1 Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh; and 2 Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn2+, plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn2+ by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn2+-specific fluorophore Zinquin that were rapidly reversed by the Zn2+ chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn2+ in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn2+ homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn2+-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn2+ that were reversed by Zn2+ supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn2+ release from thiolate clusters. This effect was restored by Zn2+ supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn2+ and suggest that the ratio of MT to T can regulate Zn2+ homeostasis in response to nitrosative stress.
endothelium; S-nitrosylation; imaging; fluorescence resonance energy transfer; Zinquin
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INTRODUCTION |
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NITRIC OXIDE (NO) IS A UBIQUITOUS signaling molecule that is known to have important biological roles in the cardiovascular, nervous, and immune systems. Although most bioregulatory targets of NO contain cysteine and/or iron at the allosteric or regulatory sites (39), other molecular reactions may contribute to the biology of NO. After iron, zinc is the most abundant intracellular metal. Virtually all intracellular zinc is associated with proteins (primarily via complex interactions with cysteines), where it is known to be an integral component of numerous metalloenzymes, structural proteins, and transcription factors. Unlike iron, zinc itself is redox inert and does not react with NO. In vitro data reveals, however, that S-nitrosylation of cysteines in zinc thiolate structures affects the activity of zinc-dependent transcription factors (3, 21, 22) and enzymes (5, 10). Furthermore, NO is capable of increasing the amount of labile Zn2+ in living cells from the hippocampus (6) and the vascular endothelium (2, 23, 35). Accordingly, it is highly plausible that zinc may play a role in aspects of NO signaling.
Metallothionein (MT), a cysteine-rich (30% of all amino acid residues), heavy-metal-binding protein, is critical to intracellular Zn2+ homeostasis, as it has the ability to bind up to seven zinc atoms per MT molecule. Although the function of MT remains unclear (31), it was originally hypothesized to affect zinc in a manner homologous to that of calmodulin for calcium (38). More recently, it has been suggested that MT acts as a sensor of cellular redox such that a shift to more-oxidizing conditions leads to release of zinc, whereas a shift to a more-reducing environment leads to binding of zinc (26). In vitro, NO is capable of interacting with MT to form either dinitrosyl iron sulfur complexes (18, 36) or S-nitrosylation to result in the NO-mediated release of cadmium (28), copper (17), or zinc (22). We recently used fluorescence resonance energy transfer (FRET) in cultured pulmonary artery endothelial cells to show that green fluorescent protein-modified MT (FRET-MT) undergoes conformational changes in the presence of NO that are consistent with the release of free metal (zinc and/or copper) from its thiolate clusters (34). Alterations in metal ion homeostasis may underlie the protective effect of MT against NO toxicity (36) or the participation of MT in NO-modified, complex physiological properties such as the myogenic reflex (34).
In the current study, we used the zinc-specific fluorophore Zinquin
(46) to image changes in intracellular labile zinc in response to S-nitrosocysteine (SNOC). SNOC caused an
increase in labile zinc in lung fibroblasts isolated from wild-type
mice but not from MT null mutant mice [MT/
]. Complementation of
MT in this latter group, however, restored the SNOC-induced increase in
labile zinc. We then used adenoviral-mediated gene-transfer protocols
to affect the ratio of MT to its apo form, thionein (T). Overexpression
of MT or FRET-MT caused an inhibition of NO-induced changes in labile
zinc or release of zinc from MT, respectively, and each effect was
reversed by growing the transfected cells in medium enriched in zinc.
This suggests that a significant component of transgenic MT or FRET-MT
was in the apo form. Collectively, the fluorescence-based assays
support a singular central role for MT in affecting NO-mediated changes
in labile zinc and suggest that the ratio of MT to T can affect
Zn2+ homeostasis in response to nitrosative stress.
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MATERIALS AND METHODS |
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MT knockout mice.
Breeding pairs of MT-I- and MT-II-deficient (MT/
) mice were
imported from Michalska and Choo (27). MT
/
mice were
bred with C57BL6 mice obtained from Jackson Immunoresearch Laboratories (West Grove, PA) to generate a parental heterozygous chimera that in
turn was backbred to wild-type C57BL6 mice. This backbreeding resulted
in
50% of offspring being heterozygous mutants. These mutants were
identified through a genotyping protocol using polymerase chain-reaction strategy on novel sites within the murine MT-II gene
that was mutated. The knockout animals were of a mixed genetic background of OLA129 and C57BL6 strains. To avoid concerns regarding the differing genetic strains, we chose to use the heterozygous mutants
rather than the wild-type animals. Fibroblasts derived from the
heterozygote embryos were shown to contain half of the MT content of
cells from wild-type animals (24), which was as expected.
Fibroblasts were isolated from lungs obtained from MT
/
and MT+/
mice via collagenase digestion and were maintained in high-glucose DMEM
(GIBCO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin,
and 100 µg/ml streptomycin at 37°C in an atmosphere with 5%
CO2.
Cultured sheep pulmonary artery endothelial cells. Sheep pulmonary artery endothelial cells (SPAECs) were cultured from sheep pulmonary arteries obtained from a nearby slaughterhouse as previously described (13). The SPAECs were grown in OptiMEM (GIBCO) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in an atmosphere with 5% CO2.
Adenoviral vectors. An E1-deleted replication-deficient adenoviral vector expressing hMT-IIA (AdV.MT) was constructed using a proprietary kit (Microbix Biosystems, Toronto, Canada) and a bacterial plasmid containing the MT insert (37). Briefly, the MT cDNA, driven by a human cytomegalovirus promoter, was subcloned into a shuttle plasmid and cotransfected into 293 cells with a complimentary plasmid carrying the remainder of the circularized viral genome (12). After recombination, plaque isolates were screened, and the selected clone was propagated in 293 cells, purified by three rounds of discontinuous CsCl step-gradient centrifugation, and desalted over a G-50 column. The titer for the AdV.MT preparation was 2.6 × 1011 plaque-forming units/ml. Infections were performed for 60 min with virus diluted in basal media to the desired multiplicity of infection. Microspectrofluorometry was performed 48 h after adenoviral infection.
We hypothesized that overexpression with the highest-titer AdV.MT would result in significant increases in T and MT. Therefore, adenovirally infected cells were grown in a zinc-enriched medium (50 µM) to shift the equilibrium toward MT, and cells were then exposed to SNOC.Cellular MT determination. Cell lysates were prepared by repetitive freezing and thawing in Tris buffer (10 mM) containing CdCl2 (10 µM). After ultrasonic dessication, the supernatant was analyzed for protein content using a modified Bradford technique, and MT was measured by a modification of a 109Cd binding assay (8). Cellular MT content was calculated (based on the assumption that 7 mol of Cd bind to 1 mol of MT) and was normalized to cellular protein.
Fluorescent microscopy. SPAECs or fibroblasts from mouse lung (MLF) were plated onto polylysine-coated glass coverslips. Cells were washed with PBS and incubated with 15-30 µM Zinquin (Toronto Research Chemicals) for 30 min at 37°C. All recordings were performed at room temperature (20-25°C). Cells were imaged using a PC-based system consisting of a Nikon Diaphot 300 microscope equipped with a quartz ×40 oil-immersion objective, a charge-couple device camera (Hamamatsu Photonics, Hamamatsu City, Japan), SimplePCI software (Compix, Cranberry, PA), and a monochromator-driven xenon light source (ASI, Eugene, OR). Zinquin was illuminated at 350 nm; light was passed through a 400-nm dichromatic mirror, and emitted fluorescence was filtered through a 510 ± 40-nm bandpass emission filter (Omega Optical, Brattleboro, VT). For analysis of images, background illumination was subtracted from the readings, and fluorescence intensity was expressed relative to baseline measurements. Cells were exposed to the NO donor SNOC, and time-dependent changes in Zinquin fluorescence were monitored as an index of labile Zn2+. Cells were also exposed to Zn2+ (in the presence of pyrithione) and the zinc-specific chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN; Sigma) as positive controls and denitrosylated SNOC for a negative control.
FRET-MT. We followed the example of the Ca2+ indicator cameleon-1 (29) and constructed a chimera in which a yellow-green fluorescent protein (GFP) variant (EYFP) and a cyan GFP variant (ECFP) were fused to the COOH and NH2 termini, respectively, of human MT-IIA (FRET-MT) (34). An E1- and E3-deleted replication-deficient adenoviral vector expressing this chimera was constructed as described for MT. We used adenoviral-mediated transfer of the FRET-MT expression vector into SPAECs and imaged these cells on a Nikon inverted microscope with a Photometrics cooled charge-couple device camera (Quantix) controlled by ISEE software (Inovision, Raleigh, NC). The cyan and yellow GFPs acted as donor and acceptor for FRET and hence revealed changes in intramolecular proximity and relative orientation of the fluorophores. By constantly monitoring the emissions ratio of the acceptor (535 nm) to the donor (480 nm) molecules, we could infer conformational changes in MT, including a relative decrease in FRET when MT was modified in such a way as to lose metal.
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RESULTS |
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Zinquin fluorescence is consistent with vesicular localization of
zinc in cultured lung fibroblasts.
We used the zinc-specific fluorophore Zinquin (4, 46) to
image labile zinc. Both MLF+/ and MLF
/
cells showed discrete areas of strong fluorescence within the cytoplasm (Fig.
1) that were colocalized to a
lipid-sensitive dye (FM-143, data not shown), which is suggestive of
vesicular storage of Zn2+. Nuclei showed only very low
amounts of diffuse fluorescence that did not change under the
conditions studied.
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NO-mediated changes in intracellular free
Zn2+ are dependent on MT.
We used live-cell imaging to examine relative changes in intracellular
free Zn2+ in response to the NO donor SNOC. When MLF+/
cells (MT concentration = [MT] = 0.07 ± 0.001 µg/mg
protein) were exposed to 2 mM SNOC, there was an immediate and gradual
increase in fluorescence above baseline levels that returned to levels
below control with the application of the zinc chelator TPEN (Fig.
2A). This response was
consistent in three separate experiments (~3-10
cells/experiment) and averaged a 20.2 ± 16.4% (SD) increase
above baseline fluorescence in response to SNOC. In contrast, there was
either no change or a small decrease in fluorescence intensity in
response to repeated applications of 2 mM SNOC (>20 cells, mean
change =
5.5 ± 1.7%) in MLFs from MT knockout mice
{MLF
/
, [MT] = 0.007 ± 0.001 µg/mg protein; Fig.
2B}. MT complementation of MLF
/
by adenoviral gene
transfer {multiplicity of infection (MOI) = 100:1; [MT] = 0.132 ± 0.021 µg/mg protein} restored the NO-induced increase in labile zinc (Fig. 2C) in 8 of 11 cells examined (average
increase = 30.9 ± 11.8%), which further confirms the
importance of MT in the response.
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NO increases TPEN-chelatable (labile)
Zn2+ in SPAECs.
Like MLF, SPAECs labeled with 15 µM Zinquin showed a punctate pattern
of fluorescence within the cytoplasm (Fig.
3A) and exhibited modest
increases in Zinquin fluorescence in response to 2 mM SNOC (Figs. 3B and 4A)
that averaged 23.8 ± 10.5% (Table
1) in 4 separate experiments (total of
35 cells). However, unlike previous reports in rat aorta endothelial
cells (2), the increases in fluorescence appeared to be
confined to the cytoplasm with no change in the nuclei. As a negative
control, treatment of cells with denitrosylated SNOC had no effect on
Zinquin fluorescence. Compared with the modest increase in fluorescence
in response to NO exposure, treatment with 100 µM zinc acetate in the
presence of 10 µM pyrithione resulted in increases in fluorescence
that ranged from 50-150% above baseline in most cells (Fig.
3C). There were notable differences in the kinetics of the
responses to both TPEN and zinc between MLF+/ and SPAECs. These
variations could reflect differences in cellular uptake of zinc or even
TPEN, competition between TPEN and Zinquin, or differing levels of MT
expression.
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Overexpression of MT inhibits NO-dependent increases in labile zinc in SPAECs. The increase in Zinquin fluorescence observed in response to SNOC in SPAECs with control levels of MT (0.02 µg/mg protein; Fig. 4A) was abolished when MT was overexpressed in large amounts by adenoviral infection with MOI equal to 100:1 or 500:1 (0.24 or 1.27 µg MT/mg protein, respectively; see Table 1 and Fig. 4B). These cells were still responsive to high concentrations of added Zn2+ with pyrithione; however, a greater concentration of zinc (200 µM vs. 100 µM) was required to achieve the same increase in fluorescence (Fig. 4B). Compared with the inhibition of the NO-induced changes in Zinquin fluorescence observed in SPAECs infected with AdV.MT at higher MOI (100:1 and 500:1), threefold increases in MT with AdV.MT MOI of 25:1 (0.06 µg/mg protein vs. 0.02 µg/mg protein before AdV.MT) had no effect on the basal response to NO that was observed in uninfected cells as was evidenced by the 28.3 ± 4.0% increase in Zinquin fluorescence upon exposure to 2 mM SNOC (see Table 1). SPAECs that were infected with a LacZ adenovirus (MOI = 500:1) showed the same response to SNOC as uninfected cells (i.e., a 20-25% increase in Zinquin fluorescence; see Table 1).
Zn2+ supplementation restores NO-dependent increase in labile Zn2+ in SPAECs that overexpress MT. We hypothesized that MT overexpression with the high titers would result in significant increases in both T and MT. Therefore, AdV.MT cells were grown in a Zn2+-enriched media to shift the equilibrium toward MT. When SPAECs that overexpressed MT were incubated for 24 h in Zn2+-supplemented media (50 µM), there was a pronounced increase in labile zinc in response to SNOC that was returned to levels below control with TPEN (see Table 1; Fig. 4D). Overnight zinc supplementation of the growing media did not induce MT in SPAECs, nor did it affect the response to SNOC in uninfected cells (see Fig. 4C and Table 1) or in SPAECs that were infected with the lowest-titer AdV.MT.
Zinc status of MT regulates FRET-MT function in SPAECs.
We used a FRET-MT chimera to further study the ability of MT/T to act
as a zinc donor or acceptor within the cell. As previously reported
(34), the calcium ionophore Br-A-23187 (1 µM) caused changes in energy transfer that were indicative of conformational changes in the FRET-MT chimera consistent with metal release (Figs. 5A and
6). We previously showed that this
Ca2+-induced change in MT function is mediated by
endothelial NO synthase (eNOS)-generated NO (34).
Overexpression of the FRET-MT chimera ([MT] = 0.41 µg/mg protein
with adenoviral MOI = 100:1, compared with [MT] = 0.16 µg/mg
protein at MOI = 25:1) significantly reduced baseline FRET and the
response to Br-A-23187, which suggests a relatively high proportion of
T under these conditions (see Figs. 5B and 6). When SPAECs
that overexpressed FRET-MT (MOI = 100:1) were incubated for
24 h in zinc-supplemented media (50 µM), baseline FRET was
significantly increased, and the response to Br-A-23187 was restored
(Figs. 5C and 6).
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DISCUSSION |
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We used fluorescent live-cell imaging and direct gene transfer to demonstrate that MT plays a critical role in the regulation of intracellular Zn2+ homeostasis by NO. The data support a role for MT in intracellular signaling events via the sequestration or release of Zn2+ by the unique thiolate clusters of the protein.
Measurement of labile zinc. Zinquin ester is a quinoline-based nonfluorescent membrane-permeable fluorophore that becomes strongly fluorescent upon specific binding to Zn2+ and can thus be used to assess the intracellular disposition of chelatable Zn2+ by monitoring fluorescence intensity. Relative changes in fluorescence intensity can be used to assess changes in labile Zn2+; however, the currently available Zinquin derivatives lack the isosbestic features that permit ratiometric measurements, and therefore cannot be used to quantify intracellular zinc concentration. In spite of this limitation, Zinquin is the most suitable fluorophore for the detection of physiologically relevant concentrations of labile Zn2+ with detection limits that range from 4 pM to 100 nM (9).
We showed that Zinquin partitions into discrete cytoplasmic domains that were colocalized to a lipid-sensitive dye (FM-143), which is suggestive of vesicular storage of labile zinc in pulmonary endothelial cells and fibroblasts. This punctate pattern of cytoplasmic fluorescence has been reported for most eukaryotic cell types studied to date (30) including cells of the central nervous system (7) and pulmonary epithelial cells (41). The pattern of intracellular fluorescence was metal dependent as shown by the pronounced increase in intensity in response to application of exogenous Zn2+ in the presence of the zinc ionophore pyrithione (see Fig. 3) and the quenching of fluorescence by TPEN. Previous reports using digital deconvolution microscopy (30) confirmed that the punctate fluorescent bodies were intracellular in nature and were not caused by Zinquin that was preferentially bound to the cell surface. The vesicle-like staining pattern was also observed under conditions that are known to inhibit endocytosis of soluble or precipitated forms of the Zinquin-zinc complex (30). Furthermore, Zinquin forms 1:1 and 1:2 Zinquin-zinc complexes with binding constants of 2.7 × 106 and 11.7 × 106 l/mol, respectively (45), which are sufficiently low to preclude Zinquin from interacting with Zn2+ bound to MT (Kd = 1.4 × 10NO-induced changes in labile Zn2+ are dependent upon MT. The present data confirmed that exogenous NO can increase labile Zn2+ in live cells as suggested previously by the NO-induced conformational changes in green fluorescent protein modified MT (34). In vitro studies using raman spectroscopy demonstrated that the NO generated by SNOC mediated Zn2+ release from MT via nitrosylation of cysteine thiol groups and subsequent disulfide formation (22). SNOC spontaneously decomposes to generate NO and disulfide with a short half-life [in the range of 2-3 min (23)]. The typical concentration of SNOC used in these experiments (2 mM) appears relatively high. However, it has been noted that the cultures are actually exposed to a burst of NO, most of which is autooxidized or has reacted with media components before reaching the target cells at the bottom of the culture dish (2). We would therefore expect the actual NO concentration to be much closer to physiological levels. It is well recognized that NO by itself does not react with thiols to yield nitrosothiol (19). Rather, NO is transformed into N2O3 and/or NO2 in the presence of O2, which can then nitrosylate MT and cause Zn2+ release and loss of thiolate groups (1, 19). The reactions of NO with cysteine redox centers of proteins are thought to occur through intermediates formed by NO and O2 and/or are facilitated by redox reactions that require the presence of oxidants (1, 19). However, the likelihood of these reactions occurring in vivo has been questioned (11, 19), and recent data suggests that NO can react directly with thiols under physiological conditions in the presence of an electron acceptor such as O2 (11).
The absence of the NO-mediated increase in labile Zn2+ in MLF from MT knockout mice and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in Zn2+ homeostasis. It has been generally accepted that MT plays an important role in preventing heavy metal toxicity (17, 32), and recent data suggests that MT is also part of a Zn2+-scavenging mechanism that enhances cell survival under conditions of extreme Zn2+ deprivation (40). An attractive idea that has gained recent momentum is that MT might function as a chaperone for supplying Zn2+ in the biosynthesis of metalloenzymes and metalloproteins (14, 16, 31, 43). Experiments in vitro indicate that such reactions are possible (26, 42) and that the glutathione redox couple modulates the transfer of zinc from MT to apoenzymes (15). These findings led Maret and Vallee (26) to propose that the specific redox properties of the sulfur ligands in the MT clusters selectively control the release and uptake of Zn2+. Thus perturbation of the redox state may permit an effective zinc transfer from MT to other proteins in vivo (43). Our data comprise the first in vivo demonstration of a potential role for the apoprotein T as a zinc-binding protein in intact cells, in that 1) nascent T, after gene transfer, did not release Zn2+ in response to NO (see Fig. 6); and 2) Zn2+ supplementation restored this phenomenon. Although it is generally assumed that the apoprotein is rapidly degraded by intracellular proteases and does not accumulate in the cell (20), the formation and transient existence of T has been demonstrated in situ (25), and the widespread presence of T has been described in tumors that were grown under zinc-deficient conditions (33). In fact, it was recently reported that T is present in liver, kidney, and brain tissue homogenates in large quantities under physiological conditions, and that T may actually be more stable than MT (44). Furthermore, it has also been shown that T can extract Zn2+ from the zinc-specific inhibitory site of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase with a resultant marked increase in activity (25). It has also been demonstrated that T can remove Zn2+ from the zinc-finger transcription factors Sp1 (47) and TF-IIIA (48) with resultant changes in DNA binding and transcriptional activity. Our data are consistent with the idea that the ratio of MT to T is a central element in the regulation of cellular Zn2+ homeostasis (25) with T acting to sequester Zn2+ and NO-mediated release from MT acting as one means of increasing intracellular free Zn2+. The in vitro data suggests that the resultant changes in labile Zn2+ could then have a modulating influence on a number of intracellular events including effects on enzyme activity (5, 15, 25) and/or the regulation of gene expression (22, 23, 47, 48). ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Dr. Bruce A. Johnson for the viral preparations and Sarah A. Tapyrik for technical assistance.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants HL-32154, GM-53789, and HL-65697 (to B. R. Pitt) and the American Heart Association (C. M. St. Croix and K. E. Dineley).
Address for reprint requests and other correspondence: C. M. St. Croix, Dept. of Environmental and Occupational Health, Graduate School of Public Health, Univ. of Pittsburgh, 3343 Forbes Ave., Pittsburgh, PA 15260 (E-mail: cls13+{at}pitt.edu).
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.
10.1152/ajplung.00267.2001
Received 17 July 2001; accepted in final form 14 September 2001.
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