1Veterans Affairs Medical Center/University of California San Diego, La Jolla, California 92161; 2Department of Pathology and Immunology, Monash University, Prahran, Victoria, Australia; and 3U538 Institut National de la Santé et de la Recherche Médicale, CHU St-Antoine, 75571 Paris Cedex 12, France
Submitted 25 July 2003 ; accepted in final form 10 December 2003
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ABSTRACT |
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calcification; dileucine motif; NPP3
Physiological calcification within bone, cartilage, and dentin is regulated by the capacity of resident cells to release vesicles that are commonly termed matrix vesicles (MVs) (2, 3). These calcification-competent cells employ MVs to modify the organization and composition of the extracellular matrix (2, 3). MVs serve to modulate matrix mineralization, a process in which their interior provides a sheltered locus for nucleation of calcium-containing mineral crystals, including hydroxyapatite (HA) (2, 3). The protein content of MVs reflects their specialized functions. Specifically, attached to the outer surface of MVs are type II and type X collagens (26, 28) and proteoglycans (45). Contained within the MV sap are matrix metalloproteinases (10) that subserve matrix modification. Certain MV constituents, including phospholipase A2 (40) and protein kinase C (41), can promote modifications of membrane lipid composition and changes in membrane integrity, including vesiculation, and can thereby affect MV genesis, nucleation of HA crystals, and extravesicular propagation of mineral formed in the MV interior. Furthermore, MVs contain ion transporters and binding proteins, as well as ectoenzymes involved in metabolism of extracellular modulators of calcification, including ATP (2, 3). In this context, nucleation of basic calcium phosphate crystals within MVs is promoted by the calcium-binding proteins annexins II, V, and VI (26, 27, 44), the Pi-generating ectoenzymes tissue-nonspecific alkaline phosphatase (TNAP) (16, 18, 27), ATPase (18), and Na+-K+-ATPase (8), and the sodium-dependent Pi transporter Pit-1/Glvr-1 (15). The enrichment of TNAP in MVs (by 12 logs) relative to the plasma membrane and whole cells is a fundamental feature of MVs that contributes substantially to MV promineralizing activity (1, 18, 22, 27). However, TNAP activity in MVs is balanced partly by the capacity of MVs to generate and retain PPi, an inhibitor of HA crystal nucleation and propagation (16, 22, 43).
NPP activity is enriched by >1 log in MVs relative to the plasma membrane and whole cells (11, 23). Prior work has shown that NPP1 is targeted to MVs in a preferential manner relative to NPP2 and NPP3 (23, 24). Furthermore, NPP1 directly antagonizes the mineralizing function of TNAP in MVs and vice versa (22). Specifically, osteoblasts of NPP1-knockout mice exhibit depressed extracellular and MV PPi levels and enhanced MV-mediated mineralization in vitro and hyperostosis in vivo, defects corrected by concurrent TNAP gene deletion (16). The primary objective of this study was to evaluate how the critical calcification-regulating NPP activity concentrates in the osteoblast plasma membrane and MVs.
NPP1 and NPP3 are class II transmembrane glycoproteins with highly homologous extracellular domains but with no significant homology in the primary sequence of their short intracellular cytoplasmic segments (7, 13). Thus we tested the hypothesis that the molecular basis for NPP1 concentration with MVs was attributable to a distinct region in the cytosolic tail of NPP1. Previous studies have identified the NPP1 cytosolic dileucine motif to be critical in basolateral membrane targeting of NPP1 in polarized epithelial cells, and NPP3 lacks such a dileucine-containing cytosolic motif (5). Results of this study uncover the signal used by osteoblasts to target and concentrate functional NPP activity to the plasma membrane and, subsequently, into MVs, where NPP1 regulates PPi concentration and mineralization.
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MATERIALS AND METHODS |
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DNA constructs and mutagenesis. We used previously described full-length wild-type NPP1 and NPP3 cDNA constructs in pcDNA3 (24). Mutagenesis at specific sites was carried out using the Quick-Change site-directed mutagenensis kit (Stratagene, La Jolla, CA), with all mutations confirmed by sequencing. Chimeric and NPP3/AAASL-LAP constructs have been described in detail elsewhere (5). To create the reporter constructs NPP1t/tm-green fluorescent protein (GFP) and NPP3t/tm-GFP, human cDNA constructs of NPP1 and NPP3, respectively, were used as templates for PCR amplification of the tail-transmembrane (t/tm) region of these molecules. The PCR products, validated by sequencing, were cloned into pEGFP-N1 vector (Clontech).
Confocal immunofluorescence microscopy. Cells were seeded at a density of 104 cells onto sterilized, poly-L-lysine-coated, 18-mm-diameter coverslips placed in 12-well culture dishes. Cells were fixed in fresh 3.5% paraformaldehyde (Electron Microscopy Sciences) in 1x PBS for 30 min and then blocked with 5% goat serum in 1x PBS. For indirect immunofluorescence, cells were incubated with primary antibodies and then with secondary Alexa Fluor 488-conjugated goat anti-mouse IgG antibodies (Molecular Probes, Eugene, OR). The wild-type NPP1, NPP1 mutants, and chimeric NPP3t/tm-NPP1 extracellular domain (ec) molecules were detected using 4H4 monoclonal antibody, which recognizes the COOH-terminal 140 amino acids of the human NPP1 protein (4). For detection of the chimera NPP1t/tm-NPP3ec and NPP3/AAASLLAP, we used a monoclonal antibody (MAb B10, anti-rat NPP3) (5). GFP fusion proteins were visualized by direct immunofluorescence after the cells were fixed with paraformaldehyde. Double staining was done to visualize NPP1 or its dileucine mutant and the vesicular proteins EEA1 (33) or LAMP2 (9), markers of early endosomes and late endosomes/lysosomes, respectively. To prevent crossover of signals, fluorescent images were acquired separately and sequentially for red and green channels. Yellow fluorescence represented colocalization. Goat polyclonal antibodies to vesicular-specific protein EEA1 and LAMP2 were obtained from Santa Cruz Biotechnology. Binding of vesicular primary antibodies was detected using Alexa Fluor 488-labeled rabbit antigoat IgG. In the case of double staining, 4H4 binding was detected using Alexa Fluor 568-conjugated rabbit anti-mouse IgG. Samples were analyzed using a Zeiss Axiovert 100M laser-scanning microscope.
Preparation and ultrastructural study of MVs from mineralizing SaOS-2 cells and mineralization assay. Stable cell lines generated after transfection were plated at a density of 3 x 106 cells per 10-cm2 plate. Four plates of cells were pooled to prepare 250 µg of extracellular matrix-associated MV fractions by a previously described method (23, 27). Briefly, cells were induced to mineralize in promineralizing medium (complete medium supplemented with 50 µg/ml ascorbate and 2.5 mM sodium phosphate). The medium was supplemented with fresh ascorbate every day and changed to fresh promineralizing medium every 3rd day. On the 10th day, the medium was aspirated, and cells were rinsed twice with 1x PBS and then digested with 2 U/ml collagenase (Worthington Biochemicals) at 37° C for 3 h. The digest was centrifuged at 30,000 g for 20 min twice to settle all the debris. The supernatant was subjected to ultracentrifugation at 300,000 g for 20 min. Finally, the MV fraction from the clear pellet was resuspended in HBSS buffer (in mM: 5.4 KCl, 0.3 Na2HPO4, 0.4 KH2PO4, 0.6 MgSO4, 137 NaCl, and 5.6 D-glucose, pH 7.4). The protein content was determined according to the manufacturer's instructions using Bradford's reagents (Bio-Rad, Hercules, CA). Primary osteoblasts were also induced to mineralize, and MVs were prepared by this method. MV samples were processed for ultrastructure study by fixation overnight at 4° C in modified Karnovsky's fixative and then in 1% OsO4 in 0.1 mM sodium cacodylate buffer, pH 7.4. Subsequent dehydration was carried out using a graded series of ethanol solutions followed by propylene oxide and infiltration with epoxy resin (Scipoxy 812, Energy Beam Sciences, Agawam, MA). After polymerization at 65° C overnight, thin sections were cut and stained with uranyl acetate (4% uranyl acetate in 50% ethanol) and then with bismuth subnitrate. Sections were examined at an accelerating voltage of 80 kV using a Zeiss EM10B electron microscope.
Assays of NPP, alkaline phosphatase, PPi measurements, mineralization, and Western blotting. Cell lysates and MVs were assayed for NPP and alkaline phosphatase activities with 1 mM p-nitrophenylthymidine 5'-monophosphate and 5 mM p-nitrophenyl phosphate, respectively (Sigma, St. Louis, MO), used as substrates (22, 23). One unit of either enzyme activity was equivalent to 1 µmol of substrate hydrolyzed per hour. A previously described (23) radiometric assay was used to determine PPi levels associated with culture media, cell extracts, and 40-µg aliquots of MVs. The values were normalized for cell DNA or MV protein concentration where indicated. To assess mineralization of cultures treated with the promineralizing media, cells were stained by incubation on day 5 with 0.5% (vol/vol) Alizarin red S solution at 23° C for 10 min. The amount of Alizarin red precipitated was quantified using a previously described cetylpyridinium-based chromogenic assay (16). The cells were washed four times with PBS before the addition of 10% (wt/vol) cetylpyridinium chloride for 10 min to release the calcium-bound Alizarin red S. The extracted solution was collected, diluted at a ratio of 1:10, and read at 570 nm optical density on a microplate reader (SpectraMAX, Molecular Devices). For corroborative visualization of the extent of mineralization, the stained cells were evaluated by light microscopy after the unbound Alizarin red S was removed by washing. Sample protein concentrations were determined by the bicinchoninic acid assay (Pierce, Rockford, IL). Proteins were subjected to SDS-PAGE in an 8% polyacrylamide gel, transferred to nitrocellulose membranes, and analyzed by Western blotting. The primary antibody 4H4 was used, and secondary anti-mouse IgG conjugated to HRP was detected by enhanced chemiluminescence.
Statistical analyses. Where indicated, error bars represent SD. Statistical analyses were performed using Student's t-test (paired 2-sample testing for means).
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RESULTS |
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To test the importance of the tail-transmembrane region of NPP1 in MV targeting, SaOS-2 cells were stably transfected with chimeras of NPP1 and NPP3 (Fig. 2, C and D) generated using rodent NPP1 and NPP3, which are highly conserved relative to their respective human forms (7, 13). These chimeras were efficiently expressed in SaOS-2 cells, and NPP activity was detected in the MVs of cells expressing the chimera bearing the NPP1t/tm region (Fig. 3B). This was not the case for the chimera bearing the NPP3t/tm region with the NPP1ec.
Next, the molecular basis for the NPP1 cytosolic tail-transmembrane domain mediating NPP1 targeting to MVs was assessed. Within the NPP1 cytosolic tail, an AAASLLAP motif is known to be the mediator of NPP1 basolateral membrane targeting in polarized cells of epithelial origin (5). This motif is conserved across species and, therefore, was evaluated for its ability to act as the signal for MV targeting. The construct wherein the AAASLLAP motif was inserted at the 5' end of the sequence encoding the cytosolic tail of NPP3 (Fig. 2E) was used to stably transfect SaOS-2 cells. Cells expressing the construct NPP3/AAASLLAP were induced to mineralize, and MVs were collected. Compared with NPP3t/tm-NPP1ec, the increased NPP activity in MVs of NPP3/AAASLLAP-expressing cells was comparable to that for NPP1t/tm-NPP3ec (Fig. 3B). Thus the AAASLLAP motif in the cytosolic tail conferred to NPP3 the de novo ability to target to MVs and concentrate there.
Dileucine cytosolic tail motif involvement in targeting NPP1 to MVs. Paired leucine residues within the AAASLLAP motif of the wild-type human NPP1 cytosolic tail at positions 4950 were mutated by substitutions to alanine, and these constructs (Fig. 2, I and J) were stably transfected. As a control, the only other leucine in the NPP1 cytosolic tail (at position 60) was substituted to alanine (Fig. 2K). Tyrosine residues in the cytoplasmic tail are potentially signals responsible for protein trafficking. Therefore, the sole tyrosine residue in the NPP1 cytosolic tail (position 75) was substituted to glycine (Fig. 2L) to test for its potential as a targeting signal. Expression of each of the site-specific mutants of the NPP1 cytosolic tail increased cellular NPP activity comparably to wild-type NPP1 in stably transfected cells (Fig. 4A). The sizes and expression levels (10- to 13-fold stimulation over empty vector transfected cells) of the mutant proteins also were grossly comparable to wild-type NPP1 (Fig. 4B). These stable cell lines were induced to mineralize, and MVs were prepared from them. Mutations in the dileucine motif at position 4950 blunted the increased NPP activity in MV preparations achieved by stable wild-type NPP1 expression (Fig. 4, C and D). In contrast, cells expressing the Leu60Ala and Tyr75Gly mutants produced MVs with increased NPP activity in a manner comparable to wild-type NPP1. Annexin V, a critical constituent of the MVs, was used as a marker and was detected in the MV preparations of all mineralizing stable cell lines (Fig. 4E).
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Subcellular localization of wild-type and mutant NPP1. The SaOS-2 stable cell lines were also analyzed by confocal immunofluorescence microscopy (Figs. 5 and 6). Use of GFP reporter constructs with the cytosolic tail-transmembrane region of NPP1 and NPP3 (schematized in Fig. 2, F and G) indicated that the tail region of NPP1 was sufficient to promote localization in the plasma membrane of SaOS-2 cells (Fig. 5B). Although punctate intracellular staining was also detected, the chimeric proteins NPP1t/tm-NPP3ec and NPP3/AAASLLAP were localized predominantly in the plasma membrane (Fig. 5, D and F). In contrast, NPP3t/tm-GFP remained in intracellular vesicles (Fig. 5C). The NPP3t/tm-NPP1ec chimera also demonstrated exclusively intracellular vesicular staining (Fig. 5E). Mutations in the dileucine cytosolic tail motif of NPP1 attenuated localization of NPP1 to the plasma membrane. Cells expressing these mutants localized NPP1 principally to apparent intracellular vesicular structures (Fig. 6, D and E). In contrast, the wild-type NPP1 and Leu60Ala and Tyr75Gly mutants comparably localized, predominantly to the plasma membrane (Fig. 6, B, C, and F). Thus the NPP1 cytosolic tail and the cytosolic tail AAASLLAP motif mediated plasma membrane localization of NPP1 in SaOS-2 cells.
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The punctate staining of the dileucine amino acid mutant colocalized with the early endosomal marker EEA1 (Fig. 7B.3). However, no significant overlap in signal was observed with LAMP-2, a late endosome/lysosomal marker (Fig. 7D.3). Figure 7, A.3 and C.3, showed that wild-type NPP1 was localized in the plasma membrane, and no colocalization with EEA1 or LAMP2 was observed. Therefore, mutations in the cytosolic dileucine motif appeared to induce aberrant NPP1 retention in early endosomes.
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Modulation of PPi levels and mineralization by NPP1 mutants. MV-associated PPi levels were 50% lower in cells expressing the NPP1 cytoplasmic tail dileucine mutants than in cells expressing wild-type NPP1 (Fig. 8A). Ultrastructural morphology of MV preparations revealed that, with NPP1Ala49Ala50 expression, there was an abundance of electron-dense inclusions within MVs, suggesting calcification (Fig. 8C), as opposed to a paucity of such inclusions in MVs of cells expressing the MV-targeted mutant NPP1Leu60Ala (Fig. 8B). Hence, the mineralizing function of NPP1 mutants was directly examined. Wild-type NPP1 expression inhibited active mineralization by SaOS-2 cells, which was assessed by staining for the calcium-binding dye Alizarin red S and by measuring precipitated Alizarin red S via a cetylpyridinium dye-binding assay (Fig. 9). NPP1 mutants that were localized to MVs increased the local PPi levels and promoted decreased mineralization, in contrast to NPP1 mutants that were not targeted to MVs. Thus the extent of targeting of NPP1 activity to MVs correlated directly with the extent of mineralization by SaOS-2 cells.
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DISCUSSION |
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The findings of this study established fundamental mechanisms in NPP1 action as a physiological calcification regulator. A prerequisite for this functional capacity of NPP1 was targeting to MVs, which is not a feature of NPP2 and NPP3 (24). A plasma membrane (and MV) targeting signal was identified in the NPP1, but not NPP3, cytosolic tail. This signal comprised the sequence motif AAASLLAP, which is conserved across species in NPP1 (7, 13). The paired leucine residues contained in this motif, at position 4950 in the NPP1 cytosolic tail, were critical residues for MV targeting. Importantly, the AAASL-LAP domain was also sufficient to confer de novo plasma membrane and MV targeting ability to NPP3 when transposed into the NPP3 cytosolic tail. This sequence motif has also been demonstrated to be responsible for basolateral localization of NPP1 in polarized epithelial cells (5). Mineralizing SaOS-2 cells expressing dileucine motif mutants of NPP1 released MVs with altered morphology and lower PPi levels, concordant with greater calcification in vitro than the wild-type NPP1-expressing cells. Although tyrosine-containing motifs are membrane-sorting signals for some proteins (25), this study determined that the cytosolic tyrosine of NPP1 was not required for NPP1 plasma membrane targeting and MV localization in osteoblasts that regulated calcification.
In this study, we observed that NPP activity was greatly reduced in MVs of NPP1-knockout mice compared with MVs from congenic wild-type cells. This finding was significant partly because NPP1 and NPP3 expression and specific activities appear roughly equivalent in primary osteoblasts, and the residual level of cell-associated NPP activity in NPP1-knockout mouse primary osteoblasts is not only substantial (50% that of wild-type cells in early culture) but also steadily increases in the primary osteoblasts as they mineralize in culture (21).
NPP1 partly regulated calcification by modulating PPi concentration in MVs. Taken together, our results suggested that calcification is normally suppressed by targeting of NPP1 to the plasma membrane and to MVs. Our results are consistent with a model in which other PPi-generating NPP family members expressed in mineralization-competent tissues are unable to compensate for loss of NPP1 because of comparatively sparse plasma membrane and/or MV localization.
Targeting to MVs of selected proteins, including annexins (26, 27), matrix metalloproteinases (10), TNAP (1, 16, 18, 22, 27), and NPP1 (11, 21, 2224), is fundamental to the specialized functions of chondrocytes and osteoblasts in regulating matrix modification and calcification. Polarized biogenesis of MVs occurs by budding from lateral plasma membrane regions of the osteoblast (31). These cells bud off MVs from areas in the plasma membrane surface-facing bone mineral in vivo (31). Classically polarized epithelial cells exhibit polarized trafficking of membrane-localized proteins, which are targeted to distinct apical or basolateral regions of the plasma membrane (23, 28). Although osteoblasts are not classically polarized in the manner of epithelial cells, they possess molecular machinery for polarized subcellular trafficking (35).
TNAP, a marker enzyme enriched in MVs, is localized on the mineral-facing lateral surface of the osteoblast plasma membrane, roughly the equivalent of "basolateral" membrane localization in polarized cell types (17, 25, 32). TNAP is glycosylphosphatidylinositol (GPI) anchored, and in epithelial cells, most GPI-anchored proteins, including TNAP, are apical (25). However, GPI-anchored proteins that typically have apical distribution in cell types such as Madin-Darby canine kidney, kidney, and intestinal epithelial cells can localize in the basolateral membrane of other polarized cell types (39, 47). Hence, the colocalization of TNAP and NPP1 in MVs likely reflects currently undefined mechanisms for concentrating MV proteins in specialized regions of the plasma membrane where MVs bud off. One mechanism distinct from apical vs. basolateral sorting mechanism that is involved in regulating MV composition is the targeting of annexins II, V, and VI to MVs enabled by Ca2+-dependent binding to phospholipid-enriched regions of the chondrocyte plasma membrane from which MVs are released by budding (26, 44).
Confocal microscopy experiments in this study revealed that NPP1 was distributed to the plasma membrane in SaOS-2 cells, whereas NPP3 was localized predominantly intracellularly. Very little staining of the plasma membrane was observed in NPP1 cytosolic tail dileucine mutant-expressing SaOS-2 cells. This is unlike the case in epithelial cells, where NPP1 and NPP3 are localized to basolateral and apical membranes, respectively (5). Furthermore, in polarized epithelial cells, NPP1 cytosolic tail dileucine motif mutation caused a shift in membrane localization, such that NPP1 become directed to the apical plasma membrane (5). The lack of an epithelial cell-equivalent "apical membrane" in SaOS-2 cells may help explain the paucity of plasma membrane localization of the NPP1 cytosolic tail dileucine mutant in osteoblastic cells in this study.
This study suggested that NPP1 had to be localized in the plasma membrane to be ultimately targeted to MVs. One of the mechanisms for sorting of membrane proteins in polarized and nonpolarized cells is transport directly from the trans-Golgi network via vesicles to distinct plasma membrane surfaces (25). Alternatively, vesicles from the trans-Golgi network can be initially directed to the basolateral membrane; then endocytosis in recycling endosomes and subsequent sorting to the appropriate plasma membrane surface occur (25, 32). In this process, adaptins mediate post-endocytic recycling of transmembrane proteins to the plasma membrane in a manner dependent on cytosolic dileucine motifs or specific tyrosine-containing motifs (6, 25, 32). In the present study, NPP1 dileucine motif mutants were retained intracellularly in early endosomal vesicles in SaOS-2 cells. One possibility is that SAOS-2 cells may lack an apical targeting pathway; thus NPP3 and the NPP1 dileucine motif mutant could conceivably reach the plasma membrane but then be internalized and retained in endosomal vesicles. Alternatively, mutations of the dileucine residues of NPP1 may have altered NPP1 interaction with adaptins or other trafficking proteins, resulting in their retention in early endosomal vesicles.
The findings in this study of a targeting function for the NPP1 dileucine motif are not the first to demonstrate a subcellular sorting role for a dileucine motif. For example, a dileucine motif is involved in targeting the human equilibrative nucleoside transporter hENT2 to the plasma membrane of renal epithelial cells (30), glycoprotein QNR-71 to melanosomes (29), and IgG Fc receptor to the basolateral face of Madin-Darby canine kidney cells (20). A dileucine motif also is involved in targeting proteins such as the epidermal growth factor receptor (19) and the cation-independent mannose 6-phosphate receptor to lysosomes (46). However, we did not observe significant localization of wild-type NPP1 in late endosomal/lysosomal vesicles, which implied that the dileucine motif serves primarily to direct NPP1 to the plasma membrane and possibly, but not definitively, further into MVs.
Targeting to the plasma membrane and concentration in MVs of ectoenzymes involved in ATP and PPi metabolism (13, 16, 18, 2124) have long been recognized to be fundamental to the specialized functions of osteoblasts and chondrocytes in calcification. More recently, attention has been directed to the capacity of MV NPP1 to generate via ATP hydrolysis the HA crystal deposition inhibitor PPi, which directly antagonizes the calcification-promoting activity of TNAP in MVs (16). Therefore, identification of the cytosolic tail dileucine motif of NPP1 as a molecular targeting motif for osteoblast plasma membrane and MV localization of NPP1 in this study provides novel understanding of where and how NPP1 needs to be delivered to exert calcification-regulating PPi-generating capacity in osteoblasts.
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ACKNOWLEDGMENTS |
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GRANTS
This study was supported by awards from the National Institutes of Health and Veterans Affairs Research Service (to R. Terkeltaub), Arthritis National Research Foundation (to S. M. Vaingankar), and National Health and Medical Research Council of Australia and Pfizer Inc. (to J. W. Goding).
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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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