From the Departments of Pharmacology,
Endocrinology, and ** Clinical Biochemistry, The Bruce Rappaport
Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel, the
Rappaport Family
Institute for Research in the Medical Sciences, 31096 Haifa, Israel,
the § Tel Aviv Community Mental Health Center, 97197 Tel
Aviv, Israel, and the ¶ Sackler Faculty of Medicine, Tel Aviv
University, 69978 Tel Aviv, Israel
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ABSTRACT |
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The peripheral-type benzodiazepine receptor (PBR) is not only widely expressed throughout the body, but it is also genetically conserved from bacteria to humans. Many functions have been attributed to it, but its primary role remains a puzzle. In the current study, we stably transfected cultures of MA-10 Leydig cells with either control or 18-kDa PBR antisense knockout plasmids. The antisense knockout vector was driven by the human enkephalin promoter, which contains two cAMP response elements, such that cAMP treatment of transfected cells could superinduce 18-kDa PBR antisense RNA transcription and, hence, down-regulate endogenous 18-kDa PBR mRNA levels. Control and knockout MA-10 cell lines were then compared at the level of receptor binding, thymidine incorporation, and steroid biosynthesis. Eighteen-kilodalton PBR knockout reduced the maximal binding capacity of tritium-labeled PBR ligands, and the affinity of receptors to the ligands remained unaltered. Additionally, 24-h accumulation of progesterone was lower in the knockout cells. Exposure of the two cell types to 8-bromo-cAMP resulted in a robust increase in steroid production. However, a complex pattern of steroid accumulation was observed, in which further progestin metabolism was indicated. The later decline in accumulated progesterone as well as the synthesis of androstenedione were different in the two cell types. At the level of cell proliferation, reduction of 18-kDa PBR mRNA showed no effect. Thus, we conclude that the 18-kDa PBR may have a more important role in steroidogenesis than in proliferation in this Leydig cell line.
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INTRODUCTION |
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Peripheral-type benzodiazepine receptors (PBR)1 are widely expressed throughout the body (1-4), yet different tissues exhibit different patterns of PBR expression (2-6). The PBR is a heterotrimer composed of the following subunits (7): an isoquinoline carboxamide-binding protein (18 kDa), a voltage-dependent anion channel (32 kDa), and an adenine nucleotide carrier (30 kDa). Diazepam-binding inhibitor is one of the putative endogenous PBR ligands (2-4, 7).
A broad spectrum of putative functions has been suggested for the PBR (8), such as regulation of steroid production (4, 8-12), involvement in cell growth and differentiation (12-15), regulation of the mitochondrial respiratory chain (8, 16, 17), regulation of heme biosynthesis (8, 18, 19), effect on the immune and phagocytic host defense response (8, 20), and modulation of voltage-dependent calcium channels (8). The first two of these functions are the most commonly suggested. The relative importance of each of these functions is still unclear. Furthermore, it is possible that these functions may show tissue specificity.
In the present study, we focused on the two major putative PBR roles, cell proliferation and steroidogenesis, in MA-10 mouse tumor Leydig cell line. Steroidogenic cells express high levels of PBR, and in such cells the PBR have been associated with steroidogenesis (4, 8-10, 12). Accumulated data suggest a role for PBR in the first step of steroidogenesis, namely the production of pregnenolone from cholesterol in the mitochondria. Papadopoulos and co-workers (9, 12) characterized the steroidogenic role of PBR in the MA-10 Leydig cell line. Other studies have demonstrated a role of PBR in the regulation of cell proliferation in different cell lines (2-4, 8, 12-15).
The 18-kDa PBR gene is composed of four exons, which can express up to two splicing variants of mRNA (21). By down-regulating mRNA levels, one can effectively reduce the amount of 18-kDa PBR protein locally expressed. One approach to depleting the 18-kDa PBR expression is the antisense knockout method (22). In the current study, we investigated the relative importance of PBR as a regulator of steroidogenesis and cell proliferation in MA-10 cells.
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EXPERIMENTAL PROCEDURES |
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Cells
The MA-10 cell line, originally cloned from the solid M548OP mouse tumor Leydig cells, was a kind gift of Dr. Mario Ascoli (Department of Pharmacology, University of Iowa College of Medicine, Iowa City, IA). MA-10 cell cultures were incubated as described previously (23). Briefly, cells were maintained in RPMI 1640 medium with L-glutamine, containing 20 mM HEPES buffer, 15% horse serum, and 50 µg/ml gentamycin sulfate (all purchased from Beit-Ha'emek Biological Industries, Beit Ha'emek, Israel). MA-10 transfected cells were grown in similar medium, except for the use of 7.5% horse serum and 7.5% fetal calf serum, instead of 15% horse serum. In the experiments in which steroidogenesis was assessed, serum-free medium was used (Dulbecco's modified Eagle's medium plus Ham's F-12 (1:1) medium, containing 15 mM HEPES buffer, 2.4 g/liter NaHCO3, 0.1 IU/ml insulin, 5 µg/ml transferrin, 0.1 mg/ml glutamine, and 50 µg/ml gentamycin (all from Beit-Ha'emek)).
Plasmid Construction
The cDNA encoding the 18-kDa PBR consists of four exons ligated within a Bluescript vector (Stratagene, La Jolla, CA). This plasmid was a kind gift from Dr. Karl E. Krueger (Georgetown University, Washington, D. C.). The antisense vectors were prepared by subcloning the 460-base pair DraI-PvuII exon 3 and 4 fragments of the 18-kDa PBR subunit gene. It was placed into the HincII site of the pENKAT12 plasmid in the antisense orientation with respect to the human enkephalin promoter (pENKPBR-A) (see Fig. 1). The human enkephalin promoter-driven chloramphenicol acetyltransferase construct (Fig. 1) showed relatively high levels of basal expression in MA-10 cells (data not shown). Furthermore, the same promoter-driven construct was inducible by cAMP analogues (24).
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Stable Transfection of MA-10 Cells
The transfection was performed as described previously (22). Briefly, cells were plated a day before transfection (5 × 105 cells/90-mm2 tissue culture dish). On the following day 15 µg of antisense knockout plasmid was routinely cotransfected with 3 µg of the neomycin-resistant plasmid pMC1 NEO poly(A) (Stratagene, La Jolla, CA). In control experiments, the plasmid pENKAT12 was used with pMC1 neo poly(A). Four hours after the transfection, the cells were subjected to glycerol shock for 90 s. The day after transfection, G418 (neomycin analogue) selection (300 µg/ml) was initiated and maintained for 3 weeks, at the end of which time cell colonies were counted and categorized by colony size. The cells were then plated and subjected to different functional assays. Control and 18-kDa PBR mRNA knockout cells were dubbed EnKat and anti-18-kDa PBR cells, respectively.
[3H]PK 11195 and [3H]Ro 5-4864 Binding Assays
[3H]PK 11195 (90 Ci/mmol; NEN Life Science Products) and [3H]Ro 5-4864 (84.5 Ci/mmol; NEN Life Science Products) were used for binding studies, as described previously (25). Cells were scraped from 90-mm2 culture dishes, washed with phosphate-buffered saline, and centrifuged at 1200 × g for 5 min. Then the cell pellets were resuspended in 1 ml of 50 mM phosphate buffer, pH 7.4, and centrifuged at 16,000 × g for 30 min.
Binding assays contained 400 µl of cell membrane (0.03-0.1 mg of
protein/ml) in the absence (total binding) or presence (nonspecific binding) of 1 µM unlabeled PK 11195 (Pharmuka
Laboratories, Gennevilliers, France) or Ro 5-4864 (Hoffmann-La Roche,
Basel, Switzerland), up to a final volume of 500 µl. After incubation
for 1 h at 4 °C, samples were filtered under vacuum over
Whatman GF/B filters and washed three times with 3 ml of phosphate
buffer. Filters were placed in vials containing 4 ml of xylene-Lumax
(3:1, v/v; Lumax was purchased from Lumac, Schaesberg, The Netherlands)
and counted for radioactivity in a -scintillation counter after
12 h.
The maximal number of binding sites (Bmax) and equilibrium dissociation constants (Kd) were calculated from saturation curves of [3H]PK 11195 and [3H]Ro 5-4864 binding, using Scatchard analysis.
RNA Isolation and Northern Blot Analysis
RNA was isolated from each of the tissues by the acid
guanidinium thiocyanate phenol chloroform method (26). RNA (10-50 µg) was electrophoresed on 1% agarose-formaldehyde gels and then transferred to Nytran-N (Schleicher & Schuell). After prehybridization for 2 h, membranes were hybridized overnight at 65 °C with
32P-labeled DNA probes (27), using a 781-base pair
EcoRI PBR cDNA fragment. To control for RNA loading
variability, membranes were subsequently reprobed with
32P-labeled glyceraldehyde-3-phosphate dehydrogenase DNA,
using a 1600-base pair PstI glyceraldehyde-3-phosphate
dehydrogenase fragment (28). After washing twice with 2 × SSC and
1% SDS for 20 min at 65 °C, membranes were exposed for 15 min to a
phosphor-imaging screen and analyzed by a phosphor imager (model BAS
1000 MacBos; Fujix, Japan), as well as to x-ray film (Kodak XAR) for
1-3 days at 80 °C.
Cell Proliferation
Cell Count-- Cells were plated in 24-well dishes (5 × 104 cells/well), trypsinized, and counted visually using a hemocytometer every 24 h over a 6-day period (with and without trypan blue staining). The count determined the doubling time and viability of EnKat and anti-PBR cells.
Thymidine Incorporation-- Cell cultures at high density were incubated for 4 days without a change of medium. Cells were then trypsinized and plated in 24-well plates (5 × 104 cells/well). One hour later, cells were exposed to a benzodiazepine receptor ligand: clonazepam (with very low affinity for PBR), Ro 5-4864 (with very high affinity for the isoquinoline carboxamide-binding protein and voltage-dependent anion channel of the PBR), or the isoquinoline carboxamide derivative PK 11195 (with high affinity for the isoquinoline carboxamide-binding protein of the PBR).
The ligands were dissolved in Me2SO. The final concentration of ligands in the wells varied over a wide range (10Steroid Biosynthesis
Cells were plated in 24-well plates (2 × 105
cells/well) and incubated overnight. On the following day, cells were
washed twice with 1 ml of serum-free medium and then incubated
overnight with 1 ml of serum-free medium. A second wash with 2 ml of
serum-free medium was performed on the following day, and cells were
incubated with 1 ml of serum-free medium for 4, 8, 12, and 24 h.
Sometimes, 1 mM of 8-bromo-cAMP (Sigma) was added to each
well. At the end of each incubation period, media were collected from
all wells, and steroids were measured by radioimmunoassay.
Progesterone, 20-dihydroprogesterone (20
- DHP), and estrone
were estimated as described by Bauminger et al. (29), and
for androstenedione determination a commercial kit (Diagnostic Systems
Laboratories, Webster, TX) was used.
Where steroid concentrations are presented (Figs. 4 and 5), n is the total number of similarly treated wells, collected in 1-4 independent experiments. When results from 2-4 experiments were combined, normalization was first conducted within each experiment: Values from individual wells were divided by the mean steroid concentration observed for EnKat cells at 8 h in the same experiment. This mean value was defined as 1, 100, or 1000, so that it would be close to the absolute value (expressed as ng/ml) of this mean.
A representative cell count (n = 6) in serum-free medium conditions was performed as a cell viability test. Cells were plated in 24-well plates (2 × 105 cells/well) and incubated overnight. On the following day, cells were washed and incubated overnight with serum-free medium. Next, cells were washed again, incubated with serum-free medium, and then trypsinized and visually counted using a hemocytometer at zero time and after 4, 8, 12, and 24 h.
Statistical Analysis
Results are expressed as mean ± S.E. Two- and one-way analysis of variance, Student-Newman-Keuls post hoc analysis, and nonparametric Mann-Whitney and Kruskal-Wallis tests were used as appropriate. The nonparametric analyses were performed whenever indicated by Bartlett's test for homogeneity of variances. A two-tailed paired t test was used to analyze the binding parameters. Statistical significance was defined as p < 0.05.
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RESULTS |
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PBR Binding Characteristics of MA-10 Transfected Cells-- PBR binding characteristics were measured by performing binding studies of the ligands [3H]PK 11195 (Fig. 2, A-C) and [3H]Ro 5-4864 (Fig. 2, D-F). As shown in Fig. 2A, reduced (50%) Bmax values of [3H]PK 11195 binding to anti-18-kDa PBR cells were obtained as compared with EnKat cells. In contrast, no significant difference in Bmax values of [3H]Ro 5-4864 binding was detected upon comparison of the anti-18-kDa PBR with the control EnKat cells (Fig. 2D).
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Effect of 18-kDa PBR Knockout on DNA Synthesis and Cell
Growth--
Involvement in cell proliferation is one of the putative
functions of PBR. We examined the effect of the mRNA 18-kDa PBR
knockout on the spontaneous proliferation of MA-10 tumor Leydig cells. When cell counts of EnKat and anti-18-kDa PBR cells were compared over
a 6-day period, no statistically significant differences between the
two cell types were observed. Both EnKat and anti-18-kDa PBR cells
exhibited a doubling time of 24 h. In other experiments (Fig.
3), thymidine incorporation was estimated
in both types of transfected cells, in the absence and presence of a
benzodiazepine receptor ligand (clonazepam, PK 11195, or Ro 5-4864)
(Fig. 3). Cells were subjected to a wide range of concentrations of the three ligands (1010 to 10
4 M)
throughout two incubation periods (16 and 26 h). First, a comparison was made between the two cell types in the absence of drugs.
For each experiment, the mean cpm/well was calculated for each of the
four control groups (two cell types and two incubation periods).
Results of four independent experiments were then combined (mean ± S.E.; n = 4). Values for anti-18-kDa PBR and EnKat
cells were, respectively, 159,460 ± 32,470 and 159,940 ± 36,740 at 16 h, and 260,730 ± 25,270 and 240,940 ± 22,640 at 26 h. Effects of the benzodiazepine receptor ligand are
presented in Fig. 3. No overt effect on the proliferation of either
EnKat or anti-18-kDa PBR cells was obtained at 16 and 26 h in the
absence or presence of any of the three ligands at concentrations up to
10
5 M. At the highest concentration
(10
4 M) of each of the PBR-specific ligands
(PK 11195 and Ro 5-4864), a significant inhibition of thymidine
incorporation was measured for both EnKat and anti-18-kDa
PBR-transfected Leydig cells. Smaller differences were measured
following treatment with 10
4 M clonazepam in
both cell populations.
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Effect of 18-kDa PBR Knockout on Steroidogenesis--
Since Leydig
cells are the main source of steroids in the testis, we examined
whether the 18-kDa PBR knockout would affect Leydig cell
steroidogenesis using the MA-10 cell model. The main steroid products
of MA-10 cells are progesterone and 20-DHP (30). Cells were
incubated in serum-free medium, as described under "Experimental
Procedures." Cells did not proliferate in this medium, as assessed by
visual cell count. The results obtained demonstrated that under basal
conditions 18-kDa PBR knockout cells secreted less progesterone and
20
-DHP than EnKat cells (Figs.
4A and
5A); however, this effect was
statistically significant only at 24 h.
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DISCUSSION |
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The approach we used to investigate the contribution of the 18-kDa PBR subunit to Leydig cell proliferation versus steroidogenesis involved creating a plasmid vector capable of producing antisense 18-kDa PBR RNA. This exogenous RNA would hybridize with endogenous 18-kDa PBR mRNA, effectively preventing it from replenishing 18-kDa PBR protein. Any important effects 18-kDa PBR might have should then be distinguishable by comparing the particular function of interest in cells containing the 18-kDa PBR antisense vector compared with parallel cultures transfected with control vectors. This study was carried out similarly to previous studies in which the focus was on other genes (22).
To determine whether MA-10 cells receiving the knockout and control vectors would express 18-kDa PBR differently, receptor-binding studies were carried out. Indeed, an approximately 50% reduction in the binding of [3H]PK 11195 was observed in cells expressing the knockout vector, compared with control. We believe that the reason the effect was not complete was that the promoter used to drive the transcription of antisense 18-kDa PBR RNA was not, in untreated MA-10 cells, as strong a promoter as others for this cell type. According to a number of studies, the isoquinoline-binding site may overlap with the benzodiazepine-binding site, but it is not identical (31-35). The latter may also reside on the 32-kDa voltage-dependent anion channel, a subunit of the PBR complex (31). To address this issue, we compared the binding of the PBR-specific ligand [3H]Ro 5-4864 in these modified cells. In our hands (see Fig. 2, D-F), no significant difference in binding was found between control and 18-kDa PBR subunit knockout cultures. This indicates that, while the 18-kDa PBR subunit is necessary for PBR isoquinoline binding, it may not be absolutely essential for PBR-specific benzodiazepine binding. This could be explained either by the overlapping benzodiazepine/isoquinoline-binding site hypothesis (as mentioned above (31-35)) and/or the presence of two benzodiazepine-binding sites in this receptor complex.
We investigated the putative role of the 18-kDa subunit of PBR in cell
proliferation using the incorporation of [3H]thymidine
into proliferating cells as our primary tool (Fig. 3). To confirm the
findings, we also counted cells of both types over five doubling
periods. Our data very clearly showed that there were no overt
differences between control and knockout cultures following any of the
treatments presented in Fig. 3, as well as without drug addition. This
would indicate that the 18-kDa PBR subunit is probably not involved in
the regulation of Leydig cell proliferation. In the same series of
experiments, a very significant inhibition of cell proliferation was
observed at the highest concentration of the drugs used
(104 M). This was observed for control as
well as knockout cultures and with PBR-specific drug treatments as well
as central benzodiazepine receptor-specific treatments. These effects
may be nonspecific, since 10
4 M is a very
high concentration. Alternatively, these data are consistent with the
putative presence of another type of benzodiazepine receptor that does
not contain the 18-kDa PBR subunit. Such a notion has already been
suggested, in theory (15).
Papadopoulos and colleagues (9, 12) have previously shown a link between PBR expression in MA-10 Leydig cells and steroidogenesis. Specifically, they claim that the PBR is involved in cholesterol transport across the mitochondrial membrane (36), a step considered to be rate-limiting in steroid biosynthesis. They showed that incubation of MA-10 cells with various benzodiazepine receptor ligands stimulated steroidogenesis; the magnitude of this stimulation correlated significantly with the drug's binding affinity to the PBR. Furthermore, the addition of PBR ligands to isolated mitochondria (but not to mitochondrial preparations lacking the outer membrane) resulted in increased pregnenolone production (9).
In another study, elimination of diazepam-binding inhibitor, allegedly
the endogenous ligand of PBR, by introduction of an antisense
oligodeoxynucleotide, entirely inhibited human chorionic gonadotropin-stimulated steroidogenesis (10). On the other hand, the
stimulatory effect of 22(R)-hydroxycholesterol, a
mitochondria-permeable cholesterol derivative, was not affected. In our
study, there was a significant reduction (about 20%) in basal
progesterone and 20-DHP levels in the medium of 18-kDa PBR knockout
cell cultures, compared with EnKat cells (Figs. 4A and
5A). This difference in progestin levels was found at
24 h.
As observed before (30), in 8-bromo-cAMP-stimulated cells progesterone
production was increased by more than 100-fold. However, the pattern of
progesterone concentrations in the medium of both cell types was
unusual in that, after an initial increase, it decreased with time.
Such findings would indicate that secreted progesterone is further
metabolized. Ascoli (30), in his characterization of the original MA-10
cell line, described a somewhat similar phenomenon. With treatment
leading to protein kinase A activation, progesterone concentration
modestly decreased between 8 and 12 h of incubation. In his study,
the formation of 20-DHP could account for most of this decrease
(30). In our study, at 24 h the decrease was much greater, and
20
-DHP decreased as well. Thus, conversion of progestins to another
metabolite(s) has to be postulated. At present, these putative
metabolites are not identified. Metabolism of progesterone to
androstenedione by 17
-hydroxylase, 17,20-lyase, and then conversion
of androstenedione to estrone, by aromatase, are relatively slow and
thus cannot be the only step involved. In any event, these conversions
of progesterone were significantly different in EnKat and the 18-kDa
PBR knockout cells; the rate of fall in progesterone concentration in
the medium was greater in the latter cells. 20
-DHP, however,
decreased at the same rate in both cell types.
The effect of 18-kDa PBR knockout on androstenedione was different from that on either progestin. Secreted androstenedione was significantly lower in anti-18-kDa PBR cells even at the earliest time point examined (4 h). This difference persisted throughout the rest of the incubation period, in which no absolute decline in concentration of the androgen was observed. Thus, here the 18-kDa PBR could be affecting androstenedione synthesis rather than metabolism. Still, a determination of a much greater array of steroid metabolites is necessary for the complete identification of the enzymatic steps affected by the knockout of 18-kDa PBR.
The PBR are not the only proteins thought to be involved in cholesterol transport in the mitochondria. Steroidogenic acute regulatory protein, a 37-kDa protein localized in the mitochondria, has recently been cloned (37). The production of this labile protein is stimulated by cAMP. The steroidogenic acute regulatory protein is apparently the long sought mediator of the acute stimulation of steroidogenesis by tropic hormones. It may be that the 18-kDa PBR act in concert with steroidogenic acute regulatory protein in steroid biosynthesis. It is possible that a very marked reduction in PBR is required for impairment of the steroidogenic response to cAMP. Thus, the modest reduction in ligand binding observed in the anti-18-kDa PBR cells is not sufficient for obtaining an unequivocal reduction in the rate of progestin production in MA-10 cells. We intend to further study the correlation between the reduction in PBR gene expression and steroidogenesis in clones of knockout MA-10 cells, in which the extent of PBR knockout is much more marked.
In summary, further studies are needed to confirm the involvement of the 18-kDa PBR subunit in MA-10 Leydig cell steroid biosynthesis; however, we have shown that cell proliferation is independent of 18-kDa PBR subunit gene expression. Other functions have been suggested to involve the 18-kDa PBR subunit, and there are cells where the PBR is expressed but steroidogenesis is not present. These data suggest that there may be other tissue-specific functions of the PBR that warrant further investigation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ada Tamir for statistical consultation and analysis and Ruth Singer for editing the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Technion V.P.R. Fund-Lawrence Neuro-Psycho-Pharmacology Research Fund.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.
§§ To whom correspondence should be addressed: Dept. of Pharmacology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O. Box 9649, 31096 Haifa, Israel. Tel.: 972-4-829-5275; Fax: 972-4-851-3145; E-mail: mgavish{at}tx.technion.ac.il.
1
The abbreviations used are: PBR, peripheral-type
benzodiazepine receptor(s); 20-DHP, 20
-dihydroprogesterone.
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REFERENCES |
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