1Department of Internal Medicine, Scott and White Clinic, and 2Department of Medical Physiology, Texas A&M University System Health Science Center College of Medicine, Temple, Texas 76508
Submitted 25 November 2002 ; accepted in final form 19 March 2003
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ABSTRACT |
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oxidative stress; cytokine; heat shock protein; cell cycle regulation
Transforming growth factor (TGF)- is a potent inhibitor of cell
growth and is a member of a superfamily of dimeric polypeptide growth factors
that are distinguished from most other cytokines by their ability to limit
cell growth (17,
26). TGF-
has diverse
effects in addition to regulation of cell growth and participates in
morphogenesis and differentiation
(18). The varied cellular
functions of this pleiotropic cytokine underlie its critical role in
pathophysiological processes such as carcinogenesis. TGF-
receptor
molecules are expressed ubiquitously, and growth inhibition has been shown to
occur in a broad range of epithelial, endothelial, and hematopoietic cells
(19).
TGF- inhibits cell division by triggering a program of
cyclin-dependent kinase (cdk) inhibitory responses that culminate in
G1 cell cycle arrest. In epithelial cells from the skin, lung, and
breast, TGF-
rapidly elevates expression of p15INK4B
(34). p15INK4B
binds to and inhibits cdk4 and cdk6 as well as displacing the
p27KIP1 protein from these complexes
(30,
43). In proliferating cells,
p27KIP1 remains bound to cdk4 and cdk6 complexes, but when
mobilized by TGF-
, p27KIP1 binds to and inhibits cdk2
(29). Prevention or inhibition
of G1 cyclin-cdk activation by TGF-
inhibits the
phosphorylation of the retinoblastoma protein
(14). In addition, E2F
activity is also impaired by TGF-
(35). In keratinocytes, colon
and ovarian epithelial cells, TGF-
additionally elevates the expression
of the p27KIP1-related inhibitor p21WAF1/CIP1, and in
mammary epithelial cells, it represses the cdk-activating phosphatase cdc25A.
Furthermore, in many cell types, TGF-
inhibits c-myc expression, which
may play a pivotal role in the loss of G1 cyclins, downregulation
of Cdc25A, or induction of the cdk inhibitor p15INK4B
(4). Thus growth inhibition by
TGF-
involves altered cellular expression of multiple cell cycle
regulatory molecules.
Proteasomal degradation controls the cellular expression of many key cell
cycle regulatory molecules including p27KIP1, which participates in
TGF- growth inhibition
(23,
27). The degradation of a
diverse range of proteins is facilitated by the presence of multiple catalytic
activities within the proteasome with the capability for the endoproteolytic
cleavage of peptide bonds on the carboxyl side of acidic, basic, and
hydrophobic residues of proteins
(32). The activities can be
differentially regulated with alterations in biological significance.
Differential regulation of these activities can occur by changes in
proteasomal subunit composition and may have biological significance
(1,
11). To determine the role of
dysregulated proteasomal function during cell growth inhibition by TGF-
,
we first characterized the effect of TGF-
on various
proteasome-associated hydrolytic activities. TGF-
specifically inhibited
a hydrolytic activity characterized and quantitated by cleavage of the peptide
7-amido-4-methyl-coumarin (AMC) substrate Cbz-Leu-Leu-Leu-AMC (z-LLL-AMC). We
next assessed putative cellular mechanisms involved in the modulation of
z-LLL-AMC activity by determining the roles of oxidative stress as well as
heat shock protein (HSP)90, an endogenous inhibitor of z-LLL-AMC hydrolysis.
Finally, to address the physiological relevance of TGF-
inhibition of
proteasomal activity, we assessed the role of z-LLL-AMC hydrolysis on cell
growth and the expression of cell cycle regulators.
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EXPERIMENTAL PROCEDURES |
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Sedimentation velocity analysis. Mz-ChA-1 cells were grown to 50100% confluence in 100 x 20-mm plates. The cells were then washed with cold phosphate-buffered saline (PBS) and lysed with 1 ml of a buffer containing 10 mM HEPES, 100 mM KCl, 5 mM MgCl2, and 0.1% Triton X-100 (pH 7.2), transferred to 12 x 75-mm borosilicate tubes, and homogenized by 20 strokes with a Tissue Tearor (Biospec, Bartlesville, OK). The homogenate was centrifuged at 21,000 g for 20 min at 4°C with a Microfuge R centrifuge (Beckman Instruments, Palo Alto, CA). The supernatant was then subjected to sucrose density gradient centrifugation with 530% sucrose in 0.02 M HEPES, pH 7.4, containing 0.15 M sodium chloride, 0.05% (wt/vol) Triton X-100, and 0.02% (wt/vol) sodium azide. After centrifugation at 100,000 g for 22 h in a Beckman LS-70M Ultracentrifuge with a SW41Ti rotor (Beckman Instruments), the gradient was separated into 12 fractions of 1 ml each. Protein content was assessed by the Bradford assay, and proteasomal activity was assessed in each fraction at 37°C for 30 min.
Measurement of cytosolic proteasomal activity. Cells were
incubated in 10% fetal bovine serum with or without TGF- for 24 h. The
cells were then washed with PBS and lysed with 1 ml of a hypotonic buffer
containing 25 mM HEPES, 5 mM MgCl2, 1 mM EGTA, and freshly added
0.5 mM PMSF, 2 µg/ml pepstatin, and 2 µg/ml leupeptin. Cells were then
homogenized by 20 strokes with a Tissue Tearor. The homogenate was centrifuged
at 21,000 g for 45 min at 4°C with a Microfuge R centrifuge. The
protein content in the supernatant cytosolic fraction was measured with the
Bradford reagent. Protease activity was assayed by adding 50 µl of
cytosolic protein (containing 4075 µg protein) to 0.5 ml of buffer
containing 25 mM HEPES, pH 7.5, 10 mM dithiothreitol, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.5 mM
PMSF, 2 µl/ml aprotinin, and 20 µM fluorescent substrate. The substrates
used to assay for proteasomal activity were z-LLL-AMC, Suc-Leu-Leu-Val-Tyr-AMC
(suc-LLVY-AMC), and Cbz-Leu-Leu-Glu-AMC (z-LLE-AMC). After incubation at
37°C for 30 min, fluorescence was measured every 5 min for 30 min with a
fluorometer (TD700; Turner Designs, Mountain View, CA) with excitation and
emission wavelengths of 360 and 480 nm, respectively. Hydrolysis of all three
substrates was linear with time for up to 3 h and directly proportional to the
amount of cytosolic extract used. For experiments involving HSP90, 20 µM
substrate in enzyme buffer was added to 10 µg of cytosolic protein in a
final volume of 200 µl in 96-well plates. Fluorescence was monitored with a
CytoFluor 4000 fluorescence plate reader (PerSeptive Biosystems, Foster City,
CA) using excitation and emission wavelengths of 360 and 460 nm, respectively.
With each experiment, standard curves were generated with AMC and proteasomal
activity was expressed as picomoles of AMC per milligram of protein per
minute.
Plasmids and transfection. Plasmids for Smad4 were
obtained from Dr. A. Roberts (National Cancer Institute, Bethesda, MD).
Mz-ChA-1 cells were transiently transfected with a Perfect Lipid transfection
kit (Invitrogen, Carlsbad, CA). Cells were seeded in 24-well plates, grown in
serum-containing medium to 4060% confluence, and then incubated for 4 h
in serum-free medium containing plasmid DNA and lipid in a ratio of 6:1. The
medium was then replaced with serum-containing medium. Cells were used 24 h
after transfection. Cells were cotransfected with cytomegalovirus
(CMV)--galactosidase (
-gal) to normalize for transfection
efficiency, and
-gal activity was assessed in cell lysates with
chlorophenol red-
-D-galactopyranoside monosodium (CPRG) and
measurement of absorbance at 575 nm.
Quantitation of reactive oxygen species generation. Quantitation of intracellular generation of reactive oxygen species was performed with the oxidant-sensitive fluorescent probe dihydroethidium. This membrane-permeant probe is oxidized within the cell to ethidium, resulting in a marked red shift in its fluorescence spectra. Moreover, the oxidized ethidium remains within the cell because it intercalates with DNA. The fluorescence of ethidium bound to DNA can then be used to quantitate the generation of reactive oxygen species in intact cells (42). H69 cells were incubated in 96-well plates (1,000 cells/well) in the presence of 20 µM dihydroethidium in Krebs-Ringers-HEPES buffer for 30 min at 37°C. Fluorescence was quantitated with excitation and emission wavelengths of 530 and 620 nm, respectively, and a fluorescence multiplate reader (CytoFluor 4000).
Immunoblot analysis. Confluent cells in culture were trypsinized and sonicated for 20 s at 4°C (sonic dismembrator; Fisher Scientific, Pittsburgh, PA) in a lysis buffer containing 50 mM Tris base, 2 mM EDTA, 100 mM NaCl, 1% NP-40, and one mini protease inhibitor cocktail tablet in 25 ml. Protein content was determined by the Bradford assay. Protein samples were separated on 412% gradient polyacrylamide gels (Novex, San Diego, CA) under reducing conditions and electroblotted to positively charged 0.45-µm nitrocellulose membrane (Millipore, Bedford, CA). The membranes were soaked for 5 min in transfer buffer (13.4 mM Tris, pH 8.3, 20% methanol, 108 mM glycine). Blots were preblocked in 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, 5% nonfat dry milk for 34 h or overnight at 4°C. HSP90 or the cdk inhibitors p16INK4A, p27KIP1, and p21WAF1/CIP1 were detected by incubating the membrane overnight at 4°C with the respective monoclonal mouse anti-human primary antibody, used at a 1:500 dilution. The primary antibodies were diluted in a solution containing 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk. The membrane was washed twice for 10 min with 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (TTBS) and then incubated with the secondary antibody, a polyclonal goat anti-mouse immunoglobulinperoxidase conjugate (Zymed, San Francisco, CA) at a 1:1,000 dilution for 60 min at 4°C. The secondary antibody was diluted in TTBS buffer. For all immunoblots, membranes were washed twice for 10 min with TTBS and then visualized with an enhanced chemiluminescence kit (ECL plus; Amersham Life Science, Little Chalfont, UK) following the manufacturer's directions.
Proliferation assay. Cells were seeded into 96-well plates (510,000 cells/well), allowed to adhere, and incubated in a final 200-µl volume of medium. Cell proliferation was assessed by a commercially available colorimetric cell proliferation assay (CellTiter 96AQueous; Promega, Madison, WI). The proliferation index was calculated as the percentage of cell number in stimulated to control cultures.
Reagents. DMEM media, fetal bovine serum, and Bradford reagent were obtained from Sigma (St. Louis, MO). PBS, CMRL, DMEM12, glutamine, and antibiotic-antimycotic mix were from GIBCO BRL (Grand Island, New York). Plasmid purification kits were obtained from Qiagen (Valencia, CA). Monoclonal mouse anti-human HSP90, p21WAF1/CIP1, p27KIP1, and p16INK4A antibodies were obtained from Pharmingen (San Diego, CA). z-LLL-AMC, z-LLE-AMC, suc-LLVY-AMC, AMC, Cbz-Leu-Leu-leucinal (z-LLL-CHO), HSP90, and U-83836E were obtained from Calbiochem-Novabiochem Corp (San Diego, CA). Protease inhibitor cocktail tablets and CPRG were obtained from Roche Molecular Biochemicals (Indianapolis, IN). All other reagents were of analytical grade from the usual commercial sources.
Statistical analysis. Data are expressed as means ± SE from at least three separate experiments performed in triplicate, unless otherwise noted. The differences between groups were analyzed with a double-sided Student's t-test when only two groups were present. For repeated measures between multiple groups, analysis was performed with ANOVA with a post hoc Bonferroni test to correct for multiple comparisons. Statistical significance was considered as P < 0.05. Statistical analyses were performed with the GBSTAT statistical software program (Dynamic Microsystems, Silver Spring, MD).
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RESULTS |
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We then assessed the effect of TGF- on hydrolysis of these
substrates. Biliary epithelial H69 cells were incubated with 10, 1, 0.1, or 0
ng/ml TGF-
for 24 h. Substrate hydrolysis was then fluorometrically
assessed in cytosolic extracts. Basal activity was 1.12, 8.33, and 14.52 nmol
AMC · mg protein-1 ·
min-1 for suc-LLVY-AMC, z-LLE-AMC, and z-LLL-AMC
hydrolysis respectively. Incubation with TGF-
did not significantly
alter the hydrolysis of suc-LLVY-AMC, and a modest increase in z-LLE-AMC was
noted only at high concentrations of TGF-
(Fig. 1, A and
B). Unexpectedly, TGF-
markedly decreased z-LLL-AMC
hydrolysis in a concentration- and time-dependent manner
(Fig. 1, C and
D). Incubation with the proteasome inhibitor lactacystin
(10 µM) for 24 h decreased z-LLLAMC hydrolysis under basal conditions to
18.7 ± 9.2% of controls and during incubation with 10 ng/ml TGF-
to 8.4 ± 3.2% of controls. The inhibitory effect of TGF-
on
z-LLL-AMC hydrolysis was completely abolished by preincubation with the
protein synthesis inhibitor cycloheximide (100 µg/ml) for 1 h, indicating
that TGF-
inhibition of z-LLL-AMC hydrolysis involves de novo protein
synthesis.
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Biliary epithelial Mz-ChA-1 cells have defective TGF--mediated
intracellular signaling that can be restored by Smad4
(44). Inhibition of z-LLL-AMC
activity was not observed in Mz-ChA-1 cells. However, partial inhibition of
z-LLL-AMC hydrolysis was observed in cells transiently transfected with
Smad4 (Fig. 2). In
combination, these studies indicate that TGF-
transcriptionally and
selectively modulates a proteasome-associated hydrolytic activity with a
substrate specificity different from that of suc-LLVY-AMC, a classic substrate
used to assay proteasomal chymotrypsin-like activity
(22).
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TGF- increases cellular oxidative stress.
TGF-
has been shown to stimulate cellular production of hydrogen
peroxide and to induce intracellular oxidative stress in several different
cell types (6,
7,
21,
31,
39,
40). Furthermore, inhibition
of z-LLL-AMC activity was described in response to metal-catalyzed oxidative
stress (5). Thus we assessed
the potential role of alterations in intracellular oxidative stress on
proteasomal function. Cellular extracts were incubated with hydrogen peroxide
for varying periods of time to increase intracellular oxidative stress
(Fig. 3A). Incubation
with hydrogen peroxide decreased z-LLL-AMC hydrolysis in a time-dependent
manner. Likewise, we observed an increase in intracellular reactive oxygen
intermediate (ROI) formation during incubation with TGF-
(Fig. 3B).
Preincubation of cells with the antioxidant U-83836E (100 µM) prevented the
loss of z-LLL-AMC activity in response to TGF-
(Fig. 3C). This
concentration of U-83836E was chosen on the basis of preliminary studies
showing effective reduction in ROI and lipid peroxidation and lack of
significant cytotoxicity (Ref.
25 and unpublished data).
These findings suggest that cellular oxidative stress may lead to inactivation
of z-LLL-AMC hydrolytic activity in response to TGF-
. A potential
mechanism of TGF-
modulation of proteasomal function may thus involve
oxidative inactivation of specific proteasomal subunits mediating the
z-LLL-AMC hydrolysis.
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Inhibition of proteasomal hydrolysis by HSP90. Intracellular
oxidative stress may increase expression of heat shock proteins. HSP90 has
been shown to inhibit proteasomal z-LLL-AMC cleavage
(41). Thus we tested the
possibility that the effects of TGF- may be mediated via an
HSP90-dependent mechanism. Hydrolysis of z-LLL-AMC, but not suc-LLVY-AMC, was
decreased in cytosolic extracts in the presence of recombinant HSP90
(Fig. 4, A and
B). Furthermore, the inhibitory effect of HSP90 on
z-LLL-AMC hydrolysis was inhibited by preincubation with 2 µM geldanamycin,
an inhibitor of the biological functions of HSP90. These observations
confirmed that HSP90 could act as a functional endogenous inhibitor in H69
cells. We next asked whether TGF-
alters expression of HSP90. However,
no significant alteration in HSP90 protein levels was observed during
incubation with TGF-
(Fig. 4,
C and D). Furthermore, incubation with
geldanamycin did not alter growth inhibition or substrate hydrolysis by
TGF-
(data not shown). In combination, these findings suggest that
although HSP90 may selectively modulate proteasomal LLL-AMC hydrolysis in
vitro, the inhibitory effect of TGF-
on this activity is mediated by an
HSP90-independent mechanism in vivo.
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TGF- increases expression of p27kip1 and
inhibits cell growth. To assess the functional consequence of proteasomal
inhibition by TGF-
, we next determined the effects of TGF-
on cell
growth and apoptosis. Consistent with the reported effects of TGF-
on a
wide variety of epithelial cell types, incubation with TGF-
decreased
H69 cell proliferation in a concentration-dependent manner
(Fig. 5A). In
contrast, incubation with IL-6, a known biliary epithelial cell mitogen,
resulted in an increase in proliferation under the same conditions. Although
TGF-
can induce apoptosis in hepatic epithelia, TGF-
did not
significantly increase nuclear fragmentation or activation of caspases-3 or -8
in H69 cells over the same range of concentrations (data not shown). Thus
growth inhibition by TGF-
involves altered cell cycle progression rather
than an increase in apoptosis. Proteasomal degradation has been implicated in
the modulation of expression of the cdk inhibitors p21WAF1/CIP1 and
p27KIP1, both of which have been implicated in cell cycle arrest by
TGF-
. Quantitative immunoblot analysis indicated that incubation with
TGF-
did not significantly alter the expression of either p16 or
p21WAF1/CIP1 but increased expression of p27KIP1
(Fig. 5B).
Preincubation with the antioxidant U-83836E decreased the effect of TGF-
on p27 expression (Fig. 6, A and
B) and on proliferation
(Fig. 6C). TGF-
was shown previously to increase p27KIP1 by altered degradation
rather than via transcriptional mechanisms
(15). Thus manipulation of
proteasomal degradation can modulate growth inhibition in response to
TGF-
. Furthermore, the direct regulation of proteasomal activity may
have functional implications for TGF-
-mediated cellular processes other
than growth inhibition.
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Modulation of proteasomal z-LLL-AMC activity by exogenous
inhibitors. The cell-permeant peptide aldehyde z-LLL-CHO, or MG132, has
been used widely in studies evaluating proteasomal degradation of a variety of
proteins including transcription factors, cell cycle regulatory proteins, and
enzymes. This inhibitor has been shown to reversibly inhibit chymotrypsin-like
proteasomal activity. As predicted on the basis of their similar structures,
z-LLL-CHO also inhibits z-LLLAMC hydrolysis. In vitro studies performed on
cytosolic extracts revealed that z-LLL-CHO potently inhibited z-LLL-AMC
hydrolysis with an IC50 of 24 nM. In contrast, z-LLL-CHO was a
weaker inhibitor of suc-LLVY-AMC or z-LLE-AMC hydrolysis (IC50 of
45 and 78 µM, respectively). Incubation of cells with z-LLLCHO resulted in
increased p27KIP1 protein expression
(Fig. 7, A and
B). Moreover, an effect on cellular growth inhibition was
also noted (Fig. 7C).
These observations provide additional support for an important role of
proteasomal activity in mediating growth arrest in response to TGF-.
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DISCUSSION |
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The signaling pathways and mechanisms responsible for direct regulation of
proteasomal function remain unknown. Several different hydrolytic activities
have been identified in human proteasomes. The overall structure of the
proteasome consists of four stacked rings, with each of the two end rings
containing seven -subunits and the two central rings containing seven
-subunits. The active sites are formed by the amino-terminal threonine
residues of the
-subunits, which face a central cavity in this
cylindrical particle (22).
Analysis of yeast mutants defective in various hydrolyzing activities has
revealed that hydrolytic activity is associated with the
-subunits. The
-subunits act as a template for correct
-subunit assembly as well
as binding regulatory complexes such as PA28 or REG and PA700, which alter
proteasomal proteolytic activities. PA28 is a potent activator that
differentially increases peptide hydrolysis by the proteasome. The peptidase
activity and substrate specificity of the 20S proteasome can be influenced by
association with these regulatory complexes. Thus perturbed proteasomal
hydrolysis may result from structural or functional alterations in either
- or
-subunits. Altered expression of the proteasome and
associated regulatory proteins has been described in some physiological
conditions such as muscle atrophy due to increased rates of global protein
degradation (3). Alterations in
the intracellular localization of the proteasome have also been observed in a
cell cycle-specific manner (2).
Thus potential mechanisms of selective regulation of z-LLL-AMC hydrolysis by
TGF-
may involve altered expression of proteasomal subunits, regulatory
proteins, or altered intracellular distribution of functionally distinct
proteasomal complexes.
Multiple studies have demonstrated a relationship between protein oxidation
and proteolysis, and the 26S proteasome has been shown to be susceptible to
oxidative inactivation (28,
37). The cellular generation
of ROI by TGF- has been shown to involve induction of NADH oxidase in
fibroblasts. A plausible mechanism for the involvement of ROI is the
inactivation of critical subunits involved in z-LLL-AMC hydrolysis.
Alternatively, TGF-
may increase the expression of a specific protein
inhibitor that selectively affects specific proteasomal components. TGF-
can directly alter the expression of the
-catalytic proteasome Z subunit
(38). However, decreased
expression of proteasomal components has not been observed during oxidative
impairment of proteasomal function
(13,
28). Thus additional studies
will be required to ascertain whether the Z subunit participates in the
selective hydrolysis of z-LLL-AMC or can be oxidatively modified.
There is considerable emerging evidence that implicates the proteasome in
antigen processing. Proteasomal inhibitors can block the generation of class I
antigenic peptides and their presentation to cytotoxic lymphocytes. Interferon
(IFN)- stimulates antigen presentation and has been shown to stimulate
the expression of proteasomal regulators PA28 or 11S (REG). IFN-
and
TNF-
also alter the proteasome subunit composition by replacing the X,
Y, and Z catalytic
-subunits with other subunits, LMP7, LMP2, and LMP10,
respectively (1,
11). These alterations in the
-catalytic subunit composition of the proteasome are expected to alter
the hydrolytic activities in a manner that would favor generation of class I
antigenic peptides. TGF-
has profound effects as a regulator of the
immune system and as a potent suppressor of immune cell activity. Indeed,
TGF-
has been implicated in a broad range of pathogenic mechanisms
involving primary effects on immune cells
(33). However, the specific
gene targets mediating TGF-
regulation of immune cell behavior remain
poorly understood. We speculate that modulation of proteasomal function may
mediate the immunoregulatory effects of TGF-
. Studies to ascertain the
role of TGF-
on proteasomal structure and function are warranted to
ascertain the potential involvement of the proteasome in immune function
regulation by TGF-
. The proteasome plays a role in numerous
intracellular processes other than cell growth regulation, and dysregulated
proteasomal function has been implicated in some disease states. Thus
identification of the precise mechanisms by which specific proteasomal
activity is regulated in physiological or pathophysiological settings should
provide new insights into diverse cellular processes.
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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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