1Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center; and 2Department of Immunology and 3Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado
Submitted 16 September 2004 ; accepted in final form 26 January 2005
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ABSTRACT |
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gene regulation; lung; fibrosis; growth factor
Insulin-like growth factor I (IGF-I), especially macrophage-derived IGF-I, has long been implicated in the pathogenesis of IPF. Originally described as alveolar macrophage-derived growth factor in the lung (4), IGF-I stimulates many responses that are consistent with its proposed role in the pathogenesis and progression of IPF, including acting as a progression factor for fibroblast proliferation (4, 21), stimulating collagen matrix synthesis (8, 9), and promoting the survival of cells of both epithelial and mesenchymal origin (5, 14, 15, 29). Additionally, IGF-I levels in bronchoalveolar lavage fluid have been shown to be significantly increased in IPF patients and patients with other fibrotic lung diseases compared with healthy control subjects (4, 11, 21). Postmortem and open lung biopsy specimens from patients with IPF exhibit elevated levels of IGF-I mRNA, and alveolar macrophages from IPF patients secrete elevated amounts of IGF-I compared with normal subjects (4, 11, 21). Previous work from our laboratory has revealed that IGF-I is also expressed by alveolar epithelial cells and interstitial macrophages in patients with IPF (27). Importantly, the degree of expression of IGF-I by interstitial macrophages was found to be closely associated with disease severity, emphasizing both the significance of interstitial macrophages and the expression of IGF-I by this cell in the progression of IPF. However, little is known about the molecular mechanisms controlling the basal expression of IGF-I in macrophages.
The rodent IGF-I gene is a single copy gene spread out over >80 kb and contains six exons (6, 23, 26). Its structure is highly conserved with the human IGF-I gene (22). Transcription can initiate from either exon 1 or 2, each being regulated by a structurally and functionally distinct promoter (1, 10, 16). Promoter 1 regulates exon 1 expression and promoter 2 regulates exon 2. Exons 1 and 2 encode alternative leader signal peptides and 5'-untranslated regions. Arkins et al. (2) have shown that macrophages exclusively use exon 1 and not exon 2 for transcription initiation, typical of extrahepatic IGF-I mRNA. The 5'-untranslated and -flanking regions of IGF-I exon 1 are highly conserved between mouse, rat, and human, with nearly 95% conservation of nucleotide sequence over this region. This high degree of homology within the 5'-untranslated region suggests similar regulatory mechanisms of IGF-I exon 1 expression between species. Promoter 1 lacks any recognizable core promoter transcriptional control elements such as TATA and CCAAT boxes (1, 10, 16). Neither does it resemble the GC-rich promoters found in housekeeping genes. A sequence resembling the "initiator" sequence in the TATA-less mouse terminal deoxynucleotidyl transferase gene (24), which directs transcription to a single site, is present. However, this does not appear to be functional in IGF-I exon 1, as evidenced by the multiple transcriptional start sites dispersed over 350 bp (1, 10, 16).
Although previous studies have shown that macrophages are an important source of IGF-I in the lung, little is known about the mechanisms that regulate IGF-I expression in macrophages. Therefore, in this study we investigated the cis-regulatory regions within the IGF-I exon 1 promoter that are required for basal expression in macrophages. We show that elements located in the 5'-untranslated region positively regulate transcriptional activity in macrophages, whereas sequences located in the 5'-flanking region confer negative transcriptional regulation.
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METHODS |
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Dulbeccos modified Eagles medium (DMEM) and fetal bovine serum were purchased from BioWhittaker (Walkersville, MD) and Atlanta Biologicals (Norcross, GA), respectively. RAW 264.7 cells were obtained from Dr. W. Murphy (University of Kansas, Kansas City, KS). HeLa cells were purchased from the American Type Culture Collection (Rockville, MD). Rat IGF-I exon 1 promoter-pGL2 luciferase constructs were kindly provided by Dr. Peter Rotwein (University of Oregon School of Medicine). The firefly luciferase reporter assay system, pGL3 Basic and Promoter luciferase reporter vector, primer extension kit, and restriction enzymes were purchased from Promega (Madison, WI). [-32P]UTP (800 Ci/mmol, 10 mCi) and [
-32P]ATP (>3,000 Ci/mmol) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Primers and oligonucleotides were synthesized by Genelink (Thornwood, NY). The mRNA isolation [poly(A) pure], in vitro transcription (MAXIscript), and RNase protection assay (RPAIII) kits were purchased from Ambion (Austin, TX). The Oligotex mRNA isolation kit was purchased from Qiagen (Valencia, CA). Opti-MEM, TRIzol reagent, and LipofectAMINE Plus reagent were purchased from GIBCO-BRL Life Technologies (Grand Island, NY). The Sp1 (sc-59), Sp3 (sc644), and Ets-1 (sc-350) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Quikchange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA).
Cell Culture
RAW 264.7 cells were seeded in six-well culture dishes at 3 x 106 cells/plate with 4 ml/well of RPMI 1640 supplemented with 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (vol/vol) FBS and cultured under a 5% CO2 atmosphere. HeLa cells were seeded in six-well culture dishes at 3 x 106 cells/plate with 4 ml/well of DMEM supplemented as above and cultured under a 5% CO2 atmosphere. Monolayers of mouse bone marrow-derived macrophages (BMDM) were prepared as previously described (30).
Construction of Reporter Vectors
The region from 1711 to +329 of the rat IGF-I exon 1 promoter/5'-untranslated region, or smaller fragments thereof, were cloned into the MluI and XhoI sites of the pGL3 vector. The sequence from XhoI to the NcoI site, the beginning of the coding region for luciferase, was removed to ensure that this region was not artifactually affecting transcription. The +1 is the most 5'-transcriptional start described by Hall et al. (10) and Peter Rotwein, the provider of the pGL2 IGF-I promoter construct used as the PCR template to make the inserts for these vectors. The +330 is the translational start site for exon 1 of IGF-I and is not included in these vectors. Site-directed mutations in the reporter constructs were made by following the manufacturers instructions for Quikchange site-directed mutagenesis (Stratagene) and were verified by sequencing.
Transfection of Reporter Constructs
Plasmid DNA was transiently transfected into cell lines using the LipofectAMINE Plus method as described in the instructions from GIBCO-BRL Life Technologies. Cells were seeded in six-well culture dishes at 0.5 x 106 cells/well for 24 h and were transfected with 0.22 pmol/well of DNA. Forty-eight hours posttransfection, cells were lysed with 400 µl of 1x passive lysis buffer, and we analyzed 30 µl of the lysate for luciferase activity by adding it to 200 µl of luciferase substrate followed by measurement in a MoonLight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) for 20 s. A portion of the cell lysate was used to determine total protein concentration using the Bradford assay. The luciferase assays were normalized to total protein.
Poly(A) RNA Isolation
Poly(A) RNA was isolated from BMDM by two methods. The first method employed was the Poly(A) Pure kit (Ambion). The manufacturers protocol was followed. The second method involved using the Oligotex kit (Qiagen) on total RNA previously isolated using the TRIzol method. The protocol of the manufacturer was followed.
Primer Extension
The primer, corresponding to the anti-sense strand of the first 30 bases of the coding region for mouse IGF-I exon 1, was end-labeled with [-32P]ATP (3,000 Ci/mmol, 10 mCi/ml). The final concentration of the primer was adjusted to 100 fmol/µl. Poly(A) RNA (0.3 µg in 5 µl of water) was mixed with 1 µl of the labeled primer and 5 µl of 2x avian myeloblastosis virus (AMV) primer extension buffer to anneal the primer to the RNA. The samples were incubated at 60°C for 30 min and then placed at room temperature for 10 min. The primer extension reaction was then completed by extending the annealed primer with the addition of 9 µl containing 1.6 µl water, 5 µl 2x AMV primer extension buffer, 1.4 µl 40 mmol sodium pyrophosphate, and 1 µl (1 unit) AMV reverse transcriptase. The reaction was mixed and incubated at 42°C for 30 min after which time 20 µl of loading dye were added. Half of the sample was electrophoresed through a 5% (29:1) denaturing (8 M urea) Tris-borate-buffered polyacrylamide gel at 350400 V. After drying the gel on Whatman paper, we visualized the primer extension products by autoradiography.
RPA
Mouse exon 1, 5'-untranslated region, and 5'-flank (the first 30 bases of the coding region and 630 bases upstream of that) were cloned into the KpnI and PstI sites of Promegas pGEM4Z vector, which contains a T7 RNA polymerase promoter downstream of the multiple cloning region. The linearized vector was agarose gel purified, and the anti-sense probe was synthesized by using the MAXIscript in vitro transcription kit from Ambion. The full-length radiolabeled probe was PAGE purified. The probe was then immediately used in the RPA for which the RPAIII kit from Ambion was employed. Briefly, 40,000 cpm of labeled probe were mixed with 0.6 µg of poly(A) RNA for each treatment condition. Two control reactions, each containing probe but yeast RNA instead of sample RNA, were included. One-tenth volume of 5 M NH4OAc and 2.5 volumes EtOH were added to all the samples, mixed, and incubated at 70°C for 15 min. The samples were then centrifuged (14,000 g, 15 min, 4°C), after which time the supernatant was discarded, and the pellets were air-dried for 5 min. The pellets were resuspended in 10 µl of hybridization buffer III, heated to 90°C for 3 min, and transferred to 42°C for incubation overnight. RNase digestion buffer III (150 µl) containing 0.375 units of RNase A and 15 units of RNase T1 were added to each sample and to one of the control samples. The other control sample received 150 µl of RNase digestion buffer III lacking RNase. All samples were incubated at 37°C for 30 min and terminated with 225 µl of RNase inactivation/precipitation III solution. Yeast RNA (2 µl, 5 mg/ml stock) were added to each tube, as carrier, along with 150 µl of EtOH, and the samples were precipitated. The resulting pellet was resuspended in 10 µl of gel loading buffer II and vortexed vigorously. The samples were incubated at 90°C for 5 min and electrophoresed through a Tris-borate-buffered 5% (29:1) denaturing (8 M urea) polyacrylamide gel at 350400 V. The gel was dried onto Whatman paper and autoradiography was performed.
Electrophoretic Mobility Shift Assay
Nuclear protein isolation.
Macrophages (1 x 107) were washed with PBS, scraped into 6 ml of PBS, and centrifuged (750 g, 10 min, 4°C). The cell pellet was resuspended in 500 µl of buffer A [10 mM HEPES, pH 7.8, containing 10 mM NaCl, 0.5 mM EDTA, 1.5 mM MgCl2, 2 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 2 mM sodium orthovanadate, and 2 mM sodium fluoride] and allowed to swell on ice for 10 min. The cells were lysed with 500 µl of buffer B [1.2% (vol/vol) Nonidet P-40 (NP-40) in buffer A], vortexed for 10 s, and centrifuged (2,500 g, 10 min, 4°C). The nuclear pellet was resuspended in 500 µl of buffer A, centrifuged (5,000 g, 5 min, 4°C), and resuspended in 50100 µl of buffer C [buffer A containing 10% (vol/vol) glycerol and 0.41 M NaCl]. The nuclei were incubated on ice for 30 min, with occasional shaking. After centrifugation (20,000 g, 10 min, 4°C), the supernatant containing the eluted nuclear proteins was stored at 70°C until use.
Probe preparation. Table 1 shows the sequences of the oligonucleotides used in the electrophoretic mobility shift assasys (EMSAs). Equimolar amounts of the sense and corresponding anti-sense oligos were annealed by boiling and gradual cooling. After radiolabeling, the probes were electrophoresed through a Tris-glycine-buffered 6% (29:1) nondenaturing polyacrylamide gel to purify the labeled probe from single-stranded oligos and unincorporated nucleotides. The labeled probes were diluted to 7,500 cpm/µl in probe dilution buffer (20 mM HEPES, pH 7.9, containing 0.1 M KCl and 1 mM MgCl2) and frozen at 20°C.
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RESULTS |
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To determine the cis-regulatory elements in the IGF-I exon 1 promoter that are required for basal expression in macrophages, we created a series of IGF-I promoter-luciferase constructs using the sequence upstream of rat IGF-I exon 1, where +1 is the most 5'-transcriptional start described by Hall et al. (10) and +330 is the translational start site. We transfected these constructs into the macrophage cell line RAW 264.7 and measured luciferase activity (Fig. 1A). The 1711 to +329 construct did not exhibit any luciferase activity above that of the empty vector. However, following sequential 5'-deletions, promoter activity increased. The 433 to +329 and 122 to +329 constructs promoted luciferase activity approximately four- and eightfold above background, respectively. These data suggest that there are suppressive elements within the distal 5'-flanking region of IGF-I that mask activity located further downstream.
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Having defined the minimal and maximal promoter regions for IGF-I in RAW 264.7 cells, we next determined whether these regions are specific to RAW 264.7 cells. We determined luciferase activity in an alveolar macrophage cell line, MH-S, and an epithelial cell line, HeLa. In the MH-S cells the pattern of expression was virtually identical to that of the RAW 264.7 cells (data not shown). IGF-I exon 1 promoter activity in the HeLa cell line was also similar to the pattern of expression in the RAW 264.7 cells (Fig. 2). However, the region from +251 to +329 directed maximal expression and also comprised the minimal promoter. The regions from +95 to +173 and +251 to +299 were not sufficient to promote luciferase activity, suggesting that the region between +299 and +329 was required in conjunction with one of these other regions to promote activity. Collectively, these data demonstrate that the region from +251 to +329 contains the minimal promoter for IGF-I exon 1, as shown in two macrophage cell lines and one epithelial cell line, and that there is no cell-specific regulation with regard to the minimal promoter. However, the region from +95 to +329 regulates maximal IGF-I exon 1 expression in macrophages but not in epithelial cells, implying possible cell-specific regulation.
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To verify that transcription could be initiated in the regions defined as the maximal and minimal promoter elements and to rule out the possibility of transfection artifact, we determined the transcriptional start site usage for the endogenous IGF-I gene in primary mouse macrophages. Others have shown, in other species and cell types, that IGF-I has multiple transcriptional start sites. Therefore, our expectation was to find multiple start sites, including a major start site within the region from +95 to +173. In addition, a minor transcriptional start site was expected in the +251 to +299 region. Figure 3A shows the results from primer extension analysis on mRNA isolated from primary macrophages. The size of the extended primer was 210 bases. Because the primer was 30 bases long (Fig. 3C), the major transcriptional start site maps to approximately +150 (180 bases upstream of the translation start site). Due to the low specific activity of the probe used for the primer extension we also used RNase protection assays on mRNA isolated from primary mouse macrophages. Figure 3B shows that several bands of various lengths were protected from RNase degradation after annealing macrophage mRNA to a radiolabeled probe containing the first 30 bases of the coding region and extending upstream another 630 bases (Fig. 3C). A major band (Fig. 3, lane 3) was observed at 215 bases, therefore mapping the major start site to approximately +145, consistent with the primer extension. A band of intermediate intensity (Fig. 3B, lane 3) was detected at 260 bases corresponding to a transcriptional start site in the +95 to +173 region, at +100. Finally, bands of weaker intensity were detected at
105 bases (Fig. 3B, lane 3), corresponding to minor transcriptional start sites located at
+255. These data thus demonstrate that the major transcriptional start site initiates in the region defined as being required for maximal promoter activity and a minor transcriptional start site lies in the region defined as the minimal promoter.
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Having defined the cis-regulatory elements required for expression of IGF-I in macrophages, we next determined whether nuclear proteins from macrophages bound these regions. Figure 4A shows the relative location and sequence for EMSA probes: +134 to +173 probe (probe 1) and +267 to +299 probe (probe 2). Incubation of these two radiolabeled probes with nuclear proteins from RAW 264.7 macrophages resulted in the formation of multiple DNA/protein complexes (Fig. 4B, left). A similar banding pattern was observed when nuclear proteins from primary macrophages were incubated with the same probes (Fig. 4B, right). To determine the specificity of binding, we added a 50-fold molar excess of unlabeled probe to the reaction. Figure 4C shows that binding to each of the radiolabeled probes was competed away by unlabeled (cold) probe. These results suggest that the cis-regions required for maximal and minimal promoter activity of IGF-I in macrophages specifically bind macrophage nuclear proteins.
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Next we investigated the composition of the complexes that bound to the +267 to +299 probe. Although degenerate, the sequence between +287 and +295 appears to be an Sp protein binding GC box (Fig. 5B). Therefore, using EMSA and supershift analysis, we examined whether Sp1 and/or Sp3 were capable of binding the +267 to +299 probe. Figure 5A shows that complex II contains Sp3, as indicated by the supershifted band and the loss of intensity of complex II. Neither the Sp1 antibody nor the Ets-1 antibody, used as a control, resulted in supershifting, suggesting that neither of these trans-acting factors bound to this probe. Because Sp3 and Sp1 bind to similar sequences, as a control we verified that the Sp1 antibody was capable of binding Sp1 and supershifting a DNA/Sp1 complex using RAW 264.7 macrophage nuclear proteins and an Sp consensus probe. Figure 5A (right) shows that, compared with complex formation in the absence of antibody, the Sp1 antibody supershifted the DNA/Sp1 complex, as evidenced by the shifting of the upper complex. Using a similar approach we also tested the Sp3 antibody with the consensus Sp probe. It can be seen that the lower complex generated from the incubation of macrophage nuclear proteins with the Sp consensus probe contains Sp3 (Fig. 5A, right). Ets-1 and nonimmune IgG antibodies were used as a controls. Thus, in conjunction with Western blot data (not shown), we show that macrophages express Sp1 and Sp3 but only Sp3 binds the +267 to +299 IGF-I probe.
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Mutation of the Sp3 Binding Site in the IGF-I Promoter Increases Transcriptional Activity in Macrophages
We next examined whether the Sp3 binding sequence was functional in the IGF-I promoter-luciferase constructs. The same mutations that are shown in Fig. 5B were made in both the +251 to +329 and +95 to +329 constructs defined to contain the cis-elements required for minimal and maximal promoter activity, respectively, in macrophages (Fig. 1B). Figure 6 shows that when either mutation was made in either construct, luciferase activity was increased (P < 0.05). These data suggest that Sp3 is acting as a transcriptional suppressor for IGF-I expression in macrophages in the region required for minimal expression.
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We next investigated the suppressive regions of the 5'-flanking region of IGF-I exon 1 located between 1711 and 122, as suggested in Fig. 1A. To define the regions responsible for suppressive activity, internal deletions of the IGF-I exon 1 promoter-luciferase constructs were engineered. The region between 855 and 122 was deleted, thereby leaving the region from 1711 to 855 juxtaposed to the region 122 to +329. As shown in Fig. 7A, the region from 1711 to 855 was sufficient to suppress luciferase activity of the 122 to +329 construct, from eightfold over control down to 0.6-fold of control. This figure also demonstrates that a construct containing the region from 1711 to 855 alone expressed less luciferase activity than the empty vector (a 90% reduction) and also less than the 1711 to +329 construct (an 83% reduction). This suggests that the 1711 to 855 region suppressed the background activity normally seen with the empty vector.
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Having shown that the regions from 1711 to 855 and from 855 to 337 suppress the IGF-I exon 1 promoter in RAW 264.7 macrophages, we next examined whether this suppressive activity was also present in other cell types. The macrophage cell lines MH-S and NR8383 and the epithelial cell lines HeLa and COS-7 were transfected with the IGF-I exon 1 constructs. The regions from 1711 to 855 and 855 to 337 suppressed luciferase activity in a similar manner in the MH-S and NR8383 macrophages as they did in the RAW 264.7 macrophages (data not shown). In contrast, luciferase activity was not suppressed in either the HeLa (Fig. 8) or COS-7 (data not shown) epithelial cell lines. In both these cell lines the 1711 to +329 construct promoted activity fivefold over the empty vector. Collectively, these data suggest that the suppressive elements in the 5'-flanking region of IGF-I exon 1 may function in a cell type-specific manner.
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DISCUSSION |
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To our knowledge, no other reports have been published using the IGF-I promoter luciferase system in macrophages. However, studies have examined transcriptional regulation of IGF-I using the luciferase reporter system in other cell types, including the liver. Pao et al. (18) identified six DNase I footprints downstream of the major transcriptional start site, and Zhu et al. (31) examined the cis-elements and trans-acting factors that were part of footprint V. It was reported that both Sp1 and Sp3 bound the sequence CCTGCCCA (31) in contrast to our finding of exclusive occupancy by Sp3. Although the reason for the discordance is unclear, our data clearly support the notion that in macrophages: 1) Sp1 is not responsible for basal regulation of IGF-I and 2) Sp3 serves to dampen the basal regulation. These conclusions are bolstered by the data that Sp1, although expressed by macrophages, does not bind the IGF-I promoter whereas Sp3 does. We have also observed that overexpression of Sp1 does not increase IGF-I promoter activity in luciferase reporter gene assays (data not shown). Mutation of the Sp3 binding site not only inhibits Sp3 binding but increases luciferase activity, consistent with the known function for Sp3 as a transcriptional repressor (3, 7).
The regulation of IGF-I in osteoblasts is qualitatively similar to the data presented here in macrophages. Pash et al. (19) reported that maximal promoter activity was observed with the 123 to +372 construct [these base numbers have been changed by 10 bases from the original report to be consistent with the numbering system used by Hall et al. (10) and herein], which includes the region required for maximal promoter activity defined in the present study (+95 to +329). In addition, McCarthy et al. (17) reported that the region from +195 to +329 is required for minimal promoter activity. Other studies have also suggested that the 5'-flanking region contains sequences that suppress IGF-I promoter activity in some cell types such as primary dermal fibroblasts (16), C6 glioma (16, 28), OVCAR-3 (28), and GH3 cells (28). Consistent with our results in HeLa cells, transfection of the longest IGF-I luciferase reporter construct into the neuroepithelioma cell line SK-N-MC resulted in a high amount of luciferase activity (10). Collectively these findings suggest that IGF-I expression via promoter 1 may be differentially regulated in different cell types.
In summary, the findings from the present study provide novel insights into both positive and negative regulation of IGF-I expression by macrophages. With the growing data to suggest that both macrophages and IGF-I may play a role in the initiation and/or progression of IPF/UIP, understanding the mechanisms by which IGF-I is regulated in macrophages is of importance. Only when basal regulation is understood can one undertake the problem of the apparent dysregulated expression of IGF-I in macrophages from IPF patients. These data open up the possibility that the 5'-flanking region of IGF-I may be a target through which IGF-I expression could be manipulated and controlled in macrophages without affecting epithelial cell expression.
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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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