2Research Service, Upstate New York Veterans Affairs Healthcare Stratton Medical Center, and 1Center for Cardiovascular Science, Albany Medical College, Albany, New York 12208
Submitted 5 November 2002 ; accepted in final form 8 October 2003
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ABSTRACT |
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decoy; edema; messenger ribonucleic acid; permeability; transcription; tumor necrosis factor-; endothelial nitric oxide synthase
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MATERIALS AND METHODS |
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All reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted.
Pulmonary Microvessel Endothelial Cell Culture
Bovine lung microvessel endothelial cells (BLMVEC) derived from fresh calf lungs were obtained at passage 4 (Vec Technologies, Rensselaer, NY). For all studies, BLMVEC were cultured from 4 to 12 passages (3, 6, 7) in culture medium containing DMEM (GIBCO-BRL, Grand Island, NY) supplemented with 20% fetal bovine serum (Hyclone Laboratories, Logan, UT), 15 µg/ml of Endothelial Cell Growth Supplement (Upstate Biotechnology, Lake Placid, NY), and 1% nonessential amino acids (GIBCO) and maintained in 5% CO2 plus humidified air at 37°C. A confluent PMEM was reached within 3-4 days.
Treatments
General. PMEM cultured in six-well plates were used for the isolation of nuclear extracts, luciferase (Luc), and -galactosidase (Gal). The lysis buffer was added to the PMEM after aspiration of the treated culture media and PBS washes.
TNF-. Highly purified recombinant human TNF-
from Escherichia coli (Calbiochem) in a stock solution of 10 µg/ml was used. The endotoxin level was <0.1 ng/µg of TNF-
as determined by standard limulus assay. We previously showed that the boiling of TNF-
for 45 min prevents all effects of TNF-
in our system (10, 13), indicating no effect of endotoxin contamination. TNF-
is used at a dose of 50 ng/ml because our previous studies indicate that 50 ng/ml induces an eNOS-mediated and ·NO-dependent increase in permeability (3, 8).
Detection of eNOS Promoter Activity
Plasmids. Plasmids containing the human eNOS promoter linked to a Luc reporter gene were characterized (40) and generously donated by Dr. William Sessa (Yale Univ., New Haven, CT). The native sequence (F1: -1600) contained 1,600 nucleotides before the 5'-transcription initiation site. Dr. Sessa's laboratory prepared a series of truncated sequences (F2: -1189, F4: -779, F5: -494, F6: -166) of the human eNOS promoter linked to the Luc reporter gene that was used to elucidate the role of the indicated cis-acting DNA elements in promoter activity (40). The efficiency of DNA expression was monitored by cotransfection with the reporter plasmid pCH-110 containing the -Gal gene driven by an active simian virus 40 promoter (Promega, Madison, WI). The Luc plasmids were grown in LB broth containing ampicillin (50 µg/ml) and purified using the EndoFree Plasmid Mega kit (Qiagen, Valencia, CA).
Transfection of plasmids. BLMVEC were seeded (2 x 105 cells/2.0 ml of culture medium) in six-well plates. At 24 h postseeding, Luc (2.3 µg/well) and Gal (0.23 µg/well) plasmids were mixed with Transfectam (12.65 µg/well; Promega) in serum-free DMEM and added immediately to the cells. The cell cultures were initially incubated with the plasmids at 37°C for 2 h, overlaid with complete media, and grown to confluence before treatment.
Luc assay. Luc and -Gal activities were measured in 96-well white microplates (no. 3917; Corning Costar, Cambridge, MA) with the Dual-Light Chemiluminescent Reporter Gene Assay System (Tropix PE Biosystems, Bedford, MA) on a Wallac 1420 Victor2 multilabel counter (EG&G Wallac, Turku, Finland) and reported as the ratio of Luc/Gal activity.
Detection of Transcription Factor DNA-Binding Activity
Nuclear extracts. Nuclear extracts were prepared with some modification of our previous method (10). BLMVEC were seeded (2 x 105 cells/2.0 ml of culture medium) in six-well plates and allowed to reach confluence in 3 days. PMEM were washed on ice twice with PBS followed by treatment with 300 µl/well of ice-cold buffer A (10 mM HEPES, 10 mM KCl, 100 µM EDTA, 100 µM EGTA, 500 µM AEBSF, 5 µg/ml antipain, 10 µg/ml antitrypsin, 10 µg/ml aprotinin, 100 µM benzamidine, 1 mM DTT, 1 µg/ml leupeptin, and 5 µg/ml pepstatin) and scraped with a plastic cell lifter. The lysates from two wells were pooled in a 1.5-ml microtube and incubated on ice for 15 min. Then, 37.5 µl of Nonidet P-40 (10% solution) were added to each microtube and vortexed for 10 s followed by centrifugation at 18,000 g for 30 s at 4°C. The pellet was resuspended in 40 µl of buffer C (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 500 µM AEBSF, 5 µg/ml antipain, 5 µg/ml antitrypsin, 5 µg/ml aprotinin, 100 µM benzamidine, 1 mM DTT, 1 µg/ml leupeptin, and 5 µg/ml pepstatin) and shaken on ice for 15 min. The suspension was centrifuged at 18,000 g for 5 min at 4°C. The supernatant was aliquoted and stored at -70°C. Protein concentration was determined using Coomassie Plus reagent (Pierce, Rockford, IL).
Oligonucleotide probes. Custom synthesized double-stranded oligonucleotides (Oligos Etc., Wilsonville, OR) derived from the human eNOS promoter (40) were
1) nucleotide -379 -[-370CACCC]-nucleotide -358
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Scrambled--370CACCC
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2) nucleotide -239 -[-231GATA]-nucleotide -219
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3) nucleotide -196 -[-186CACCC]-nucleotide -176
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Scrambled--186CACCC
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EMSA. Oligonucleotides were end labeled using [-32P]ATP and T4 polynucleotide kinase, and the unincorporated label was removed using ProbeQuant G-50 Micro Columns (Amersham Pharmacia Biotech, Piscataway, NJ). The oligonucleotide's final specific activity was 50,000-100,000 cpm/µl. The reaction mixture was composed of nuclear extract (4 µg) and binding buffer [10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 0.5 mM EDTA, 0.5 mM DTT, 4% glycerol, and 0.05 mg/ml poly(dI-dC)·poly(dI-dC)] in a total volume of 20 µl and incubated for 10 min with or without specific, nonspecific, or mutant cold competitors.
-32P oligonucleotide cognate (1 µl) was added to the mixture, and the mixture was incubated for 20 min, loaded on 6% polyacrylamide gels, and electrophoresed at 125 V for 3.5 h with 0.5x TBE buffer (National Diagnostics, Atlanta, GA). For supershift assays, 4 µg of antibody or nonimmune rabbit IgG were added to the reaction mixture and incubated at room temperature for 30 min before
-32P oligonucleotide binding. This mixture was loaded on 4% polyacrylamide gels and electrophoresed at 200 V for 3 h with 0.5x TBE buffer. All binding reactions and electrophoresis were performed at 4°C on prechilled gels unless specified otherwise. Gels were dried overnight and autoradiographed at -70°C (overnight or until an adequate signal was developed). The following supershift antibodies were obtained from Santa Cruz Biotechnology: GATA-1 (M-20), GATA-2 (H-116), GATA-2/3 (C-20), GATA-4 (C-20), GATA-5 (M-20), GATA-6 (C-20), Sp1 (PEP-2), Sp3 (D-20), and GKLF (M-19, which cross-reacts with multiple KLFs).
It had been determined in preliminary studies that 4 µg of nuclear protein was within the linear range of a curve, indicating that the increase in GATA- and CACCC-binding vs. an increase in sample protein was similar between control and TNF- groups.
Quantification of Autoradiographs
The autoradiographs were digitized and band optical density was quantified with Sigma Scan Pro as in our previous studies (SPSS Scientific Software, San Rafael, CA) (10, 14). Each band was analyzed by fill area (manual threshold 100) multiplied by intensity to give total intensity units.
Assay of Cell Viability
Trypan blue exclusion. BLMVEC were seeded (2 x 105 cells/ml of culture medium) and grown until confluent in 35-mm well dishes. Following respective drug procedures, PMEM were washed with PBS (2 ml) followed by treatment with 0.05% trypsin (0.2 ml) for 1 min at 37°C. The cells were resuspended in PBS (1 ml). An aliquot of cell suspension (50 µl) was combined with 0.08% trypan blue (50 µl) for 3 min, and then 10 µl of the mixture was counted for total cell number using a hemocytometer. Cell viability was defined by the following formula: cell viability = (cells excluding trypan blue/total cells) x 100.
Statistics
A one-way analysis of variance was used to compare values among the treatments. If significance among treatments was noted, a Bonferroni multiple comparison test was used to determine significant differences among the groups (30). A t-test was used when appropriate. Each PMEM well and flask represented a single experiment. There were 5-10 separate experiments per group in all studies if not otherwise specified. All data were reported as means ± SE. Significance was at P < 0.05.
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RESULTS |
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We previously showed that TNF does not affect cell viability (3, 7, 10). In the present study, treatment with eNOS promoter plasmids ± TNF (50 ng/ml) did not affect PMEM viability. The trypan blue exclusion was similar among the groups (e.g., control: 97.1 ± 0.5% vs. TNF: 95.0 ± 0.5%).
TNF- Induces a Decrease in the Activity of the Human eNOS Promoter: a Potential Role For Cis-Acting DNA Elements -370CACCC, -231GATA, and -186CACCC
We used the wild-type (i.e., F1) and truncated (i.e., F2, F4, F5, and F6) expression plasmids to explore the potential cis-acting DNA elements that affect expression in response to TNF-. Figure 1 shows the mean values of the Luc/Gal activity ratios from PMEM that were initially transfected with F1, F2, F4, F5, and F6 expression plasmids and then incubated either 0.5 or 4 h with TNF-
or control media. Figure 1 demonstrates that the constitutive Luc/Gal ratio decreased to similar levels in the F2 and F4 control groups, which then decreased further to similar levels in the F5 and F6 control groups. Figure 1 also demonstrates that a 0.5-h exposure to TNF-
had no significant effect on the Luc/Gal ratio, whereas a 4-h exposure to TNF-
caused a significant decrease in the Luc/Gal ratio compared with the respective control in the F1 through F5 groups but not in the F6 group. The results of Fig. 1 confirm the data of Zhang et al. (40), indicating constitutive regulation of the eNOS promoter that spans many potential transcription factor binding sites. Moreover, we demonstrate that the response to TNF-
disappears in the transition from the F5 to the F6 construct, which indicates that the cis-acting DNA elements -370CACCC, -231GATA, and -186CACCC found on the F5 promoter fragment may be involved in the effect of TNF-
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TNF- Induces a Change in the Nuclear Protein Binding of the DNA Element -370CACCC
EMSA gels were used to verify that the potential cis-acting DNA elements, as identified above, exhibit binding to nuclear protein that is affected by TNF-. Figure 2A shows the mean values of EMSAs of nuclear lysates isolated from PMEM incubated for 0.5 and 4 h with TNF-
or control media. Figure 2A demonstrates that there was a time-dependent increase in mean band density of the -370CACCC probe in PMEM exposed to TNF-
for 4 h compared with the respective control group. Figure 2B is a representative EMSA gel that demonstrates this TNF-induced increase in binding activity as well as the binding specificity. The addition of 50-fold molar excess unlabeled -370CACCC oligonucleotide decreased the band density to an undetectable level, whereas 50-fold molar excess unlabeled -370 scrambled, Sp1 consensus, and -186CACCC oligonucleotides had no effect.
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EMSA cold competitor and supershift methodologies were used to elucidate which member of the GT/CACC box family of transcription factor proteins may be binding to -370CACCC. Figure 3 shows EMSA gels representative of supershift and competitor experiments using the -370CACCC probe. Figure 3A indicates specificity in the uppermost band by competition with 50-fold molar excess cold -370CACCC and that no competition of binding was observed in the presence of an equivalent excess of cold Sp1 consensus oligonucleotide. In addition, a portion of the -370CACCC probe was shifted upward, along with a significant reduction in band intensity at the normal position, in the sample treated with anti-Sp3 antibody, whereas nonimmune IgG, anti-Sp1, and anti-GKLF had no effect. Figure 3B indicates an increase in density of the -370CACCC probe in the 4-h TNF--treated group relative to control, similar to Fig. 2B. The -370CACCC probe again shifted upward in the samples treated with anti-Sp3, along with almost complete disappearance of probe at the normal shift band position, whereas IgG had no effect.
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TNF- Induces a Change in the Nuclear Protein Binding of the DNA Element -231GATA
Figure 4 demonstrates a time-dependent decrease in mean band density with both -231GATA and Santa Cruz (sc)-GATA (GATA consensus) probes following TNF- treatment that was significantly different at 4 h compared with control. The TNF-
-induced decrease was greater as measured with the sc-GATA probe than with the -231GATA probe. Figure 4B is a representative EMSA gel that demonstrates this TNF-induced decrease in binding activity as well as the differential binding characteristics between -231GATA and sc-GATA. Both the left and right halves of the gel, probed with 32P--231GATA and 32P-sc-GATA, respectively, reveal a marked decrease in the GATA binding activity, primarily in the uppermost band, in lysates from cells treated with TNF for 4 h. The left half of the gel shows that the addition of 50-fold molar excess cold -231GATA as well as cold sc-GATA oligonucleotide decreased the lane densities of the 32P--231GATA probe to almost undetectable levels, whereas cold mutant -231GATA oligonucleotide had no effect. The right half, probed with 32P-sc-GATA, demonstrates a difference in the competition pattern between 50-fold molar excess cold -231GATA and sc-GATA oligonucleotides. Cold sc-GATA competed strongly with all bands, and cold mutant sc-GATA had no effect. Both cold -231GATA and sc-GATA oligos decreased the uppermost band completely, whereas -231GATA exhibited little competition with the lower two bands. The GATA consensus oligonucleotide (sc-GATA) contains dual GATA sites and purportedly binds all six known GATA isotypes, whereas -231GATA may be more isotype specific.
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EMSA supershift methodology was used to determine which member of the GATA family of transcription factor proteins may be binding to -231GATA. Figure 5 shows representative EMSAs of experiments using the -231GATA and sc-GATA probes. Figure 5 indicates there was a decrease, relative to control, in both -231GATA and sc-GATA probe densities in the 4-h TNF--treated group, similar to Fig. 4. Both -231GATA and sc-GATA probes were shifted upward in samples treated with anti-GATA-4. An almost complete elimination of the normal -231GATA bands occurred, whereas sc-GATA was attenuated primarily in the uppermost band. Samples probed with sc-GATA were only faintly supershifted with anti-GATA-6, although with no observable reduction of probe at the normal band positions. The control IgG and anti-GATA-5 had no effect on the migration response of -231GATA and sc-GATA probes. In addition, attempts to supershift samples with antibodies toward GATA-1 through -3 were unsuccessful with -231GATA or sc-GATA probes (data not shown).
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TNF- Has No Effect On the Nuclear Protein Binding of the DNA Element -186CACCC
Figure 6A demonstrates that there was no change in mean density of the -186CACCC probe bands in either the 0.5- or 4-h TNF--treated groups compared with their respective control groups. Figure 6B is a representative EMSA gel that demonstrates the binding characteristics of the -186CACCC probe and the effect on binding activity of 4-h TNF-
treatment relative to control. Figure 6B shows that the addition of 50-fold molar excess cold -186CACCC oligonucleotide decreased the density of the second band from the top to almost undetectable levels, whereas the cold -186scrambled oligonucleotide had no effect.
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The combined results derived from Figs. 2, 3, 4, 5, 6 indicate that the DNA sites -370CACCC and -231GATA from the F5 region of the eNOS promoter bind the transcription factors Sp3 and GATA-4, respectively, and are candidates for the TNF- response elements that decrease the activity of the eNOS promoter.
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DISCUSSION |
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The literature indicates that indeed eNOS is dynamically regulated because there is an increase in eNOS promoter activity and/or expression after treatment with lysophosphatidylcholine (5), cyclosporine (27), estrogen (16), vascular endothelial growth factor (2), an interferon-/TNF-
/lipopolysaccharide mixture (22), and shear stress (41). To our knowledge, the assessment of eNOS gene promoter activity in response to TNF-
using pulmonary microvessel endothelium has never been investigated. TNF-
increases the generation of ·NO in pulmonary endothelium (3, 8). However, TNF-
suppresses the expression of eNOS mRNA and eNOS protein in both systemic and pulmonary endothelium (1, 29, 38, 39). Thus the study of the modulation of eNOS-DNA promoter activity is important because of the existence of a possible control mechanism for eNOS-mediated generation of ·NO and the resultant endothelial injury in response to TNF-
(3). Moreover, ·NO mediates, at least in part, the vasoreactivity and lung injury during the systemic inflammation syndrome associated with shock and other vascular disorders (9, 12, 25). This study is the first to demonstrate that TNF decreases eNOS promoter activity.
We used a series of truncated eNOS promoters linked to a Luc reporter plasmid that was initially developed and characterized by Zhang et al. (40). The present study indicates that TNF- caused a decrease in the expression of the F1, F2, F4, and F5 eNOS promoter-Luc plasmids but did not affect expression of the F6 plasmid. Thus the TNF-
-induced decrease in eNOS promoter activity is dependent on the promoter region lost between F6 and F5. Two of the three characterized cis-acting DNA-binding sites located in this region are -370CACCC and -186CACCC. TNF-
caused an increase in nuclear protein binding to the 32P-labeled -370CACCC oligonucleotide probe despite no change for the 32P-labeled -186CACCC probe. The 32P--370CACCC and 32P--186CACCC probe specificity was verified because 1) protein binding was prevented by competition with their respective unlabeled counterparts, 2) unlabeled -186CACCC probe did not compete with 32P--370CACCC protein binding, and 3) their respective cold scrambled oligonucleotides did not compete. Thus it is probable that the nucleotides flanking the CACCC sites also determine the binding to the -370CACCC and -186CACCC probes. The CACCC binding proteins belong to the Sp and the KLF family of zinc finger proteins that have a highly conserved COOH-terminal region with three zinc fingers and a divergent NH2-terminal domain rich in proline (23, 33, 36, 37, 40). In the present study, supershift assays verified that the -370CACCC oligonucleotide of the eNOS promoter specifically binds to the Sp3 transcription factor protein.
Another cis-acting DNA-binding site, identified by Zhang et al. (40), which is located in the promoter region lost between F6 and F5, is -231GATA. TNF- caused a decrease in nuclear protein binding to both the 32P-labeled sc-GATA and -231GATA oligonucleotide probes. The 32P--231GATA and 32P-sc-GATA probe specificity was verified because 1) protein binding was prevented by competition with their respective unlabeled counterparts, 2) the sc-GATA and -231GATA probes exhibited cross-competition of protein binding with each other, and 3) their respective unlabeled mutant oligonucleotides did not compete. The greater TNF-
-induced decrease in the EMSA seen with the sc-GATA probe may be due to the differences between the probes, which are 1) the nucleotides flanking the actual GATA protein binding site and 2) the presence of dual binding sites in the sc-GATA probe as opposed to one in the -231GATA probe. Yet, the cold sc-GATA probe effectively competed out binding to the 32P-labeled -231GATA oligonucleotide probe, and the cold -231GATA probe competed out binding to the uppermost 32P-sc-GATA band. This uppermost band also exhibited the most profound decrease in response to TNF-
and was completely supershifted with anti-GATA-4. In the present study, the supershift assay verified that the -231GATA probe of the eNOS promoter can specifically bind to the GATA-4 transcription factor protein.
The identity of the specific transcription factors, out of 25 potential Sp/KLF and GATA candidates combined, is noted by the effect of TNF on Sp3 and GATA-4 binding in the present investigation. The roles of Sp3 and GATA-binding proteins in the eNOS promoter response to TNF-
have not been previously studied. Sp3 exists in three isoforms, which all contain a repression domain and exhibit different levels of expression of two possible activation domains, and has been described as both an activator and a repressor (28). The TNF-
-induced increase in Sp3 binding to -370CACCC and concomitant decrease in eNOS promoter activity may indicate an inhibitory role for an Sp3 isoform in this study. It is interesting to note that in our EMSAs of -370CACCC, there is a single, crisp, specific band that does not compete with Sp1 consensus and yet is supershifted by anti-Sp3, indicating perhaps an Sp3 isoform that preferentially binds GT/CACC-box over GC-box sites.
There are six members of the GATA-binding protein group of transcription factors that are all characterized by a cysteine-Zn finger region adjacent to a basic domain (11, 21, 24, 26, 32). Minami et al. (24) showed that the transforming growth factor-1-mediated inhibition of the flk1/KDR gene (i.e., the VEGF receptor) is mediated by reduced activity of GATA-1/2, which is similar to previous findings that TNF-
induces a decrease in eNOS mRNA (3), and presently, the decreased activity of GATA-4. Presently, our ongoing investigation in progress using a mutated -231GATA (TGATA
TCTAA) eNOS promoter and a -370CACCC decoy oligonucleotide (data not shown) indicates that TNF induces a decrease in eNOS promoter activity, which is dependent on the dual change in activity of both -231GATA and -370CACCC. To our knowledge, there are no data in the literature that indicate the regulation of, or a role for, the GATA- and CACCC-binding proteins in TNF-
-induced changes in endothelial homeostasis.
There are many signaling pathways that link TNF- to the activity of the two putative transcription factors GATA-4 and Sp3. We have documented the activation of PKC-
and the generation of reactive oxygen and nitrogen species in pulmonary endothelial cells in response to TNF-
(7, 8, 10, 13). The literature indicates a role for phosphorylation events because PKC-
/PKC-
expression enhances transcription of eNOS (20), phosphorylation of GATA-4 mediates transcription of endothelin-1 (26), and phosphatase inhibition enhances Sp1 activity (36). A role of reactive oxygen species is also noted because homocysteine (i.e., an antioxidant) affects angiotensin-induced GATA-4 activation (31), and GATA-2 mediates suppression of the erythropoietin gene, possibly via H2O2 (32).
In summary, our data indicate that TNF- causes the suppression of eNOS promoter activity in pulmonary microvessel endothelial cells. Moreover, Sp3 and GATA-4 are putative transcription factors that mediate the TNF-induced decrease on the eNOS promoter.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by the Department of Veterans Affairs Research Incentive Fund Program (to A. Johnson), the Charitable Leadership Foundation Medical Technology Acceleration Program (to A. Johnson), and National Heart, Lung, and Blood Institute Grant RO1-HL-59901 (to A. Johnson).
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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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