Both aldosterone and glucocorticoids have been shown to inhibit iNOS activity and hence to decrease levels of NO without effecting iNOS mRNA expression (5, 8, 23, 30). Because corticosteroids are the principal physiological regulators of transepithelial Na+ transport, we reasoned that one effect of aldosterone and glucocorticoids is to decrease iNOS activity and thereby reduce NO inhibition of ENaC activity in Na+-reabsorbing epithelia. This model of NOS regulation has been reported in the neuronal isoform. Phosphorylation of nNOS by calmodulin kinase at S847 reportedly inhibits nNOS activity (17, 29). Because iNOS and nNOS share 57% homology, we hypothesized that corticosteroids might also mediate the phosphorylation of iNOS, possibly through serum- and glucocorticoid-inducible kinase (SGK1), to decrease NO production in Na+-transporting epithelia, which would otherwise inhibit ENaC function.
SGK1 was first described as an immediate, early induced transcript in mammary epithelial cells by serum and glucocorticoids (44). It also has been shown that aldosterone increases the level of SGK1 expression within minutes in A6 distal nephron cell lines (6) and mammalian cortical collecting duct (CCD) cells (33). Moreover, knockdown of kinase activity by dominant negative SGK1 (18) or antisense SGK1 (34) expression substantially decreases ENaC activity in renal epithelia, and SGK1-null mice experience impaired Na+ retention when fed a low-salt diet (45). It has been proposed that SGK1 positively regulates ENaC through direct interaction of its PY motif with the ubiquitin ligase Nedd4-2, which would lead to the eventual degradation of surface ENaC proteins (9, 39). However, because SGK1 is expressed in a wide range of tissues and several pathways may lead to the regulation of amiloride-sensitive Na+ transport, additional SGK1 effectors may be involved in SGK1's signal transduction cascade, leading to the upregulation of ENaC activity. Therefore, our current study examined the physiological interaction between SGK1 and iNOS to determine whether the NO inhibition of amiloride-sensitive Na+ channel activity and corticosteroid inhibition of NOS were mediated by SGK1.
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METHODS |
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Primary mouse alveolar type II (ATII) cells were isolated and maintained as described previously (24). All procedures involving animals were reviewed and approved by our Institutional Animal Care and Use Committee. Briefly, 3-mo-old BALB/c mice were anesthetized with pentobarbital sodium and then killed after the lungs were perfused with PBS. Lungs were removed from the animal, and subsequently enzyme digested with dispase and 0.1 mg/ml DNAse in DMEM. Dispersed cells were then passed through a 20-µm nylon mesh and purified using the differential adherence technique (10).
Electrophysiological measurements.
With the use of patch-clamp techniques, cell-attached recordings were established on the apical membrane of A6 cells and grown on permeable supports. Polished micropipettes were pulled from filamented borosilicate glass capillaries (TW-150; World Precision Instruments) with a two-stage vertical puller (Narishige, Tokyo, Japan). The resistance of fire-polished pipettes were between 5 and 10 M when filled with pipette solution containing (in mM) 96 NaCl, 3.4 KCl, 0.8 CaCl2, 0.8 MgCl2, and 10 HEPES, pH 7.4. Under the above culture conditions, a high-resistance seal (>20 G
) was usually formed after slight negative pressure was applied to the patch membrane. Channel currents were sampled at 5 kHz with a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Union City, CA) and filtered at 1 kHz with a low-pass Bessel filter. Data were recorded using a computer equipped with AxoScope8 software (Molecular Devices). The open probability (Po) of the channels was calculated using FETCHAN in pCLAMP6 software. Experiments were conducted at 2223°C.
A6 and M1 cells were grown to confluence on Transwell-permeable supports (Corning, Acton, MA). After 20 days in culture, the potential difference (PD) and transepithelial resistance (RTE) across cell monolayers were measured using an epithelial voltohmeter equipped with stick electrodes (World Precision Instruments, Sarasota, FL). The equivalent short-circuit current (Isc) was calculated according to Ohm's law (Isc = PD/RTE) and then corrected for the surface area of the Transwell insert.
Immunoprecipitation of iNOS and Western blot analysis. A6, M1, and ATII cells were rinsed three times with PBS before being lysed with 600 µl of lysis buffer (150 mM NaCl, 10 mM NaPO4, pH 7.4, 0.1% SDS, 1% Nonidet P-40, 0.25% Na+-deoxycholate, and freshly prepared 1x protease inhibitor cocktail), and protein concentration was determined using bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL). iNOS immunoprecipitations were performed with rabbit polyclonal iNOS antibody (Upstate, Lake Placid, NY). To coimmunoprecipitate iNOS with SGK1, 3 µl of rabbit polyclonal anti-SGK1 antibody were incubated as described previously (46) with the cell lysate overnight at 4°C. The next day, immunoprecipitated protein complexes were immobilized with ImmunoPure protein A beads (Pierce Chemical), electrophoresed on a 7.5% acrylamide gel under denaturing conditions, and then transferred to Protran nitrocellulose membrane (Schleicher & Schuell, Keene, NH). The membrane was blocked in TBST buffer (10 mM Tris, pH 7.5, 70 mM NaCl, and 0.1% Tween) with 5% dry milk and then incubated with 1 µg/ml mouse monoclonal anti-iNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. IgG-horseradish peroxidase (HRP)-labeled secondary antibody (KPL, Gaithersburg, MD) was added at a concentration of 1 µg/10 ml TBST and incubated for another 1 h at room temperature. After being washed thoroughly, HRP signal was detected using the enhanced chemiluminescence substrate and Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ).
Immunocytochemistry. Primary mouse ATII cells were allowed to adhere overnight on 1-mm round poly-D-lysine-coated coverslips (Becton Dickinson Labware, Bedford, MA) in growth medium and then were fixed with 4% paraformaldehyde for 10 min at room temperature. A 1:1,000 dilution of polyclonal rabbit anti-SGK1 and mouse monoclonal iNOS antibody (see above) was diluted in Ca2+- and MgCl-free PBS with 3% horse serum and 1% BSA and then was applied to the cells for 1 h at room temperature. The cells were then washed three times with blocking buffer, followed by application to the cells of a 1:10,000 mix of Alexa Fluor 488-labeled goat anti-rabbit and Alexa Fluor 568-labeled goat anti-mouse antibodies (Molecular Probes, Eugene, OR) for an additional 1 h at room temperature. Cells were washed and fixed again and then mounted onto a slide with antifade reagent (Molecular Probes). Subcellular localization of SGK1 and iNOS was analyzed using standard confocal microscopy, and 1-µm-thick sections throughout the cell were sequentially imaged. The emission data for SGK1 and iNOS were collected separately and subsequently superimposed using LSM 5 Image Browser software (Carl Zeiss, Thornwood, NY)
In vitro SGK1 kinase assay.
Active SGK1 enzyme (160, S422D) was purchased from Upstate, and the kinase assay procedures were performed as recommended by the manufacturer. Briefly, in a 50-µl reaction volume, 25 ng of SGK1, 10 µCi [
-32P]ATP, 10 µM cold ATP, and PKA/PKC inhibitors were incubated at 30°C for 10 min with either hiNOS enzyme (Alexis Biochemicals, San Diego, CA) or iNOS oligopeptides custom produced by Sigma Genosys (The Woodlands, TX) as described in the text. Subsequent to incubation, a 35-µl aliquot of the reaction was transferred to phosphocellulose squares, washed, and read in scintillation liquid. Data were recorded as counts per minute (cpm), which indicate
-32P incorporation into iNOS substrate. Assay buffer was substituted for hiNOS or oligopeptides in negative background control groups.
32P labeling. Confluent M1 cells were serum and hormone deprived for 72 h and then rinsed twice with sodium phosphate-free DMEM (Invitrogen). The cells were then labeled with 0.5 mCi 32P Pi (Amersham Biosciences) for 6 h and then treated with 1.5 µg of aldosterone (or remained in serum- and hormone-free medium as control) for an additional 4 h. Radioactively labeled lysates were then immunoprecipitated using anti-iNOS or SGK1 antibody, separated on 7.5% denaturing gel, and transferred to nitrocellulose using the procedures described above. Quantification of 32P-labeled iNOS protein was enhanced with the use of Molecular Dynamics PhosphorScreen (Sunnyvale, CA) and quantified using ImageQuant software obtained from Amersham Biosciences.
Measurement of NO release. Measurement of NO release was performed on freshly isolated ATII cells. Immediately after isolation, ATII cells were seeded onto Costar Transwell 12-mm inserts (Corning) at confluent densities. NO release was determined by measuring NO and its oxidation products, NO2 and NO3, from the culture medium as described in (27). Briefly, after incubation with or without aldosterone overnight, culture medium was collected and injected into a vessel containing 0.8% NaCl3 in 1 N HCl at 95°C. NO was detected using a chemiluminescence NO analyzer (model 280; Sievers, Boulder, CO), and standard curves were generated using 0.110 µM NaNO3 in serum- and hormone-free DMEM.
Statistical evaluation. Statistical analyses were performed using Student's t-test, with statistical significance defined as P < 0.05. Data are expressed as means ± SE. ANOVA among multiple parameters was performed using the Holm t-test, which allows sequential comparison of unadjusted P values.
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RESULTS |
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Several previous studies have shown opposing data regarding the effect of NO on Na+ transport. For example, infusion of substances such as acetylcholine (which causes NO release into the renal artery) increases urinary volume and decreases Na+ absorption in in vivo animal models (35). Application of acetylcholine and NO donors such as spermine NONOate and nitroglycerin to M1 cells also has shown that NO directly decreases net Na+ flux in mouse CCD cell lines (40). However, other studies have reported that sodium nitroprusside release of NO fails to change net Na+ flux (19) or that NO can even stimulate amiloride-sensitive Na+ channels in rat CCD cells (32). Because such differences could be attributed to differences in the properties of the cell types tested or even the specific NO donor used, we tested the effect of PAPA-NONOate on both the A6 Xenopus distal renal cells and in the mouse M1 CCD cell lines to better understand NO's role in regulating ENaC. Both cell lines are model systems for studying amiloride-sensitive Na+ transport. The NO donor PAPA-NONOate is a zwitterion capable of rapidly releasing 2 M NO per mole of parent compound. The half-life of PAPA-NONOate at room temperature is 76.6 min in PBS and culture medium (20). We found that very low (50 nM-3 µM) concentrations of PAPA-NONOate were effective in decreasing Isc values in both A6 and M1 cell lines within 1 min, and persisted for at least 10 min without significant change. Figure 1 shows the effect of on Isc 5 min after applying 1.5 µM PAPA-NONOate to the apical membrane of both A6 and M1 cell lines. As a control for the NO donor compound, we allowed the same concentration of PAPA-NONOate to expire at room temperature overnight and then applied the expired PAPA-NONOate to similarly maintained A6 and M1 cells. This inactivated compound is not capable of donating NO. In this way, we could determine whether an effect on Na+ transport was due to the release of NO or to the metabolites of the parent compound. The data shown in Fig. 1, A and B, left, are expressed as %Isc decrease (from pretreatment Isc measurements) after 1.5 µM active PAPA-NONOate or inactivated compound was added to the apical membrane. Active PAPA-NONOate significantly decreased the Isc in A6 cells from an average value of 8.23 ± 0.68 µA/cm2 to 5.91 ± 0.62 µA/cm2, an
29% decrease in Isc, n = 20 (Fig. 1A, left). Application of the inactivated PAPA-NONOate did not substantially affect Isc values of A6 cells. The
3% decrease (n = 12) after applying the control compound did not cause a significant decrease in Na+ current compared with pretreatment Isc values (Fig. 1A, right). However, in A6 cells, the percent current decrease in 1.5 µM PAPA-NONOate vs. inactive compound was statistically significant (P < 0.0005). Similarly, 1.5 µM PAPA-NONOate significantly decreased amiloride-sensitive Isc of M1 cells by 44%, from 10.63 ± 0.60 µA/cm2 to 6.0 ± 0.34 µA/cm2, n = 12 (Fig. 1B, left). The Isc values of M1 cells declined by 19% in the control studies, n = 12 (Fig. 1B, right). Again, statistical comparison between the 1.5 µM NONOate-treated M1 group vs. control compound showed a significant decrease in the changes in current (P < 0.0005).
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NO decreases the Po of ENaC in A6 cells. When applied to the apical surface of A6 cells, 1.5 µM PAPA-NONOate decreased the Po of ENaC from 0.186 ± 0.043 to 0.045 ± 0.009 (P < 0.05) without significantly changing the unitary current of the channel (Fig. 2, A and B). The top trace in Fig. 2A is a representative cell-attached single-channel recording from a renal A6 cell before application of PAPA-NONOate showing typical ENaC activity. The bottom trace shown in Fig. 2A shows the same single-channel recording 3 min after application of NO donor, with an apparent decrease in ENaC activity. The Po was calculated from seven independently performed patch-clamp studies, and average values are shown in Fig. 2B. In a separate study, similar to the transepithelial Isc studies described above, we also added 1.5 µM inactivated PAPA-NONOate after an initial control recording period. Again, we found that the parent NO donor and its metabolites were not responsible for the NO-induced decrease in ENaC Po. Inactivated PAPA-NONOate did not substantially decrease Po values in A6 cells (Fig. 2C).
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SGK1 associates with iNOS in coimmunoprecipitation studies and in vivo.
The lung and kidney express high levels of iNOS mRNA and protein in response to cell injury (25). Consequently, under some circumstances, very high levels of NO can be produced. Even in unstimulated conditions, however, measurable quantities of NO are produced from iNOS (16). This implies that in the absence of any NOS inhibitor, ENaC Po will always be reduced to a greater or lesser extent by NO. Often, when it is necessary to increase Na+ transport, new channels are inserted in the apical membrane of Na+-transporting epithelial cells. If the Po of the newly inserted channels is reduced by endogenous NO, then Na+ transport will be limited even though there are new apical transporters. Aldosterone increases Na+ transport, and one mechanism by which it produces this increase is by increasing the number of apical channels. The increase is produced by an activation of SGK1. After promoting the insertion of new channels, however, it makes sense that the Po of the new channels would not be inhibited by NO. Therefore, we examined whether SGK1 might also increase Po by inhibiting NOS and NO production while promoting an increase in the number of channels. To test this hypothesis, we first studied whether SGK1, an important regulator of Na+ transport, is associated with iNOS in Na+-transporting epithelia. Our model for SGK1 regulation of ENaC activity presumed that SGK1 decreases iNOS activity via direct phosphorylation of iNOS to maintain low levels of NO. Inhibition of iNOS is a plausible mechanism for normal ENaC function because pharmacologically inhibiting iNOS with the use of N-nitro-L-arginine methyl ester (37) and iNOS/ mice (22) renders these mice iNOS deficient and hypertensive.
We first showed that iNOS is easily detectable in Na+-transporting epithelia and coimmunoprecipitates with SGK1. In each 7.5% PAGE Western blot analysis assay, the left bands in Fig. 3A show iNOS immunoprecipitated from A6 cell lysate (Fig. 3A, 1), M1 lysate (Fig. 3A, 2), and primary ATII cell lysate (Fig. 3A, 3) using anti-iNOS antibody. These immunoreactive bands serve as the positive signal control for the coimmunoprecipitation of iNOS with SGK1 in the right lanes of each respective blot in Fig. 3A. With the use of the same experimental protocol used for 13 in Fig. 3A, iNOS protein did not coimmunoprecipitate with rabbit polyclonal anti-GAPDH antibody from A6, M1, and ATII cell lysate (Fig. 3A, negative control).
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SGK1 phosphorylates iNOS in in vitro kinase assays.
To demonstrate that SGK1 is capable of phosphorylating iNOS, we performed in vitro kinase assays in which active SGK1 enzyme was incubated with iNOS protein in the presence of radioactively labeled [-32P]ATP. Figure 4A shows that iNOS is phosphorylated by SGK1 at high levels similar to those of the positive SGK-tide control (28, 36). Because SGK1 is a Ser/Thr kinase, we next identified which amino acid residues on iNOS are specifically phosphorylated by SGK1.
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Our current data show that SGK1 is capable of phosphorylating iNOS and that the two proteins are closely associated in ATII cells freshly obtained from mouse alveolar epithelium. Below, we describe a novel aldosterone-dependent, posttranslational modification of iNOS in M1 CCD cells.
Aldosterone alters in vivo phosphorylation of iNOS and decreases NO production in Na+-transporting epithelia.
First, we metabolically labeled M1 cells with 32P Pi in the presence or absence of 1.5 µM aldosterone. Subsequently, we immunoprecipitated iNOS from 32P-labeled cells and subjected the immunoprecipitate to SDS-PAGE using a 7.5% gel. The outlined region in Fig. 5A highlights 32P-labeled iNOS protein (left) and its corresponding pixel intensity profile (middle). Figure 5A, right, shows the results of Western blot analysis performed to confirm that the 32P-labeled band in the left column was indeed immunoreactive with anti-iNOS antibody. The bands in the autoradiogram and Western blot analysis overlap precisely when the images are superimposed. Similarly, Fig. 5B shows that iNOS protein, which coimmunoprecipitated with anti-SGK1 antibody, from cells grown without aldosterone (left) exhibited less phosphorylated iNOS protein compared with M1 cells grown with aldosterone (right) aldosterone. The average pixel intensities of phosphorylated iNOS from the autoradiogram are expressed as percent control in Fig. 5C. Aldosterone increased the level of 32P-labeled iNOS in M1 cells 300% above control levels, regardless of whether iNOS protein was directly immunoprecipitated using anti-iNOS antibody (Fig. 5A) or coimmunoprecipitated with polyclonal SGK1 antibody (Fig. 5B).
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DISCUSSION |
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Our findings in the M1 CCD and A6 distal nephron cell lines are similar to findings in two other independent groups. Stoos and colleagues (40, 41) showed that acetylcholine-induced NO release from endothelial cells, as well as addition of the NO donor spermine NONOate, inhibited Na+ reabsorption in CCD cells. Rückes-Nilges et al. (38) reported that 1 mM of sodium nitroprusside clearly inhibited amiloride-sensitive Na+ reabsorption in Xenopus kidney A6 distal nephron cell lines. However, in their study, the NO donors sodium nitroprusside and spermine NONOate did not alter either the amiloride-sensitive or the amiloride-insensitive portions of Isc in primary cultures of human nasal epithelial cells. It is becoming apparent that perhaps the efficiency of NO release from different NO donors may effect ion transport differently. The effect of NO also may depend on the cell type examined. For example, our research to date also includes the effect of NO donors S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) on ENaC activity in primary cultured adult rat ATII cells. Application of 100 µM GSNO and SNAP to patch-clamp recording solution did not significantly alter ENaC Po in ATII cells. This finding is similar to the results obtained by Rückes-Nilges et al. (38), who showed that NO released from spermine NONOate had no inhibitory potency on the human nasal epithelium. The different effects of NO donors in renal epithelial cells and in the lung epithelium may pertain to the slow half-lives of the donors used in the studies. GSNO and SNAP have slow half-lives of 38 ± 5 h, and the half-life of NO release at room temperature is
4 h for spermine NONOate. Furthermore, the total amount of NO release from SNAP donor corresponds to <2% of the total SNAP present (12). It has been shown that slow release of NO over a long period of time has less potent biological effects than the same amount of NO released rapidly (26). Perhaps the slow half-lives and inefficient release of NO may account for the inability of GSNO, SNAP, and spermine NONOate to alter Na+ transport properties in lung epithelial cells.
SGK1, which is important in mediating both early and late phases of aldosterone activity (1, 6, 33), also may be the key regulator of NO production and iNOS activity after corticosteroid stimulation. Our in vitro kinase assays identified iNOS as a novel SGK1 substrate. Specifically, miNOS oligopeptides that were 20 (peptide 1; S733) and 13 (peptide 2; S903) amino acid residues long were phosphorylated by SGK1. Shorter, nine-amino acid peptide sequences surrounding hiNOS S114, S749, S909, S917, and S965 were not phosphorylated effectively by SGK1 in our kinase assays. Our results suggest that longer peptide sequences may be required for appropriate protein folding and kinase phosphorylation. Interestingly, S903 in peptide 2 is preceded by a Pro residue at the 4 position. Prolines can act as structural disruptors for -helices or as a turning point for
-sheets. This may explain the 12.88 ± 5.28% [
-32P]ATP incorporation into peptide 2. Because iNOS does not fully express the traditionally conserved SGK1 phosphorylation sequence (RxRxxS/T), perhaps protein folding is particularly crucial for appropriate Ser phosphorylation by SGK1.
Figure 7 summarizes and illustrates our model of aldosterone-regulated Na+ transport, involving the SGK1, iNOS, and NO components. In the absence of aldosterone or after direct application of PAPA-NONOate, NO immediately inhibits ENaC by reducing ENaC Po without altering the unitary current or apparent channel density. However, aldosterone-induced increases in the expression of SGK1 may lead to the phosphorylation and hence inactivation of iNOS protein. Indeed, our studies have shown that local production of NO decreases after aldosterone treatment, which may be an important mechanism involved in controlling ENaC activity.
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Contrary to these findings, Guo et al. (15) reported a cGMP-independent mechanism of ENaC regulation by NO donors. Although they, too, reported that NO generated by PAPA-NONOate inhibited 60% of the amiloride-sensitive Isc in cultured ATII monolayers, they also reported that the NO-induced decrease in alveolar Isc was not accompanied by an increase in intracellular cGMP levels. Alternatively, the inhibitory effect of NO on ENaC may occur through direct interaction of NO with the channel or with other ENaC-regulatory proteins. DuVall et al. (11) recently suggested that direct nitration or nitrosylation of key Tyr residues on the outer borders of the transmembrane domain (TM) of -ENaC subunit (Y134 and Y137 in TM1; Y482, Y484, and Y485 in TM2) may alter ENaC activity.
Overall, the present data support an inhibitory effect of NO on ENaC activity in both M1 CCD and A6 epithelial cell lines. In addition, we have shown that the mechanisms by which aldosterone regulates ENaC function include phosphorylation of iNOS and decreased synthesis of NO, possibly through the SGK1 signaling pathway.
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GRANTS |
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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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