Divisions of 1 Nephrology and 2 Endocrinology, Department of Medicine, Mount Sinai/University Health Network, University of Toronto, Toronto, Ontario, Canada M5G 2C4
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ABSTRACT |
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Effects of hyperglycemia on glomerular cells may be
mediated by glucose entry into the hexosamine pathway, and mesangial cell (MC) expression of the hexosamine pathway rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) is increased in
diabetic glomerulosclerosis. We hypothesized that GFAT activity would
be an important determinant of gene expression in glomerular MC. When
overexpressed in primary MC, GFAT produced a two- to threefold increase
in the activity of plasminogen activator inhibitor-1 (PAI-1) promoter.
There was a 1.4-fold increase in PAI-1 promoter activity in cells
exposed to high glucose (20 mM), whereas in MC overexpressing GFAT,
exposure to high glucose caused a 3.5- to 4-fold increase in promoter
activity. PAI-1 promoter activation was dependent on GFAT enzyme
activity because o-diazoacetyly-L-serine and
6-diazo-5-oxonorleucine, inhibitors of GFAT enzyme
activity, abrogated the activation of PAI-1 promoter in MC
overexpressing GFAT. Glucosamine, which is downstream of GFAT in the
hexosamine pathway, produced a 2.5-fold increase in the PAI-1 promoter
activity. In addition to increasing the mRNA levels for transforming
growth factor-1 (TGF-
1), GFAT overexpression also increased mRNA
levels for the TGF-
type I and type II receptors.
TGF-
-neutralizing antibody did not normalize PAI-1 promoter activity
in MC exposed to glucosamine or those overexpressing GFAT. We conclude
that GFAT expression and activity are important determinants of gene expression in MC and that flux through the hexosamine pathway activates
expression of genes implicated in vascular injury pathways.
glutamine:fructose-6-phosphate amidotransferase; plasminogen activator inhibitor-1; diabetic nephropathy; gene expression
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INTRODUCTION |
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THE MECHANISMS
RESPONSIBLE for diabetic glomerulosclerosis have not been fully
elucidated, but clinical trials, like the Diabetes Control and
Complications Trial (DCCT), have shown that glycemic control is a key
determinant of diabetic microvascular injury (1). Diabetic
nephropathy is characterized pathologically by expansion of the
glomerular mesangium, and at the cellular level, de novo generation of
diacylglycerol and activation of protein kinase C (PKC) by high glucose
has been linked to expression of extracellular matrix protein genes in
glomerular mesangial cells (MC) (14). However, more
recently, Kolm-Litty and co-workers (28) have suggested
that the glucose-induced increases in transforming growth factor-1
(TGF-
1) expression is dependent, at least in part, on glucose flux
through the hexosamine pathway.
Under physiological conditions, a small percentage (1-3%)
of glucose entering cells is shunted through the hexosamine pathway (21, 33). In the first step of the pathway (Fig.
1), fructose-6-phosphate is converted to
glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) (20, 32). Glucose flux through the hexosamine pathway plays
an important role in the development of insulin resistance
(33). Cultured cells that overexpress GFAT develop insulin
resistance in the absence of hyperglycemia (6, 12), and
transgenic mice that overexpress GFAT in skeletal muscle and adipose
tissue are insulin resistant (23, 41). We hypothesized
that enhancing flux of metabolites through the hexosamine pathway in MC
by increasing GFAT expression might also activate genes implicated in
vascular injury.
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To test this hypothesis we first studied the effects of GFAT
overexpression on activation of the plasminogen activator inhibitor-1 (PAI-1) promoter in primary cultured MC. PAI-1 is the major
physiological inhibitor of tissue plasminogen activator and urokinase
(31), and increased PAI-1 plasma levels have been
associated with coronary artery disease and vasculopathy in patients
with diabetes mellitus (5, 44). To further link GFAT
overexpression to vascular injury, we studied the effect of GFAT
overexpression on mRNA levels for TGF- type I and type II receptors
because TGF-
1 has been implicated in the pathogenesis of diabetic
nephropathy (42, 43).
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EXPERIMENTAL PROCEDURES |
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Materials
D-Glucose, D-glucosamine, o-diazoacetyly-L-serine (azaserine), 6-diazo-5-oxonorleucine (DON), o-nitrophenyl-Preparation and Culture of MC
Primary MC were obtained from male Sprague-Dawley rats as described (25). The cells were cultured (37°C, 95% air-5% CO2) in DMEM supplemented with FBS (20%), penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (2 mM). Cells were used between passages 14 and 20.Plasmids
Plasmid PL12, containing the human PAI-1 promoter (Transient Transfection of MC
Twenty-four hours before transfection, MC (1.5 × 105 cells/well) were plated onto six-well plastic plates (Sarstedt). The following day, transfection was carried out by using effectene (Qiagen) as described by the manufacturer. Cells (70-80% confluent) were transfected with 0.05 µg pCMV-Assay of Luciferase and -Galactosidase Activity
ATP Assay
Cellular ATP content was determined by using a bioluminescent kit (Sigma-Aldrich). Media was aspirated, wells were washed twice with PBS, and assay for cellular ATP content was performed according to the manufacturer's specification. Cellular ATP was expressed as micromoles ATP per milligram cell protein.RNA Isolation and Semiquantitative RT-PCR
Total RNA from MC was isolated by the single-step method of Chomczynski and Sacchi (8) as published previously (25, 42, 43). Isolated RNA was stored in diethyl pyrocarbonate-treated water atSemiquantitative RT-PCR was performed as previously reported (25,
42, 43). For -actin the sense primer corresponded to bp
331-354 and the antisense to bp 550-57. The TGF-
1 sense primer corresponded to bp 1143-1169 and the anti-sense to bp
1521-1547. The TGF-
type I receptor sense primer corresponded
to bp 426-445 and the antisense to bp 651-671 (GenBank
accession no. L26110), whereas the TGF-
type II receptor sense
primer corresponded to bp 818-837 and the antisense to bp
1129-1146. GFAT sense primer corresponded to 1034-1053,
whereas the antisense primer was 1515-1534 of human GFAT gene
(GenBank accession no. M90516). The specific primer sequences were the following.
-Actin
5' AAC CCT AAG GCC AAC CGT GAA AAG 3'
TGF-1
5' CGA GGT GAC CTG GGC ATC CAT GAC 3'
TGF- type I receptor
5' TGG TCC AGT CTG CTT CGT CT 3'
TGF- type II receptor
5' CCG TGG CTG TCA AGA TCT TC 3'
GFAT 5' AGC TGT GCA AAC ACT CCA GA3'
3'TTA CGA CCA GGA CTC TAA CC 5' For amplification, 2.5 µl of the RT product were mixed with 7.5 µl of PCR mix containing 0.1 µM of each of the primer pairs and 2 U of Taq polymerase. The sample was placed onto a Perkin-Elmer DNA thermal cycler (model 480) and heated to 94°C for 4 min before the application of temperature cycles. For TGF-Statistical Analysis
Statistical analyses were performed with the INSTAT statistical package (GraphPad Software, San Diego, CA). The difference between means was analyzed by using the Bonferroni multiple comparison test. Significance was defined as P < 0.05. ![]() |
RESULTS |
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Overexpression of GFAT in MC Activates the PAI-1 Promoter
To determine whether GFAT overexpression would activate the PAI-1 promoter, MC were cotransfected with the PAI-1 promoter-luciferase construct (PL12) and plasmid pCIS (empty vector) or pCIS containing the human GFAT gene (pCIS-GFAT), and growth arrested in 0.5% FBS for 48 h in 5.6 mM glucose. We observed that transient transfection of pCIS-GFAT led to a fourfold increase in the steady-state mRNA levels for GFAT compared with cells transfected with the empty vector, pCIS (Fig. 2). In addition, cotransfection of GFAT in the presence of the GFAT inhibitor azaserine did not influence the level of expression in the MC (Fig. 2).
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Transient transfection of pCIS-GFAT led to a 2.5- to 3-fold increase in
luciferase activity in MC maintained in 5.6 mM glucose, compared with
cells transfected with pCIS (Fig. 3;
P < 0.001, n = 4). To determine
whether the increase in PAI-1 promoter activity in MC transfected with
pCIS-GFAT was dependent on an increase in GFAT activity, luciferase
activity was measured in cell lysates from MC transfected with
pCIS-GFAT in the presence of two GFAT inhibitors, azaserine and DON.
Inhibition of GFAT activity abrogated the effect of pCIS-GFAT
transfection on PAI-1 promoter activity, as illustrated in Fig. 3.
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High Glucose Further Activates the PAI-1 Promoter in MC Transfected With GFAT
Previous studies have shown that increased GFAT sensitizes cells to the effects of glucose, specifically their response to insulin (12). We hypothesized that high glucose might lead to a further increase in PAI-1 promoter activity in cells transfected with pCIS-GFAT. To test this hypothesis, we compared luciferase activity in MC transiently transfected with either pCIS or pCIS-GFAT in 5.6 and 30 mM glucose.In MC cotransfected with pCIS and PL12, then exposed to varying glucose
concentrations, luciferase activity increased by ~35% in 20 and 30 mM glucose compared with physiological glucose concentration (Fig.
4A; P < 0.05, n = 4). Cotransfection with pCIS-GFAT and PL12 led to a
2.8-fold increase in luciferase activity in 5.6 mM glucose that was
further augmented by 30 mM glucose so that MC transfected with
pCIS-GFAT and exposed to 30 mM glucose exhibited a 3.5- to 4-fold
increase in the luciferase activity (P < 0.001, n = 4). High-glucose-induced PAI-1 promoter activity in
pCIS-GFAT-transfected cells was not an osmotic effect because the
addition of mannitol (24.4 mM) to the media containing glucose (5.6 mM)
did not further increase PAI-1 promoter activity in
pCIS-GFAT-transfected cells (Fig. 4B).
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Overexpression of GFAT Increases
TGF- Receptor mRNA Levels in
MC
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GFAT Overexpression Does Not Influence Cellular ATP
A recent report (24) suggests that the impact of glucosamine on cells, including induction of insulin resistance, may be related to depletion of intracellular ATP. To determine whether overexpression had any impact on cellular ATP levels, we compared ATP levels of lysate from untransfected cell, cells transfected with pCIS, and cells transfected with pCIS-GFAT. We have found previously found that exposure of MC to glucosamine did not affect cellular ATP levels (18). Similarly, there was no difference in cellular ATP levels between untransfected cells or cells transfected with pCIS or pCIS-GFAT (Fig. 6).
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PAI-1 Promoter-Luciferase Reporter Activity in Cells
Overexpressing GFAT is Unrelated to Cellular Protein
Content or -Galactosidase Activity
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Glucosamine Activates the PAI-1 Promoter in MC
To further implicate flux through the hexosamine pathway in the activation of the PAI-1 promoter by GFAT overexpression, we studied the effect of glucosamine on the PAI-1 promoter because glucosamine is downstream of the rate-limiting enzyme GFAT in the hexosamine pathway (Fig. 1). Primary cultured MC were transiently transfected with the plasmid PL12. MC were growth arrested in 0.5% FBS and exposed to 1-10 mM glucosamine. Luciferase activity was assayed in cell lysates after 48 h and compared with MC in 5.6 mM glucose. As illustrated in Fig. 7A, there was a dose-dependent effect of glucosamine on activation of the PAI-1 promoter, and 10 mM glucosamine led to a twofold increase in luciferase activity (P < 0.02 vs. 5.5 mM glucose, n = 3). Similarly, as illustrated in Fig. 7B, when cells were exposed to glucosamine there was a 2.3-fold increase in mRNA for TGF-
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The PAI-1 promoter construct PL12 used in these studies is TGF-1
responsive. Because Kolm-Litty and co-workers (28) have reported that flux through the hexosamine pathway increases TGF-
1 production by MC, we sought to determine whether activation of the
PAI-1 promoter by GFAT overexpression and/or glucosamine was due to
autocrine stimulation by TGF-
1. As expected, Fig.
8A shows that the PL12 promoter construct is TGF-
1 responsive and that coincubation with a pan-specific neutralizing antibody to TGF-
can
prevent promoter activation. In these studies, 5 ng/ml of recombinant
TGF-
1 led to a threefold increase in luciferase activity after
48 h, and 30 µg/ml of neutralizing antibody normalized
luciferase activity. In contrast, TGF-
-neutralizing antibody did not
normalize luciferase activity in MC overexpressing GFAT or in MC
exposed to 10 mM glucosamine (Fig. 8, B and C).
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DISCUSSION |
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One-third of diabetic patients will develop kidney disease characterized by progressive mesangial matrix expansion, proteinuria, and declining glomerular filtration rate, but the mechanisms responsible for diabetic glomerular injury remain poorly understood. There is a relationship between blood glucose control and microalbuminuria, and the DCCT has shown that the blood glucose level is a key determinant of microvascular injury in diabetic patients (1). At the level of the MC, high glucose concentrations have been linked to increased expression of extracellular matrix protein genes like collagen I, collagen IV, and fibronectin via a number of pathways (15). For example, the nonenzymatic formation of extracellular advanced glycosylation end products can influence cell signaling and gene expression in MC without even requiring entry of glucose into the cell (4, 9). De novo synthesis of diacylglycerol after glucose enters the MC is critical in the response to high glucose because specific PKC isoforms are activated (14, 15), and increased flux through the polyol pathway in high glucose may further facilitate PKC activation (19). More recently, Kolm-Litty and co-workers (28) have suggested that glucose flux through the hexosamine pathway (Fig. 1) is important in the pathogenesis of diabetic glomerulopathy (28).
In the hexosamine pathway, fructose-6-phosphate is first converted to glucosamine-6-phosphate by the rate-limiting enzyme GFAT (33). Under normal physiological conditions, only a small percentage (1-3%) of glucose entering cells is shunted through the hexosamine pathway (21, 33). The end product of the hexosamine pathway, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), is a substrate for O-glycosylation of intracellular proteins (22, 30, 39, 40). Although GFAT activity is low in the kidney compared with other tissues like the liver, fat, and skeletal muscle (38), recent in vivo studies of human kidney biopsy samples have revealed that GFAT immunostaining increases in MC of glomeruli exhibiting diabetic glomerulosclerosis (36). A twofold increase in GFAT activity is sufficient to induce insulin resistance in rat-1 fibroblasts (11, 12), and GFAT overexpression sensitizes adipocytes and tissues to glucose-induced insulin resistance so that insulin responsiveness is impaired at lower concentrations of glucose in cells that overexpress GFAT (12). These studies lead us to speculate that increased GFAT activity might be an important determinant of gene expression in diabetic glomerulus. To test our hypothesis, we sought to determine whether GFAT overexpression was sufficient to activate gene expression in primary MC. We chose to study the effect of GFAT overexpression on activation of the PAI-1 promoter because PAI-1 is the major physiological inhibitor of tissue plasminogen activator and urokinase (5, 31, 44). An increase in PAI-1 is predicted to decrease extracellular matrix protein degradation by reducing plasmin activation and thus may contribute to the accumulation of extracellular matrix protein in the glomerulus. Moreover, increased PAI-1 plasma levels have been associated with vasculopathy in patients with diabetes mellitus (5, 44).
Our first major observation was that transient overexpression of GFAT with pCIS-GFAT activated the PAI-1 promoter in primary cultured MC in comparison to cells transfected with the empty vector, pCIS. The increase in PAI-1 promoter activity was dependent on GFAT activity because both of the GFAT inhibitors, azaserine and DON, prevented the increase in PAI-1 promoter activity in pCIS-GFAT-transfected cells. Neither inhibitor affected GFAT expression. All of the experiments were first performed in 5.6 mM glucose, and therefore our results are similar to the studies of Crook and co-workers (11, 12) on insulin resistance in rat-1 fibroblasts, in that GFAT overexpression is sufficient to activate the PAI-I promoter.
High glucose activated the PAI-1 promoter (Fig. 4A), and we also observed a further increase in PAI-1 promoter activity in MC overexpressing GFAT after exposure to 30 mM glucose (Fig. 4B). Again, this response was dependent on GFAT and was not due to an unexpected effect of the transfection protocol because cells transfected with pCIS did not exhibit a similar increase in PAI-1 promoter activity when they were exposed to 30 mM glucose. The response was also dependent on glucose because an osmotic stimulus with mannitol did not reproduce the effect of 30 mM glucose (Fig. 4B). These findings suggest that overexpression of GFAT sensitizes MC to effects of high glucose.
PAI-1 transcription is highly induced by TGF-1 (7, 35).
The PL12 promoter construct used in this study contains the promoter
region from +699 to +54, and this region has several TGF-
1-responsive elements, including an activator protein-1 site and
two CAGA boxes (7, 35). Because glucosamine induces
TGF-
1 expression (13, 28), it was possible that
activation of the PAI-1 promoter in our experiments was due to
autocrine effect of TGF-
1. Therefore, we coincubated transfected MC
with a pan-TGF-
-neutralizing antibody to inhibit autocrine
stimulation by TGF-
1. The concentration of antibody was determined
by studying TGF-
1-induction of our PAI-1 promoter construct (PL12).
The TGF-
-neutralizing antibody completely inhibited the activation
of the PAI-1 promoter by recombinant TGF-
1, whereas activation by
glucosamine or by overexpression of GFAT was only partially blocked by
the neutralizing antibody (Fig. 8). These results suggest that
receptor-mediated TGF-
1 activity was only partially responsible for
activation of the PAI-1 promoter in cells exposed to glucosamine or
those in which GFAT was overexpressed. Thus in our primary MC,
overexpression of GFAT or provision of the downstream substrate
glucosamine activates the PAI-1 promoter independently of autocrine or
paracrine TGF-
1 activity. We have made similar observations in MC
cotransfected with a dominant-negative TGF-type II receptor construct
(18).
The present study did not address the mechanism responsible for the effect of flux through the hexosamine pathway on activation of the PAI-1 promoter; however, flux through the hexosamine pathway is believed to exert an effect on gene expression by increasing intracellular concentrations of GlcNAc and UDP-GlcNAc, which are substrates for the O-glycosylation of proteins (22, 39, 40, 45). The intracellular levels of O-linked glycosylated proteins correlate with GFAT activity, and blockade of GFAT activity or inhibition of GFAT expression with antisense oligonucleotides lowers the intracellular levels of O-GlcNAc-modified proteins (39, 40). The posttranslational modification of serine residues in transcription factors by O-glycosylation can affect the activity of the transcription factors. For example, O-glycosylation of Sp1 stabilizes the protein and prevents proteosomal degradation (20). There are Sp1 consensus sequences in the PAI-1 promoter, and we have found that site-directed mutagenesis of two adjacent Sp1 binding sites in the PAI-1 promoter attenuates glucosamine-induced activation of the PAI-1 promoter (18).
Recently, Hresko and others (24) reported that glucosamine-induced insulin resistance in fat cells was secondary to depletion of cellular ATP. In the present study, there was no difference in cellular ATP for untransfected MC, cells transfected with the empty vector, or those overexpressing GFAT (Fig. 6). We have also found that glucosamine did not affect cellular ATP levels (18), and our observations of a lack of effect of glucoasmine on cellular ATP is supported by other studies (10).
A number of studies have implicated TGF-1 in the pathogenesis of
diabetic nephropathy (3, 48, 50), and recent studies have
shown that glucosamine increases TGF-
1 in porcine MC and that flux
through the hexosamine pathway may be responsible for glucose-induced
increases in TGF-
1 expression in MC (28). In addition,
glucosamine activates TGF-
promoter-luciferase reporter in various
cells in culture (13). TGF-
1 influences extracellular matrix protein synthesis by interacting with specific cell surface receptors, TGF-
type II and TGF-
type I receptors (32, 34, 47), but the effect of GFAT overexpression on TGF-
1 and
TGF-
receptor expression in primary cultured MC has not been
defined. We hypothesized that increased flux through the hexosamine
pathway in MC overexpressing GFAT might stimulate expression of
TGF-
1 type I and type II receptors, in addition to TGF-
1, in MC.
The rationale for these studies was further supported by the
observations that expression of TGF-
type I and type II receptors is
commonly increased after injury in a variety of tissues including skin (17), vascular endothelium (46), and
glomerulus (37, 49).
Our third major observation was that GFAT overexpression was sufficient
to increase TGF- receptors and TGF-1 expression in MC in
physiological glucose concentrations (5.6 mM glucose). Interestingly, GFAT expression is increased in dermal wound healing (36),
a setting in which TGF-
receptor expression also increases
(17). Although the mechanism underlying the effect of GFAT
overexpression on TGF-
receptors and TGF-
1 expression was not
determined, like the PAI-1 promoter, Sp1-regulatory elements are
present in the promoters of all three of these genes (2, 16, 26,
27). It is also possible that other regulatory elements
are responsible for the effect of the hexosamine pathway on the
expression of these genes. For example, the AP-1 transcription
factor(s) can be O-glycosylated, and Kolm-Litty and
co-workers (29) have also recently reported that
glucosamine activates PKC, which may also contribute to these changes
in gene expression.
High glucose produced a modest activation of the PAI-1 promoter, but we
were unable to demonstrate a consistent effect of high glucose on the
mRNA levels for TGF-1 and type I and type II receptors. This
observation does not rule out an impact of glucose on mRNA for TGF-
1
and type I and type II in our primary MC. Instead, we believe that it
reflects low flux through the hexosamine pathway in these primary
cells. This conclusion is further supported by the finding that in
cells overexpressing GFAT that there is further augmentation of PAI-1
promoter activity in high glucose.
In summary, our findings support the general hypothesis that metabolic pathways implicated in insulin resistance may also be important in the development of diabetic complications. More specifically, our findings suggest that the rate-limiting enzyme in the hexosamine pathway, GFAT, sensitizes MC to the effects of high glucose by regulating flux through the hexosamine pathway and, through this action, the hexosamine pathway may contribute to diabetic glomerular injury by influencing expression of genes implicated in vascular injury.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. J. Quon for generously providing the plasmids (pCIS, pCIS-GFAT and pCIS-GFP).
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FOOTNOTES |
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This work was supported by a grant from the Juvenile Diabetes Foundation and the Medical Research Council of Canada (to J. W. Scholey and I. G. Fantus). L. James is supported by a Research Fellowship from the Kidney Foundation of Canada-Medical Research Council Partnership Program.
Address for reprint requests and other correspondence: J. W. Scholey, Div. of Nephrology, Toronto General Div., Univ. Health Network, Eaton Bldg. 13-243, 200 Elizabeth St., Toronto, Ontario, Canada M5G 2C4 (E-mail: james.scholey{at}utoronto.ca).
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.
Received 24 November 2000; accepted in final form 20 June 2000.
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