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Introduction
Reactive oxygen species (ROS) is known to be involved
in the pathological processes of many cardiovascular
diseases, such as atherosclerosis, hypertension, and
restenosis. Our previous study in cultured rat vascular
smooth muscle cells (VSMC) showed that the intracellular
increase of superoxide anion by exposure to LY83583 led to
an early transient (10 min) and late sustained activation (120
min) of extracellular signal-regulated kinase (ERK) 1/2, and
stimulated VSMC proliferation[1]. Further experiments
demonstrated that the early phase was directly activated by ROS
and the late was mediated via secreted oxidative-stressed
factors involving heat shock protein 90
(HSP90)[1,2]. However, the mechanism of HSP90 mediating ERK1/2 activity needs
to be explored further.
HSP90 is known as a molecular chaperone and
associates with various proteins, including transcription factors
and protein kinases, and plays an important role in the
activity[3,4],
stability[5-7], and intracellular
distribution[8] of its
associated partner molecules. It has been reported that
HSP90 could associate with mitogen-activated protein
kinases, such as MAPK/ MAK/ MRK overlapping kinase
(MOK)[5] and Raf[6,8,9] which are up-streams of ERK1/2. In
addition, HSP90 could also promote the solubility of its
partner molecules and enhance the nuclear translocation of its
partner mol-ecules.
ERK1/2 is ubiquitous cytosolic
serine-threonine kinase and mediates cell growth and proliferation. ERK1/2 is
activated by various stresses and growth stimuli and is involved
in signaling transduction from cytoplasm into cell
nuclei[5,11]. It is well known that the phosphorylation of ERK2 promotes
its dimerization and nuclear translocation, and the increase
of ERK1/2 solubility facilitates the nuclear translocation of
itself[12]. Once entering cell nuclei, phosphor-ERK1/2
activates transcription factors Elk-1. Thus, the solubility level
and nuclear-translocation ability of phosphor-ERK1/2 are 2
determinant events in cell growth[13].
Therefore, we propose that HSP90 acts as a molecular
chaperone, mediates late-phase ERK1/2 activation and cell
proliferation by associating with phosphor-ERK1/2, and
increases the solubility and nuclear translocation of
phosphor-ERK1/2.
Materials and methods
Materials Sprague-Dawley (SD) rats (150 g) were
obtained from the Experimental Animal Center, Nanhua
University (Hengyang, China, SPF grade, Certificate
No SCXK Xiang 2004-0011). LY83583 was obtained from Cayman (Ann
Arbor, MI, USA) and geldanamycin was from Alomone (Jerusalem, Israel). Both protein A agarose and
3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
were from Sigma (St Louis, MO, USA). Antirat ERK1/2
(C-14), phosphor-ERK1/2 (E-4, Thr202/Tyr204), HSP90 (H-114),
and β-actin (C-2) were from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Fluorescein isothiocynate (FITC) or
horseradish peroxidase (HRP)-conjugated goat antimouse or
rabbit second antibody was from Boster (Wuhan, China).
Dulbecco's modified Eagle's medium (DMEM), fetal bovine
serum (FBS), and 0.25% trypsin-EDTA solution were from
Gibco (New Zealand, NY, USA).
Cell culture and experiment grouping Rat VSMC were
isolated from the aorta of 150 g male SD rats and cultured by
an expellant method originally described by Campbell and
Campbell[14]. The culture medium was DMEM containing 10
mmol/L sodium pyruvate supplemented with 20% FBS. Then
the cells were maintained in 10% FBS and up to passage 12
when used. After confluence, the cells were inoculated on
100 mm dishes. The cells were
growth-arrested by incubation in 0.1%
FBS/DMEM for 24 h prior to
use. VSMC were identified with an anti-actin antibody by
immunohistochemical stain (S-P). First, the VSMC were treated with LY83583
[6-anilinoquinoline-5,8-quinoli-nedione, produce ROS, 1
µmol/L, dissolved in phosphate-buffered saline (PBS)] for different
times (0, 5, 10, 30, 60, 90, 120, and 180 min) to determine the
late peak of phosphor-ERK1/2. Then different treatments
were classified as the following groups: control group (PBS
120 min), LY group (1 µmol/L LY83583 for 120 min), Gel+
DMSO+LY group (pretreated with geldanamycin which was
dissolved in DMSO for 30 min, then treated with 1 µmol/L
LY83583 for 120 min), and the DMSO+LY group (pretreated
with DMSO, the vehicle of geldanamycin, for 30 min, then
treated with 1 µmol/L LY83583 for 120 min).
Extraction of the total, soluble, insoluble, and nuclear
protein The cells were harvested following 2 quick rinses in
PBS (pH 7.4) in ice-cold lysis buffer (50 mmol/L Tris-base, pH
7.4, 150 mmol/L NaCl, 10% glycerol, 1 mmol/L
O,O'-Bis (2-aminoethyl)
ethyleneglycol-N,N,N',N'
-tetraacetic acid (EGTA), 1 mmol/L Na-or-thovanadate, 5 µmol/L
ZnCl2, 100 mmol/L NaF, 10 µg/mL aprotinin, 1 µg/mL leupeptin, 1
µmol/L phenylmethylsulfonyl fluoride, and 1% Triton X-100) and
homogenized by pulling through a 21 Ga needle 20 times.
The detergent was omitted from the lysate buffer by
centrifugation at 10 000×g at 4
oC for 20 min and the protein concentration of the supernatant was
determined[16].
The growth-arrested VSMC were divided into 4 groups
as described before. The total, soluble, insoluble and nuclear
protein was extracted as described
previously[2]. In brief, the cells were scraped in a buffer containing 137 mmol/L NaCl, 2.7
mmol/L KCl, 4.3 mmol/L Na2HPO4,
1.4 mmol/L KH2PO4, 20
µg/mL leupeptin, 1 mmol/L sodium orthovanadate, and 400
µmol/L phenylmethylsulfonyl fluoride, and frozen at
-20 oC for 4 times, then centrifuged at
17 000×g for 60 min. The supernatants were recovered as soluble fractions, and
the precipitates were redissolved by adding diluted (5:7) SDS
sample buffer and then boiled for 5 min to obtain insoluble
fractions.
The nuclear extracts were prepared as described by
Backlund et al[16]. In brief, the cells were harvested
in ice-cold PBS and pelleted by brief centrifugation
(1000×g). The supernatant was discarded and the cell pellet was
washed once in hypotonic buffer [10 mmol/L
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.9, 1.5 mmol/L
MgCl2, 10 mmol/L KCl, 0.2 mmol/L phenylmethylsulfonyl
fluoride, and 0.5 mmol/L 1,4-dithiothreitol (DTT)]. The cells
were resuspended in hypotonic buffer and allowed to swell
on ice for 10 min before homogenization in a glass Dounce
homogenizer with 14 up-and-down strokes, using a B-type
pestle. The nuclei were collected by
centrifugation at 3300×g for 15 min and resuspended in 200 µL low
salt buffer (20 mmol/L HEPES, pH 7.9, 25% glycerol, 1.5 mmol/L
MgCl2, 10 mmol/L KCl, 0.2 mmol/L EDTA, 0.2 mmol/L
phenylmethyl-sulfonyl fluoride, and 0.5 mmol/L DTT). KCl (2.5 mol/L) was
added dropwise to a final concentration of 0.4 mmol/L, and
the nuclei were incubated for 30 min with continuous gentle
mixing. The extracted nuclei were pelleted by centrifugation
at 25 000×g for 30 min at 4
oC. The resulting supernatant (nuclear extract) was aliquoted. The protein concentration
was determined by bicinchoninic acid (BCA) protein assay.
All of the lysates were immediately frozen at -80
oC.
Western blotting analysis and immunoprecipitation
For Western blotting, the cell lysates containing equal amounts
of proteins were added with 5×sample buffer (0.31 mol/L Tris
base, pH 6.8, 2.5% SDS, 50% glycerol, and 0.125%
bromophenol blue) and boiled for 3 min. The protein was resolved on
10% SDS-PAGE. The protein was transferred to
polyvinylid-ene fluoride (PVDF) film. Then the film were blocked in 5%
skim milk in Tris-buffered saline Tween (TTBS) (50 mmol/L
Tris-HCl, pH 8.3, 200 mmol/L NaCl, and 0.05% Tween-20).
The filters were incubated with anti-rat HSP90 (1:1000),
ERK1/2 (1:1200), and phosphor-ERK1/2 (1:1500) antibodies for 120
min at 37 oC or 4 oC overnight, detected by
HRP-conjugated second antibodies, and then detected by an enhanced
chemiluminescence reagent[17].
For immunoprecipitation, total cell lysates containing
equal amounts of proteins were incubated with an anti-HSP90
antibody for 45 min at 4 oC . The antibody-protein complexes
were incubated with protein A ¡ª agarose for 20 min at 4
oC , and the antibody-protein complexes that were bound to the
beads were pelleted at 2000×g for 2 min. The beads were
washed 3 times with lysis buffer and once with PBS,
resuspended in the sample buffer, and immediately frozen at
-80 oC [18].
Immunofluorescence The prepared cells were washed
in PBS 3 times for 5 min, permeabilized with 0.01% Triton
X-100 in PBS for 1 min and 100% methanol for 3 min at
-20 oC , and then blocked with 1% goat serum and PBS (pH
7.5) for 30 min . The cells were incubated with a
phosphor-ERK1/2 antibody in 1% goat serum and PBS (pH 7.5) for 60
min. After washing with PBS (pH 7.5) 3 times for 10 min, the
cells were incubated with the FITC second antibody in 1%
goat serum and PBS (pH 7.5) for 60 min and washed with PBS
(pH 7.5) 3 times for 10 min[15].
Cell counting and MTT assay For determining number
of living cells, a modified MTT assay was performed as
described previously before[1]. Briefly, VSMC
(1×105
cells/mL) were grown in 96-well plates for 24 h. After
incuba-tion with agonists for 2 h, cells were treated with MTT (0.5
mg/mL) for 4 h at 37 oC. The cell culture medium was removed,
and cells were lysed by addition of 100 μL of isoamyl alcohol.
The metabolized MTT was evaluated by optical dencity
(OD) in an enzyme-linked immuno-sorbent assay reader at 570 nm.
The proliferation ratio=(cell counting or
OD of the experimental group_cell counting or
OD of the control group)/cell counting or
OD of control group×100%.
Statistical analysis All data were expressed as mean±SD.
The statistical analysis of the data was performed using
ANOVA as appropriate. Values with P<0.05 were considered
to be statistically significant.
Results
Oxidative stress increased cytosolic HSP90 in rat
VSMC Brief exposure of VSMC to LY83583 stimulated
ERK1/2 activity with peaks at 10 and 120 min. The late peak was
responsible for secreted oxidative stress-induced factors in
the conditioned medium that included HSP90, cyclophilin A,
and cyclophilin B[1]. In this present experi-ment, the VSMC
were exposed to LY83583 for 0_180 min. Figure 1 shows that
1 µmol/L LY83583 treatment stimulated ERK1/2 activity and
HSP90 expression in the total cell lysates. The ERK1/2
activation peaked at 10 and 120 min, respectively. However, the
LY83583-induced HSP90 expression peaked only at 120 min,
which was consistent with the late peak of
phosphor-ERK1/2 (Figure 1).
HSP90 mediates LY83583-induced ERK1/2 activation
To examine the role of cytosolic HSP90 in LY83583-induced
ERK1/2 activation, the VSMC were pretreated with a specific
HSP90 inhibitor geldanamycin (1, 5, and 10 µmol/L) for 30
min and then exposed to LY83583 for 120 min. The data
showed that LY83583 significantly increased total phosphor-
ERK1/2 by 2.5-fold, and geldanamycin inhibited the effect of
LY83583 in a dose-dependent manner (Figure 2).
LY83583 increased HSP90 binding with
phosphor-ERK1/2 To identify cytosolic HSP90 as a molecular
chaperone binding with ERK1/2 or phosphor-ERK1/2, the total cell
lysates were co-immunoprecipitated with an anti-HSP90
antibody and blotted with anti-ERK1/2 and
phosphor-ERK1/2 antibodies, respectively. We observed that LY83583
significantly increased HSP90 association with phosphor-ERK1/2
(1.8-fold), but not with ERK1/2, as compared with the control.
Pretreatment with geldanamycin attenuated the effects of
LY83583 (Figure 3).
Roles of HSP90 in increasing the solubility of
phosphor-ERK1/2 To further explore the effects of HSP90 on
phosphor-ERK1/2, we collected the soluble and insoluble
extracts of VSMC treated with LY83583 or/and geldanamycin,
and analyzed the levels of ERK1/2 and phosphor-ERK1/2 by
Western blotting. As shown in Figure 4, LY83583 obviously
increased the phosphor-ERK1/2 level of soluble extracts and
decreased the phosphor-ERK1/2 level of insoluble extracts
(Figure 4, 5). Geldanamycin attenuated the effect of LY83583.
Roles of HSP90 in enhancing the nuclear translocation
of phosphor-ERK1/2 It has been reported that an increase
of phosphor-ERK1/2 solubility might enhance its function.
In the present study, we investigated the effect of HSP90 on
the nuclear translocation of phosphor-ERK1/2. The nuclear
extracts were harvested from VSMC treated with LY83583
or/and geldanamycin, and the level of phosphor-ERK1/2 was
measured by Western blotting. The results showed that
LY83583 increased the level of phosphor-ERK1/2 in nuclei
by 7.6-fold as compared with the control, and geldanamycin
abolished the effect of LY83583 (Figure 6). An
immunofluorescence analysis demonstrated that the obvious nuclear
translocation of phosphor-ERK1/2 was observed in
LY83583-treated VSMC as compared with untreated VSMC.
Pretreatment with geldanamycin reduced the LY83583-stimulated
nuclear translocation of phosphor-ERK1/2 (Figure 7). DMSO
alone had no effect on the solubility and nuclear
translocation of phosphor-ERK1/2 (Figures 5, 7).
HSP90 mediates VSMC proliferation induced by
oxidative stress To further confirm the relationship between the
increased HSP90 expression and oxidative stress-stimulated
cell proliferation, we observed the effects of HSP90 inhibitors,
geldanamycin and radicicol (another HSP90 inhibitor
without influence on ROS), on LY83583-induced VSMC. MTT
assay and cell counting showed (Table 1) that LY83583
treatment accelerated VSMC proliferation by 90%. Geldanamycin
and radicicol significantly inhibited LY83583-stimulated cell
growth.
Discussion
HSP90 is known as a molecular chaperone and is
associated with various proteins, such as
pp60v-src[19], Raf-1[20],
Cdk4[21], and Casein kinase II
(CK2)[22]. Our previous studies demonstrated that oxidative stress increased HSP90
secretion and promoted VSMC growth[1,2]. Interestingly, the
LY83583-induced generation of ROS peaked at 15 min and
returned to baseline by 120 min[1,2]. However, the
LY83583-induced activation of ERK1/2 showed 2 peaks at 10 min (early
peak) and 120 min (late peak), respectively. Obviously, the
late peak is not directly related with ROS induced by LY83583.
Further experiments demonstrated that the late peak was
mediated via secreted oxidative-stressed factors, which
involve HSP90, cyclophilin A, and cyclophilin
B[1,2]. However, the role of HSP90 in ERK1/2 activity needs to be further
investigated. In the present study, we demonstrated that
HSP90 was associated with phosphor-ERK1/2 when VSMC
were challenged to oxidative stress. The association of
phosphor-ERK1/2 with HSP90 promoted the solubility and nuclear
translocation of phosphor-ERK1/2, indicating that HSP90
participated in the process of intracellular ERK1/2 activation.
Geldanamycin, a benzoquinone ansamycin, can
specifically bind to HSP90[23] in vitro
and in vivo and inhibit the function of HSP90. It also interferes indirectly with the
functions of the other protein molecules with which HSP90
associates[24], often resulting in the disruption of
HSP90-containing multimolecular
complexes[9]. Miyata et
al[5] demonstrated that HSP90 regulated stability and solubility of intracellular
protein MOK by binding with it, and geldanamycin disrupted
the effect of HSP90. Our data here showed that geldanamycin
treatment altered the chaperone function of HSP90 by
attenuating the interaction between HSP90 and
phosphor-ERK1/2, abolishing oxidative stress-induced activation and
decreasing the solubility and nuclear translocation of
phosphor-ERK1/2.
The solubility of activated ERK1/2 is essential for their
signal transduction. It has been reported that HSP90
increases the solubility of MOK, a novel member of
mitogen-activated protein kinases
(MAPK)[5]. Our experiments showed that oxidative stress increased the soluble
phosphor-ERK1/2 and decreased insoluble phosphor-ERK1/2.
Geldanamycin abolished the effects of oxidative stress,
indicating that the solubility of phosphor-ERK1/2 requires the
participation of HSP90.
Phosphor-ERK1/2 enters into nuclei to display its
func-tion. The specific associations of non-catalytic proteins are
important for catalytic subunits recognizing substrate or
cellular localization. In the present experiment, we found
that oxidative stress stimulated phosphor-ERK1/2 entering
cell nuclei, and geldanamycin blocked the effect of oxidative
stress.
Those results indicated that HSP90 could regulate
ERK1/2 activity by promoting ERK1/2 phosphorylation,
increasing the interaction of itself with
phosphor-ERK1/2, and enhancing the solubility and nuclear translocation of
phosphor-ERK1/2. It has been reported that HSP90 can bind with MOK,
a member of MAPK and bind with mitogen-activated protein
kinase kinase (MAPKK) kinases-Raf, which decrease the
degradation and increase the phosphorylation of MOK and
Raf[4_6]. Therefore, we propose that HSP90 might increase
phosphor-ERK1/2 in 2 ways: first, HSP90 delays the
degradation of phosphor-ERK1/2 by binding with it. Second,
HSP90 increases the phosphorylation of ERK1/2 by binding
with MAPKK and affecting MAPKK translocation.
It is well known that oxidative stress stimulates VSMC
proliferation by activating the signal transduction pathway
of growth factors, such as MAPK. Since HSP90 mediated
the activation of ERK1/2 induced by oxidative stress, we
propose that the function inhibition of HSP90 will inhibit cell
proliferation. Our experiments showed that geldanamycin,
radicicol (another HSP90 inhibitor), and PD98059 (an
inhibitor of ERK1/2 activation) all significantly blocked the effects
of oxidative stress on VSMC proliferation.
In summary, HSP90 can mediate the oxidative
stress-stimulated, late-phase activation of ERK1/2 and VSMC
proliferation by promoting the ERK1/2 phosphorylation, the
association with phosphor-ERK1/2, and the solubility and
nuclear translocation of phosphor-ERK1/2.
Acknowledgements
The authors are indebted to Dr Zhi-hong ZHOU (Institute
of Molecular and Cell Biology, Singapore) for her valuable
help in the preparation of the manuscript.
References
1 Liao DF, Jin ZG, Bass AS, Daum G, Gygi SP, Aebersold R,
et al. Purification and identification of secreted oxidative stress-
induced factors from vascular smooth muscle cells. J Biol Chem
2000; 275: 189_96.
2 Jin ZG, Melaragno MG, Liao DF, Yan C, Haendeler J, Suh YA,
et al. Cyclophilin A is a secreted growth factor induced by oxidative
stress. Circ Res 2000; 87: 789_96
3 Bamberger CM, Wald M, Bamberger AM, Schulte HM. Inhibition
of mineralocorticoid and glucocorticoid receptor function by the
heat shock protein 90-binding agent geldanamycin. Mol Cell
Endocrinol 1997; 131: 233_40.
4 Grammatikakis N, Lin JH, Grammatikakis A, Tsichlis PN, Cochran
BH. p50(cdc37) acting in concert with HSP90 is required for
Raf-1 function. Mol Cell Biol 1999; 19: 1661_72.
5 Miyata Y, Ikawa Y, Shibuya M, Nishida E. Specific association of
a set of molecular chaperons including HSP90 and Cdc37 with
MOK, a member of the mitogen-activated protein kinase
superfamily. J Biol Chem 2001; 276: 21 841_8.
6 Stancato LF, Silverstein AM, Owens-Grillo JK, Chow YH, Jove
R, Pratt WB. The HSP90-binding antibiotic geldanamycin
decreases Raf levels and epidermal growth factor signaling without
disrupting formation of signaling complexes or reducing the
specific enzymatic activity of Raf kinase. J Biol Chem 1997; 272:
4013_20.
7 Akagami M, Morrison P, Welch WJ. Benzoquinoid ansamycins
(herbimycin A and geldanamycin) interfere with the maturation
of growth factor receptor tyrosine kinases. Cell Stress
Chaperones 1999; 4: 19_28.
8 Prima V, Depoix C, Masselot B, Formstecher P, Lefebvre
P. Alteration of the glucocorticoid receptor subcellular localization
by non steroidal compounds. J Steroid Biochem Mol Biol 2000;
72: 1_12.
9 Schulte TW, Blagosklonny MV, Ingui C, Neckers L. Disruption
of the Raf-1-HSP90 molecular complex results in destabilization
of Raf-1 and loss of Raf-1-Ras association. J Biol Chem 1995;
270: 24 585_8.
10 Stancato LF, Chow YH, Hutchison KA, Perdew GH, Jove R,
Pratt WB. Raf exists in a native heterocomplex with HSP90 and
p50 that can be reconstituted in a cell-free system. J Biol Chem
1993; 268: 21 711-6.
11 Cobb MH, Goldsmith EJ. How MAP kinases are regulated. J Biol
Chem 1995; 270: 14 843-6.
12 Khokhlatchev AV, Canagarajah B, Wilsbacher J, Robinson M,
Atkinson M, Goldsmith E, et al. Phosphorylation of the MAP
kinase ERK2 promotes its homodimerization and nuclear
translocation. Cell 1998; 93: 605_15.
13 Marshall CJ. Specificity of receptor tyrosine kinase signaling:
transient versus sustained extracellular signal-regulated kinase
activation. Cell 1995; 80: 179_85.
14 Campbell JH, Campbell GR. Culture techniques and their
applications to studies of vascular smooth muscle. Clin Sci 1993; 85:
501_12.
15 Setalo G Jr, Singh M, Guan X, Toran-Allerand CD.
Estradiol-induced phosphorylation of ERK1/2 in explants of the mouse
cerebral cortex: the roles of heat shock protein 90 (HSP90) and
MEK2. J Neurobiol 2002; 50: 1_12.
16 Backlund M, Johansson I, Mkrtchian S, Sundberg MI. Signal
transduction-mediated activation of the aryl hydrocarbon receptor in
rat hepatoma H4IIE cells. J Biol Chem 1997; 272: 31 755_63.
17 Teruel T, Hernandez R, Lorenzo M. Ceramide mediates insulin
resistance by tumor necrosis factor-alpha in brown adipocytes by
maintaining Akt in an inactive dephosphorylated state. Diabetes
2001; 50: 2563_71.
18 Wu LX, Xu JH, Huang XW, Zhang KZ, Wen CX, Chen YZ.
Down-regulation of p210bcr/abl by curcumin involves disrupting
molecular chaperone functions of HSP90. Acta Pharmacol Sin
2006; 27: 694_9.
19 Perdew GH, Wiegand H, Vanden Heuvel JP, Mitchell C, Singh SS.
A 50 kilodalton protein associated with raf and pp60(v-src)
protein kinases is a mammalian homolog of the cell cycle control
protein cdc37. Biochemistry 1997; 36: 3600_7.
20 Stancato LF, Chow YH, Hutchison KA, Perdew GH, Jove R,
Pratt WB. Raf exists in a native heterocomplex with HSP90 and
p50 that can be reconstituted in a cell-free system. J Biol Chem
1993; 268: 21 711_6.
21 Lamphere L, Fiore F, Xu X, Brizuela L, Keezer S, Sardet
C, et al. Interaction between Cdc37 and Cdk4 in human cells. Oncogene
1997; 14: 1999_2004.
22 Kimura Y, Rutherford SL, Miyata Y, Yahara I, Freeman BC, Yue
L, et al. Cdc37 is a molecular chaperone with specific functions
in signal transduction. Genes Dev 1997; 11: 1775_85.
23 Whitesell L, Mimnaugh EG, De Costa B, Myers CE, Neckers LM.
Inhibition of heat shock protein HSP90-pp60v-src heteroprotein
complex formation by benzoquinone ansamycins: essential role
for stress proteins in oncogenic transformation. Proc Natl Acad
Sci USA 1994; 91: 8324_8.
24 Segnitz B, Gehring U. The function of steroid hormone
receptors is inhibited by the HSP90-specific compound geldanamycin.
J Biol Chem 1997; 272: 18 694_701.
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