Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
Submitted 10 December 2002 ; accepted in final form 1 April 2003
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ABSTRACT |
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rat pancreatic islets; -cell; signaling; pertussis toxin; G proteins
The inhibitory pathways act in contradictory fashion on many of the same
sites as the stimulatory pathways
(30). For example, 1)
inhibitors activate the ATP-sensitive K (KATP) channel, repolarize
or hyperpolarize the cell membrane, and thus inhibit the action of glucose
that depolarizes the membrane via closure of this channel; 2)
inhibitors decrease the activity of the L-type Ca2+ channel by two
means, indirectly by the activation of the KATP channel and
hyperpolarization, and directly by inhibiting the channel itself; 3)
inhibitors decrease the activity of adenylyl cyclase and lower cAMP levels,
thus reducing the effect of cAMP to potentiate insulin secretion. However, the
most dominant inhibitory mechanism (4) is at a "distal"
site in stimulus-secretion coupling. This distal site is at a crucial, late
step in exocytosis, beyond the elevation of intracellular Ca2+ and
beyond the potentiating actions of cAMP and diacylglycerol. Information on the
G protein interactions with these four major inhibitory sites is limited. It
was reported that activation of KATP channels was due to
-subunits of Gi and/or Go proteins
(43). The
-subunits of
Gi-2 and Gi-3 mediate the decreased activity of adenylyl
cyclase in the
-cell
(18).
In this study, we have taken advantage of two well-known inhibitors of
protein acylation, 2-bromopalmitate (2-BrP)
(8,
20,
36,
39) and cerulenin
(11,
29,
31,
41), to investigate the distal
inhibitory mechanisms of insulin secretion. By mediating protein-membrane and
protein-protein interactions, protein acylation is thought to be important for
cell signaling, e.g., in the T cell
(25,
26,
34,
39) and -cell
(6,
31,
41,
44). We found that inhibitors
of protein acylation blocked the distal inhibitory effects of NE,
somatostatin, galanin, and PGE2. The data suggest that the distal
mechanism of inhibition of insulin secretion involves the acylation of a
protein or proteins that are critical to the normal process of exocytosis.
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MATERIALS AND METHODS |
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Measurements of insulin release. Insulin release was measured under static incubation conditions at 37°C with batches of five size-matched islets per tube. When Ca2+ was present in the buffer, islets were preincubated in regular KRBH buffer for 60 min. The islets were then incubated in the presence and absence of test agents (as we will describe) for 15 or 60 min. Under Ca2+-free conditions, the islets were washed three times with Ca2+-free KRBH buffer containing 1 mM EGTA (Ca2+-free KRBH-EGTA buffer) and preincubated in Ca2+-free KRBH-EGTA buffer for 60 min. They were then incubated in fresh Ca2+-free KRBH-EGTA buffer in the presence and absence of test agents for 60 min. At the end of the incubations, samples were taken and kept at -20°C until radioimmunoassayed for insulin using a charcoal separation method. Insulin secretion was expressed as fractional release, the percentage of the insulin content of the islets that was released over 15 or 60 min (15, 41). When exogenous free fatty acids (FFAs) and their analogs were used, the concentrations of the FFAs were estimated from the molar ratios of the fatty acids and FFA-free BSA, as reported in the literature (16, 18). In the last series of experiments (on the effects of arachidonic and eicosapentaenoic acids), the FFA-free BSA concentration was changed from 0.5 to 0.4%, because the new batch of BSA appeared to bind 2-BrP differently from the old batch, and the complete blockade of NE action by 2-BrP was obtained at 0.4%.
Palmitate oxidation measurement. Palmitate oxidation was measured as previously reported with minor modifications (5, 41). Briefly, groups of 15 islets (in duplicate) were incubated at 37°C for 1 h in 200 µl of KRB buffer, containing 0.2 µCi [1-14C]palmitate, 0.8 mM L-carnitine, and other components as indicated. At the end of the incubation, islet metabolism was terminated by adding 200 µl of 0.5 M HCl to the incubation mixture. 14CO2 was collected overnight in 300 µl of 1 M NaOH and measured by liquid scintillation spectrometry. One batch of islets was used for each experiment, so that all of the determinations were paired.
Measurement of palmitate incorporation into lipids. This procedure was performed as previously described with minor modification (1, 5). Briefly, groups of 3050 islets were incubated at 37°C for 1 h in 200 µl of KRB buffer, containing 0.2 µCi [1-14C]palmitate, 0.8 mM L-carnitine, and other components as indicated. At the end of the incubations, the incubation media were removed, and the islets were washed twice with 1 ml of ice-cold PBS. Two hundred microliters of 0.2 M NaCl were added to the islet pellet, and the mixture was immediately frozen in liquid N2. After the islet pellet was thawed, 750 µl of chloroform-methanol (2:1) and 50 µl of 0.1 N KOH were added. After vigorous vortexing for 3 min, the phases were separated by centrifugation at 2,000 g for 20 min. The top aqueous layer was removed, and the bottom lipid-soluble layer was washed once with 200 µl of methanol-water-chloroform (48:47:3). Two hundred microliters of the lipid-soluble phase were added to 5 ml of BioSafe2 scintillation mixture, and incorporation of radiolabel into lipids was quantified by liquid scintillation spectrometry.
Measurement of cAMP. This measurement was performed as previously described with minor modification (9, 13, 40). Briefly, groups of 20 islets were preincubated at 37°C for 1 h in 200 µl of KRBH buffer, in the presence or absence of cerulenin as indicated. The islets were then incubated for 15 min in 200 µl of KRBH containing 5.6 mM Glc, 50 µM IBMX, 6 µM forskolin (Fsk), and 10 µM propranolol, with or without testing reagents as indicated. The incubation was terminated by addition of 300 µl of 0.2 N HCl. The tubes were then placed in boiling water for 15 min with occasional vortexing. cAMP was determined by the Biotrak cAMP enzymeimmunoassay (EIA) system.
Materials. Glucose, 2-BrP, palmitate, 16-hydroxy palmitate (16-OH palmitate), 2-hydroxy myristate (2-OH myristate), FFA-free BSA, NE, forskolin, 12-O-tetradecanoylphorbol 13-acetate (TPA), L-carnitine, somatostatin, galanin, and PGE2 were obtained from Sigma (St. Louis, MO). Cerulenin was purchased from Fluka (Milwaukee, WI). [1-14C]palmitate was obtained from New England Nuclear (Boston, MA). The Biotrak cAMP EIA system was from Amersham Pharmacia Biotech (Piscataway, NJ).
Statistical methods. All data are shown as means ± SE. Statistical significance was evaluated by two-way ANOVA or t-tests as appropriate. Differences were considered significant at P < 0.05.
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RESULTS |
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Experiments were next carried out under normal conditions in the presence
of Ca2+. As shown in
Fig. 2, insulin secretion was
stimulated by 11.1 mM glucose, and this was not affected by 20 µM 2-BrP
(control, 1.98 ± 0.41 vs. 2-BrP, 2.12 ± 0.47% of content/h;
= -0.14 ± 0.37; P = 0.73), a finding that is
consistent with previous studies
(32,
45). NE inhibited
glucose-stimulated insulin secretion completely (1.98 ± 0.41 vs. 0.58
± 0.09% of content/h;
= 1.40 ± 0.10; P <
0.001) in the absence of 2-BrP but incompletely (1.98 ± 0.41 vs. 1.01
± 0.17% of content/h;
= 0.97 ± 0.06; P <
0.05) in the presence of 2-BrP. Thus the blocking effect of 2-BrP on the
inhibitory action of NE in the presence of extracellular
Ca2+ was less than that in the absence of extracellular
Ca2+. Under Ca2+-free conditions,
the only functional inhibitory mechanism for NE to inhibit insulin secretion
is at the distal site, and this is completely blocked by 2-BrP. In the
presence of extracellular Ca2+, the inhibitory effect of
NE seen when the islets are exposed to 2-BrP could be exerted via activation
of the KATP channels, reduced Ca2+ influx,
and inhibition of adenylyl cyclase. These combined data suggest that only the
distal inhibitory action of NE is blocked by 2-BrP.
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To exclude the possibility that the action of 2-BrP was due to nonspecific
effects rather than inhibition of protein acylation, we examined the effects
of palmitate and 16-OH palmitate under the same
Ca2+-free conditions as we tested 2-BrP. These two
compounds are structurally similar to 2-BrP but do not block protein acylation
(39) as does 2-BrP. Insulin
secretion was not changed by either 20 µM palmitate (control, 2.64 ±
0.49 vs. palmitate, 2.72 ± 0.33% of content/h; = -0.08 ±
0.39; P = 0.85) or 20 µM 16-OH palmitate (control, 2.29 ±
0.26 vs. 16-OH palmitate, 2.48 ± 0.38% of content/h;
= -0.19
± 0.15; P = 0.21). It was inhibited equally by NE in the
absence and presence of 20 µM palmitate (control, 0.74 ± 0.16 vs.
palmitate, 0.51 ± 0.06% of content/h;
= 0.23 ± 0.11;
P = 0.12), and in the absence and presence of 20 µM 16-OH
palmitate (control, 0.78 ± 0.11 vs. 16-OH palmitate, 0.92 ±
0.12% of content/h;
= -0.14 ± 0.17; P = 0.31).
To distinguish between the two most common forms of protein acylation,
palmitoylation and myristoylation, we examined the effect of 2-OH myristate,
which blocks myristoylation but not palmitoylation
(19). Under
Ca2+-free conditions, insulin secretion was stimulated
by 11.1 mM glucose in the presence of TPA and Fsk and was completely inhibited
by NE (2.23 ± 0.37 vs. 0.79 ± 0.10% of content/h; = 1.44
± 0.43; P < 0.05). In the presence of 20 µM 2-OH
myristate, glucose-stimulated secretion was not altered by 2-OH myristate
(control, 2.23 ± 0.37 vs. 2-OH myristate, 2.58 ± 0.39% of
content/h;
= -0.35 ± 0.40; P = 0.44) and was still
completely inhibited by NE (2.58 ± 0.39 vs. 0.93 ± 0.18% of
content/h;
= 1.65 ± 0.51; P < 0.05). Hence, NE
inhibited glucose-stimulated insulin secretion to a similar extent in the
absence and presence of 20 µM 2-OH myristate (0.79 ± 0.10 vs. 0.93
± 0.18% of content/h;
= -0.14 ± 0.11; P =
0.23). Therefore, myristoylation does not appear to be involved in the
mechanism of inhibition.
Effect of cerulenin to block the inhibition of insulin secretion by
NE. The effects of cerulenin, a well-known inhibitor of protein
acylation, are shown in Fig. 3.
In this series of experiments, insulin secretion was evoked by a depolarizing
concentration of 50 mM KCl in the presence of TPA and Fsk. We used KCl because
nutrient-stimulated, but not non-nutrient-stimulated, insulin secretion is
inhibited by cerulenin (41).
Fsk was used to keep intracellular cAMP at supermaximal levels and, like TPA,
to maximize the secretory response. In these experiments, cerulenin was
included only in the preincubation period. In the absence of cerulenin, NE
inhibited the stimulated insulin secretion by 96% (P < 0.001).
Cerulenin blocked the inhibitory action of NE in a concentration-dependent
manner. The IC50 for cerulenin to block the action of NE was 10
µg/ml (45 µM), and complete blockade was seen at 100 µg/ml (450
µM). In separate experiments, basal insulin secretion in the presence of
2.8 mM glucose was not significantly affected by 100 µg/ml cerulenin
(control, 0.40 ± 0.08 vs. cerulenin, 0.48 ± 0.10% of content/15
min; = -0.08 ± 0.04; P = 0.16), nor was insulin
secretion stimulated by the combination of KCl, TPA, and forskolin (control,
0.96 ± 0.23 vs. cerulenin, 1.14 ± 0.25% of content/15 min;
= -0.18 ± 0.32; P = 0.63). The fact that 100 µg/ml
cerulenin did not alter either basal or KCl-stimulated insulin secretion
suggests that cerulenin affects neither the islet's ability to secrete insulin
nor its membrane integrity to permit leakage of insulin. Under these
experimental conditions, in the presence of 50 mM KCl and 6 µM Fsk, the
effects of NE on the KATP channel to hyperpolarize the cell and to
lower cAMP levels are bypassed. Therefore, the data presented so far with
2-BrP and cerulenin, two structurally different inhibitors of protein
acylation, strongly suggest that protein acylation is involved in the distal
inhibitory action of NE.
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Effects of 2-BrP on the inhibition of insulin secretion by somatostatin, galanin, and PGE2 under Ca2+-free conditions. Three other physiological inhibitors of insulin secretion, somatostatin, galanin, and PGE2, were tested to see whether the effect of 2-BrP was specific for NE or whether it blocked the effects of other inhibitors also. As seen in Fig. 4, under Ca2+-free conditions, 1 µM somatostatin, 100 nM galanin, and 10 µM PGE2 inhibited glucose-stimulated insulin secretion by 73% (P < 0.01), 61% (P < 0.01), and 66% (P < 0.01), respectively. In contrast, in the presence of 20 µM 2-BrP, these inhibitors had no effect on insulin secretion; their inhibitory actions were completely blocked. Thus the effect of 2-BrP is a general effect against several inhibitors.
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Effects of NE, 2-BrP, and cerulenin on palmitate metabolism. It
has been reported that NE reduced the oxidation of palmitate as well as the
incorporation of glucose into phospholipids and neutral lipids in the presence
of palmitate through an 2-adrenergic mechanism
(35). 2-BrP and cerulenin have
also been reported to affect lipid metabolism
(11,
17). Consequently, we examined
the effects of NE, 2-BrP, and cerulenin on lipid metabolism to determine
whether a change in metabolism was a contributory factor in the inhibitory
effects of 2-BrP and cerulenin on the action of NE. As expected, and as shown
in Table 1, palmitate oxidation
was significantly lower in the presence of 11.1 mM glucose than in the
presence of 2.8 mM glucose (0.38 ± 0.03 vs. 0.51 ± 0.04 pmol
· islet-1 · h-1;
= -0.13 ± 0.02; P < 0.01). Palmitate oxidation was
not significantly affected by NE at either 2.8 mM or 11.1 mM glucose.
Similarly, NE did not significantly change [1-14C]palmitate
incorporation into cellular lipids at either 2.8 mM or 11.1 mM glucose.
Glucose at 11.1 mM increased [1-14C]palmitate incorporation into
lipid by 20% (P < 0.001) relative to incorporation in the presence
of 2.8 mM glucose. Furthermore, both 2-BrP and cerulenin decreased 11.1 mM
glucose-induced palmitate oxidation by 66% in the absence and presence of NE.
However, the effects of 2-BrP and cerulenin on [1-14C]palmitate
incorporation were different. Cerulenin had no effect, whereas 2-BrP increased
11.1 mM glucose-induced palmitate esterification.
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Effects of NE, 2-BrP, and cerulenin on cAMP production. The data
so far demonstrated that the distal site of NE action was blocked by 2-BrP and
cerulenin. However, it was not clear whether 2-BrP and cerulenin acted at any
of the other sites of NE action. Therefore, we examined their effects on cAMP
production of islets. As shown in Table
2, 10 µM NE significantly decreased Fsk-stimulated cAMP
production from 580 ± 57 to 275 ± 52 fmol ·
islet-1 · 15 min-1, a 53%
inhibition (P < 0.01). More importantly, in the presence of 20
µM 2-BrP or 100 µg/ml cerulenin, NE was still able to suppress cAMP
production (Table 2). This
demonstrates that neither 2-BrP nor cerulenin blocks the binding of NE to the
2-adrenergic receptor, receptor activation of related G
proteins, and G protein inhibition of adenylyl cyclase.
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Effect of 2-BrP on the inhibition of insulin secretion by high
concentrations of NE. On the basis of these data on cAMP production, the
displacement of NE from the 2-adrenergic receptor appears
not to contribute to the blocking effect of 2-BrP on NE action. To strengthen
this point, we further tested the effect of 2-BrP in the presence of a very
high NE concentration (100 µM) under Ca2+-free
conditions. As shown in Fig. 5,
insulin secretion was stimulated by 11.1 mM glucose in the presence of TPA and
Fsk, and this was not affected by 2-BrP (control, 3.65 ± 0.45 vs.
2-BrP, 3.18 ± 0.35% of content/h;
= -0.47 ± 0.34;
P = 0.27). This stimulated insulin secretion was completely inhibited
by NE at both 10 µM (3.65 ± 0.45 vs. 0.73 ± 0.08% of
content/h;
= 2.92 ± 0.52; P < 0.01) and 100 µM
(3.65 ± 0.45 vs. 0.86 ± 0.19% of content/h;
= 2.78
± 0.62; P < 0.01). However, in the presence of 2-BrP,
insulin secretion was not affected by NE at either 10 µM (3.65 ±
0.45 vs. 2.89 ± 0.21% of content/h;
= 0.76 ± 0.48;
P = 0.40) or 100 µM (3.65 ± 0.45 vs. 3.44 ± 0.62% of
content/h;
= 0.21 ± 0.99; P = 0.73). Therefore,
increasing the NE concentration, even to 100 µM, did not reinstate its
inhibitory action on insulin secretion.
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Effects of etomoxir and polyunsaturated fatty acids on the inhibition
of insulin secretion. 2-BrP has been used extensively to suppress
carnitine palmitoyltransferase I (CPT I) activity. To determine whether the
effects of 2-BrP to block the inhibitory effect of NE could be related to this
suppression of CPT I activity, we examined the effects of another CPT I
inhibitor, etomoxir (45). In
paired experiments shown in Fig.
5, etomoxir was used under exactly the same conditions as was
2-BrP. In contrast to 2-BrP, insulin secretion was inhibited by NE to a
similar extent in the absence and presence of etomoxir (0.73 ± 0.08 vs.
1.06 ± 0.17% of content/h; = -0.33 ± 0.14; P =
0.10). Hence, inhibition of CPT I activity is not a contributory factor in the
effects of 2-BrP on NE action.
In further control experiments, we asked whether polyunsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, could mimic the effect of 2-BrP on NE-mediated inhibition of insulin secretion. Both arachidonic acid and eicosapentaenoic acid interfere with T cell signal transduction, an action that is thought to be due to the inhibition of protein palmitoylation (39). Under Ca2+-free conditions, NE failed to inhibit insulin secretion in the presence of 0.5 mM arachidonic acid or eicosapentaenoic acid (data not shown).
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DISCUSSION |
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Inhibition of protein acylation by 2-BrP and cerulenin is considered responsible for the effects shown in this study. The reasons for this follow. 1) If the suppression of CPT I activity (23) were the reason for 2-BrP to block the inhibitory action of NE, etomoxir would be expected to mimic this effect, because etomoxir is also a CPT I inhibitor. Instead, etomoxir had no effect on the action of NE. Additionally, cerulenin is not a CPT I inhibitor. Furthermore, whereas both 20 µM 2-BrP and 100 µg/ml cerulenin inhibited palmitate oxidation of 11.1 mM glucose, only 2-BrP significantly increased 11.1 mM glucose-induced palmitate incorporation into lipids. Hence, the different influences on palmitate metabolism by 2-BrP and cerulenin suggest that palmitate metabolism has no significant role in the effects of 2-BrP and cerulenin on the action of NE. 2) The similarities between the concentration-response characteristics of cerulenin to block the action of NE in this study and the concentration-response characteristics for the inhibition of protein acylation in other reports (11, 29, 41) suggest that cerulenin is functioning under our conditions as an inhibitor of protein acylation. 3) The short incubation time of our experimental conditions precludes the long-term effects of cerulenin and 2-BrP. These include the effects of cerulenin on apoptosis, cell growth, DNA, RNA, and protein synthesis (10, 11, 21, 22, 29) and the effects of 2-BrP on the levels of insulin and uncoupling protein-2 mRNA (3, 38).
Because of the fast, reversible nature of inhibition of insulin secretion by most physiological inhibitors, the rapidly reversible palmitoylation reaction would be more important for modulating this function (7) than the relatively slower myristoylation reaction. This is in accord with our observation that 2-OH myristate, which blocks myristoylation but not palmitoylation (19), had no influence on the inhibitory action of NE.
There is indirect evidence to indicate that NE inactivates long-chain acyl-CoA synthetase and other enzymes of glycerolipid synthesis (35). However, in the present study, we did not find any effects of NE on palmitate oxidation. Nor did we find any effects on esterification at low or high concentrations of glucose. Moreover, 2-BrP was still able to increase [1-14C]palmitate incorporation into cellular lipid at 11.1 mM glucose even in the presence of NE. Thus our data provide no evidence for an effect of NE on palmitate metabolism under our experimental conditions.
With respect to the other actions of NE in the -cell, neither 2-BrP
nor cerulenin affected the action of NE to decrease cAMP production. This
indicates that inhibitors of protein acylation do not block the interaction of
NE at the
2-adrenergic receptor, related G protein
activation, or inhibition of adenylyl cyclase. This further excludes the
possibility that the effects of 2-BrP and cerulenin are due to nonspecific
effects.
In a study using capacitance measurements in mouse pancreatic -cells,
activation of calcineurin was proposed as the distal mechanism for NE,
galanin, and somatostatin to inhibit insulin secretion
(24). Calcineurin is a
Ca2+- and calmodulin-dependent protein phosphatase
(42). However, NE-induced
inhibition of insulin secretion was unaffected by blockade of calcineurin in
INS-1 (12),
HC-9, and
HIT cell lines (L. M. Shen and G. W. G. Sharp, unpublished observations).
Moreover, under stringent Ca2+-free conditions that
presumably exclude the Ca2+-dependent activity of
calcineurin, NE completely blocks stimulated insulin secretion
(13). Additionally, under
these Ca2+-free conditions, 2-BrP still effectively
blocked the distal inhibitory effects of NE, somatostatin, galanin, and
PGE2. Calcineurin does not appear to be involved in the mechanism
of the distal inhibition of insulin secretion. PTX-sensitive Gi and
Go proteins can exert their inhibitory effects on insulin secretion
via two possible mechanisms: 1) direct action of the Gi
and Go subunits (
and/or
) on proteins critical
for exocytosis; 2) activation of an enzyme or protein mediator of the
inhibition by the
- and/or
-subunits. Relevant to the
latter, our data suggest that distal inhibition of insulin secretion is
achieved by activation of a specific protein acyltransferase.
It is known that, regarding inhibition of insulin secretion,
2-adrenergic receptor, Gi
1 and
Go
, SNAP-25, and synaptotagmin are palmitoylated, whereas
Gi
1 and Go
are myristoylated
(2,
4,
28,
33,
37). Finally, on the basis of
our finding that inhibitors of protein acylation blocked the distal site of
inhibitors of insulin secretion, we propose that the inhibitors of insulin
secretion, acting via PTX-sensitive G proteins, activate a specific protein
acyltransferase that results in acylation of a protein or proteins critical to
exocytosis. This particular acylation and subsequent disruption of essential
and precise interactions of the exocytotic machinery would block insulin
secretion. It should be noted that protein acyltransferase would be a novel
target for G protein activation.
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DISCLOSURES |
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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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