Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026
Submitted 21 August 2003 ; accepted in final form 18 November 2003
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ABSTRACT |
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aldosterone; sodium depletion; distal colon; amiloride; serum- and glucocorticoid-induced kinase 1
These observations imply that the early aldosterone response (29), which takes place within the first 2 h of administration of the hormone, differs in these tissues. In the cortical collecting tubule of the kidney, aldosterone may increase ENaC activity by translocation of channels to the apical membrane (19), by reducing the rate of endocytosis (10), or by activating previously silent channels (14). In the colon, however, aldosterone must first induce synthesis of new ENaC channels.
Recently, it has been proposed that serum- and glucocorticoid-induced kinase 1 (sgk1) mediates the early increase in sodium reabsorption in aldosterone-responsive segments of the renal tubule and distal colon (13, 30). This notion is based on an observation made in cultured cells in which aldosterone induces a rapid (<30 min) and large increase in the abundance of sgk1 mRNA and protein (2, 9). Previous studies (6, 26) have shown that aldosterone also increases the levels of sgk1 mRNA and protein in various tissues. However, in contrast to cultured cells, in which basal levels are very low or undetectable, the tissues so far examined exhibit significant levels of basal expression (3).
The goals of this study were 1) to examine the distribution of sgk1 in the rat intestinal epithelia and 2) to examine the correlation of aldosterone-mediated induction of sgk1 and ENaC subunits in the distal colon.
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MATERIALS AND METHODS |
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Plasmids. Full-length sgk1 cDNA was obtained from mouse poly(A+) mRNA by RT-PCR. The PCR product was ligated to pcDNA3.1-TOPO vector (Invitrogen) and sequenced at the Yale-Keck Foundation DNA sequencing facility. A plasmid construct containing protein kinase B (PKB; Akt) full-length cDNA and tagged with the hemagglutinin antigen (HA) epitope was obtained from Dr. A. Bennett (Yale University, New Haven, CT).
Antibodies. A rabbit polyclonal antibody directed against mouse sgk1 (S299-S404) glutathione S-transferase (GST)-fusion protein has been previously described (3). Rabbit polyclonal antibodies for - and
-subunits of rat ENaC have also been described previously (11). A new rabbit polyclonal anti-
-ENaC antibody was raised against a GST-fusion protein encompassing amino acids 310345 from the rat sequence. All three ENaC subunit antibodies were purified in affinity columns of maltose fusion proteins (New England Biolabs) made with the corresponding sequences used for the immunization of rabbits: E10F77 for
-subunit, G559E636 for
-subunit, and 310345 for
-subunit.
A polyclonal anti-Akt antibody was obtained from Cell Signaling Technology. This antibody detects Akt1, -2, and -3 proteins independently of their phosphorylation status and does not cross-react with related kinases. Monoclonal anti-HA antibody was from Santa Cruz Biotechnology.
Reagents. For immunonofluorescence, the secondary antibody was AlexaFluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR). For immunostaining of polysacharides in the distal colon and filamentous actin we used wheat germ agglutinin and phalloidin conjugated to Texas Red-X (Molecular Probes), respectively. Nuclei were visualized with 4',6-diamidino-2-phenylindole (DAPI) by mixing with the mounting solution at a concentration of 5 µg/ml.
Cell culture and transfections. Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection and grown in a 1:1 mixture of DMEM/F-12 medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Cells were transfected with Lipofectamine 2000 (Invitrogen) following instructions from the manufacturer. For immunofluorescent experiments, cells were grown on glass coverslips. After transfection, cells were incubated for 24 h to allow recovery and expression of the protein of interest.
Animal treatments. Animals were divided into four groups: 1) sham adrenalectomized (ADX) rats served as the control group, 2) ADX rats were bilaterally adrenalectomized and 0.9% of NaCl was given in drinking water, 3) 1 wk of low-salt diet (ICN, Cleveland OH), and 4) single subcutaneous injection of aldosterone (0.002 mg/kg) followed by euthanasia after 4 h. Adrenalectomy was performed as previously described (27). Briefly, Sprague-Dawley rats (Charles River, Boston, MA) weighing 200250 g were anesthetized by ether inhalation for surgical preparations. Animals in each group were given pentobarbital (1,000 mg/kg) and killed by decapitation according to protocols approved by the Institutional Animal Care and Use Committee. The intestine was immediately removed, and the luminal contents were cleansed with cold PBS. The intestine was dissected in 4-cm segments corresponding to jejunum, ileum, and distal colon. Jejunum was taken 10 cm after the stomach, ileum was taken as the segment preceding the cecum, and the colon was the most distal segment that could be dissected from the gut.
Extraction of RNA and Northern blot analysis. Total RNA was extracted from tissues using TRIzol reagent (Invitrogen). After the concentration of RNA was measured at 260 and 280 nm optical density, 15 µg from each sample were size-fractionated by electrophoresis in formaldehyde agarose gels containing ethidium bromide. RNA was transferred to nylon membranes (Hybond-N; Amersham) and cross-linked by UV light. Membranes were prehybridized with 50% formamide, 5x sodium chloride-sodium phosphate-EDTA, 2x Denhardt's reagent, 0.1% SDS, and 100 µg denatured salmon sperm DNA at 42°C for 2 h. A denatured radiolabeled probe (whole coding sequence of sgk1 labeled with [-32P]dCTP by random priming, specific activity >2 x 108 counts·min-1·µg-1) was added and incubated for an additional 14 h. Filters were washed to maximal stringency of 0.2x SSC, and 0.1% SDS at 68°C. Membranes were exposed to X-ray film at -80°C with an intensifying screen.
Western blot analysis. CHO cells were lysed in a Triton X-100-containing buffer. After clarification, equal volumes of the lysate were resolved on SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Blots of samples run in parallel were probed with anti-sgk, anti-Akt, and anti-HA antibodies at 1:10,000, 1:2,000 and 1:1,000 dilutions, respectively. The secondary antibodies were anti-rabbit or anti-mouse conjugated with peroxidase (Sigma) at 1:10,000 dilution. Signals were developed with ECL+ (Amersham Pharmacia Biotech).
Processing of rat tissues. Segments of intestine were cut open, and the mucosa was scraped with glass slides. In addition, samples of whole brain, liver, and skeletal muscle were separately homogenized in a glass/Teflon homogenizer in 1 ml of 10 mM HEPES, pH 7.4, 1.7% (wt/vol) sucrose, and complete protease inhibitors (Roche). Homogenates were centrifuged at 1,000 g for 10 min at 4°C. After collection of the supernatants, the sucrose concentration was brought to 8% (wt/vol) and the samples were centrifuged at 250,000 g for 30 min at 4°C in an Optima-TLX ultracentrifuge (Beckman). The supernatant and pellet were assayed for protein concentration with BCA protein assay kit (Pierce). Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Immobilon-P). Membranes were blocked with 5% nonfat dry milk in TBST (20 mM Tris, pH 7.6, 120 mM NaCl, 0.1% Tween) for 30 min. Rabbit anti-sgk antibody (1:8,000) or purified anti--,
- or
-ENaC (1:300) were incubated for 2 h at room temperature. After three 15-min washes with TBST, a 1:10,000 dilution of horseradish peroxidase-labeled anti-rabbit secondary antibody (Sigma) was incubated for 1 h at room temperature. Membranes were washed three times with TBST, and signals were developed with ECL+ (Amersham Pharmacia Biotech) according to the manufacturer's protocols and exposed to BioMax MR film (Eastman Kodak). Signals were quantified by scanning densitometry of X-ray films with a GS-800 Calibrated Densitometer (BioRad).
Indirect immunofluorescence microscopy. CHO cells grown on coverslips and transiently transfected with sgk1 or Akt cDNAs were fixed with 4% formaldehyde solution made in PBS. After several washes with PBS, cells were permeabilized with the same buffer containing 0.3% Triton X-100 and 0.1% BSA. Nonspecific antibody-binding sites were then blocked by incubation in a solution of 16% filtered goat serum, 0.3% Triton X-100, 150 mM NaCl, and 20 mM NaPi, pH 7.4. Cells were then incubated overnight with 1:1,000 dilution of anti-sgk antibody or 1:50 dilution of anti-Akt antibody in blocking solution. After washes, cells were incubated for 1 h with anti-rabbit secondary antibody conjugated to AlexaFluor 488 (Molecular Probes) diluted 1:200 in blocking solution.
For immunofluorescent microscopy of tissues, rats were anesthetized with pentobarbital sodium (65 mg/kg ip) and perfused via the left ventricle with HBSS with drainage from the severed inferior vena cava until the kidneys were thoroughly blanched. Rats were then perfusion fixed with paraformaldehyde/lysine/sodium periodate (PLP; 2%, 75 mM, and 10 mM, respectively). Distal colon was excised and further fixed in PLP overnight at 4°C. Fixed tissues were washed four times with PBS, infiltrated with 30% sucrose in PBS, frozen in liquid nitrogen, and cut into 10-µm thick sections with a Leica CM3050S cryostat. For indirect immunofluorescence, sections were preincubated at room temperature in PBS for 10 min and in 1% bovine serum albumin in PBS for 15 min and were incubated at room temperature for 12 h with primary antibody as indicated. Sgk1 antibody (1:2,000) required 0.1% SDS in the incubation medium. -,
-, and
-Rat ENaC antibodies were used at a dilution of 1:500. In some cases, peptide antigens were included in the incubation mix at 12 µg/ml. After being washed with PBS, sections were incubated for 1 h with fluorophore-conjugated secondary antibodies (1015 µg/ml), again washed for three 5-min washes in PBS, and mounted in 50% glycerol in PBS, pH 7.5, containing 2% n-propyl-gallate as an antiquenching agent. Sections were examined with a Zeiss Axiophot. Digital images were acquired with AxioCam HRm and compiled with Adobe Photoshop 7.0.
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RESULTS |
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We have generated and previously characterized the sgk antibody used in this work (3). However, the question has been raised of possible cross-reactivity with other members of the AGC family. The antibody was raised with a GST-fusion protein containing the mouse sgk1 sequence from residues S299S404. When this segment of the sgk1 protein was compared with all other proteins in the GenBank using the BLAST program (National Center for Biotechnology Information, National Institutes of Health), in addition to the other isoforms of sgk1, -2, and -3, the only other protein that shared significant homology (40%) was Akt (PKB).
We tested whether our anti-sgk antibody cross-reacts with Akt by overexpressing sgk1 or Akt in transiently transfected CHO cells. Fig. 1A shows the results of Western blots examined with anti-sgk antibody. Cells transfected with sgk1 cDNA revealed a band of the expected molecular weight, but control and cells transfected with Akt were negative. To demonstrate that the absence of signal in the Western blot was not due to the lack of expression of Akt, we stripped the membrane and reprobed it with a commercial Akt antibody. With the anti-Akt antibody, a band was revealed in all samples but was strongest in the one transfected with Akt cDNA, suggesting that the antibody recognizes the endogenous and the transfected Akt proteins. This was confirmed by rebloting the membrane with an anti-HA monoclonal antibody. Only cells transfected with Akt were positive. The upper band seen in all lanes is nonspecific for the monoclonal anti-HA antibody.
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The possibility of cross-reactivity was also examined by immunofluorescence microscopy. The anti-sgk1 antibody strongly reacted with CHO cells transfected with sgk1 but not with cells transfected with Akt, whereas the opposite immunoreactivity was detected with anti-Akt antibody (Fig. 1B). In summary, these experiments show that our anti-sgk antibody does not detect Akt1, whether endogenous or overexpressed.
Expression of sgk1 in rat intestine
Because it has been previously shown that sgk1 associates to the microsomal fraction (3, 7), samples from various tissues were separated into microsomes (pellet) and soluble fraction (supernatant) before analysis by Western blotting. Figure 2 shows a representative Western blot probed with anti-sgk1 antibody. The tissues examined were jejunum, ileum, distal colon, whole brain, liver, and skeletal muscle. Signals were detected in the pellet of jejunum, ileum, and distal colon and were much weaker in brain. All soluble fractions were negative. The antibody recognizes a band of 53 kDa molecular mass, which corresponds to sgk1. Because sgk2 and -3 exhibit slower electrophoretic mobility when run on similar gels (3), the results indicate that sgk1 is the predominant isoform expressed in rat intestine.
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Distribution of Sgk1 in Distal Colon
To determine the sites of sgk1 expression, we performed indirect immunohistochemistry of distal colon from control rats. Figure 3A shows sgk1 immunoreactivity (green) predominantly in the absorptive cells of the surface epithelium. A weaker signal was observed in the crypts. Goblet cells and brush border were stained with wheat germ agglutinin conjugated to Texas Red-X (Fig. 3B), and the nuclei were stained with DAPI (Fig. 3C). The overlay of images in Fig. 3, AC is shown in Fig. 3D. A similar experiment in which the anti-sgk1 antibody was mixed and preincubated with the cognate fusion protein before addition to the colon section completely abolished the fluorescent signal (not shown).
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It has been shown previously that the cellular localization of sgk1 changes according to the phase of the cell cycle. In a mammary tumor cell line, sgk1 is cytoplasmic in G1 and nuclear in S and G2/M phases of the cell cycle (8). In the ovary, on treatment with follicle stimulating hormone, sgk1 resides in the nucleus of proliferating granular cells, whereas in terminally differentiated luteal cells, sgk1 is localized in the cytoplasm (1). Because there is a high cell turnover in the crypts, we were particularly interested in determining whether the subcellular localization of sgk1 was different in crypts vs. the surface epithelium. Fig. 3D, however, did not reveal any significant colocalization of DAPI and sgk1 in the nuclei of the mucosal cells.
It can be observed in Fig. 3 that the signal of sgk1 is more prominent in the basolateral side of epithelial cells than toward the apical membrane. To further define the subcellular localization of sgk, we costained sections of distal colon from sodium-depleted rats with Texas Red-phalloidin and either anti-sgk antibody (Fig. 4A) or anti--subunit of ENaC antibody (Fig. 4B). Phalloidin binds to filamentous actin and intensely stains the brush border of the colonic epithelium; thus we used it here to localize proteins closely associated to the apical membrane. Figure 4A shows that phalloidin (red) does not colocalize with sgk1 (green) but does colocalize with the apical
-subunit of ENaC, giving an intense overlapping signal (yellow) along the apical membrane of the absorptive epithelium. Deeper in the crypts, in which apical expression of ENaC is low, the brush border contains only phalloidin.
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Effect of Steroids on the Levels of Expression of Sgk1 in the Distal Colon
We examined the level of expression of sgk1 in aldosterone-responsive and -nonresponsive segments of the intestine, such as the distal colon and the small intestine, respectively. Analysis was conducted at the levels of RNA and protein.
Rats were submitted to several treatments to alter the circulating levels of glucocorticoids and/or aldosterone. Four conditions were examined: 1) a control group representing rats fed normal chow; 2) sodium depletion with concomitant increases in the levels of circulating aldosterone was induced by feeding a low-salt diet for 1 wk; 3) bilateral adrenalectomy (ADX) with supplemental salt in the drinking water was used to reduce levels of glucocorticoids and aldosterone; and 4) 4 h after injection of a single dose of aldosterone administered in the peritoneum. After the respective treatments, the mucosa of the distal colon and small intestine were scraped and homogenized in TRIzol for extraction of RNA or in homogenization buffer for protein isolation.
Total RNAs were analyzed by Northern blotting using a radiolabeled sgk1 probe. Figure 5, shows the Northern blots of distal colon and small intestine and the same samples of total RNA stained with ethidium bromide. The Northern blot from colon was exposed to film for 24 h, whereas that from small intestine was exposed for 48 h. The results indicate expression of sgk1 mRNA in distal colon and small intestine of control animals, after treatment with low salt for a week, and after single injection of aldosterone. Bilateral adrenalectomy decreased the levels of expression in both distal colon and small intestine.
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To evaluate the effects of aldosterone on sgk1 protein expression, the tissues were homogenized in the absence of detergent. Microsomes and soluble fractions were examined by Western blot analysis. Figure 6 shows sgk1 expression in the epithelia of the distal colon and small intestine of the control group and experimental treatments. Sgk1 protein was detected in control animals, low-salt diet, and single dose of aldosterone with similar abundance. Adrenalectomy decreased the level of expression in both small intestine and colon.
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Effect of Steroids on the Levels of Expression of ENaC Protein in Distal Colon
To correlate expression of sgk1 and ENaC in the distal colon under conditions of high and low aldosterone levels, we performed Western blot analysis with antibodies for the -,
-, and
-subunits of ENaC. Equal amounts of protein were loaded in each lane, and the blot was sequentially probed with anti-
-, -
-, and -
-specific antibodies. The
-,
-, and
-subunits were detected as bands of expected molecular weights in animals treated with a single injection of aldosterone or fed a low-salt diet (Fig. 7). In contrast, the three subunits were not detected in control and ADX animals.
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Similarly, sections of distal colon stained with specific ENaC antibodies for the three subunits did not display immunoreactivity in control and ADX rats, but it was detected in the superficial epithelium of sodium-depleted animals (Fig. 8).
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DISCUSSION |
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Analysis of sgk1 expression in the rat intestine revealed a broader distribution than the one predicted by the distribution of mineralocorticoid receptors, i.e., restricted to the distal colon. Sgk1 was expressed in the epithelium of distal colon but also in jejunum and ileum. Localization reported here is consistent with a previous publication (17) that showed expression of mRNA sgk1 by in situ hybridization in the gut of mouse embryos from late stages of development.
Because sgk1 has been shown to modulate the activity of ENaC, it was of special interest to examine whether these proteins exhibit spatial and temporal relationships in the distal colon. Regarding distribution, we found that sgk1 and the subunits of ENaC are expressed predominantly in the absorptive epithelium of distal colon, whereas in crypts and goblet cells, the expression was weaker (Figs. 3, 4, and 8). At the subcellular level, both proteins associated with the microsomal fraction, but there were differences in the localization. ENaC was detected in the apical membrane closely associated with a brush-border marker (phalloidin) (Fig. 4). In contrast, the signal from sgk1 was distributed predominantly toward the basolateral membrane and was excluded from the apical brush border (Figs. 3 and 4). These results also indicate that migration of sgk1 with the pellet of nondetergent cellular homogenates is not due to tethering to actin microfilaments. Although these experiments do not rule out a small fraction of sgk1 being expressed at the apical membrane, we can conclude that most of the sgk1 is away from the apical membrane. Ideally, to demonstrate colocalization of ENaC and sgk1 at the apical membrane, it would have been preferable to costain sections with antibodies for these two proteins; however, our antibodies for ENaC and sgk1 are both raised in rabbits, precluding the experiments.
Currently, we do not know the functional meaning of the subcellular localization of sgk1 in absorptive epithelia. Sgk1 may be retained close to the basolateral membrane for activation by extracellular signals or proximity to its immediate phosphorylation targets, or, alternatively, it may serve to prevent sgk1 from reaching its targets until arrival of the appropriate stimuli. Once activated, a fraction of sgk1 may be released to interact with apical proteins. If this fraction is small, it will be difficult to detect with the immunochemical techniques used in our studies.
We also observed dissociation of expression of sgk1 and ENaC subunits in the distal colon. In basal conditions, sgk1 mRNA and protein levels were abundant, but ENaC subunits were low and difficult to detect by Western blotting. Sodium depletion did not increase sgk1, whereas it produced a robust induction of the three subunits of ENaC. Although previous studies (4, 12, 22, 23) have shown ENaC mRNA in the distal colon of animals fed a normal chow, there is no expression of protein or it is expressed at very low levels. Hence, the absence of electrogenic amiloride-sensitive sodium transport in the distal colon in basal conditions (12, 15) is likely due to expression of few channels and not to the presence of nonfunctional (i.e., silent) channels as is the case in the distal tubule of the kidney (14, 20). Our findings are also consistent with the time course of induction of amiloride-sensitive sodium transport by aldosterone. The distal colon does not exhibit an early phase of the aldosterone response, but the
2-h lag period before a gradual increase in ENaC activity that is detected (12) reflects the time required for de novo synthesis and not of translocation or activation of preformed channels.
Results in the literature regarding the magnitude of sgk1 induction by aldosterone are controversial, in part, because the experiments have been conducted under different conditions. Some studies were done in ADX animals with or without glucocorticoid receptor blockers, others in adrenal-intact rats (6, 26). Some studies examined the whole colon, whereas others examined only the distal segment. Doses of administered aldosterone varied widely. Even in a single study (26), different sets of experiments yielded different results (2.5-fold vs. 3.9-fold change in sgk1 mRNA). Finally, the way the data have been reported (normalized to different standards) not only obscures but makes it difficult to assess the actual change in sgk1 levels (6, 26). However, despite all these discrepancies, there are common findings. First, sgk1 is expressed at significant levels in the distal colon and kidney of adrenal intact rats. Second, normal physiological concentrations of glucocorticoids maintain sgk1 expression in both aldosterone-sensitive and -insensitive tissues. Third, high levels of aldosterone also increase sgk1 expression in some tissues.
Results indicate that in the distal colon, sgk1 does not behave as a strongly aldosterone-induced gene, which is in contrast to ENaC. In the distal colon of control rats, subunits of ENaC are undetectable by standard methods of Western blot analysis and immunohistochemistry but are strongly induced by acute (4 h) and chronic (low-salt diet) physiological increases of aldosterone.
What are the physiological implications of these findings? Sgk1 has a wide distribution in absorptive epithelia, including intestine and kidney. Not all of these tissues express ENaC or respond to aldosterone, indicating that sgk1 operates in an aldosterone-independent manner in many epithelia. The robust expression of sgk1 protein under basal conditions suggests that sgk1 has a constitutive role in a variety of absorptive epithelia. The initial report (9) of modulation of ENaC activity by sgk1 has been followed by multiple studies extending the effects of sgk1 to many other channels and transporters: the Na+-K+-2Cl- cotransporter (16), Na+/H+ exchanger (34, 35), Na+-K+-ATPase (25), CFTR (31), and the K+ channel of the outer medulla of the kidney (33). Some of these proteins are also expressed in various regions of the intestinal mucosa together with distinct intestinal transporters. Although we did not test the effects of sgk1 on the latter, the results raise the possibility that some of the intestinal transporters may also be modulated by sgk1 activity. However, the mechanism(s) by which sgk1 exerts its effects on ion channels and transporters has not yet been elucidated.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-54062 and National Heart, Lung, and Blood Institute Grant PO-HL-55007-01 to C. Canessa. D. Alvarez de la Rosa was supported by a Training Fellowship from the National Kidney Foundation. N. Hernandez was supported by Short-Term Research Training for Minority Students National Heart, Lung, and Blood Institute Grant HL-07722.
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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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REFERENCES |
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