Department of Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas 75708-3154
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ABSTRACT |
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Surfactant protein
B (SP-B) is essential for the maintenance of biophysical properties and
physiological function of pulmonary surfactant. Tumor necrosis
factor- (TNF-
), an important mediator of lung inflammation,
inhibits surfactant phospholipid and surfactant protein synthesis in
the lung. In the present study, we investigated the TNF-
inhibition
of rabbit SP-B promoter activity in a human lung adenocarcinoma cell
line (NCI-H441). Deletion experiments indicated that the TNF-
response elements are located within
236 bp of SP-B 5'-flanking DNA.
The TNF-
response region contained binding sites for nuclear
factor-
B (NF-
B), Sp1/Sp3, thyroid transcription factor (TTF)-1,
and hepatocyte nuclear factor (HNF)-3 transcription factors.
Inhibitors of NF-
B activation such as dexamethasone and
N-tosyl-L-phenylalanine chloromethyl ketone and
mutation of the NF-
B element did not reverse TNF-
inhibition of
SP-B promoter, indicating that TNF-
inhibition of SP-B promoter activity occurs independently of NF-
B activation. TNF-
treatment decreased the binding activities of TTF-1 and HNF-3 elements without altering the nuclear levels of TTF-1 and HNF-3
proteins.
Pretreatment of cells with okadaic acid reversed TNF-
inhibition of
SP-B promoter activity. Taken together these data indicated that in
NCI-H441 cells 1) TNF-
inhibition of SP-B promoter
activity may be caused by decreased binding activities of TTF-1 and
HNF-3 elements, 2) the decreased binding activities of TTF-1
and HNF-3
are not due to decreased nuclear levels of the proteins,
and 3) okadaic acid-sensitive phosphatases may be involved
in mediating TNF-
inhibition of SP-B promoter activity.
lung; respiratory distress syndrome; gene regulation; cytokines
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INTRODUCTION |
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SURFACTANT, A COMPLEX of lipids and proteins, is synthesized and secreted by type II epithelial cells of the lung. Surfactant maintains alveolar integrity through reduction of surface tension at the alveolar air-tissue interface (11) and plays important roles in host defense in the lung (36). Deficiency of surfactant is associated with the development of respiratory distress syndrome in preterm infants (2). Surfactant-associated proteins (SP), SP-A, SP-B, and SP-C, serve important roles in the biophysical properties, metabolism, and physiological function of surfactant (37). SP-B is particularly essential for the stabilization of the phospholipid monolayer formed on the alveolar surface (8). Deficiency of SP-B as a result of mutation in the SP-B gene causes fatal respiratory failure in infants with congenital alveolar proteinosis (25), and targeted disruption of the SP-B gene causes abnormalities in surfactant metabolism and respiratory failure in newborn mice (7). Heterozygous mice expressing 50% of normal SP-B levels displayed decreased lung compliance and air trapping, indicating that less than normal levels of SP-B may contribute to lung dysfunction (6). SP-B mRNA is developmentally and hormonally regulated in the lung. In the adult lung, SP-B mRNA is expressed in a cell type-specific manner by the alveolar type II and bronchiolar (Clara) epithelial cells (26, 39).
Adult respiratory distress syndrome (ARDS) is a life-threatening
disease with a mortality rate of >50% (10). Abnormal
surfactant function due to alterations in surfactant composition and
metabolism may contribute to the pathophysiology of lung dysfunction in
ARDS (19). The levels of total phospholipids, SP-A, and
SP-B in bronchoalveolar lavage material are significantly reduced in
ARDS patients and patients at risk for ARDS compared with those in
normal individuals (12, 13). Tumor necrosis factor-
(TNF-
), an early-response cytokine, is an important mediator of lung
inflammation (17, 35) and is present at high levels in the
blood and alveolar lining fluid in ARDS (16).
TNF- is thought to cause lung injury and subsequent respiratory
failure by increasing lung epithelial permeability and sequestration and activation of neutrophils (35). TNF-
also inhibits
surfactant phospholipid synthesis (3) and surfactant
protein gene expression (38) that can potentially
contribute to lung injury and respiratory failure in ARDS patients.
TNF-
decreased SP-A and SP-B mRNA levels in NCI-H441 cells
(38), a human pulmonary adenocarcinoma cell line with
characteristics of bronchiolar epithelial cells (Clara cells), and SP-B
mRNA content in the mouse lung (27). Mechanisms by which
TNF-
decreases SP-B mRNA expression are not well understood. In
NCI-H441 cells, TNF-
was found to act at the posttranscriptional level to decrease SP-B mRNA expression and the 3'-untranslated region
of SP-B mRNA was implicated to contain cis-acting elements necessary for destabilization of mRNA (28). The role of
transcriptional mechanisms in mediating the inhibitory effects of
TNF-
on SP-B mRNA expression is not known.
In the present study, we found that TNF- inhibited SP-B promoter
activity in NCI-H441 cells, indicating that transcriptional mechanisms
have important roles in the TNF-
downregulation of SP-B gene
expression. The objective of our investigation was to identify and
characterize cis-DNA elements and interacting transcription factors that are required for TNF-
downregulation of SP-B promoter activity. Deletion experiments showed that the SP-B minimal promoter (
236/+39 bp) (22) was inhibited by TNF-
and further
deletion of 5' DNA to
140 bp did not alter the response of the
promoter. DNA binding activities of thyroid transcription factor
(TTF)-1 and hepatocyte nuclear factor (HNF)-3 but not of Sp1 elements were decreased in nuclear extracts of TNF-
-treated cells. Our studies also showed that a nuclear factor-
B (NF-
B) element
(
150/
141 bp) in the SP-B promoter was not important for TNF-
inhibition of SP-B promoter activity. These data suggested that TNF-
inhibition of SP-B promoter activity is mediated through the
downregulation of binding activities of TTF-1 and HNF-3 elements.
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MATERIALS AND METHODS |
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Cell culture and transfections. NCI-H441 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml) at 37°C in a humidified atmosphere of 5% CO2 and air.
Plasmids were amplified in Escherichia coli Top10F' strain (Invitrogen, Carlsbad, CA) and purified by anion-exchange chromatography (QIAGEN) according to the manufacturer's protocol. Plasmid DNA was quantified by measuring absorbance at 260 nm, and its quality was verified by agarose gel electrophoresis and ethidium bromide staining. At least two independent preparations of plasmids were used for transfection. Plasmid DNAs were transiently transfected into cells by liposome-mediated DNA transfer using LipofectAMINE (GIBCO BRL) as described previously (22). Cells were cotransfected with aNuclear extract preparation.
Nuclear extracts from cells were prepared according to Schreiber et al.
(32). Typically, cells from a confluent 75-cm2
flask were used for the preparation of the extract, and all steps were
performed on ice or at 4°C. In brief, cells were incubated in 400 µl of hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM
MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM
phenylmethylsulfonyl fluoride, 2 µg/ml each of leupeptin and
aprotinin, and 0.5 mg/ml benzamidine) for 15 min and then lysed by the
addition of 0.6% Nonidet P-40. Cell lysate was centrifuged at
3,000 rpm for 5 min to pellet the nuclei, and the cytosolic fraction
was removed, adjusted to 25% with respect to glycerol, and stored at
80°C. Nuclei were extracted by incubating in 100 µl of extraction
buffer (20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml each of leupeptin and aprotinin, and 0.5 µg/ml benzamidine) for 30 min followed by centrifugation at 14,000 rpm for 10 min. After centrifugation, the supernatant was divided into aliquots into
chilled tubes, frozen in liquid nitrogen, and stored at
80°C. The
protein concentration of nuclear extract was determined by Bradford's
method using Bio-Rad protein assay reagent (4).
Plasmid constructions and site-directed mutagenesis. The construction of plasmids containing different lengths of rabbit SP-B 5'-flanking DNA linked to the CAT gene has been described previously (21, 22). In all SP-B-CAT promoter constructs, the 3' end point of SP-B 5'-flanking DNA was at +39 bp.
Nucleotides in the NF-Electrophoretic mobility shift assays.
Synthetic double-stranded oligonucleotides were annealed by heating 10 µM of single-stranded oligonucleotides in 10 mM Tris · HCl,
pH 7.5, containing 10 mM MgCl2 and 50 mM NaCl at 95°C for 5 min and then allowed to cool to room temperature over a period of 60 min. Double-stranded oligonucleotides were end labeled with [-32P]ATP and T4 polynucleotide kinase.
Electrophoretic mobility shift assays (EMSA) were performed by
incubating 0.5 or 1.0 ng (3-10 × 104 cpm) of the
oligonucleotide probe with 5 µg of nuclear protein in 20 µl of
binding buffer [13 mM HEPES, pH 7.9, 13% glycerol, 80 mM KCl, 5 mM
MgCl2, 1 mM dithiothreitol, 1 mM EDTA, and 0.2 or 0.5 µg
of poly(dI-dC) as nonspecific competitor] for 20 min at 30°C.
Nuclear proteins were incubated in binding buffer for 20 min at 30°C
and then incubated with the labeled probe. Competition experiments were
performed by addition of the indicated molar excess of cold wild-type
or mutant oligonucleotides before addition of the labeled probe. The
coding strand sequences of oligonucleotides (transcription factor
binding sites are underlined and mutated nucleotides are shown in bold)
used are as follows: Spl (
130 bp),
138- GCTGGGAAGGGGCTGGTTCAAAACA-114; Sp1 (
35 bp),
50-TCCCATGCTCCCGCCCCCAGCTAT-28; TTF-1 (
112 and
102 bp),
118-TCAAAACACCTGGAGGGCTCTCCAGGACAAG-90; HNF-3
(
88 bp),
96-GACAAAGGCAAACACTGAGGTC-75; NF-
B (wild type),
155-AGGGCAGGAATGCCC CTGCTG-135; NF-
B (mutant),
158-AGGAGGGCCACGCTGCCCCTGCT-136; and NF-
B (consensus),
5'-AGTTGAGGGGACTTTCCCAGGC-3'.
CAT and -galactosidase assays.
CAT activity of cell extracts was determined by the liquid
scintillation counting assay (33) using
[14C]chloramphenicol and n-butyryl coenzyme A
as described previously (22).
-Galactosidase activity
was determined by the chemiluminescent assay using Galacto-Light Plus
(TROPIX, Bedford, MA) substrate according to the recommended protocol.
CAT activities were normalized to cotransfected
-galactosidase
activity or protein content of cell extracts.
Immunoblotting.
Nuclear and cytosolic proteins were separated by SDS-PAGE on 10% gels
and electrophoretically transferred to nitrocellulose membranes. The
blots were probed with polyclonal antibodies to TTF-1, HNF-3, and
Sp1 proteins and developed with an enhanced chemiluminescence detection
system kit (Amersham Life Sciences, Piscataway, NJ).
Measurement of lactate dehydrogenase activity.
The effect of TNF- on cell toxicity was determined by measuring the
lactate dehydrogenase (LDH) activity of culture medium. LDH activity
was measured with CytoTox nonradioactive cytotoxicity assay kit
(Promega, Madison, WI) according to the manufacturer's protocol.
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RESULTS |
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Effect of TNF- on SP-B promoter activity.
We determined the effect of TNF-
on SP-B promoter activity by
transient transfection of SP-B-CAT promoter construct containing
2,176/+39 bp of SP-B 5'-flanking DNA into NCI-H441 cells. Results showed that TNF-
acted in a dose-dependent manner to decrease SP-B
promoter activity (Fig. 1). At a
concentration of 5 ng/ml and higher, TNF-
decreased SP-B promoter
activity by 60-70% after 48 h of incubation. Measurement of
LDH in cell culture medium indicated that TNF-
at concentrations of
5 and 25 ng/ml did not have any significant cytotoxic effects on the
cells.
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Deletion mapping of SP-B 5'-flanking region for identification of
TNF- response sequence.
We used deletion mapping to identify SP-B genomic regions that are
important for TNF-
inhibition of SP-B promoter activity. SP-B-CAT
constructs containing SP-B 5'-flanking DNA deleted at the 5'-end were
transiently transfected into NCI-H441 cells, and the effect of TNF-
(25 ng/ml) on CAT activity was determined after 48 h of incubation
(Fig. 2). Results showed that the minimal SP-B promoter,
236/+39 bp, was inhibited by TNF-
and further deletion of 5'-flanking DNA to
140 bp did not alter response of the
promoter. These data indicated that TNF-
-responsive elements are
located within
236 bp of SP-B 5'-flanking DNA.
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Functional role of NF-B binding site in TNF-
inhibition of
SP-B promoter activity.
Deletion experiments showed that the minimal SP-B promoter was
inhibited by TNF-
, and further deletion to
140 bp did not alter
response of the promoter. These data suggested that the SP-B promoter
regions
236/+39 and
140/+39 bp contain cis-DNA elements
necessary for TNF-
inhibition. TNF-
regulation of a variety of
genes is mediated through activation of the transcription factor
NF-
B (9). We sought to determine the role of NF-
B in
TNF-
inhibition of SP-B promoter activity.
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Functional role of Sp1, TTF-1, and HNF-3 elements in TNF-
inhibition of SP-B promoter activity.
Our studies have shown that Sp1, TTF-1, and HNF-3 elements are
necessary for SP-B promoter function and act in a combinatorial manner
to maintain promoter activity (23). Because we found that
the NF-
B element does not have a functional role in TNF-
inhibition of SP-B promoter activity, we investigated whether alterations in the binding activities of Sp1, TTF-1, and HNF-3 elements
are important for TNF-
inhibition of SP-B promoter activity.
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Effects of mutations in TTF-1 and HNF-3 binding sites on TNF-
inhibition of SP-B promoter activity.
TNF-
reduced DNA-binding activities of TTF-1 and HNF-3 elements in
nuclear extracts of NCI-H441 cells, suggesting that TTF-1 and HNF-3
elements have important roles in TNF-
inhibition of SP-B promoter
activity. To further assess the role of these elements in TNF-
inhibition of SP-B promoter, we determined the effects of mutations of
these elements on TNF-
inhibition of SP-B promoter activity.
Consistent with our previous findings, mutations of TTF-1 and HNF-3
elements caused 90% inhibition of SP-B promoter activity
(23). Results showed that whereas TNF-
inhibited
wild-type SP-B promoter by >50% (control = 100, treated = 48 ± 4.17, n = 4; one-tailed P = 0.0007 by one-sample t-test), it had significantly less
effect on the mutant promoter (control = 9.9 ± 2.94, treated = 7.1 ± 1.4, n = 4; two-tailed
P > 0.4 by unpaired t-test and Mann-Whitney test).
Effect of TNF- on the intracellular translocation of TTF-1 and
HNF-3.
It was recently reported that phorbol ester inhibition of SP-B promoter
activity in NCI-H441 cells is caused by decreased nuclear levels of
TTF-1 and HNF-3 factors as a result of cytosolic trapping
(34). To determine whether TNF-
inhibition of SP-B promoter activity is caused by changes in the intracellular
translocation of TTF-1 and HNF-3, we investigated TTF-1 and HNF-3 DNA
binding activities in the cytosolic and nuclear extracts of cells
treated with and without TNF-
. Results showed that TTF-1 and HNF-3
binding activities were reduced in nuclear extracts of treated cells
and no binding activity could be detected in cytosolic fractions (Fig. 6). This suggested that decreased DNA
binding activities are not caused by changes in the intracellular
translocations of transcription factors.
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Effects of TNF- on the levels of TTF-1, HNF-3, and Sp1 proteins.
To determine whether reduced DNA binding activities of TTF-1 and HNF-3
are the result of reduced levels of TTF-1 and HNF-3 proteins, we
determined the levels of TTF-1, HNF-3, and Sp1 in H441 cells treated
with and without TNF-
by immunoblotting analysis. Analysis of the
levels of the transcription factors in the nuclear and cytosolic
fractions of cells showed that TNF-
treatment did not alter the
levels of TTF-1, HNF-3
, and Sp1 in the nuclear or cytosolic
fractions (Fig. 7). In contrast to TTF-1,
which was not detected in the cytosolic fraction, HNF-3
and Sp1 were
detected in the cytosolic fractions of control and treated cells.
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Effect of okadaic acid on TNF- inhibition of SP-B promoter
activity.
TNF-
decreased DNA binding activities of TTF-1 and HNF-3 elements
without altering the levels of immunoreactive TTF-1 and HNF-3 proteins.
This suggested that the reduced DNA binding activities of TTF-1 and
HNF-3 could be due to factors that influence the binding affinities of
the proteins. Phosphorylation is a widely recognized mechanism by which
DNA binding activities of transcription factors are regulated
(15). TTF-1 contains seven serine phosphorylation sites
and is phosphorylated in vitro by protein kinase C (42). cAMP-dependent protein kinase A phosphorylation of TTF-1 has been implicated in the activation of SP-B (40) and SP-A
(20) gene expression in lung epithelial cells. To
determine whether TNF-
inhibition of SP-B promoter activity is the
result of alterations in the phosphorylation status of key
transcription factors, we investigated the effect of okadaic acid, a
protein phosphatase inhibitor, on TNF-
inhibition of SP-B promoter
activity. Results indicated that pretreatment of cells with okadaic
acid for 2 h nearly reversed TNF-
inhibition of SP-B promoter
activity (Fig. 8).
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DISCUSSION |
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TNF- has been implicated to have a major role in the
pathogenesis of ARDS (17, 35). Reduced levels of
surfactant, including surfactant proteins that occur in ARDS, could be
a contributing factor leading to the development of lung dysfunction in
ARDS (19). TNF-
decreases expression of SP-B mRNA in
NCI-H441 cells in vitro (39) and in mouse lung in vivo
(27). Decreased SP-B mRNA stability in TNF-
-treated
NCI-H441 cells has been implicated to result in decreased mRNA levels
(28). In the present study, we found that TNF-
decreased SP-B promoter activity, indicating that transcriptional
mechanisms also contribute to the downregulation of SP-B gene
expression by TNF-
.
Deletion mapping of SP-B 5'-flanking region showed that SP-B promoter
constructs containing 236/+39 and
140/+39 bp were inhibited by
TNF-
, indicating that TNF-
response sequences are located within
236 or
140 bp of SP-B 5'-flanking DNA. TNF-
has both stimulatory
and inhibitory effects on gene expression, and its effects are often
mediated through the activation of transcription factor NF-
B
(9). A number of studies have shown that specifically the
NF-
B p50/p50 homodimer functions as a transcriptional repressor (5, 18, 31). The minimal SP-B promoter contained a NF-
B binding site (
150/
141 bp) that is recognized by c-Rel, p50, and p65
proteins. Analysis of the functional role of NF-
B in TNF-
downregulation of SP-B promoter activity through the use of inhibitors
of NF-
B activation such as dexamethasone and TPCK and mutation of
the NF-
B site showed that NF-
B does not have a role in TNF-
downregulation of the SP-B promoter. These data are consistent with
recent findings that showed TNF-
and phorbol ester inhibition of
SP-A and SP-B mRNA accumulation in NCI-H441 cells occurs independently
of NF-
B activation (29). The function of the NF-
B
binding site in SP-B promoter function is unclear. Mutation of the
NF-
B binding site caused an ~40% decrease in SP-B promoter
activity (21), suggesting that it may be important for the
basal activity of the SP-B promoter.
Our data indicated that TNF- inhibited SP-B promoter activity
independently of NF-
B activation, suggesting the involvement of
other factors. We investigated the involvement of Sp1, TTF-1, and HNF-3
factors in TNF-
inhibition of the SP-B promoter. Sp1, TTF-1, and
HNF-3 are essential for SP-B promoter activity and function in a
cooperative or combinatorial manner to activate the promoter
(23). We found that the binding activities of TTF-1 and
HNF-3 elements were reduced by 40% in TNF-
-treated cells, suggesting that TNF-
inhibition of SP-B promoter activity might be
caused by decreased binding activities of TTF-1 and HNF-3 elements.
TNF- treatment did not alter the nuclear levels of immunoreactive
TTF-1 and HNF-3 proteins. This suggested that the decreased binding
activities of TTF-1 and HNF-3 elements in TNF-
-treated cells could
be the result of alterations in the binding affinities rather than the
levels of factors. These results indicated that the action of TNF-
to decrease SP-B promoter activity is different from that of phorbol
ester. Phorbol ester inhibition of human SP-B promoter activity in H441
cells was suggested to be caused by cytosolic trapping of TTF-1 and
HNF-3 proteins (34).
Okadaic acid nearly reversed TNF- inhibition of SP-B promoter
activity, implying that okadaic acid-sensitive phosphatases may have
important roles in TNF-
inhibition of SP-B gene expression. It
remains to be determined whether TTF-1 and HNF-3 proteins serve as
targets for phosphatases activated by TNF-
. TNF-
treatment of
neonatal rat cardiac myocytes resulted in a concentration-dependent increase in type 2A protein phosphatase activity, indicating the potential role of TNF-
in the control of protein function by phosphorylation (41). TTF-1 contains seven serine
phosphorylation sites and is phosphorylated by protein kinase C in
vitro (42). cAMP induction of SP-A promoter activity in
type II epithelial cells is mediated by increased binding of TTF-1 to
the promoter as a result of enhanced phosphorylation (20).
Similarly protein kinase A activation of human SP-B promoter activity
in NCI-H441 cells is mediated by phosphorylation of TTF-1
(40). HNF-3
and -
are phosphorylated in Hep G2
cells, but whether phosphorylation influences their DNA binding and
transactivation capabilities is not known (30).
In summary, our studies have shown that TNF- inhibited SP-B promoter
activity in H441 cells by decreasing the DNA binding activities of
TTF-1 and HNF-3 without affecting their abundance. Pretreatment
of cells with okadaic acid reversed TNF-
inhibition of SP-B promoter
activity, implying that alterations in the phosphorylation status of
key transcription factors may be necessary for inhibition of
promoter activity. Our studies have also shown that TNF-
downregulation of SP-B promoter activity occurs independently of
NF-
B activation. Although our results have indicated that TNF-
inhibition of SP-B promoter activity is the result of decreased binding
activities of TTF-1 and HNF-3 elements, the involvement of other
unidentified factors cannot be ruled out.
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ACKNOWLEDGEMENTS |
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This research was supported by the National Heart, Lung, and Blood Institute Grant HL-48048.
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FOOTNOTES |
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Address for reprint requests and other correspondence: V. Boggaram, Dept. of Molecular Biology, Univ. of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75710 (E-mail: vijay{at}uthct.edu).
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 1 November 1999; accepted in final form 22 May 2000.
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