2Division of Endocrinology, Diabetes, and Hypertension, Department of Medicine and Membrane Biology Program, Harvard Medical School, Boston, Massachusetts; and 1Osteoporosis and Bone Metabolic Unit, Department of Clinical Biochemistry and Endocrinology, Copenhagen University Hospital Hvidovre, Denmark
Submitted 19 October 2004 ; accepted in final form 15 January 2005
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ABSTRACT |
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G protein-coupled receptor; casr; Leydig cells; humoral hypercalcemia of malignancy; dominant negative; 4-amino-5-methylamino-2',7'-difluorofluorescein diacetete; inducible nitric oxide synthase
NO is produced by three isoforms of the enzyme NO synthase (NOS). These are neuronal (n), inducible (i), and endothelial (e)NOS (1). iNOS is classically upregulated in inflammation by endotoxin, interferon-, TNF-
, and IL-1
(36). iNOS is constitutively active and is the major producer of NO. Importantly, its activity is independent of intracellular calcium (Ca
) and calmodulin, which distinguishes it from n- and eNOS. iNOS acts as a homodimer, a process that can be prevented by interaction with kalirein, a peptide biosynthetic enzyme present in secretory granules (25). Currently, specific iNOS inhibitors are in clinical trials for the treatment of hypotensive shock (36).
NO production has been reported in cultured rat Leydig cells expressing iNOS mRNA, induced by IL-1 (30). In this context, NO functions as a negative regulator of Leydig cell testosterone production in vitro (10, 35). Furthermore, suppression of serum testosterone levels by agents promoting NO production and enhancement by administration of a general NOS inhibitor, L-NAME, in rats showed the role of NO as a negative modulator of steroidogenesis in Leydig cells. Immunohistochemistry of rat testis detected iNOS in both normal and inflamed Leydig cells (23). These reports reveal an important role for NO in the physiology and pathophysiology of the Leydig cell.
Extracellular calcium (Ca) can regulate NO production in vivo and in vitro (4, 16, 27). nNOS and eNOS are activated by opening of calcium channels, resulting in calcium influx. However, it is possible that calcium also acts by an extracellular "sensing" mechanism. One such mechanism is the calcium-sensing receptor (CaR), a G protein-coupled receptor (GPCR) that responds to very small changes in Ca
in tissues involved in Ca
homeostasis. The CaR is the mediator of calcium-induced inhibition of parathyroid hormone release and has been shown to have a variety of other functions, including the regulation of the cell cycle, peptide secretion, and apoptosis (33). We recently showed that the CaR upregulates promalignant cellular functions, such as parathyroid hormone-related protein (PTHrP) transcription and release, proliferation, protection against apoptosis, and expression of pituitary tumor transforming gene (PTTG) in rat primary Leydig cancer cells (H-500) in primary culture (8, 31, 32, 34). The H-500 model is a well-established xenotransplantable rat Leydig cell cancer model for the syndrome of humoral hypercalcemia of malignancy (24). Using this model, we tested our hypothesis that stimulating the Leydig cancer cells with high calcium would increase NO production through higher expression of iNOS. Furthermore, we hypothesized that the resultant high calcium-induced iNOS response would be mediated through the CaR.
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MATERIALS AND METHODS |
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A rabbit polyclonal antiserum against iNOS was purchased from BD Transduction Laboratories (Lexington, KY). An inhibitor of NOS, L-NAME, was obtained from Calbiochem-Novabiochem (San Diego, CA), and a selective iNOS inhibitor, 1400W, was obtained from Biomol (Plymouth Meeting, PA). The enhanced chemiluminescence kit, Supersignal, was purchased from Pierce (Rockford, IL). Protease inhibitors were obtained from Boehringer Mannheim (Mannheim, Germany), and other reagents were from Sigma Chemical (St. Louis, MO).
Cell Culture
The Rice H-500 rat Leydig cell tumor was obtained from the National Cancer Institute-Frederick Cancer Research and Development Center DCT Tumor Repository (Frederick, MD). Male Fischer 344 rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 200220 g (10 wk of age) were used for tumor implantation. A fragment of the H-500 tumor or dispersed H-500 cells (106/rat) was implanted or injected subcutaneously, respectively, in each rat, and the tumors were allowed to grow for 814 days. The encapsulated tumor was then excised, rinsed several times with cell culture medium, minced into small pieces, and dispersed by repeated pipetting and several passages through a 22-gauge needle. Dispersed H-500 cells were subsequently plated in RPMI 1640 medium supplemented with 10% FBS and 100 U/ml penicillin-100 µg/ml streptomycin and grown at 37°C in a humidified 5% CO2 atmosphere. Cells were passaged every 45 days using 0.05% trypsin-0.53 mM EDTA and used for experimentation within the first 10 passages. All cell culture reagents were purchased from GIBCO-BRL (Grand Island, NY), with the exception of FBS, which was obtained from Gemini Bio-Products (Calabasas, CA). Rats were handled in accordance with local institutional guidelines.
Western Blotting
For the determination of iNOS protein levels, monolayers of H-500 cells were grown on six-well plates. Cells were incubated for 4, 18, or 40 h in serum-free, calcium-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl2. At the end of the incubation period, the medium was removed, the cells were washed two times with ice-cold PBS containing 1 mM sodium vanadate and 25 mM NaF, and then 100 µl ice-cold lysis buffer was added (20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25 mM NaF, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 50 mM glycerophosphate, and a cocktail of protease inhibitors). The protease inhibitors were aprotinin, leupeptin, soybean trypsin inhibitor, pepstatin, and calpain inhibitor (10 µg/ml each), all from frozen stocks, and 100 µg/ml Pefabloc. The sodium vanadate, NaF, and Pefabloc were freshly prepared on the day of the experiment. The cells were scraped in the lysis buffer, sonicated for 5 s, and then centrifuged at 6,000 rpm for 5 min at 4°C. The supernatants were frozen at 20°C. After being thawed, equal amounts of supernatant protein (20 µg) were separated by SDS-PAGE. The separated proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell) and incubated with blocking solution (10 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and 0.25% BSA) containing 5% dry milk for at least 1 h at room temperature. iNOS protein levels were detected by immunoblotting using an 18-h incubation at 4°C with a 1:2,000 dilution of a rabbit polyclonal antiserum specific for iNOS (Transduction Laboratory). Blots were washed for five 15-min periods at room temperature (1% PBS, 1% Triton X-100, and 0.3% dry milk) and then incubated for 1 h with a secondary goat anti-rabbit, peroxidase-linked antiserum (1:2,000) in blocking solution. Blots were then washed again (3 x 15 min). Bands were visualized by chemiluminescence according to the manufacturer's protocol (Supersignal; Pierce Chemical). The same membrane was used after stripping (Restore Western Blot Stripping; Pierce) to measure -actin. Protein concentrations were measured with the Micro BCA protein kit (Pierce).
Northern Blot Analysis
To study whether high Ca affects the expression of iNOS mRNA, we performed Northern blot analysis as described previously (7). In brief, cellular RNA was isolated (9) using the Tri-Zol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. The RNA recovered was quantitated by spectrophotometry, and aliquots of 20 µg total RNA from H-500 cells incubated at low (0.5 mM) or high Ca
(7.5 mM) concentrations in serum-free, calcium-free DMEM containing 4 mM L-glutamine, 0.2% BSA, and 0.5 mM CaCl2 were loaded on a formaldehyde-agarose gel after denaturation. The gel was stained with ethidium bromide to visualize RNA standards and ribosomal RNA to document equal loading of RNA from the various experimental samples. The RNA was then blotted on nylon membranes (Duralon; Stratagene, La Jolla, CA). An iNOS cDNA probe was prepared by one-step RT-PCR using 2 µg total RNA derived from H-500 cells using the following primers: 5'-TGC TAT TCC CAG CCC AAC AAC-3' (iNOS sense, 120140) and 5'-TTT TGC CTC TTT GAA GGA GCC-3' (iNOS antisense, 486466). The PCR product was then subcloned into the TOPO TA cloning kit (Invitrogen) following the manufacturer's instructions and sequenced to confirm its homology with the corresponding region of the rat iNOS mRNA (accession no. NM_012611.1). The plasmid containing iNOS cDNA was digested with EcoR I, and the insert was 32P labeled. Blots were hybridized with a cDNA probe for iNOS and washed under high-stringency conditions as described previously (21). Equal loading was confirmed by stripping and then reprobing the membranes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Specific radioactive signals were analyzed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) with the ImageQuant program.
Infecting H-500 Cells with CaR Constructs in Recombinant Adeno-associated Virus
High-efficiency gene transfer into H-500 cells was accomplished using a recombinant adeno-associated virus (rAAV)-based method. The CaR sequence with a naturally occurring, dominant-negative mutation (R185Q), and the same vector containing the cDNA for the -galactosidase gene (BG) were under the control of a cytomegalovirus immediate-early promoter element and were packaged as previously described (38). The BG served as the control for nonspecific effects of rAAV infection. Cells were seeded (1,000 cells/well) in 96-well plates in 0.1 ml of growth medium and cultured overnight. About 1,000 virus particles/cell (as optimized by pilot studies) were used to infect each well. Cells were washed one time with serum-free
-minimal essential medium. Virus particles were then added, and the culture was incubated for 90 min in serum-free medium at 37°C in a cell-culture incubator. Equal volumes of RPMI 1640 containing 20% serum were added to the cells to achieve a final serum concentration of 10%. The cells were then cultured for 48 h, and experiments with low and high calcium concentrations were performed as described in subsequent sections.
Quantitative Real-time PCR
To amplify iNOS and GAPDH cDNA, sense and antisense oligonucleotide primers were designed based on the published cDNA sequences using Primer Express version 2.0.0 (Applied Biosystems, Foster City, CA). Oligonucleotides were obtained from Genosys (Woodlands, TX). The sequences of the primers were as follows: 5'-GAT TCA GTG GT CCA ACC TGC A-3' (iNOS sense, 621641), 5'-CGA CCT GAT GTT GCC ACT GTT-3' (iNOS antisense, 738718; iNOS accession no. NM_012611.1), 5'-TTC AAT GGC ACA GTC AAG GC-3' (GAPDH sense), and 5'-TCA CCC CAT TTG ATG TTA GCG-3' (GAPDH antisense; GAPDH accession no. M17701). cDNA was synthesized with the Omniscript RT Kit (Qiagen, Valencia, CA) using 2 µg total RNA in a 20 µl reaction volume. For real-time PCR, the cDNA was amplified using an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). The double-strand DNA-specific dye SYBR Green I incorporated in the PCR reaction buffer QuantiTech SYBR PCR (Qiagen) to allow for quantitative detection of the PCR product in a 25-µl reaction volume. The temperature profile of the reaction was 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. An internal housekeeping gene control, GAPDH, was used to normalize differences in RNA isolation, RNA degradation, and the efficiencies of the RT. The size of the PCR product was first verified on a 1.5% agarose gel, followed by melting curve analysis thereafter.
Biological NO Imaging by 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate
Cells were plated on coverslips in a six-well plate. After 72 h at 7080% confluence, the cells were challenged with 0.5 or 7.5 mM calcium for 18 h in serum-free, calcium-free DMEM containing 4 mM L-glutamine and 0.2% BSA. The cells were then washed two times with RPMI 1640 medium without phenol red. The cells were then loaded for 1 h with 1 ml RPMI 1640 (without phenol red) containing 10 µM 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM diacetate; Molecular Probes, Eugene, OR), 0.1% pluronic acid, and 1 mM probenecid at 37°C in a 5% CO2 incubator with no light. Finally, the coverslips were washed two times in RPMI 1640 medium without phenol red.
Direct visualization of NO production. The coverslips incubated in 37°C RPMI 1640 without phenol red were placed horizontally under the microscope lens. Photomicrographs with the fluorescent NO indicator were acquired with a laser scanning confocal microscope (Leica TCS-NT, Heidelberg, Germany) equipped with an argon-krypton laser at an excitation wavelength of 488 nm and a bandpass filter for 500550 nm (29). Simultaneous visualization of cell morphology by differential interference contrast microscopy was performed to confirm equal cell numbers on the coverslips. The fluorescence images were obtained as a 1,024 x 1,024 pixel frame. All other settings, including scanning speed, pinhole diameter, and voltage gain, remained the same for all experiments. The images were stored on magneto-optical storage devices.
Measurement of NO by fluorometry in cell populations. The coverslips were placed diagonally in thermostatted quartz cuvettes containing 37°C RPMI 1640 medium without phenol red. Excitation monochrometers were centered at 490 nm, and emission light was collected at 520 ± 40 nm through a wide-band emission filter.
Microarray
Total RNA was quantified by measuring ultraviolet absorption ratio at 260/280 nm and checked for quality using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Preparation of the biotin-labeled cRNA target was performed using the BioRobot 9604 (Qiagen) and a PTC-225 DNA Engine Tetrad Cycler (MJ Research, Boston, MA). Single-stranded cDNA was prepared from 2 µg total RNA using a T7-(dT) 24-oligonucleotide primer and Superscript II RNaseH-RT (200 U/µl). Included in this reaction was a mixture of six bacterial RNAs of known concentration for use as positive controls (2.5 pg/ml araB/entF, 8.33 pg/ml fixB/gnd, and 25 pg/ml hisB/leuB). Double-stranded cDNA was then generated with Escherichia coli DNA polymerase I (10 U/µl) and RNase H (2 U/µl). After purification using a Qiagen QIAquick purification kit, the double-stranded cDNA served as a template to prepare biotin-labeled cRNA via in vitro transcription, performed in the presence of biotinylated nucleotides. The labeled cRNA transcripts were purified using RNEasy columns (Qiagen) and assessed for quantity and quality using the same methods described above. The biotin-labeled cRNA was then randomly fragmented by incubating 2 µg of the sample in the presence of magnesium for 20 min at 94°C. This resulted in fragmented target with a size range between 100 and 200 bases.
Hybridization and scanning. The biotinylated cRNA target was hybridized to two ADME-Rat Expression Bioarrays (Motorola Life Sciences). For each array, 2 µg of the fragmented target cRNA was added to 260 µl of hybridization buffer, denatured, injected in hybridization chambers, sealed, and incubated for 18 h at 37°C while shaking at 300 rpm. Each array was rinsed in a stringent 46°C wash in 0.75x TNT TrisoHCl, NaCl, and Tween-20 solution for 60 min, followed by streptavidin labeling for 30 min (RT) and then the 0.75x TNT and 0.05% TNT in series. The slides were spin-dried in an Eppendorf 5810R centrifuge (2,000 rpm for 3 min; swinging bucket rotor). Processed arrays were scanned using an Axon GenePix Scanner, and array images were acquired and analyzed using CodeLink Expression Analysis Software.
Motorola cDNA chip data analysis. The "normalized intensity" probe data generated by the CodeLink Expression Analysis Software (Amersham) were exported into Microsoft Excel. The data were then separated into the following two classes: high dose and low dose. The Excel function TTEST (low-dose data, high-dose data, 2, 2) was applied to each gene probe. Probes with P values greater than the P value threshold (PVAL-THRESH) of 0.05 were eliminated. The Excel function AVERAGE (high dose data)/AVERAGE (low dose data) was applied next. Probes with ratios between the RATIO-THRESH of 2.0 and 1/RATIO-THRESH of 0.5 were removed. The remaining probes were candidates for significantly changed mRNAs. These genes demonstrated acceptable P values and exhibited at least a twofold change between the high- and low-dose calcium treatments.
Statistics
Data are presented as means ± SE of the indicated number of experiments. Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test or Student's t-test when appropriate. A P value of <0.05 was taken to indicate a statistically significant difference.
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RESULTS |
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We next sought to determine whether the Ca-induced increase in iNOS mRNA was mediated by the CaR. In cells infected with rAAV expressing BG, Ca
stimulated iNOS mRNA in a dose-responsive fashion, with 5.87 ± 3.67- and 19.62 ± 4.37-fold increases at 3.5 and 7.5 mM, respectively, compared with 0.5 mM calcium (P < 0.05). Infecting H-500 cells with a dominant-negative CaR (R185Q) via rAAV substantially reduced the stimulation of iNOS mRNA by Ca
compared with cells infected with BG. The difference between BG and dominant negative was significant at 7.5 mM calcium (P < 0.05; Fig. 4).
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DISCUSSION |
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The mediatory role of the CaR in our study was proved by infecting the H-500 cells with the dominant-negative R185Q CaR and comparing the effect of high Ca on iNOS mRNA with that in cells infected with the control rAAV, expressing BG, a protein approximately the same size as the CaR. Introducing the dominant-negative CaR in the cell produced a downward and rightward shift in the calcium iNOS concentration-response curve, similar to the effect of this mutant CaR on the response of the wild-type CaR to Ca
in transiently transfected HEK 293 cells (2). Similar inhibitory effects of the dominant-negative CaR results were seen in H-500 cells when examining PTHrP release and PTTG, a protooncogene, in response to calcium (32, 34). The effect of the CaR on iNOS is not a nonspecific event caused by activation of any GPCR, since activation of a functional G
q-11-coupled purinergic receptor in these cells by ADP
S (a nondegradable form of ADP) failed to alter iNOS expression in the H-500 cells. Activation of ADP receptors (like the CaR, a GPCR coupled to G
q-11) has been reported to upregulate the level of iNOS mRNA in human gingival epithelial cells (22). In the same cells, additional studies showed that IL-15 enhanced iNOS expression at both the mRNA and protein levels, similar to our study (39). However, another study found that IL-4 and interferon (IFN)-
increased iNOS enzyme activity and protein expression in B cell chronic lymphocytic leukemia (B-CLL) cells during in vitro culture (19). IFN-
, like calcium in our study, but not IL-4, increased iNOS mRNA expression in the cultured B-CLL cells, suggesting that IL-4-mediated changes of iNOS protein expression occurred at the posttranscriptional level. In macrophages, endotoxin-mediated NO synthesis has been shown to be dependent on heterotrimeric Gq protein signal transduction (17). In addition, another GPCR agonist, thrombin, whose cognate receptor is linked to G
13, has been found to regulate iNOS in endothelial cells (14). Therefore, multiple hormones, including those activating GPCRs, regulate iNOS mRNA expression in a complex and tissue-specific manner.
The time course for the effect of calcium on iNOS upregulation was sustained, suggesting that iNOS upregulation could be functionally important. Therefore, we evaluated real-time NO production after stimulating the cells with low or high calcium overnight. Our photomicrographs and the spectofluorometric data show that NO production is upregulated. The DAF dyes were developed using the same principle as the fura dyes, i.e., the dye is bound to an ester group that is cleaved off by cytosolic esterases (15). Intracellular DAF bound to NO and not to other substrates changes the DAF to aromatic diamines, thereby inducing a chemical transformation and a resultant change in the fluorescence of the dye, which is measured by exciting the cells at 490 nm and measuring emission at 500550 nm. Shortly after the discovery of the DAF dyes, a report indicated that calcium changed the fluorescence of the DAF-NO complex (5). This was later amended by Suzuki et al. (28), who showed that calcium changes NO release by the NO donors and not the DAF-NO complex fluorescence. In our study, we washed and incubated H-500 cells treated with low and high calcium in RPMI 1640 media without phenol red containing the same low level of Ca(0.5 mM), so that the calcium levels were the same at the time of the experimental readings.
Immunohistochemistry and mRNA data obtained using RT-PCR have shown that iNOS is constitutively present in the Leydig cells of adult rats (23). iNOS expression was upregulated by lipopolysaccharide, an inflammation-inducing substance. An NOS inhibitor decreased serum testosterone and testicular interstitial fluid formation without changing the level of luteinizing hormone. Therefore, those authors suggested that iNOS was important in the autocrine or paracrine regulation of the testicular vasculature, Leydig cell steroidogenesis, and spermatogenesis in normal rat testis. We report here that high Ca substantially increased NO production in Leydig cancer cells. We speculate, therefore, that the increase in iNOS production caused by high Ca
will be important in regulating testosterone production and growth of the tumor. In H-500 cells, we have shown that the CaR induces proliferation and protection against apoptosis (31). However, high calcium-induced proliferation in H-500 cells was not altered by the competitive NOS inhibitor L-NAME or the iNOS inhibitor 1400W (data not shown). The increased NO production by iNOS could change the rate of mutagenesis or the production of testosterone in the nonmalignant Leydig cells, which could be the subject of future study. In support of the first hypothesis, NO produced by iNOS in B-CLL cells has been reported to be an important factor in the leukemic cells' resistance to apoptosis (3). Furthermore, the protective effect of NO against apoptosis has been proposed to take place through scavenging of reactive oxygen species (ROS) in prostate cancer (37). Data support the hypothesis that it is the balance between NO and ROS that decides the protective or destructive capacity of NO (20).
The intracellular downstream mediators of the CaR identified thus far include G proteins, PLC, protein kinase C, mitogen-activated kinases, and transactivation of the epidermal growth factor receptor (13, 40). The data presented here suggest that the CaR may also use NO as a second messenger. Although the CaR is ubiquitously expressed in the body, its most important functions are regulation of PTH secretion and renal calcium excretion (33). Serendipitously, earlier data showed that nitroprusside, an NO donor, abolished PTH release and adenosine 3',5'-monophosphate accumulation in dispersed bovine parathyroid cells (11, 12) . The inhibition of PTH release was observed at all calcium concentrations, and the calcium chelator EGTA failed to prevent the inhibition. Therefore, we speculate that regulation of NO production by the CaR may not be limited to cancer cells, such as H-500 cells.
One can pose a reasonable question regarding the design of this study: how can we be sure that the elevated Ca concentration is not acting by raising Ca
levels? Two factors argue against this suggestion. First, Ca
is not a cofactor or known regulator of iNOS (unlike e- and nNOS). Second and more importantly, Ca
does not increase when H-500 Leydig cells are exposed to high Ca
concentrations (32), although it should be acknowledged that these measurements were made on coverslips, while single cell measurement may be more sensitive for detecting small changes in Ca
. That activation of the CaR does not lead to Ca
spikes was also reported in intact gastric mucosa cells (6).
In conclusion, we show here for the first time that calcium, acting via the CaR, induces upregulation of iNOS at the protein and transcript levels. In H-500 Leydig cancer cells, no nonspecific effect of G protein activation on iNOS was observed, since ADP, whose receptor is functionally active in these Leydig cells, failed to upregulate transcript for this enzyme. The time course revealed a rapid and sustained increase in iNOS transcript, which lasted at least 40 h. Indeed, NO production was upregulated in cells stimulated with high calcium overnight. The NO upregulation induced by the CaR may be a promalignant feature important in regulation of testosterone production in Leydig cells and in tumor growth more generally.
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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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REFERENCES |
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