2Geriatrics Research and Education Center, 3South Texas Veterans Health Care System, Audie L. Murphy Memorial Hospital Division, and 1Department of Medicine, The University of Texas Health Science Center, San Antonio, Texas 78229-3900
Submitted 25 November 2002 ; accepted in final form 21 March 2003
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ABSTRACT |
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reactive oxygen species; Rac1; arachidonic acid; protein synthesis
In this study, we explored the role of Rac1 activation and Nox4 in MCs and provide the first evidence that in these cells ANG II activates Akt/PKB via stimulation of the GTPase Rac1. We show that a Nox4-containing NAD(P)H oxidase is present in MCs and that ROS derived from Nox4 contribute to ANG II-induced protein synthesis. We propose that PLA2-mediated generation of AA is responsible for ANG II-induced Rac1 activation. In turn, Rac1 regulates Akt/PKB activity through generation of ROS by stimulation of a Nox4-based NAD(P)H oxidase.
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MATERIALS AND METHODS |
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Antisense (AS) oligonucleotides were designed near the ATG start codon of rat Nox4 (5'-AGCTCCTCCAGGACAGCGCC-3'). AS and the corresponding sense (S) oligonucleotides were synthesized as phosphorothiolated oligonucleotides and purified by high-performance liquid chromatography (Advanced Nucleic Acid Core Facility, University of Texas Health Science Center at San Antonio, TX). AS and sense oligonucleotides for Nox4 were transfected by electroporation as described above.
Northern blot analysis. Total RNA was isolated from MCs by using RNAzol B method (Cinna Biotex), separated electrophoretically on formaldehyde-agarose gels, transferred to a gene screen membrane, and hybridized with full-lengh mouse Nox4 cDNA as described (34). Nox4 cDNA was a kind gift of Dr. K.-H. Krause (Geneva University Hospitals, Geneva, Switzerland).
Subcellular fractionation and measurement of Rac1 distribution. Subcellular fractionation was performed as previously described (14). The cytosolic and membrane fractions were subjected to 12.5% SDS-polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% low-fat milk in Tris-buffered saline and then incubated with a mouse monoclonal anti-Rac1 (1:1,000 dilution) antibody (Upstate Biotechnology). The Rac1 antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse IgG, and bands were visualized by enhanced chemiluminescence.
Measurement of Rac1 activity. Measurement of Rac1 activity was performed by affinity precipitation according to the modified method of Benard et al. (4). MCs were grown to near confluency in 100-mm dishes and were serum deprived for 48 h. Cells were then stimulated with 1 µM ANG II or 30 µM AA for the indicated times and lysed with the ice-cold cell lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin) at 4°C for 30 min. Cell lysates were then centrifuged for 30 min at 10,000 g at 4°C, and the supernatant was incubated with 30 µg of the p21-binding domain of p21-activated kinase (PAK-1) linked to glutathione S-transferase (GST-PAK-1-PBD) on glutathione-agarose beads (Upstate Biotechnology) for 30 min at 4°C. The beads were then washed in the cell lysis buffer, and the bound proteins were eluted in Laemmli sample buffer and separated by 12.5% SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed as described above for the measurement of Rac1 distribution.
Immunoprecipitation, Akt/PKB activity assay, and immunoblotting.
MCs were grown in 60- or 100-mm dishes and serum deprived for 48 h. All
incubations were carried out in serum-free RPMI 1640 at 37°C for a
specified duration. The cells were lysed in radioimmune precipitation buffer
[20 mmol/l Tris · HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml
aprotinin, 20 µg/ml leupeptin, 1% NP-40] at 4°C for 30 min. The cell
lysates were centrifuged at 10,000 g for 30 min at 4°C. Protein
was determined in the cleared supernatant using the Bio-Rad method.
Immunoprecipitation and Akt/PKB activity assay were performed as described
(15). For immunoblotting,
rabbit polyclonal anti-Akt1/PKB (Cell Signaling Technology; 1:1,000)
was used.
Measurement of production in intact MCs. Measurement of
released into the media of
MCs was performed by detection of ferricytochrome c reduction, as
described by Johnston (22).
Medium from growth-arrested MCs grown in six-well plates (2 x
106 cells/well) was aspirated and replaced with 1 ml of Hanks'
balanced salt solution without phenol red containing 80 µM cytochrome
c with or without 1 µM ANG II or 30 µM AA. At the end of the
incubation, the medium was removed and centrifuged for 2 min at 10,000
g at 4°C to stop the reaction. The optical density was measured
by spectrophotometry at 550 nm and converted to nanomoles of cytochrome
c reduced using the extinction coefficient
E550 = 21.0 x 103 M/cm. The
reduction of cytochrome c that was inhibitable by pretreatment with
superoxide dismutase (SOD; 50 µg/ml) represents
release.
NADPH oxidase assay. NADPH oxidase activity was measured by the
lucigenin-enhanced chemiluminescence method
(18). MCs were washed five
times in ice-cold phosphate-buffered saline and were scraped from the plate in
the same solution followed by centrifugation at 800 g at 4°C for
10 min. The cell pellets were resuspended in lysis buffer containing 20 mM
KH2PO4, pH 7.0, 1 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, and 0.5 µg/ml leupeptin. Cell suspensions
were homogenized with 100 strokes in a Dounce homogenizer on ice, and aliquots
of the homogenates were used immediately. To start the assay, 100 µl of
homogenate were added into 900 µl of 50 mM phosphate buffer, pH 7.0,
containing 1 mM EGTA, 150 mM sucrose, 5 µM lucigenin as the electron
acceptor, and 100 µM NADPH as an electron donor in the presence or absence
of 30 µM AA. Photon emission in terms of relative light units was measured
either every minute for 12 min or every 30 s for 10 min in a luminometer.
There was no measurable activity in the absence of NADPH. A buffer blank
(<5% of the cell signal) was subtracted from each reading before
calculation of the data.
production was expressed as relative chemiluminescence (light) units per
milligram of protein. Protein content was measured using the Bio-Rad protein
assay reagent.
Detection of intracellular ROS. The peroxide-sensitive fluorescent probe 2',7'-dichlorodihydrofluorescin diacetate (Molecular Probes) was used to assess the generation of intracellular ROS as described previously (15). This compound is converted by intracellular esterases to 2',7'-dichlorodihydrofluorescin, which is then oxidized by H2O2 to the highly fluorescent 2',7'-dichlorodihydrofluorescein (DCF). Differential interference contrast images were obtained simultaneously using an Olympus inverted microscope with a x40 Aplanfluo objective and an Olympus fluoview confocal laser-scanning attachment. DCF fluorescence was measured with an excitation wavelength of 488 nm light, and its emission was detected using a 510- to 550-nm band-pass filter.
Protein synthesis. [3H]Leucine incorporation into trichloroacetic acid-insoluble material was used to assess protein synthesis as described (15).
Statistical analysis. Results are expressed as means ± SE. Statistical significance was assessed by Student's unpaired t-test. Significance was determined as probability (P) <0.05.
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RESULTS |
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To evaluate the role of PLA2 in the activation of Rac1 by ANG II, we examined the effect of two structurally unrelated PLA2 inhibitors, mepacrine and aristolochic acid. Preincubation of MCs with the inhibitors markedly reduced Rac1 binding to PAK-1-PBD induced by ANG II (Fig. 1B). Furthermore, the direct addition of AA (30 µM) to MCs resulted in a twofold increase in Rac1 translocation to the membrane fraction with a maximum effect at 5 min (Fig. 1C, top). Treatment of MCs with AA induced a time-dependent increase in the amount of affinity-purified Rac1-GTP, which was detectable within 1 min and maximal at 2.55 min (Fig. 1C, bottom). The time course of activation of Rac1 by AA correlated well with the kinetics of Rac1 activation by ANG II. Collectively, these data indicate that the effect of ANG II on Rac1 activation is mediated by AA via activation of PLA2.
Rac1 mediates ANG II-induced Akt/PKB activation in MCs. The ability of ANG II to stimulate both Rac1 and Akt/PKB via an AA/PLA2-dependent mechanism suggests that the small GTPase Rac1 may mediate the effects of ANG II on Akt/PKB. Therefore, we assessed the effect of dominant negative form of Rac1 (N17Rac1) on Akt/PKB activation. Expression of Myc-tagged dominant negative N17Rac1 in MCs suppressed both ANG II- and AA-induced activation of Akt/PKB (Fig. 2A). The role of Rac1 in Akt/PKB activation is supported by the observation that constitutively active Rac1 (L61Rac1) is sufficient to fully activate Akt/PKB (Fig. 2B). Immunoblotting of the cell lysates using anti-Myc antibody confirms expression of the mutant protein. Collectively, these data demonstrate that the small GTPase Rac1 regulates Akt/PKB activation in response to ANG II and AA.
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Rac1 mediates the increase in production of ROS in response to ANG II
and AA. To position Rac1 and ROS in the signal transduction pathway
engaged by ANG II to regulate Akt/PKB activity, we first compared the ability
of ANG II and AA to influence the rate of
generation in
vector-transfected and dominant negative N17Rac1-transfected MCs by measuring
the SOD-inhibitable reduction of ferricytochrome c. As shown in
Fig. 3A, treatment of
vector-transfected cells with 1 µM ANG II or 30 µM AA resulted in a
rapid and time-dependent increase in
generation, which reached a
plateau at 510 min, corresponding to a four- to fivefold increase over
control values. A significant increase in
generation was observed as
early as 1 min after exposure to ANG II or AA. The time course of ANG II- and
AA-induced
generation
paralleled that of ANG II- and AA-induced Rac1 activation. Expression of
N17Rac1 mutant in MCs markedly reduced ANG II- and AA-stimulated
generation
(Fig. 3A).
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We next evaluated the effect of AA on NADPH-dependent superoxide-producing
activity in MCs transfected with the N17Rac1 mutant. With the use of
lucigenin-enhanced chemiluminescence, we found that in vector-transfected
cells addition of 30 µM AA to MC homogenates induced a time-dependent
eightfold increase in NADPH-driven
generation
(Fig. 3B). Expression
of N17Rac1 nearly abolished the activation of NADPH oxidase by AA.
Preincubation of homogenates with diphenyleneiodonium (DPI) abrogated
AA-stimulated NADPH oxidase activity. In addition, SOD (50 µg/ml) inhibited
AA-induced photoemission, thereby confirming identity of the product as
(Fig. 3B). These data
indicate that activation of superoxide-producing NADPH oxidase by AA is most
likely mediated via Rac1.
Dismutation of
spontaneously, or enzymatically, by SOD produces H2O2.
The production of intracellular H2O2 by vector- or
N17Rac1-transfected MCs in response to ANG II or AA treatment was demonstrated
with a fluorescence-based assay. ANG II- and AA-induced
H2O2 production was significantly blocked by the N17Rac1
mutant (Fig. 3C). The
role of Rac1 in generation of ROS is confirmed by the observation that
expression of constitutively active Rac1, L61Rac1, led to an increase in
intracellular ROS (Fig.
3C). To determine whether H2O2 is
the source of the fluorescence, cells were preincubated with catalase, an
enzyme that specifically metabolizes H2O2 to
H2O and O2. Catalase completely blocked the ANG II- and
AA-stimulated increase in DCF fluorescence, suggesting that intracellular
H2O2 is primarily responsible for the fluorescence
signal (Fig. 3C).
Collectively, these data demonstrate that the rapid release of ROS elicited by
ANG II and AA is mediated by Rac1 activation of a superoxide-generating
NAD(P)H oxidase in MCs.
A Nox4-containing NAD(P)H oxidase is implicated in ANG II- and
AA-induced ROS generation in MCs. It has been proposed that
superoxide-producing NAD(P)H oxidases similar to the phagocyte NADPH oxidase
exist in nonphagocytic cells
(11,
16,
17,
19,
23,
46). The subsequent search for
nonphagocyte NAD(P)H oxidases led to the discovery of the Nox family of
gp91phox homologues
(25). Because the
gp91phox homologue Nox4 is highly expressed in the kidney
(12,
33), we determined whether a
Nox4-based oxidase may account for the ROS generation in response to ANG II
and AA. Northern blot analysis reveals that a 3.1-kb Nox4 transcript is highly
expressed in rat MCs (Fig.
4A, left). Transfection of MCs with
phosphorothioate-modified AS oligonucleotides but not S oligonucleotides for
Nox4 markedly decreased Nox4 mRNA expression
(Fig. 4A,
right). AS also caused a significant decrease in basal
NADPH-dependent superoxide-producing activity
(Fig. 4B), suggesting
that the NADPH oxidase activity in MCs is at least partly due to Nox4. We next
assessed the role of Nox4 in ANG II- and AA-induced ROS production.
Transfection of AS Nox4 completely inhibited both ANG II- and AA-stimulated
superoxide-specific ferricytochrome c reduction, whereas S Nox4 had
no effect (Fig. 4C).
Of note, as described above for the NADPH-dependent
generation, basal production
of
in intact cells was also
decreased by AS Nox4 treatment, suggesting that the Nox4-containing oxidase is
the major source of ROS in MCs. Similarly, ANG II- and AA-stimulated ROS
generation as measured by 2',7'-dichlorodihydrofluorescin
diacetate fluorescence-based assay was significantly reduced in MCs
transfected with AS Nox4 (Fig.
4D). Conversely, fluorescence was not affected by
transfection of MCs with S Nox4. Together, these results indicate that Nox4 is
required for an increase of ROS production by ANG II and AA in MCs.
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Nox4 mediates ANG II-induced redox-dependent Akt/PKB activation and protein synthesis in MCs. We examined the effect of Nox4 AS oligonucleotides on activation of the AA-mediated redox-dependent activation of Akt/PKB. Transfection of MCs with AS Nox4, but not S Nox4, prevented activation of Akt/PKB in response to ANG II or AA (Fig. 5A). Moreover, transfection of MCs with AS Nox4 but not S Nox4 blocked Rac1-induced Akt/PKB activation (Fig. 5B), indicating that Nox4 is a downstream target of Rac1. These data support a role for Nox4 in the redox signaling cascade triggered by ANG II and mediated by AA and Rac1 to activate Akt/PKB.
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To assess the role of Nox4 in protein synthesis, we tested the effect of AS Nox4 on ANG II- and AA-stimulated [3H]leucine incorporation. As shown in Fig. 6, AS Nox4 but not S Nox4 significantly reduced stimulation of protein synthesis by ANG II and AA.
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DISCUSSION |
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The cellular mechanisms by which ANG II exerts its biological activities
are not completely defined. Involvement of small GTPases of the Rho family in
ANG II signaling is suggested by several studies that describe a critical role
for Rho in ANG II-induced vascular smooth muscle cell hypertrophy
(1,
45) and for Rac1 in PAK and
c-Jun NH2-terminal kinase activation
(28,
31). Although ANG II is known
to activate Akt/PKB in many cell types
(15,
38), the small G protein Rac
has not been implicated in the activation of Akt/PKB by ANG II. Here, we
demonstrate that ANG II stimulates Rac1 and that inhibition of Rac1 by
expression of dominant negative Rac1 blocked ANG II-induced Akt/PKB
activation, indicating a role for the small G protein Rac1. Our findings are
in contrast to other studies in endothelial cells
(41), COS cells
(24), or neuronal cells
(39), demonstrating that Rac1
and Akt/PKB are activated in parallel, downstream of a phosphoinositide
3-kinase (PI3-K)-regulated pathway. However, we recently showed that in MCs
ANG II-induced activation of Akt/PKB is PI3-K independent
(15). On the other hand, Rac
has recently been implicated as an upstream activator of Akt/PKB with
stimulation of the T cell antigen receptor
(13) and the Toll-like
receptor (2). These data
demonstrating that PLA2 inhibitors mepacrine and aristolochic acid
abrogated ANG II-induced Rac1 activation and that AA mimics the stimulatory
effect of ANG II on Rac1 indicate that AA acts as an upstream activator of
Rac1 in the cascade linking PLA2-coupled ANG II receptor to the
small GTPase. In neutrophils, AA induces the translocation of the GTP-bound
active form of Rac from cytosol to membrane
(30). Furthermore, Chuang et
al. (9) found that AA can
disrupt the binding of Rac to the GDP dissociation inhibitor RhoGDI (which
maintained Rac1 in its inactive form) leading to Rac activation. Rac1
activation appears to be agonist dependent as well as cell specific. In
fibroblasts and in response to tumor necrosis factor-, Rac acts
upstream of cytosolic PLA2
(43,
44). It is reasonable to
assume that differences in Rac1 effector pathways, as well as in the nature of
the isoform of PLA2 implicated in MCs, at least in part, may
account for these differences.
Akt/PKB is a target of ROS in MCs and ROS play a role as second messengers
mediating the stimulatory effect of ANG II and AA on Akt/PKB
(15). To determine if ROS act
downstream of Rac1, we introduced a dominant negative N17Rac1 into MCs, which
resulted in a marked decrease in the level of ROS produced in response to ANG
II and AA. Moreover, constitutively active L61Rac1 markedly increases ROS
generation even in the absence of ANG II or AA treatment. Therefore, one
function of small G protein Rac is to regulate redox-dependent signal
transduction pathways. Examples of such a sequential pathway in which an
agonist or other stimuli lead to activation of small GTPase Rac and
subsequently to the generation of ROS are well documented
(32,
3537,
40,
43,
44). However, it is currently
unclear exactly how Rac regulates the production of ROS in nonphagocytic
cells. Importantly, we show that stimulation of superoxide-producing NADPH
oxidase activity by AA occurs through Rac1, revealing the existence of a
functional link between Rac1 and NADPH-dependent ROS generation. In phagocytic
cells, Rac proteins are involved in the assembly of the NADPH oxidase system,
responsible for transferring electrons from NADPH to molecular oxygen with the
subsequent production of ,
which is then rapidly dismutated spontaneously or enzymatically to
H2O2. The phagocyte oxidase consists of two plasma
membrane-associated proteins, gp91phox (the catalytic
subunit) and p22phox, which comprise flavocytochrome
b558, and two cytosolic factors,
p47phox and p67phox
(10,
27). Because many of the
components of the NADPH oxidase system, such as p47phox,
p67phox, and p22phox, appear to be
expressed in a variety of ROS-producing nonphagocytic cell types including MCs
(19,
23), it is tempting to
speculate that there exists an NAD(P)H oxidase enzyme complex similar to the
phagocyte oxidase whose activity may be regulated by Rac. However, the
apparent absence in these cells of gp91phox, the major
electron transport subunit of the enzyme, has cast doubt on this presumption.
The recent identification in nonphagocytic cells of novel
gp91phox homologues termed Nox proteins provides a
possible explanation for this paradox
(25). The present study
indicates that Nox4, the gp91phox homologue, is highly
expressed in MCs and that impairment of its function with AS oligonucleotides
inhibits NADPH oxidase activity, providing the first identification of the
catalytic subunit of NAD(P)H oxidase in MCs. Moreover, Nox4 is clearly
required for ANG II- and AA-induced superoxide generation and Akt/PKB
activation. Furthermore, Nox4 mediates the stimulation of Akt/PKB by Rac1.
These data support the concept that a Nox4-based oxidase is coupled to ANG II
redox signaling in MCs.
Although the oxidases of the Nox family are proposed to play a role in a
variety of biological processes such as cell growth and angiogenesis, hypoxic
response, immune function, bone resorption, and proton transport
(12,
19,
25,
33,
46), their bona fide functions
are largely unknown. The molecular mechanisms implicated in MC growth are
poorly understood. We previously described that stimulation of protein
synthesis in response to ANG II is mediated via a DPI-sensitive
redox-dependent signaling cascade involving Akt/PKB activation in MCs
(15). In this study, we report
that inhibition of Nox4 by AS oligonucleotide treatment markedly reduced ANG
II-induced protein synthesis, a critical step in MC hypertrophy. We also find
that impairment of Nox4 function inhibited protein synthesis in response to
AA, indicating that Nox4 is also a downstream target of AA in this pathway.
These observations provide the first evidence that Nox4 contributes to protein
synthesis. Nox4 has been proposed to be a key actor in events as diverse as
oxygen sensing in the kidney
(12,
33), bone resorption
(46), cell growth inhibition
in Nox4-overexpressing fibroblasts
(12,
33), and cell growth induction
in melanoma cells (7). The
incomplete inhibition of ANG II-stimulated protein synthesis, in contrast to
abolition of ANG II-/AA-induced superoxide production and Akt/PKB activation
to Nox4 AS oligonucleotides, suggests the involvement of additional
redox-insensitive signaling mechanisms in the regulation of protein synthesis
by ANG II. For instance, redox-dependent activation of p38-mitogen-activated
protein kinase and Akt/PKB by ANG II is not sufficient for vascular smooth
muscle cell hypertrophy, but rather requires parallel, independent activation
of the redox-insensitive extracellular signal-regulated kinases 1 and 2
(19,
26). Alternatively, other
non-Nox4 oxidases may be involved. Interestingly, in vascular smooth muscle
cells, at least two NAD(P)H oxidases are expressed: a Nox1-based oxidase and a
Nox4-based oxidase (26,
32). It has been proposed that
the different Nox proteins mediate different phases of the response to ANG II
(26,
32). This study and our recent
observations are consistent with the existence of a Rac1- and Nox4-dependent
Akt/PKB activation pathway leading to stimulation of protein synthesis: ANG II
PLA2
AA
Rac1
Nox4
ROS
Akt/PKB
protein synthesis (Fig.
7). The precise mechanism by which ANG II, Nox4, and Akt/PKB
stimulate protein synthesis remains to be determined. Protein synthesis is a
critical step during cell hypertrophy. Cell cycle proteins have been
incriminated in cell hypertrophy
(21,
42). The cyclin kinase
inhibitor p27kip1 mediates ANG II-induced hypertrophy in
tubular epithelial cells (21)
and glucose-induced hypertrophy in MCs
(42). In addition,
platelet-derived growth factor (PDGF) inhibits p27kip1,
resulting in enhanced DNA synthesis in MCs
(8). The effect of PDGF on
p27kip1 is due to sustained PI3-K-dependent activation of
Akt/PKB (8). It is important to
emphasize that activation of Akt/PKB by ANG II is not sufficient to induce
proliferation of MCs (15),
suggesting that the extent and temporal activation of this signaling pathway
regulate a different biological activity in MCs. Thus activation of Akt/PKB
may result in hypertrophy or proliferation depending on the nature of the
agonist and target cells. The hypertrophic effect of ANG II more likely
triggers additional specific signaling cascade(s) concomitantly with Akt/PKB
activation. The nature and redox sensitivity of these pathways, as well as
their impact on cell cycle events, remain to be elucidated.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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