Program in Membrane Biology and Renal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Submitted 20 July 2004 ; accepted in final form 12 January 2005
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ABSTRACT |
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lysosome; trafficking; vasopressin receptor in LLC-PK1 cells
In all cases, the internalized receptor-ligand complex is first delivered to early endosomes (EE), in which the acidic pH is sufficient to facilitate the dissociation of many ligands. Some receptors that lose their ligands are recycled by vesicular carriers that initially transfer receptors to a perinuclear tubulovesicular compartment, the recycling endosome, followed by the return of the transferred receptors to the cell surface. Alternatively, cargo receptors may be delivered from the early endosome into the late endosome or multivesicular body. This compartment can recycle the receptors directly to the plasma membrane or sort them into either the trans-Golgi network (TGN) or lysosomes (33, 36, 42, 45). For example, the epidermal growth factor (EGF) receptor and its ligand are both delivered to lysosomes for degradation (13, 14, 20). Furthermore, the -opioid receptor appears to be degraded by the proteasome (54).
The V2R is a "slow recycling" GPCR that regulates water reabsorption by renal collecting duct epithelial cells. V2R is phosphorylated upon activation by vasopressin (VP) binding. Activated V2R then binds to -arrestin, and the complex is internalized via clathrin-mediated endocytosis (6, 41). Unlike the
2AR, internalized V2R fails to recycle rapidly and V2R forms a stable complex with
-arrestin throughout the internalization pathway (23, 40). Innamorati et al. (26) showed that prolonged association of
-arrestin with the V2R could be responsible for intracellular retention but not the final destination of the receptor, in contrast to the idea that stable binding of
-arrestin directs internalized receptors to lysosomes (8). Although mechanisms involving agonist-induced GPCR endocytosis have been characterized extensively, less is known about the intracellular pathways and proteins involved in fast and slow GPCR recycling. An earlier study (34) showed that the VP ligand is delivered to lysosomes after V2R binding, as are many other ligands that are internalized by receptor-mediated endocytosis; but the fate of the actual V2R was not followed in that earlier report.
Therefore, to study the endocytosis and recycling pathways followed by the V2R itself, we established stably transfected LLC-PK1a epithelial cell lines expressing a V2R-green fluorescent protein (V2R-GFP) chimera. Our immunofluorescence, biochemical, and ligand binding data show that much of the V2R that is internalized after VP addition to cells enters a lysosomal degradation compartment. The reestablishment of baseline levels of VP binding sites (V2R) at the cell surface requires de novo protein synthesis, providing a partial explanation for the slow recycling pathway previously reported for this receptor.
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MATERIALS AND METHODS |
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Construction of wild-type V2R-GFP. GFP was attached to the carboxy terminus of the V2R. The TGA stop codon after the carboxy-terminal serine was replaced using site-directed mutagenesis with an in-frame CGG sequence. The entire V2R-containing cassette was subcloned into the 5'-Xho1 and 3'-BamHI sites of the pEGFP-N1 vector (Clontech, Palo Alto, CA). The fidelity of the construct was confirmed by performing sequence analysis at the Massachusetts General Hospital Core DNA Sequencing Facility.
Cell culture and transfection. LLC-PK1a cells, a variant of the native LLC-PK1 cell line, which expresses only very low levels of endogenous V2R (6), were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 10% heat-inactivated FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). This cell line was provided by Dr. Steven Krane (Arthritis Unit, Massachusetts General Hospital, Boston, MA). To obtain stable cell lines expressing wild-type V2R-GFP (LLC-V2R-GFP) or GFP alone (LLC-GFP), LLC-PK1a cells were plated at a density of 150,000 cells/60-mm dish 20 h before transfection. For transfection, Lipofectamine (15 µl) with 4 µg of plasmid DNA was added to the cells, incubated at 37°C for 4 h, and washed once with serum-free DMEM. After 1420 days of selection in medium containing 1 mg/ml geneticin (G418), resistant colonies were isolated with cloning rings and transferred to separate culture dishes for expansion and analysis of their [3H]-VP binding abilities. Several clones were isolated, and their [3H]-VP binding activities were characterized. All clones shared similar characteristics in terms of V2R biology, although the absolute number of receptors expressed differed among the different clones.
[3H]-VP binding to LLC-V2R-GFP cells. [3H]-VP binding assays were performed in 48-well plates. LLC-V2R-GFP cells were plated at a density of 30,000 cells 48 h before the binding assay. Briefly, 0.25 ml of ice-chilled phosphate-buffered saline (PBS), pH 7.4, containing 0.9 mM CaCl2, 0.9 mM MgCl2, 3.5 mM KCl, 1 mg/ml glucose, 1 mM tyrosine, 1 mM phenylalanine, and 0.5% BSA containing the appropriate dilution of [3H]-VP (NEN, Boston, MA) was added to each well. Incubation was performed for 3 h at 4°C. Nonspecific binding was determined in the presence of excess unlabeled VP (1 µM). Incubations were stopped by performing two rinses with ice-cold PBS at pH 7.4. Cells were solubilized in 500 µl of NaOH (0.1 N) and transferred to scintillation vials. After 12 h, 5 ml of scintillation fluid (Optic-Fluor; Packard, Groningen, The Netherlands) were added. The bound radioactivity was determined using a liquid scintillation analyzer (TriCarb 2200 CA; Packard).
Recovery of cell surface [3H]-VP binding sites on LLC-V2R-GFP cells after direct downregulation was studied. Cells were grown in Transwell cell culture filter chambers. They were plated at a density of 100,000 cells/filter (day 1) and grown to confluence (106 cells/filter, day 6). The cells were incubated for 1 h at 37°C with VP (1 µM) diluted in DMEM. After ligand removal by three acid washes (in mM: 50 sodium citrate, 0.2 NaH2PO4, and 90 NaCl, pH 5), the pH was neutralized by three washes with cold PBS, pH 7.4, and then the cold medium was replaced by warmed cell culture medium (DMEM supplemented with 10% heat-inactivated FBS). Cells were incubated for different times at 37°C before binding assays as described above. Briefly, after the recovery incubation, cells were incubated for 3 h at 4°C with [3H]-VP (9 nM) on the basolateral side. Nonspecific binding was determined in the presence of 1 µM unlabeled VP. Incubations were stopped by rinses with ice-cold PBS at pH 7.4. The bound radioactivity in solubilized cells in NaOH (0.1 N) was determined using a liquid scintillation analyzer.
cAMP assays. Briefly, LLC-V2R-GFP cells were grown in 96-well plates until confluence was reached. The cells were pretreated for 15 min with the phosphodiesterase inhibitor IBMX (1 mM), followed by incubation with different concentrations of VP for 10 min at 37°C. The intracellular levels of cAMP were measured with the BioTrak kit (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (5). Each intracellular cAMP assay was performed in triplicate.
Immunofluorescence. LLC-V2R-GFP cells were plated on 12 x 12-mm glass coverslips (Fisher Scientific, Pittsburgh, PA). The cells were incubated with or without VP (1 µM) at either 37°C or 20°C for either 1 or 4 h, respectively. Most of the experiments were performed in duplicate with or without cycloheximide (10 µg/ml) present in the medium to determine the potential contribution of newly synthesized vs. recycling V2R-GFP. After treatment, cells were fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), and 5% sucrose for 20 min at room temperature. The cells were washed three times in PBS and then used for immunocytochemistry.
To examine the recovery of cell surface V2R-GFP fluorescence on LLC-V2R-GFP cells, the cells were incubated for 1 h at 37°C in the presence or absence of VP (1 µM) diluted in DMEM. After incubation, the cells were washed three times with a solution containing (in mM) 50 sodium citrate, 0.2 NaH2PO4, and 90 NaCl, pH 5, and then washed three more times with cold PBS, pH 7.4. The cold medium was replaced by warmed cell culture medium and incubated for 7 h at 37°C before being fixed as described above and used for immunocytochemistry.
Immunocytochemistry was performed using several antibodies that recognize different intracellular compartments. Primary antibodies were applied to cells permeabilized with 1% SDS for 4 min at room temperature as an antigen retrieval step (11). Golgi cisternae and associated vesicles were identified using an anti--subunit of coat protein coatomer (anti-
-COP) antibody (0.01 µg/ml; Sigma), and the trans-Golgi network (TGN) was labeled using either an anti-clathrin antibody (5 µg/ml) or an anti-P230 (a TGN protein marker) antibody (5 µg/ml). A secondary antibody, Cy3-conjugated donkey anti-mouse (1.5 µg/ml), was applied for 1 h at room temperature. Coverslips were mounted on slides with Vectashield medium (Vector Laboratories, Burlingame, CA). Localization of both GFP fusion proteins and compartments marked by antibodies were visualized using a Bio-Rad Radiance 2000 confocal microscope.
In addition to these antibodies, we also used a fluorescent tracer to study the intracellular localization of V2R-GFP. Cells were preincubated for 30 min with Lysotracker (500 nM; Molecular Probes, Eugene, OR), a lysosomal marker, before the VP or cold treatment. Vesicles containing GFP-V2R were then compared with the distribution of Lysotracker-labeled vesicles. After incubation, the cells were fixed and visualized as described above.
The effect of cycloheximide on another protein trafficking exocytosis pathway was also examined using LLC-AQP2 cells, an LLC-PK1 cell line that stably expresses c-myc-tagged aquaporin 2 (28). These cells were incubated for 6 h in the presence or absence of cycloheximide (10 µg/ml). After incubation, VP (1 µM) and forskolin (10 µM) were added to the incubation medium for 10 min to induce AQP2 plasma membrane expression as previously described (28). Treated cells were fixed and stained with a monoclonal anti-c-myc antibody as previously reported (5).
Fluorescence intensity of the perinuclear patch that appeared after incubation at 20°C was quantified using IP Lab Spectrum software (Scanalytics, Vienna, VA) on fluorescence microscopic images. The patch was outlined using the freehand drawing tool available as part of the software package, and the average pixel intensity of the resulting region of interest was obtained for each cell. The mean pixel intensity is the average of values from 30 different cells in each condition. This quantification is representative of at least of three independent experiments. Statistical analyses were performed using the unpaired or paired Students t-test when applicable. Difference were considered significant at P < 0.05.
Protein extraction. Confluent LLC-V2R-GFP and LLC-GFP cells were incubated at 37°C for 6 h in the absence or presence of VP (1 µM) with or without preexposure for 30 min to the protein synthesis blocker cycloheximide (10 µg/ml); a lysosomal protein degradation inhibitor, chloroquine (10 µM); or lactacystin (6 µM), a proteasome inhibitor. After treatment, cells were lysed for 20 min at 4°C in radioimmunoprecipitation assay (RIPA) buffer containing 50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS supplemented with protease inhibitors (Roche, Mannheim, Germany). Protein concentrations were measured by performing a bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL) before Western blot analysis.
Endoglycosidase digestion of solubilized material. Proteins from either LLC-V2R-GFP or LLC-GFP cells were extracted in RIPA buffer as described above. Solubilized proteins (650 µg) were incubated in the presence of either N-glycosidase F (PNGase F, 25 U; New England Biolabs, Boston, MA) or a mixture containing both neuraminidase (10 mU) and O-glycosidase (0.5 mU) (Roche, Indianapolis, IN). Some solubilized protein aliquots were incubated simultaneously with all enzymes. Endoglycosidase digestion was performed at 37°C for 24 h in a final volume of 250 µl. It was terminated by addition of denaturing buffer and incubation at 70°C for 10 min. The resulting material was immediately analyzed using SDS-PAGE.
SDS-PAGE and Western blot analysis. Protein samples were separated using 12% Bis-Tris-PAGE (Invitrogen, Carlsbad, CA) and then transferred to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), followed by Western blot analysis. Membranes were blocked by incubation overnight with blotting solution (PBS, pH 7.4, 0.05% Tween 20, and 5% nonfat dry milk). Membranes were first incubated for 1 h with a polyclonal rabbit anti-GFP antibody (0.4 µg/ml; Molecular Probes), then with Amdex goat anti-rabbit IgG-horseradish peroxidase (1:100,000 dilution; Amersham, Little Chalfont, UK). Signals were detected using the protocol described in the Western Lightning Chemiluminescence Reagent Plus system (PerkinElmer Life Sciences, Boston, MA). Identified protein bands were quantified using a video densitometer and Kodak 1D software (Kodak, New Haven, CT).
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RESULTS |
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Two protein degradation inhibitors were incubated with VP to study this degradation hypothesis further. Incubation with lactacystin, a proteasome degradation inhibitor, showed no inhibitory effect on V2R-GFP degradation in VP-stimulated cells (Fig. 4, lanes 3 and 4). In contrast, inhibition of V2R-GFP degradation was observed in the presence of chloroquine, a lysosomal degradation inhibitor (Fig. 4, lanes 5 and 6). Densitometric analysis showed that in the presence of both VP and lactacystin or with VP alone, the 57- to 68-kDa upper band was reduced by 78 ± 8% (n = 3; mean ± SE). No reduction in band intensity was detectable when VP was added together with chloroquine (n = 3). These data indicate that receptor degradation that occurred during VP stimulation, represented by the relative change in the 57- to 68-kDa vs. 46-kDa band intensities, was abolished by lysosomal degradation inhibitors.
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To confirm that other cellular trafficking processes were still intact after several hours of protein synthesis inhibition, we have repeated our previously published result (27) that VP was able to stimulate plasma membrane insertion of AQP2 in LLC-PK1 cells exposed to cycloheximide for 6 h (Fig. 10C). Furthermore, we treated LLC-V2R-GFP cells for 10 h with cycloheximide after VP treatment and then washed out the drug. After a further 12-h incubation in the absence of VP, plasma membrane levels of the receptor were completely restored to control values (data not shown). These data indicate that cycloheximide treatment for several hours does not irreversibly damage protein trafficking pathways in LLC-PK1 cells.
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DISCUSSION |
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To dissect the intracellular pathways and reveal fate of the V2R after ligand-induced internalization, a V2R-GFP chimeric construct was stably expressed in LLC-PK1a epithelial cells, a subtype of LLC-PK1 cells that expresses a very low amount of endogenous V2R (6). The introduction of GFP at the carboxy terminus of V2R produces a fully functional V2R-GFP chimera as found with other GPCRs (8, 17, 50). In contrast, an NH2 terminus GFP-V2R chimera was not inserted into the plasma membrane (data not shown). Western blot analysis using anti-GFP antibodies allowed us to detect protein bands between 57 and 68 kDa corresponding to the chimeric protein, similar to those described in Madin-Darby canine kidney or human embryonic kidney (HEK)-293 cells (3, 22), as well as two additional bands at 4652 kDa that were much weaker than the 57- to 68-kDa smear under baseline conditions. The 57- to 68-kDa smear corresponds to the intact N- and O-glycosylated V2R-GFP chimeric protein (4, 24, 44), the band at 52 kDa is a nonglycosylated form of V2R-GFP, and the lower molecular mass 46-kDa band probably represents a nonglycosylated degradation product (see below). This 46-kDa band was detectable at low levels even in the absence of VP stimulation, reflecting a low level of intracellular V2R trafficking and degradation observed under nonstimulated conditions.
The chimeric V2R-GFP binds VP normally, initiates a cAMP signaling response, and internalizes into a perinuclear location after agonist stimulation. The resulting reduction of cell surface [3H]-VP binding sites in LLC-V2R-GFP cells is fully restored to prestimulation levels only several hours after agonist removal as previously described for the wild-type V2R (23, 26). After internalization, V2R-GFP passes through an early endosome-antigen 1 positive endosomal compartment as expected (data not shown) and ultimately becomes predominantly colocalized with the lysosomal marker Lysotracker. In parallel, Western blot analysis data showed that the intensity of the 57- to 68-kDa band representing intact V2R-GFP was considerably reduced 4 h after exposure of cells to VP, while the intensity of the lower molecular mass 46-kDa band was significantly increased. This VP-induced shift in band density was completely prevented by the lysosomal inhibitor chloroquine but was not affected by lactacystin, a proteasome inhibitor. Taken together, these data suggest that after internalization, a significant quantity of V2R is trafficked to lysosomes for degradation. However, the degradation process may be initiated in late endosomes, which also contain proteases and are a site of significant proteolysis (9, 51). Furthermore, a potential contribution of previously described cell surface proteases to this cleavage event cannot be excluded (32).
This interpretation leads to the conclusion that complete restoration of cell surface V2R levels after downregulation would require de novo receptor synthesis. This hypothesis is supported by two distinct but convergent sets of data from our study. When cells were exposed to VP in the presence of cycloheximide to block protein synthesis, the rate of recovery of the cell surface receptor pool was inhibited significantly between 2 and 6 h after VP washout. After VP washout for 2 h, an additional 20% of the prestimulation level of cell surface receptor was restored, despite the presence of cycloheximide. However, no subsequent increase in this level was detected after inhibition of protein synthesis, in contrast to untreated cells, in which levels were restored to 75% of the nonstimulated value after 6 h. These data indicate that while the early recovery (20% of total recovery) could be due to recycling V2R or to the membrane insertion of a pool of preexisting intracellular V2R, the bulk (80%) of the restoration process is due to the insertion of newly synthesized V2R into the cell surface. The more rapidly inserted pool may be important for maintaining at least a partial VP response of collecting duct principal cells under physiological conditions of fluctuating VP levels.
An earlier study in isolated rat collecting ducts concluded that the V2R recycles rapidly, in contrast to several other reports that it is a slow recycler (30). Furthermore, the reappearance of cell surface VP binding sites was not inhibited by cycloheximide in this earlier report (30). However, in addition to the differences in cell type and the use of a V2R-GFP construct in the present study, there are several other methodological differences between the studies that make a direct comparison of the results difficult. First, the exposure to cycloheximide was brief and would not have inhibited the processing and packaging of V2R that had already been synthesized. Approximately 30 min are required for newly synthesized proteins to be transported through post-endoplasmic reticulum compartments, including the Golgi, and for an effect of cycloheximide on membrane protein insertion to become apparent. Thus the packaging and export to the cell surface of this presynthesized pool of V2R could explain the reappearance of cell surface binding sites that were measured in the previous study. The presence of a rapidly inserted pool of the V2R was also detected in the present study in LLC-PK1 cells. Approximately 20% of total cell surface binding sites were restored within 60 min of VP washout, and this reappearance was not inhibited by cycloheximide. Second, the molecular mass and Bmax of the collecting duct receptors examined by Kim et al. (30) are similar to those of smooth muscle and hepatocyte vasopressin V1 receptors, which are known to be expressed in collecting duct principal cells (1, 18, 55) and to which the VP ligand would also bind. Cycloheximide has been reported to have no effect on rapid V1 receptor recycling in smooth muscle cells (10) or in hepatocytes (16). Third, the internalization studies of Kim et al. (30) were performed at 24°C. In the present study, we were unable to detect V2R degradation when cells were incubated at 20°C, implying that this process is temperature dependent. Lysosomal degradation of the EGF receptor is also inhibited by incubation at 25°C (48).
The de novo synthesis hypothesis is also supported by our data derived from the use of a 20°C temperature block to dissect the pathway of intracellular V2R trafficking. Under these conditions, V2R-GFP accumulates during a 4-h period in a compact perinuclear patch even in the absence of VP stimulation. This accumulation is significantly reduced by cycloheximide, indicating that de novo protein synthesis is required for its appearance. Previous studies from several laboratories, including our own, have shown that this low-temperature treatment causes a block in the export of proteins from the TGN (27, 38). In the present study, V2R-GFP in the perinuclear patch colocalized with the TGN markers clathrin and P230, but less so with the Golgi vesicle/cisternal marker -COP. Thus both newly synthesized proteins and any proteins that recycle through the TGN could contribute to the formation of the patch. It is known, for example, that the TGN protein furin recycles between the plasma membrane and the TGN via a recycling endosome compartment, indicating that these two compartments are interconnected at least in some recycling pathways (35, 49). After VP treatment at 20°C, the patch was larger and brighter than it was in the absence of VP, indicating that it contains internalized V2R in addition to newly synthesized V2R-GFP. The level of resolution of our images was not sufficient to determine whether both newly synthesized and internalized V2R were located in the same vesicles within the patch, however. The internalized V2R in the patch showed only a very partial colocalization with Lysotracker and was not degraded under low-temperature conditions, indicating that it had been blocked in a prelysosomal compartment. Whether internalized V2R-GFP is blocked in the TGN or in another closely related, temperature-sensitive compartment such as the recycling endosome is unclear.
The conclusion that the lysosome appears to be the final destination of much of the internalized V2R-GFP contrasts with the results of a previous study showing that internalized receptors remained intact for several hours in a transferrin, Rab11-positive recycling endosome compartment in the perinuclear region of HEK-293 cells (25). The difference in cell types may be one explanation for this difference, but it is also probable that in contrast to V2R-GFP, the degraded V2R was not detectable in the previous study. Furthermore, the amount of degradation product may be proportional to the duration and strength of the agonist incubation, which also differed between the two studies. An earlier report showed that the VP ligand is directed to lysosomes after internalization but that trafficking of the V2R itself was not directly followed (34). The appearance of an internalized fluorescent VP analog in large, lysosome-like intracellular vesicles in collecting duct principal cells has also been described (31), although no definite identification of the vesicles was attempted. Clear identification of the nonlysosomal perinuclear compartment that contains internalized V2R, and newly synthesized V2R is complicated by the dynamic interaction between the numerous vesicular compartments involved in the trafficking of the receptor. Furthermore, many "specific" markers, including Rab11, may be found in more than one compartment (12, 46, 53). Further dissection of the juxtanuclear compartments involved in V2R processing will require extensive double-labeling studies at the electron microscopy (EM) level.
The information for receptor targeting to either recycling or lysosomal degradation pathways may reside in the cytoplasmic tail of the receptor (52). A recent report suggests that the presence of a cluster of serines in the tail of a GPCR appears to mediate stable binding of arrestin and to dictate trafficking of the internalized receptor to the lysosome (40). The hypothesis that arrestin may target GPCRs to lysosomes is also supported by the subcellular localization of -arrestin in multivesicular bodies (2). Furthermore, VP stimulation leads to rapid,
-arrestin-dependent ubiquitination of the V2R and increased degradation (37). Whether these mechanisms are also involved in the lysosomal targeting of internalized V2R remains to be determined. One question that is unanswered by our present data is the physiological reason for V2R receptor degradation in its slow recycling pathway. It is possible that the association between VP and its receptor has evolved to withstand the unusually harsh environment that exists in much of the renal medulla. Not only is the interstitium in which receptor-ligand binding occurs hypertonic with respect to urea and NaCl but also its pH is significantly more acidic than plasma (29). Thus V2R-ligand binding may be at least partially resistant to the pH-induced dissociation that normally occurs for many receptor-ligand pairs in the acidic endosomal compartment (39). After receptor-ligand dissociation, receptors are often recycled back to the plasma membrane, while the ligand is targeted to lysosomes for degradation. If this uncoupling does not occur in the case of the VP-V2R interaction, both may then be subject to lysosomal degradation. Thus the apparently inefficient process of V2R degradation, rather than recycling, that occurs after internalization may be the price that the kidney pays to maintain body fluid homeostasis under the influence of the antidiuretic hormone VP. While the cortical environment of the kidney is more "friendly" with respect to pH and tonicity, the need for the V2R to function in the medulla may have contributed to the evolution of its binding characteristics.
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GRANTS |
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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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