Departments of 1 Medicine and 2 Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267; and 3 Department of Medicine, University of North Carolina, Chapel Hill North Carolina 27599
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ABSTRACT |
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Thickening of airway mucus and lung
dysfunction in cystic fibrosis (CF) results, at least in part, from
abnormal secretion of Cl and HCO3
across the tracheal epithelium. The mechanism of the defect in HCO3
secretion is ill defined; however, a lack of
apical Cl
/HCO3
exchange may exist in
CF. To test this hypothesis, we examined the expression of
Cl
/HCO3
exchangers in tracheal
epithelial cells exhibiting physiological features prototypical of
cystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosis
transmembrane conductance regulator (CFTR)] or normal trachea (CFT-1
cells transfected with functional wild-type CFTR, termed CFT-WT). Cells
were grown on coverslips and were loaded with the pH-sensitive dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and
intracellular pH was monitored. Cl
/HCO3
exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1
cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited by
DIDS. The mRNA expression of the ubiquitous basolateral AE-2
Cl
/HCO3
exchanger remained unchanged.
However, mRNA encoding DRA, recently shown to be a
Cl
/HCO3
exchanger (Melvin JE, Park K,
Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:
22855-22861, 1999.) was abundantly expressed in cells expressing
functional CFTR but not in cells that lacked CFTR or that expressed
mutant CFTR. In conclusion, CFTR induces the mRNA expression of
"downregulated in adenoma" (DRA) and, as a result, upregulates the
apical Cl
/HCO3
exchanger activity in
tracheal cells. We propose that the tracheal HCO3
secretion defect in patients with CF is partly due to the
downregulation of the apical Cl
/HCO3
exchange activity mediated by DRA.
cystic fibrosis transmembrane conductance regulator; cystic fibrosis; trachea; bicarbonate secretion; downregulated in adenoma; chloride ion/bicarbonate exchange
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INTRODUCTION |
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CYSTIC FIBROSIS
(CF), an autosomal recessive disease, presents with defective fluid and
electrolyte secretion in secretory epithelia (24,
25, 37). CF results from mutational
inactivation of a cAMP-sensitive Cl channel (cystic
fibrosis transmembrane conductance regulator, CFTR) with resultant
functional impairments in the respiratory, pancreatic, hepatobiliary,
and genitourinary systems (21, 26). With
respect to the lung, respiratory dysfunction is thought to result
primarily from the thickening of airway mucus, leading to increased
infection and respiratory failure (27). This thickened mucus may be a result of abnormal secretion of Cl
and
HCO3
across the tracheal epithelium, altering the ion
composition and, as a result, the viscosity of the airway surface
liquid (5, 15, 33).
Although gene therapy has been regarded as an appealing potential therapeutic option in the treatment of the CF defect, it has not been completely successful in reversing the defect (1). An alternative hope is that a better understanding of the physiological role(s) of CFTR in various organs will lead to novel strategies capable of correcting the functional defect in the absence of functional CFTR.
The currently accepted model of tracheal HCO3
secretion suggests that 1) intracellular
HCO3
accumulates due to basolateral diffusion of
CO2 and subsequent action of carbonic anhydrase, and
2) G protein-coupled receptors activate cAMP-sensitive CFTR,
which then secretes HCO3
(34). According
to this model, CFTR or a highly homologous transporter directly
mediates HCO3
secretion into the tracheal lumen.
Whether CFTR carries HCO3
directly or regulates other
HCO3
transporting processes remains speculative
(13, 23).
In addition to the currently recognized isoforms of anion exchangers
(AEs) that have Cl/HCO3
exchange
activity (16), the "downregulated in adenoma" (DRA) protein, which has been shown to mediate sulfate, oxalate, and Cl
transport in Xenopus oocytes
(32), was recently found to have Cl
/HCO3
exchange activity
(19). DRA was originally cloned via subtractive hybridization in a colon cDNA library and was found to be expressed in
normal colon but not in adenocarcinomas (28). Its function was not known at the time of its cloning. Subsequently, it was found
that patients with congenital Cl
diarrhea, who lack
apical Cl
/HCO3
exchange activity, had
null mutations in DRA (11, 12). Taken together with recent functional studies (19), it is clear
that DRA mediates Cl
/HCO3
exchanger activity.
To characterize the functional and molecular mechanism(s) of
HCO3 secretion across the tracheal epithelium, an
attempt was made to characterize HCO3
transporters in
tracheal epithelial cells. We specifically tested the possibility that
a Cl
/HCO3
exchanger is expressed in
tracheal epithelial cells and may be regulated by CFTR.
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MATERIALS AND METHODS |
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Cell lines.
CFT-1 cells are derived from a primary culture of tracheal epithelial
cells isolated from a patient with CF (homozygous F508 mutation) and
were cultured as previously described (38). Stably transfected CFT-1 cells bearing functional CFTR (termed CFT-WT) were
cultured in a similar fashion (20). CFT-1 cells
transfected with F508 CFTR cDNA (termed CFT-MT) were used as a
control for cells transfected with the functional CFTR to determine the
role of the transfection procedure per se on the expression of ion transporters.
Cell pH measurement.
Changes in intracellular pH (pHi) were monitored
using the pH-sensitive fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF;
see Refs. 2 and 3). Cells were grown to confluence on a glass
coverslip, incubated in the presence of 5 µM BCECF in a solution
consisting of 115 mM tetraethylammonium (TMA), 25 mM
KHCO3 or choline HCO3, and 10 mM HEPES at pH
7.4, and gassed with 5% CO2-95% O2. The
monolayer was then perfused with the appropriate solutions in a
thermostatically controlled holding chamber (37°C) in a Delta Scan
dual excitation spectrofluorometer (PTI, South Brunswick, NJ). The
Cl and HCO3
-containing solution
contained (in mM) 115 TMA-Cl, 25 mM KHCO3, 0.8 K2HPO4, 0.2 KH2PO4, 1 CaCl2, 1 MgCl2, and 10 HEPES. For
HCO3-free solution, KHCO3 was replaced with an
isosmolar concentration of TMA-Cl. The Cl
-free,
HCO3
-containing solution contained (in mM) 115 mM
N-methyl-D-glucamine (NMDG)-gluconate, 25 mM
KHCO3, 0.8 K2HPO4, 0.2 KH2PO4, 1 calcium gluconate, 1 magnesium
gluconate, and 10 HEPES. For HCO3-free solution,
KHCO3 was replaced with an isosmolar concentration of NMDG-gluconate. The fluorescence ratio at excitation wavelengths of 500 and 450 nm was used to determine pHi values. Calibration curves were established by the KCl/nigericin technique.
HCO3
-free or HCO3
-containing
solutions were used to determine the HCO3
dependence
of the transporter. To examine the
Cl
/HCO3
exchanger activity, cells were
switched to a Cl
-free medium. This maneuver results in
cell alkalinization due to reversal of
Cl
/HCO3
exchanger. Upon pHi
stabilization in Cl-free medium, cells were switched back to the
Cl
-containing solution. This resulted in rapid cell
acidification back to baseline due to activation of the
Cl
/HCO3
exchanger. The initial rate of
cell acidification was used as the rate of
Cl
/HCO3
exchanger activity. This rate
of change in pHi was used to compile the summary data.
RNA isolation and Northern blot hybridization.
Total cellular RNA was extracted from CFT-1, CFT-WT, and CFT-MT cells
according to the established methods, quantitated
spectrophotometrically, and stored at 80°C. Total RNA samples (30 µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel,
transferred to Magna NT nylon membranes, cross-linked by ultraviolet
light, and baked (36). Hybridization was performed
according to Church and Gilbert (7). The membranes were
washed, blotted dry, and exposed to a PhosphorImager screen (Molecular
Dynamics, Sunnyvale, CA). A 32P-labeled specific 400-bp
cDNA (EcoR I-EcoR I fragment) from the mouse DRA
cDNA was used as a specific probe. For AE2, a 1.6-kb rat cDNA (codons
456-1002) was used.
Materials. [32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma Chemical (St. Louis, MO). The RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes (Eugene, Oregon).
Statistical analyses. Values are expressed as means ± SE. The significance of difference between mean values was examined using ANOVA. P < 0.05 was considered statistically significant.
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RESULTS |
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In the first series of experiments, we examined the presence of
Cl/HCO3
exchanger activity in CFT-1
cells in the presence or absence of HCO3
.
Representative tracings are shown in Fig.
1A and demonstrate that, in
the presence of HCO3
, CFT-1 cells mildly alkalinized
upon removal of Cl
and acidified back to baseline upon
switching to the Cl
-containing solution. A summary of
multiple experiments is shown in Fig. 1B. These results
indicate that low levels of Cl
/HCO3
exchange activity are present in CFT-1 cells.
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In the next series of experiments, the presence of
Cl/HCO3
exchange activity in CFT-WT
cells was examined in the presence or absence of
HCO3
. Representative tracings in Fig.
2A demonstrate successive cell alkalinization and acidification upon removal and addition of Cl
, respectively. A summary of multiple experiments,
shown in Fig. 2B, indicates the presence of an active
Cl
/HCO3
exchanger in CFT-WT cells.
Interestingly, the CFT-WT cells also showed significant cell
alkalinization (upon removal of Cl
) and cell
acidification (upon switching back to the Cl
-containing
solution) in the absence of HCO3
in the media (Fig.
2). These data are consistent with the presence of
Cl
/base (i.e., Cl
/OH
)
exchange.
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Comparison of the results in Figs. 1 and 2 indicates that
Cl/HCO3
exchange activity is enhanced
in cells transfected with functional CFTR. The experiments in CFT-1 and
CFT-WT cells were performed on separate days. To verify the
reproducibility of the results, Cl
/HCO3
exchange activity was compared in CFT-1 and CFT-WT cells on the same
day. Representative tracings are shown in Fig.
3A and demonstrate enhanced
Cl
/HCO3
exchange in cells transfected
with functional CFTR. A summary of the results of multiple experiments,
Fig. 3B, demonstrates an approximately threefold increase in
Cl
/HCO3
exchange in CFT-WT cells.
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The inhibitory effect of the disulfonic stilbene DIDS on
Cl/HCO3
exchange activity was examined
in CFT-WT cells. The results of several experiments are summarized in
Fig. 4A and indicate that Cl
/HCO3
exchange is inhibited by
~52% in the presence of 500 µM DIDS in CFT-WT. The AE activity in
CFT-1 cells showed 62% inhibition by 300 µM DIDS (Fig.
4B). The lack of complete inhibition of
Cl
/HCO3
exchange activity by DIDS could
be due to the possibility that the tracheal AE is not as sensitive as
the known AEs to the inhibitory effect of disulfonic stilbenes.
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The effect of cAMP stimulation on
Cl/HCO3
exchange was examined in CFT-WT
cells. Cells were exposed to 10 nM forskolin at 5 min before switching
to the Cl
-free media and were kept exposed for the
duration of the experiment. As shown in Fig.
5A (representative tracings)
and Fig. 5B (summary of multiple experiments),
Cl
/HCO3
exchange was not stimulated by
forskolin.
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The purpose of the next series of experiments was to determine the
molecular identity of the AE that is responsible for enhanced Cl/HCO3
exchange in cells transfected
with functional CFTR. In the first series of experiments, expression of
the ubiquitous basolateral AE2 Cl
/HCO3
exchanger was examined by Northern hybridization. As shown in Fig.
6, AE2 mRNA expression was the same in
cells with functional CFTR as in cells that lack a functional CFTR.
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Because AE2 mRNA levels remained unchanged in cells with functional
CFTR, we entertained the possibility that other AE(s) may be
responsible for enhanced Cl/HCO3
exchange in cells with functional CFTR. Toward this end, the mRNA
expression of DRA, an anion transporter with
Cl
/HCO3
exchanger activity
(4, 10, 19) that is defective in
congenital Cl
diarrhea, was examined. Northern
hybridization (Fig. 7) demonstrated that
DRA mRNA is expressed abundantly in functional CFTR-bearing tracheal
epithelial cells but could not be detected in either CFT-1 cells or in
CFT-1 cells that are transfected with a mutant CFTR (CFT-MT cells). To
determine whether DRA is expressed in native tracheal tissue, RNA was
extracted from normal mouse trachea and was used for Northern
hybridization. The results showed that mouse trachea expresses DRA mRNA
(Fig. 8).
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DISCUSSION |
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The functional and molecular expression of
Cl/HCO3
exchangers was examined in
validated human tracheal epithelial cells that exhibit physiological
features prototypical of CF (CFT-MT) and normal tracheal epithelium
(CFT-WT). The results demonstrated the presence of
Cl
/HCO3
exchanger activity in both
CFT-1 and CFT-WT cells (Figs. 1 and 2).
Cl
/HCO3
exchanger activity was
significantly enhanced in tracheal epithelial cells transfected with
functional CFTR (Fig. 3). Cl
/HCO3
exchange was inhibited by DIDS (Fig. 4) and was not stimulated by
forskolin (Fig. 5). Northern hybridizations indicated the induction of
DRA in the wild-type CFTR-bearing tracheal epithelial cells (Fig. 7),
whereas the mRNA expression of AE2 remained unaffected (Fig. 6).
Patients with CF demonstrate a significant impairment of
Cl and HCO3
secretion in their trachea
(5, 15, 33). The defective Cl
secretion is due to decreased activity of CFTR, which
is a cAMP-sensitive Cl
channel. The cellular mechanism of
the HCO3
secretion defect in the trachea of CF
patients, however, is less well understood. The currently accepted
model of tracheal HCO3
secretion (34)
suggests that HCO3
is secreted via a cAMP-sensitive
electrogenic transporter. This could be consistent with direct
secretion of HCO3
via CFTR (34). The
results of studies in cultured 3T3 cells transfected with functional
CFTR, however, have been contradictory; HCO3
transport via CFTR was demonstrated by one group (22) but
not the other (18). Studies in HEK293 cells transfected
with wild-type CFTR failed to demonstrate any HCO3
transport by CFTR (18). Recent data from our laboratory
indicate that CFTR does not carry HCO3
in cultured
pancreatic duct cells (31). Taken together, these results
indicate that CFTR does not play a significant role in the direct
transport of HCO3
.
Enhanced Cl/HCO3
exchange in tracheal
epithelial cells stably expressing a functional CFTR is intriguing
(Fig. 7). Functionally, these results are consistent with indirect but
tight coupling between luminal Cl
(secreted via CFTR) and
the apical Cl
/HCO3
exchanger and
provide a basis for enhanced HCO3
secretion in
tracheal epithelial cells expressing functional CFTR. These studies do
not conflict with the results of earlier studies demonstrating
increased HCO3
secretion in tracheal epithelial cells
transfected with functional CFTR (34). Indeed, our studies
extend those observations by indicating that CFTR indirectly increases
HCO3
secretion by upregulating the apical
Cl
/HCO3
exchanger. Our results are also
in agreement with recent studies in cultured cells (18)
and the duct fragments from the mouse submandibular gland
(17) indicating enhancement of
Cl
/HCO3
exchanger activity by
functional CFTR. The molecular identity of the
Cl
/HCO3
exchanger that was upregulated
by CFTR, however, was not examined in those studies (17,
18).
The novel aspect of our finding is the induction of the AE DRA in tracheal epithelial cells expressing functional CFTR (Fig. 7). DRA was originally found to be expressed in colon, cecum, and small intestine (12, 28). It is localized to the apical domain of the colonic cell membrane (6). Although RNase protection assay failed to identify DRA expression in lung (32), very low levels of DRA mRNA in mouse lung were observed in our studies (unpublished results). It is plausible that the very weak DRA mRNA signal in the lung is originating from the tracheal epithelial cells, which constitute a minority of the cell population in the lung samples.
In addition to being a Cl channel, CFTR is also a
regulator of other ion transporters. Whether CFTR-mediated enhanced
expression of Cl
/HCO3
exchanger DRA
requires Cl
transport by CFTR remains unknown. One
plausible, although not very likely, mechanism is that functional CFTR
increases intracellular HCO3
by increasing the
basolateral NBC-driven HCO3
uptake secondary to
membrane depolarization (22, 31). The increased intracellular HCO3
concentration can then
increase the expression and activity of DRA. Another plasusible
mechanism is that CFTR increases the expression of DRA via direct
interaction [i.e., via its nuclear-binding domains (NBD)]. As such,
it would be the presence of CFTR in the plasma membrane, and not its
Cl
channel activity, that regulates DRA. Studies are
under way to test these hypotheses. Alternatively, it is possible that
the downregulation of DRA in CF tracheal cells results from the
upregulation of chemical mediators that are increased in CF cells.
Studies have shown that cultured
F508 CF human bronchial gland cells express elevated levels of proinflammatory cytokines compared with
non-CF bronchial cells (35). Whether the upregulation of these cytokines could mediate the downregulation of DRA remains speculative. Patients with CF are known to express elevated levels of
proinflammatory cytokines in their trachea (35). It is
plausible that these cytokines can downregulate DRA.
Patients with CF show a HCO3 secretion defect in the
intestine (9). Studies in the CF mouse duodenum indicate
impaired apical HCO3
secretion (29),
confirming the important role that CFTR plays in HCO3
secretion in the intestine. Although some investigators have proposed a
likely direct role for CFTR in HCO3
secretion
(29), this has not been demonstrated by others
(17, 18, 31). Whether decreased
HCO3
secretion in the intestine of CF patients or
knockout animals is due to the downregulation of the apical
Cl
/HCO3
exchanger DRA remains unclear.
Northern hybridizations on the RNA isolated from CF and wild-type mice
intestine showed decreased expression of DRA in the cecum, proximal
colon, and distal colon (data not shown). Whether a similar process
occurs in the small intestine needs further investigation. Using
voltage-clamped Ussing chambers, Clarke and Harline (8)
demonstrated that CFTR stimulation increases apical
HCO3
secretion, along with Cl
, in mouse
small intestine. Enhanced HCO3
secretion was
associated with the generation of a current, suggesting that the bulk
of HCO3
secretion was mediated via an electrogenic
pathway. A luminal Cl
/HCO3
exchanger
also contributed to HCO3
secretion (8).
In mice with mutant CFTR, both mechanisms of HCO3
secretion were significantly diminished. It was
proposed that HCO3
secretion in small intestine
involves electrogenic secretion via CFTR HCO3
conductance and electroneutral secretion via a CFTR-dependent Cl
/HCO3
exchange process
(8).
The lack of an effect by cAMP on
Cl/HCO3
exchange in tracheal epithelial
cells needs further clarification. cAMP activates CFTR in tracheal
epithelial cells, leading to increased Cl
secretion. The
results of the current studies are consistent with the possibility that
the Cl
that is secreted via CFTR is recycled back via the
apical Cl
/HCO3
exchanger. Lack of
stimulation of Cl
/HCO3
exchange by cAMP
should not affect the coordinated actions of the apical
Cl
/HCO3
exchanger and CFTR in mediating
HCO3
secretion in tracheal epithelial cells. It is
likely that DRA is a high-capacity transporter and, given its high
level of expression in CFT-WT cells, it could easily recycle the
Cl
that is secreted via CFTR in response to cAMP.
In addition to trachea, pancreatic duct cells also demonstrate impaired
HCO3 secretion in CF. Recent investigations indicate
that the mechanism of HCO3
transport in the
pancreatic duct cells is distinct from the tracheal epithelial cells.
Studies in the guinea pig pancreatic duct cells demonstrated that
removal of luminal Cl
or addition of DIDS only partially
inhibited secretin-stimulated HCO3
secretion (<25%;
see Ref. 14), strongly suggesting that the apical
Cl
/HCO3
exchanger does not play a major
role in agonist-stimulated HCO3
secretion. Recent
studies from our laboratory indicate that HCO3
uptake
across the basolateral membranes of pancreatic duct cells is mediated
via the NBC (30). We further observed that cAMP potentiates NBC activity through membrane depolarization that results
from the activation of CFTR-mediated Cl
secretion
(30). Based on those studies, we proposed that the defect
in agonist-stimulated ductal HCO3
secretion in
patients with CF is predominantly due to decreased NBC-driven
HCO3
entry at the basolateral membrane secondary to
the lack of a sufficient electrogenic driving force in the absence of
functional CFTR (30). Interestingly,
HCO3
uptake across the basolateral membrane of
tracheal epithelial cells occurs predominantly via a
Na+-HCO3
cotransporter distinct from
NBC-1 (unpublished observations), which is the dominant basolateral
transporter that mediates the uptake of HCO3
in
pancreatic duct cells. The schematic diagram in Fig.
9 illustrates the interaction of CFTR
with the Cl
/HCO3
exchanger in tracheal
epithelial cells and demonstrates the proposed mechanism of
HCO3
uptake across the basolateral membrane.
HCO3
enters the cells via an NBC distinct from NBC-1
(unpublished results) and is secreted at the apical membrane via the
Cl
/HCO3
exchanger.
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In conclusion, CFTR induces the expression of DRA along with
Cl/HCO3
exchanger activity in tracheal
epithelial cells. The specific mechanism of this induction (direct
interaction with CFTR or indirect interaction by alterations in the
cell or luminal Cl
concentration) remains unknown. We
propose that the tracheal HCO3
secretion defect in
patients with CF is partly due to the downregulation of the apical
Cl
/HCO3
exchanger activity mediated by DRA.
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ACKNOWLEDGEMENTS |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789 and DK-54430 (to M. Soleimani) and DK-50594 (to G. E. Shull) and by a grant from the Dialysis Clinic Incorporated (to M. Soleimani).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Soleimani, Dept. of Medicine, Univ. of Cincinnati, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.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. §1734 solely to indicate this fact.
Received 21 September 1999; accepted in final form 20 January 2000.
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