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(Received for publication, July 11,
1995; and in revised form, July 28, 1995) From the
Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is
widely distributed in the central nervous system and periphery.
Pharmacological studies have suggested that there are at least three
receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been
reported previously. Here we report the isolation by expression cloning
of a cDNA encoding a human NPY receptor displaying a pharmacology
typical of a Y2 receptor. COS-7 cells transfected with the cDNA express
high affinity binding sites for NPY, peptide YY, and
NPY
Neuropeptide Y (NPY) ( Heterogeneity
among NPY receptors has been observed. Based on the rank order of
potency of NPY and related peptides to displace The NPY Y1 receptor has been cloned from rat
forebrain(4) , human fetal brain(5) , bovine
hypothalamus, (
The CHO cells stably expressing the NPY receptor
(CHO-hY2) in a 24-well tissue culture plate (5
Neuropeptide Y is a peptide hormone with diverse and
important physiological effects in the central nervous system and
periphery that are mediated through multiple receptor subtypes. In
order to gain insight into the molecular mechanisms associated with
these effects, we undertook the cloning of a human NPY Y2 receptor. A
plasmid cDNA expression library was prepared from poly(A) Binding of To characterize the pharmacology of the NPY receptor encoded by
pNPYR-398 cDNA,
Figure 1:
Pharmacological
characterization of a human NPY receptor expressed in COS-7 cells.
Displacement of specific binding of
Fig. 2shows the
nucleotide sequence of the pNPYR-398 cDNA and the deduced amino acid
sequence for the encoded NPY Y2 receptor. The nucleotide sequence
surrounding the initiation codon agrees well with Kozak's
consensus sequences(16) . The receptor is 381 amino acids in
length and has a molecular mass of approximately 42 kDa. Hydrophobicity
analyses determined according to the method of Kyte and Doolittle (17) suggest the existence of seven transmembrane-spanning
regions. There is a single putative N-linked glycosylation
site in the N-terminal region. There is an aspartic acid in the
putative second transmembrane region that is conserved in many
G-protein-coupled receptors and frequently involved with signal
transduction(18) . In the C-terminal tail there is a cysteine
residue that might be involved in palmitolyation (19) as well
as several serine residues that might be involved in regulatory
phosphorylation. A long polyadenylation signal was found at the 3` end
of the clone, suggesting that the cDNA clone encompasses most of the
mRNA species observed in the Northern blot (Fig. 3).
Figure 2:
Nucleic acid and amino acid sequence of a
human Y2 receptor. The nucleotide sequence of clone pNPYR-398 and the
deduced amino acid sequence for the encoded human NPY receptor, subtype
2 (Y2). The deduced amino acid sequence is shown under the nucleic acid
sequence. The putative transmembrane regions I-VII are underlined; the borders of the regions were assigned based on
hydrophobicity profile and comparison to the Y1 sequence. The asterisk (*) indicates a potential N-glycosylation
site; the ``+'' indicates a potential palmitolyation
site.
Figure 3:
Northern hybridization. Northern blot
analysis of human brain sections probed with a 1.9-kb fragment of
pNPYR-398 cDNA that contained the entire open reading frame of the Y2
receptor. A, lane1, amygdala; lane2, caudate nucleus; lane3, corpus
callosum; lane4, hippocampus; lane5, hypothalamus; lane 6, substantia nigra; lane7, subthalamic nucleus, lane 8,
thalamus. B, lane1, cerebellum; lane2, cortex; lane 3, medulla; lane4, spinal cord; lane 5,
occipital pole; lane 6, frontal lobe; lane7, temporal lobe; lane8, putamen.
Molecular size markers are indicated in kb. The blots were also probed
with
Comparison of the amino acid sequence of this NPY receptor to that
of the human Y1 receptor (5) reveals significant differences.
Overall, the two receptors are 31% identical at the amino acid level.
Their sequences show the greatest similarity in the first intracellular
loop and the second and sixth transmembrane regions, being 63%, 61%,
and 50% identical, respectively. On the nucleotide level, the coding
regions of the two NPY receptors were 47% identical. This relatively
low homology to the Y1 receptor may explain why cloning with Y1 probes
has not been fruitful. A search of the GenBank(TM) data base
revealed that the Y2 nucleic acid sequence showed some similarity to
the NK-2R neurokinin A receptor and two putative opioid
receptors(20, 21, 22) . The Y2 receptor is
believed to be a common presynaptic receptor (2) and to be the
predominant NPY receptor in the brain(23) . To study the
distribution of the mRNA corresponding to the Y2 receptor encoded by
pNPYR-398, Northern blots of poly(A) Southern blot analysis to human genomic DNA (Fig. 4) suggests that there is a single Y2 receptor gene. Since
the Y2 receptor is known to be expressed in the periphery (1) these data support the hypothesis that either there is
another form of the Y2 receptor in the periphery that is very different
from the described here or the Y2 mRNA is present at very low levels.
Figure 4:
Southern hybridization. Southern blot of
human genomic DNA probed with the 1.9-kb fragment of Y2 receptor cDNA
described in Fig. 3and washed under high stringency conditions.
Each lane has approximately 12 µg of DNA. Molecular size markers
are indicated in kb.
The truncated fragment of the pNPYR-398 clone containing the Y2 open
reading frame described above was subcloned into pcDNA3 and transfected
into CHO cells for stable expression. Competition binding experiments
demonstrated a pharmacology identical to that seen in the COS-7 cells (Table 1). In neuroblastoma cells, the Y2 receptor appears to
couple to multiple second messenger systems including the inhibition of
forskolin-stimulated cAMP accumulation and calcium
mobilization(24, 25) . To examine the functional
coupling of the recombinant Y2 receptor to second messenger systems,
the effect of NPY, [Leu
Figure 5:
Functional coupling of the recombinant Y2
receptor to second messenger systems. A, inhibition of
forskolin (10 µM)-stimulated cAMP accumulation by NPY
analogs in CHO-hY2 cells: lane 1, vehicle; lane 2,
forskolin (10 µM); lane 3, NPY; lane 4,
[Leu
Activation of
the CHO-hY2 cells with the NPY analogs NPY In summary, while NPY appears to be an important
mediator of a wide variety of physiological functions, a clear
understanding of how these functions are mediated has been hampered by
the lack of cloned NPY receptors. The present cloning and
pharmacological characterization of pNPYR-398 that encodes a human NPY
Y2 receptor should facilitate the elucidation of the structure,
function, regulation, and diversity of neuropeptide Y receptors as well
as the identification of Y2 receptor-specific antagonists.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
U32500[GenBank].
Volume 270,
Number 39,
Issue of September 29, pp. 22661-22664, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, whereas
[Leu
,Pro
]NPY binds with lower
affinity. The receptor is 381 amino acids in length and has seven
putative transmembrane regions typical of G-protein-coupled receptors.
Comparison of the amino acid sequence of this Y2 receptor to that of
the human Y1 receptor indicates that the two receptors are 31%
identical at the amino acid level. Northern blot analyses reveal a
single 4-kilobase mRNA species and indicate that the messenger RNA is
present in many areas of the central nervous system. NPY induced
calcium mobilization and inhibited forskolin-stimulated cAMP
accumulation in Chinese hamster ovary cells that stably express the Y2
receptor cDNA, indicating that the recombinant Y2 receptor is
functionally coupled to second messenger systems.
)is a 36-amino acid peptide
amide that is widely distributed in the central and peripheral nervous
systems. It belongs to a family of homologous peptides including the
gut peptide YY (PYY) and pancreatic polypeptide. NPY has been highly
conserved throughout evolution and is therefore thought to be an
important hormone/neurotransmitter. Centrally its effects include blood
pressure regulation, memory enhancement, anxiolysis/sedation, and
increased food intake, and in the periphery it affects vascular and
other smooth muscle activity, intestinal electrolyte secretion, and
urinary sodium excretion(1, 2) .I-NPY
binding, NPY receptors have been classified into at least three
receptor subtypes, Y1, Y2, and Y3(3) . Both the Y1 and Y2
subtypes have high affinity for NPY and PYY. The Y1 subtype has a high
affinity for the NPY analog
[Leu
,Pro
]NPY and low affinity for
C-terminal fragments of NPY such as NPY
. In
contrast, the Y2 receptor subtype has high affinity for
NPY
and low affinity for
[Leu
,Pro
]NPY. The Y3 subtype has a
low affinity for PYY. The existence of a fourth receptor, Y1a or Y4,
that is important in the feeding response has been hypothesized (2) .
)and murine genomic DNA(6) . It
belongs to the superfamily of G-protein-coupled receptors and appears
to couple to more than one second messenger
systems(5, 7) . To date the cloning of a Y2 receptor
has not been reported. Attempts to isolate a Y2 cDNA using homology to
the Y1 cDNA have not been successful. Here we report the isolation by
expression cloning (8) of a cDNA encoding a human NPY receptor
displaying a pharmacology typical of a Y2 receptor. The clone has been
functionally expressed in CHO cells where its activation causes
mobilization of calcium and inhibition of forskolin-stimulated cAMP
accumulation.
Expression Cloning
An oriented expression cDNA
library was prepared from SMS-KAN mRNA in the pcDNA1 vector.
Approximately 2.5 
10
COS-7 cells were seeded on to
single-well Labteks(TM) (Nunc) and transfected with 2 µg of
plasmid DNA prepared from pools of bacterial clones from the library
using Lipofectamine(TM) (Life Technologies, Inc.). Three days later
the cells were washed twice with binding buffer A (Dulbecco's
modified Eagle's medium containing 1% BSA). Following incubation
with 100 pMI-PYY (2200 Ci
mmol
, DuPont NEN) in buffer A at 37 °C for 2 h,
they were washed three times, air-dried, and autoradiographed. The one
positive pool detected during the primary screening was subdivided and
screened similarly until a pure clone was isolated.
Stable Expression of NPY-Y2 Receptor in Chinese Hamster
Ovary (CHO) Cells
A BamHI and XbaI fragment of
the pNPYR-398 clone, which contained the entire open reading frame for
the receptor, was subcloned into the pcDNA3 vector cut with the same
enzymes. This resulting construct was linearized with PvuI and
introduced into the CHO cells by electroporation (9) using a
Bio-Rad Gene Pulser(TM). After 48 h the cells were placed in
selection medium (Ham's F-12 media containing 10% fetal bovine
serum, 50 units/ml penicillin G, 50 µg/ml streptomycin, 2 mML-glutamine, and 300 µg/ml G418). Clones were picked
after 3 weeks, expanded, and assayed for NPY receptor expression using
a radioligand binding assay.Radioligand Binding
COS-7 cells (5
10
cells/assay), transfected with the purified pNPYR-398
clone were incubated in a 96-well microtiter plate in 100 µl of
binding buffer B (50 mM Hepes, pH 7.4, 5 mM
MgCl, 1 mM CaCl, 0.5% BSA, 1 mg/ml of
pepstatin, leupeptin, and aprotinin, 0.1% bacitracin, 5 mg/ml N-p-tosyl-L-lysine
chloromethyl ketone, and 1 mM phenylmethylsulfonyl fluoride)
containing 100 pM
I-NPY (2200 Ci/mmol, DuPont
NEN), with or without competitor, at room temperature with shaking. The
reaction was terminated with the addition of 50 µl of the reaction
mix into 600 µl of ice-cold binding buffer B. The quenched reaction
mixture was centrifuged at 4 °C for 20 min, the supernatant removed
and the pellets were counted in a
counter (Cobra, Packard
Instrument Co.).
10 cells/well) were incubated in 250 µl of binding buffer C
(Dulbecco's modified Eagle's medium containing 0.1% BSA)
with 100 pMI-PYY, with or without competitor,
at room temperature for 1 h. The cells were then washed twice with 0.5
ml of ice-cold buffer C, suspended in 0.5 M NaOH, transferred
to a counting tube, and counted in a
counter. The competition
binding and saturation binding data were analyzed by logistic.fit and
hyperbola.fit programs of Sigma Plot, respectively.
Sequencing
The nucleotide sequence of both strands
of the cDNA insert was determined by the dideoxy chain termination
method (10) on an Applied Biosystems model 373A automated
sequencer. The sequence data were analyzed using the GCG sequence
analysis software package(11) .Northern Blot Analysis
Human brain multiple tissue
Northern blots (2 µg of poly(A) RNA/lane) were
purchased from Clontech. The blots were probed using standard molecular
biology techniques(12) . The BamHI/XbaI
fragment of the pNPYR-398 cDNA clone described above was labeled to a
specific activity of 5
10
cpm/µg with
[
-P]dCTP by random priming to be used as a
probe. Hybridization was at 42 °C in 5
SSPE, 10
Denhardt's solution, 100 µg/ml denatured salmon sperm, 50%
formamide, and 2% SDS for 24 h with shaking. The membranes were washed
twice in 2 SSC and 0.05% SDS at room temperature, followed by
one wash in 0.1 SSC and 0.1% SDS at 50 °C for 40 min and
autoradiographed.Southern Blot Analysis
Approximately 12 µg of
human genomic DNA digested with BamHI or EcoRI were
loaded on an 0.8% agarose gel in TAE, electrophoresed at about
1.75-2.0 V/cm for approximately 36 h and transferred to a
nitrocellulose membrane. Probe was prepared as described for the
Northern blot. Hybridization was overnight at 37 °C in 4
SSC, 1 Denhardt's solution, 7 mM Tris-Cl, pH
7.4, 40% formamide, 10% dextran sulfate, and 100 µg/ml sheared
herring sperm DNA with shaking. The membrane was washed thoroughly in 2
SSC and 0.1% SDS at room temperature, followed by two 15-min
washes in 0.1 SSC and 0.1% SDS at 65 °C and
autoradiography.cAMP Assays
CHO-hY2 in six-well plates (1
10
cells/well) were preincubated for 10 min at 37 °C in
media (Ham's F-12 containing 10% fetal bovine serum) containing
100 µM 3-isobutyl-1-methylxanthine. They were then treated
with media (basal), forskolin (10 µM), peptide, or
forskolin plus peptide (100 nM) for 10 min at 37 °C. The
reaction was quenched by incubation in 1 N HCl for 30 min at 4
°C. Cellular debris was removed by centrifugation, and the samples
were lyophilized. The cAMP content was determined by radioimmune assay
(DuPont NEN). For experiments in which G
was
inhibited, cells were incubated overnight in media containing 100 ng/ml
pertussis toxin.Calcium Mobilization Assay
CHO-hY2 cells (1
10 cells/ml) were washed twice with
phosphate-buffered saline and loaded with Fura-2AM (Molecular Devices)
in buffer D (Hank's balanced salt solution, 1 mM
CaCl, 1 mM MgCl, 25 mM Hepes,
pH 7.5, and 0.2% BSA) for 45 min at 37 °C. The cells were then
washed with buffer D, suspended at 3 10 cells/ml,
and placed in a Spex spectrofluorometer with submersible magnetic
stirrer attachment. A stable base line was established at 340 nm and 490 nm
before the NPY
(1
µM), NPY
(1 µM), MCP-1
(5 µM), or galanin (5 µM) was added and the
fluorescence measured. Fluorescence maxima and minima were determined
using Triton X-100 (1%) and EGTA (10 mM), respectively.
Calcium mobilized was calculated according to McCormack and
Cobbold(13) ; the K
value used was 224
nmol/liter.
RNA isolated from SMS-KAN cells, a human neuroblastoma cell line
that expresses high levels of Y2 receptor(14) . Plasmid library
DNA was transfected into COS-7 cells that are null for NPY binding; the
cells were assayed for NPY receptor expression by
I-PYY
binding. Detection was by autoradiography. A single pool (pool 398) was
identified that had increased levels of
I-PYY binding.
This pool was subdivided and the process was repeated until a pure cDNA
clone (pNPYR-398) was isolated.
I-NPY and
I-PYY to COS-7 cells transfected with pNPYR-398 was
saturable and high affinity, with K
values of 0.58
and 0.27 nM, respectively. Specific binding to COS-7 cells
transfected with vector (pcDNA1) was negligible. Scatchard analysis
suggests that I-NPY bound to a single class of binding
sites in COS-7 cells transfected with pNPYR-398 cDNA (data not shown).
I-NPY (100 pM) binding to COS-7
cells transfected with the recombinant plasmid was competed with
various NPY analogs (Fig. 1). PYY, NPY, and NPY
were the most effective competitors of
I-NPY
binding, followed by the Y2-specific agonist NPY
(Table 1). The Y1-specific agonist
[Leu
,Pro
]NPY inhibited
I-NPY binding only at high concentration with an
IC
of 0.5 µM.
[D-
Trp]NPY, a competitive antagonist of
NPY in rat hypothalamus(15) , inhibited
I-NPY
binding with an IC
of 2.9 µM. The rank order
of potency of NPY and related peptides to COS-7 cells expressing the
NPY receptor cDNA is PYY
NPY NPY >
NPY
> NPY
[Leu
,Pro
]NPY >
[D-Trp
]NPY. To further confirm the
characteristics of pNPYR-398 NPY receptor, the potency of the NPY
analogs to this receptor in competing for
I-PYY to COS-7
cells transfected with pNPYR-398 was studied. The results were similar
to those described for
I-NPY binding (Table 1).
These data are consistent with the previously reported pharmacology for
the Y2 subtype of NPY receptors(3) .
I-NPY to membranes
from COS-7 cells transfected with pNPYR-398. Binding was determined in
the presence of the indicated concentrations of PYY (
), NPY
(), NPY
(
), NPY (
), [Leu,Pro
]NPY
(
), and [D-
Trp]NPY
(
).
P-labeled actin cDNA as a
standard.
RNA from several
parts of the brain were hybridized with a 1.9-kb fragment of the Y2
cDNA that contained the entire receptor open reading frame. A single
4-kb mRNA transcript was detected in the amygdala, caudate nucleus,
corpus callosum, hippocampus, hypothalamus, and subthalamic nucleus (Fig. 3A), cortex, medulla, occipital pole, and frontal
lobe (Fig. 3B). No hybridization was detected in RNA
from peripheral tissues (data not shown). This suggests that either
another form of the Y2 receptor is found in the periphery or that the
Y2 mRNA transcript is present at levels undetectable by Northern
analysis. In situ hybridization studies are in progress to
address this question.
,Pro
]NPY,
and NPY
on cAMP and calcium mobilization was
studied in the CHO-hY2 cells. Activation with 100 nM NPY,
[Leu
,Pro
]NPY, and
NPY
inhibited forskolin (10 µM)
stimulated cAMP accumulation by 72%, 7%, and 71%, respectively (Fig. 5A). When the cells were pretreated with
pertussis toxin (100 ng/ml), the peptides did not inhibit the
accumulation of forskolin-stimulated cAMP, suggesting that the Y2
receptor couples to G
. In addition, NPY did not
inhibit the accumulation of forskolin-stimulated cAMP in CHO cells
transfected with the pcDNA3 vector (data not shown).
,Pro
]NPY; lane 5,
NPY
, (lanes 3-5, peptides at 1
µM); lane 6, forskolin plus NPY; lane 7,
forskolin plus [Leu
,Pro
]NPY; lane 8, forskolin plus NPY
(lanes
6-8, forskolin at 10 µM and peptides at 100
nM). The cAMP values are expressed as a percentage of the cAMP
level observed in the presence of forskolin (100%). B. Calcium
mobilization by NPY analogs in CHO-hY2 cells: lane1,
NPY
(1 µM); lane2,
NPY
(1 µM); lane3, MCP-1 (5 µM); lane 4, galanin (5
µM). Three independent assays were performed for each
ligand tested. The data represent an average of the two best
experiments.
(1
µM) and NPY
(1 µM)
resulted in a 13-14-fold increase in intracellular calcium (Fig. 5B). Neither MCP-1 (5 µM) or galanin
(5 µM) elicited a change in intracellular calcium levels.
Although calcium mobilization is frequently associated with
G-protein-coupled receptors linked to phospholipase C(26) ,
this is not necessarily the case(27) . The specific G-protein
mediating the increase of intracellular calcium described here is not
known at this time.
))
We thank Drs. Michele Agler, Kenneth Carlson, Zahra
Fathi, Stanley Krystek, Eric Parker, and Maria Webb for critical
discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. Diez, J. Koistinaho, S. J. Dearmond, D. Groth, S. B. Prusiner, and T. Hokfelt Marked decrease of neuropeptide Y Y2 receptor binding sites in the hippocampus in murine prion disease PNAS, November 25, 1997; 94(24): 13267 - 13272. [Abstract] [Full Text] [PDF]
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M. Jackerott and L.-I. Larsson Immunocytochemical Localization of the NPY/PYY Y1 Receptor in the Developing Pancreas Endocrinology, November 1, 1997; 138(11): 5013 - 5018. [Abstract] [Full Text] [PDF]
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C. J. Small, D. G. A. Morgan, K. Meeran, M. M. Heath, I. Gunn, C. M. B. Edwards, J. Gardiner, G. M. Taylor, J. D. Hurley, M. Rossi, et al. Peptide analogue studies of the hypothalamic neuropeptide Y receptor mediating pituitary adrenocorticotrophic hormone release PNAS, October 14, 1997; 94(21): 11686 - 11691. [Abstract] [Full Text] [PDF]
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 C. A. Blaze, P. J. Mannon, S. R. Vigna, A. R. Kherani, and B. A. Benjamin Peptide YY receptor distribution and subtype in the kidney: effect on renal hemodynamics and function in rats Am J Physiol Renal Physiol, October 1, 1997; 273(4): F545 - F553. [Abstract] [Full Text] [PDF]
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 X. Chen, D. A. Dimaggio, S. P. Han, and T. C. Westfall Autoreceptor-induced inhibition of neuropeptide Y release from PC-12 cells is mediated by Y2 receptors Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1737 - H1744. [Abstract] [Full Text] [PDF]
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S. M. Leupen, L. M. Besecke, and J. E. Levine Neuropeptide Y Y1-Receptor Stimulation Is Required for Physiological Amplification of Preovulatory Luteinizing Hormone Surges Endocrinology, July 1, 1997; 138(7): 2735 - 2739. [Abstract] [Full Text] [PDF]
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S. Sheriff, William. T. Chance, Josef. E. Fischer, and A. Balasubramaniam Neuropeptide Y Treatment and Food Deprivation Increase Cyclic AMP Response Element-Binding in Rat Hypothalamus Mol. Pharmacol., April 1, 1997; 51(4): 597 - 604. [Abstract] [Full Text]
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E. Grouzmann, T. Buclin, M. Martire, C. Cannizzaro, B. Dorner, A. Razaname, and M. Mutter Characterization of a Selective Antagonist of Neuropeptide Y at the Y2 Receptor. SYNTHESIS AND PHARMACOLOGICAL EVALUATION OF A Y2 ANTAGONIST J. Biol. Chem., March 21, 1997; 272(12): 7699 - 7706. [Abstract] [Full Text] [PDF]
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P. M. Rose, J. S. Lynch, S. T. Frazier, S. M. Fisher, W. Chung, P. Battaglino, Z. Fathi, R. Leibel, and P. Fernandes Molecular Genetic Analysis of a Human Neuropeptide Y Receptor. THE HUMAN HOMOLOG OF THE MURINE "Y5" RECEPTOR MAY BE A PSEUDOGENE J. Biol. Chem., February 7, 1997; 272(6): 3622 - 3627. [Abstract] [Full Text] [PDF]
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