1 Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808; 2 Department of Basic Pharmaceutical Sciences, West Virginia University Health Sciences Center, Morgantown, West Virginia 26506; and 3 Department of Pharmaceutical Chemistry, Songkla University, Songkla 90110, Thailand
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ABSTRACT |
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The present study
investigated transcriptional inactivation of TNF- gene by nuclear
factor-binding oligonucleotides (ON) and their effects on pulmonary
inflammatory responses in mice. PCR-based gene mutation and gel shift
assays were used to identify specific cis-acting elements
necessary for nuclear factor binding and transactivation of TNF-
gene by lipopolysaccharide (LPS). LPS inducibility of TNF-
was shown
to require transcriptional activation by NF-
B at multiple binding
sites, including the
850 (
1),
655 (
2), and
510 (
3)
sites, whereas the
210 (
4) site had no effect. Maximum
inducibility was associated with the activation of
3 site. The
sequence-specific, double-stranded ON targeting this site was most
effective in inhibiting TNF-
activity induced by LPS. The inhibitory
effect of ON on TNF-
bioactivity was also investigated using a
murine lung inflammation model. Pretreatment of mice with ON, but not
its mutated sequence, inhibited LPS-induced inflammatory neutrophil
influx and TNF-
production by lung cells. Effective inhibition by ON
in this model was shown to require a liposomal agent for efficient
cellular delivery of the ON. Together, our results indicate that
transcriptional inactivation of TNF-
gene can be achieved by using
ON that compete for nuclear factor binding to TNF-
gene promoter.
This gene inhibition approach may be used as a research tool or as
potential therapeutic modality for diseases with etiology dependent on
aberrant gene expression.
tumor necrosis factor-
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INTRODUCTION |
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ABERRANT ACTIVATION AND
EXPRESSION of genes are associated with the development of many
human diseases. Most genes are quiescent or have minimal activity in
affecting physiological processes. However, in certain pathological
conditions, these genes are abruptly turned on by a preexisting genetic
switch, causing them to overexpress. An activation of the TNF- gene
by nuclear transcription factors is one such example of the
uncontrolled genetic switch. Several nuclear transcription factors,
including NF-
B, AP1, nuclear factor of activated T cells (NF-AT),
Erg-1, cAMP response element binding protein, C/EBP
, and Ets have
been shown to be involved in the transcriptional activation of TNF-
(8, 19, 20, 25, 30, 31, 44). The activation of the
TNF-
gene by different transcription factors is dependent on the
nature of stimulation (39) and on cell type (19,
38). For example, NF-
B, but not AP1 or AP2, is involved in
the activation of TNF-
transcription of LPS-stimulated monocytes
(43), whereas NF-AT, not NF-
B, plays a role in phorbol 12-myristate 13-acetate (PMA)-stimulated T cells
(38).
Because TNF- plays an important role in the pathogenesis of a
variety of inflammatory and immune diseases, this cytokine has been
identified as a key target for pharmacological manipulation (18,
33, 36, 41, 45). TNF-
is produced principally by macrophages
and acts on a variety of immune and nonimmune cells to initiate and
amplify inflammatory response (36). The expression of
TNF-
is regulated at different levels, transcriptional and posttranscriptional (4). At the transcriptional level,
TNF-
is regulated primarily by NF-
B, which acts in synergy with
other transcription factors such as AP-1 and C/EBP (8,
43). Several high- affinity DNA-binding motifs for NF-
B have
been found on the TNF-
promoter (8, 35). Mutational
analysis has also shown that these sites are essential for gene
induction (9). Such observations provide the basis that
blocking the action of NF-
B alone would be sufficient to inhibit
TNF-
gene expression.
NF-B belongs to a superfamily of protein dimers frequently composed
of two DNA-binding subunits, NF-
B1 (p50) and RelA (p65) (1, 2). It is normally kept in an inactive form in the
cytoplasm by attachment of the inhibitory subunit I
B. The activation
of NF-
B is accomplished by phosphorylation of the I
B by specific I
B kinases, which triggers a complete degradation of the inhibitor (37). The activated NF-
B is then translocated into the
nucleus where it binds to the promoter region of target gene and
activates its transcription. Because the interaction between NF-
B
and its gene target is sequence specific, we hypothesize that
oligonucleotides (ON) carrying the same base sequences as those of the
NF-
B recognition sites may be used to selectively inhibit the
transcriptional activation of a target gene. To test this hypothesis,
we first identified specific DNA regulatory elements on the TNF-
gene promoter that are involved in NF-
B binding and transcriptional
activation of TNF-
. Because previous studies have shown that not all
NF-
B binding sites are required for TNF-
activation (8,
35), and because promoter-binding activities may not necessarily
reflect the resulting gene expression, we therefore determined the
relative contribution of each specific NF-
B-binding domain on
TNF-
gene expression. On the basis of the information obtained, we
designed specific ON that contain the sequence most critically required for TNF-
gene activation. We tested these ON for their inhibitory effect on TNF-
expression in both in vitro and in vivo murine inflammation lung model.
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MATERIALS AND METHODS |
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Cells and reagents.
The mouse macrophage cell line RAW 264.7 was obtained from American
Type Culture Collection (Rockville, MD). The cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal
bovine serum, 2 mM glutamine, and 100 U/ml penicillin-streptomycin. Specific antibodies against NF-B p50 and p65 subunits were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used in the supershift assay. The liposomal agent
N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) was obtained from Boehringer Mannheim
(Indianapolis, IN), and LPS (Escherichia coli 0111:B4, 1 endotoxin unit/µg) was from Sigma Chemical (St. Louis, MO).
ON containing different NF-
B-binding sites of the murine TNF-
gene promoter were synthesized according to the underlined DNA
sequences shown in Fig. 1A.
They were named
1,
2,
3, and
4, respectively, based on
their NF-
B binding sequences. Normal phosphodiester and
nuclease-resistant phosphorothioate ON containing two repeated
sequences of the
3 motif and their mutated sequences were also
synthesized and used in gene inhibition studies (see sequences in Fig.
1B). Before use, all ON were annealed with their
complementary strands to generate double-stranded DNA. Annealing was
achieved by heating the ON to 100°C for 10 min and then cooling to
room temperature for 3 h.
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Animals and bronchoalveolar lavage.
Male BALB/c mice, 4-6 wk old, were obtained from Jackson
Laboratories (Bar Harbor, ME). They were acclimated in an American Association for Accreditation of Laboratory Animal Care-approved facility for at least 1 wk before use. The mice were fed water and food
ad libitum. Intratracheal instillations into mice were performed
according to an established method (10). The protocol was
approved by the Animal Care and Use Committee of West Virginia University. Mice were anesthetized with a mixture of ketamine and
xylazine (45 and 8 mg/kg ip, respectively) and challenged by
aspiration. The animals were placed on a board in a supine position.
The animals' tongues were extended with lined forceps, and 50 µl of
the test solution were placed on the back of the tongue. At indicated
times after treatment, mice were euthanized with an intraperitoneal
injection of 0.25 ml of pentobarbital sodium (EUTHA-6; Western Medical
Supply, Arcadia, CA), and bronchoalveolar lavage (BAL) was
performed. A tracheal cannula was inserted, and the lungs were lavaged
through the cannula using ice-cold PBS. Five lavages of 0.8 ml each
were collected. BAL cells were isolated by centrifugation at 500 g for 10 min, and the supernatants were collected and used
for TNF- measurements. The cell pellets were resuspended in 1 ml of
HEPES buffer (10 mM HEPES, 145 mM NaCl, 5.0 mM KCl, 1.0 mM
CaCl2, and 5.5 mM D-glucose, pH 7.4) and placed on ice. Cell counts and differentials were then determined using a
Coulter Multisizer II and AccuComp software (Coulter Electronics, Hialeah, FL).
Point mutation of TNF- promoter and gene transfection.
PCR-based DNA mutation procedure was used to generate point mutations
of the four NF-
B binding sites on the TNF-
gene promoter. The
four NF-
B binding sites are indicated in boldface lettering in Fig.
1A. PCR primers used for the mutation of
B sites were listed in Fig. 1B. The wild-type
863/
18 promoter
fragment was first generated and used as a template for subsequent
generation of the mutated promoter fragments. The promoter DNAs were
inserted into pCR2.1-TOPO cloning vector (Invitrogen, Carlsbad, CA) and ligated by HindIII/XhoI double digestion. The
inserts in the right orientation were cloned at the
HindIII/XhoI sites in the pGL3-basic luciferase
vector (Promega, Madison, WI). These mutated reporter plasmids were
named
1m,
2m,
3m, and
4m, respectively. For gene
transfection studies, the plasmids were individually introduced into
RAW cells with the aid of the liposomal agent DOTAP. In these experiments, cells were plated on a 12-well plate
(106/well) and allowed to grow for 24 h before
transfection. The plasmid DNA (1 µg/ml) was diluted in DMEM and mixed
with DOTAP (10 µg/ml) for 15-20 min. Cells were then incubated
in this mixture medium for 4 h at 37°C. After transfection, the
medium was replaced with a growth medium containing 10% fetal bovine
serum, and the cells were cultured for an additional 48 h before
the level of reporter gene expression was determined.
Assays of luciferase activity and TNF- protein expression.
Luciferase activity was measured by enzyme-dependent light production
using a luciferase assay kit (Promega). After each experiment, cells
were washed and incubated at room temperature for 10 min in 250 µl of
lysis buffer (Promega). Ten-microliter samples were then taken and
loaded into an automated luminometer (Bio-Rad, Hercules, CA). At the
time of measurement, 100 µl of luciferase substrate were
automatically injected into each sample, and total luminescence was
measured over a 20-s time interval. Output is quantitated as relative
light units per microgram of protein of the sample. For analysis of
TNF-
protein, cell-free supernatants were used. TNF-
levels were
determined using a TNF-
ELISA kit (R&D Systems, Minneapolis, MN)
according to the manufacturer's instructions.
Electrophoretic mobility shift assay.
To detect NF-B binding activity, nuclear protein extracts were first
prepared as follows. Cells were treated with 500 µl of lysis
buffer (50 mM KCl, 0.5% Nonidet P-40, 25 mM HEPES, 1 mM PMSF, 10 mg/ml
leupeptin, 20 µl/ml aprotinin, and 100 mM DTT) on ice for 4 min.
Nuclei were pelleted by centrifugation at 14,000 rpm for 1 min and were
resuspended in 300 µl of extraction buffer (500 mM KCl, 10%
glycerol, 25 mM HEPES, 1 mM PMSF, 1 µl/ml leupeptin, 20 µg/ml
aprotinin, and 100 µM DTT). After being centrifuged at 14,000 rpm for
5 min, the supernatant was harvested and stored at
70°C. The
protein concentration was determined using BCA protein assay reagent
(Pierce, Rockford, IL).
LPS stimulation and ON inhibition studies.
For in vitro studies, RAW cells were plated on 96-well plates
(105 cells/well) and preincubated for 12 h at 37°C
with 1 µM ON in serum-free DMEM. After being preincubated, the cells
were treated with LPS (1 µg/ml) at 37°C for 6 h. After the
treatment, cell culture supernatants were collected and used for
TNF- protein assay. The cell pellets were harvested and used for
protein extraction and nuclear factor binding assay. For in vivo
studies, mice were treated via an intratracheal instillation with 50 µl of the test solution containing 30 µg of LPS. In studies
designed to assess the inhibitory effects of ON, mice were pretreated
intratracheally with ON (1-100 µg), either alone or in
combination with DOTAP (100 nmol), for 2 h and then challenged
with LPS (30 µg). At various times after treatment, mice were killed,
and BAL was performed. BAL cells were isolated by centrifugation as
earlier described and used for cell counts and differentials. The
supernatants were collected and used for TNF-
measurements. For lung
histological studies, a separate group of animals was similarly treated
but not subjected to BAL. After death, the lungs were inflated with 10% formalin solution instilled through the trachea for 2 h and then fixed with buffered 10% formalin solution for 24 h. After being embedded in paraffin, the samples were sectioned, mounted on
glass slides, and stained with hematoxylin and eosin (H&E) for light
microscopic examination.
Statistical analysis. Each study group consisted of four experiments. Statistical analysis between study groups was performed with paired two-tailed Student's t-test. The level of significance was P < 0.05.
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RESULTS |
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Maximum LPS inducibility of TNF- promoter requires NF-
B
activation at the
3 site.
It has been reported that there are four NF-
B binding sites in the
1 kb region of murine TNF-
promoter (8, 35). These
B binding sites are depicted in Fig. 1A. With the use of
point mutation assays, we further evaluated the role of specific
B sites on LPS-inducible promoter activity of TNF-
. The four
B sites in the
863 region of TNF-
promoter were individually mutated by PCR. The four mutated and wild-type promoters (
836/
18) were obtained and named
1m,
2m,
3m,
4m, and
863WT,
respectively. These promoters were cloned into the T/A cloning
vector and then subcloned into the pGL-3 basic vector at
HindIII/XhoI sites. The promoter activity was
determined by luciferase assay using transiently transfected macrophage
RAW 264.7 cells. The results showed that the plasmid containing
wild-type promoter had a strongly LPS-inducible promoter activity,
whereas those containing
1m,
2m, or
3m had reduced promoter
responsiveness (Fig. 2A). It
was noted that mutation of the
3 site led to a very strong reduction
in both the basic and inducible promoter activities, whereas mutation
of the
4 site had no effect on the promoter activity.
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Inhibition of TNF- expression in RAW cells by site-specific ON.
The identification of the
3 site as the most critical site for LPS
induction of TNF-
suggests the potential utility of
3-containing ON as an effective and specific inhibitor of TNF-
expression in cell
systems. To test this possibility, two ON, each containing two repeated
3 sequences (to increase the NF-
B binding capability) but with
different chemical modifications, were synthesized. The first ON
contains a naturally occurring phosphodiester backbone (PD), whereas
the second ON contains a nuclease-resistant phosphorothioate backbone
(PT). The two ON were tested for their inhibitory effect on LPS-induced
TNF-
expression in RAW cells. Figure
3A showed that both PD and PT
were effective in inhibiting TNF-
protein expression, whereas their
mutated sequences (mPD and mPT) had no effects, thus suggesting the
specificity of the inhibitory effect. The observation that PD was as
effective as PT also suggests the relative stability of this ON to
nuclease digestion under the experimental conditions.
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Inhibition of LPS-induced pulmonary inflammation in mice by ON
inhibitors.
LPS-induced pulmonary inflammation is associated with an increased
production of TNF- and sequestered pulmonary neutrophils (6,
10). In the present study, mice were treated with LPS intratracheally (30 µg/mouse), and the levels of TNF-
and
infiltrating neutrophils in BAL fluids were determined. Figure
4A shows that LPS treatment
caused a rapid increase in TNF-
level with a peak response at 6 h. Neutrophil cell count also increased with a peak response at 24 h. Treatment of mice with saline control had no significant effects on
both TNF-
and neutrophil cell count at all times (results not
shown). To test the effect of ON inhibitors on lung inflammatory
response, mice were pretreated intratracheally with varying amounts of
ON inhibitors or their mutated sequences (1-10 µg/mouse) and
then challenged with LPS (30 µg/mouse). Figure 5, A and B, shows
that the ON inhibitors PD and PT, when used alone, had relatively minor
effects on LPS-induced TNF-
production and neutrophil
influx. No inhibitory effects were observed with the control
mPD or mPT. Increasing the amount of ON inhibitors beyond 100 µg/mouse did not result in improved inhibitory effects. Because ON
are known to be taken up poorly by cells due to their hydrophilic
nature and are relatively unstable due to nuclease digestion (32,
34), it is, therefore, possible that poor cellular uptake or
enzymatic instability of these compounds, coupled with rapid clearance
from the lung (22, 28), may be responsible for the in vivo
inefficiency. Because the results of this study showed that the
nuclease-resistant PT did not give better inhibitory effects compared
with the nuclease-sensitive PD, it is therefore more likely that poor
cellular uptake and/or rapid clearance may be the key contributor(s) of
ON inefficiency.
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DISCUSSION |
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The use of sequence-specific ON as inhibitors of gene expression
provides a powerful tool for elucidating the role of a particular gene
and allows specific therapeutic intervention when that gene is
overexpressed (32, 34). The strong binding affinity of ON
to their targets makes these compounds potentially effective and
specific against pathological gene expression. Inhibition of gene
expression by double-stranded transcription factor-binding ON (also
called "decoy" ON) has previously been reported (5) and has increasingly been investigated as a new therapeutic strategy for the treatment of various diseases (see Ref. 24 for
review). In general, the use of ON-based therapeutics requires that two conditions be met: the identification of an appropriate target and the
use of an efficient and specific means for inhibition. In several
inflammatory and immune disorders, an overexpression of the early
response cytokine TNF- has been shown to play a pivotal role in the
induction and progression of the disease (23). Therefore,
suppression of this cytokine represents a logical therapeutic approach
for treating disease.
We have shown in this study that it is possible to inhibit TNF- gene
expression by utilizing ON that bind specifically and competitively to
the regulatory protein NF-
B. Our approach was based on the
identification of specific target sequences on the TNF-
promoter
that are required for NF-
B binding and transactivation of the
TNF-
gene. Several NF-
B binding sites with various
transcriptional activities were identified on the TNF-
promoter. The
3 (
510) site was the most critical site for LPS inducibility of
TNF-
expression. This conclusion was supported by our gene mutation and EMSA studies, which indicated that mutation of the
3 site abolished LPS-induced TNF-
promoter activity (Fig. 2A)
and that ON containing the
3 sequence was most effective in
inhibiting NF-
B binding activity (Fig. 2B). These results
are consistent with previous gene deletion experiments that
demonstrated that the promoter region spanning the nucleotide
655 to
427 was required for maximum induction of mouse TNF-
gene
(35). In a separate study, however, Drouet et al.
(12) reported that all four
B sites of the TNF-
gene
promoter were roughly equal in importance regarding their LPS
inducibility as determined by gene mutation assay. The basis for this
discrepancy is not clear but may be due to differences in specific
point mutations of the
B motifs, cellular sources, and treatment
conditions in the two studies. It is important to note that individual
B sites normally act in concert with other
B sites as well as
other protein binding regions; therefore, their relative activity and
contribution are generally interdependent.
Although the role of NF-B in the regulation of mouse TNF-
gene
has been established, its role in human TNF-
gene remains a subject
of controversy, partly because the high-affinity
3 (
510) site in
the mouse promoter is absent in the human gene (21).
Previous studies of the inducibility of human TNF-
gene promoter by
PMA failed to indicate a role for NF-
B (13, 16). However, subsequent studies showed that both NF-
B and non-NF-
B nuclear proteins are required for maximum induction of the human TNF-
gene by LPS and to a lesser extent by PMA (14,
21). Comparative studies of the similarities and differences
between human and mouse TNF-
promoters and their responses to LPS
have been reported (21).
With the use of a supershift assay, we further demonstrated in this
study that the DNA-NF-B complexes induced by LPS in mouse RAW cells
consisted of the p65/p50 heterodimer and the p50/p50 homodimer. The p65
has previously been shown to provide a trans-acting domain
for NF-
B activation, whereas the p50 acts as a repressor in the
transcription (7). The ability of the
3 ON (PD and PT)
to inhibit the NF-
B complex formation (Fig. 3C) supports our findings of the inhibitory effect of
3 ON on TNF-
expression (Fig. 3A).
With the use of a murine lung inflammation model, we also demonstrated
that the 3 ON could inhibit LPS-induced TNF-
production and
inflammatory neutrophil influx (Fig. 5). This inhibition was sequence
specific because ON carrying mutated
3 sequence (mPD and mPT) had no
effects. Although the inhibitory effect of
3 ON on lung inflammation
can be attributed to the blockage of TNF-
, other possible
mechanisms, such as blockage of other NF-
B-dependent genes, may also
be involved. In this study, the inhibitory effects of
3 ON were
shown to require a liposomal delivery agent, DOTAP, for efficient
inhibition. Previous studies have shown that DNA, when given alone via
pulmonary administration, is rapidly cleared from the lung (22,
28). However, when codelivered with liposomes, the DNA remains
in the lung for an extended period and to a greater level before being
washed out of the capillary bed by normal blood flow (22,
28). Thus it appears that the retention time of the DNA or other
drug molecules in the lung is likely to play a critical role in
determining therapeutic efficacy. With regard to ON, it has been
reported that ON, due to their polyanionic nature, poorly permeate the
cells to reach their intracellular target sites (17, 42).
Several research groups (3, 29, 33, 40) also observed that
in the absence of appropriate delivery systems, ON exhibited weak or no
biological activity, whereas in the presence of delivery systems, e.g.,
liposomes, ON showed strong activity. In agreement with these findings,
our results showed that coadministration of the ON with the liposomal
agent DOTAP greatly enhanced the inhibitory activities of ON. The DOTAP itself exhibited no inhibitory effects, indicating that this agent has
no direct effect on lung cells but likely acts by increasing the
cellular uptake and/or retention time of ON in the lung. Furthermore, both PD and PT ON were similarly effective when codelivered with DOTAP,
suggesting a stability-enhancing effect of DOTAP on the PD ON. It is
interesting to note that unlike the in vivo inhibitory effect, the
effect of ON in vitro did not require the liposomal delivery agent.
However, such an effect required a prolonged incubation of the cells
with ON, i.e., 12 h before LPS stimulation, in serum-free medium
to minimize degradation (15). A short-term incubation with
ON, i.e., <2 h, did not result in any significant reduction in the
cellular TNF-
response (results not shown). Likewise, a long-term
pretreatment of mice with ON (12 h, without liposome) before LPS
stimulation did not result in an improved inhibitory effect of the ON,
presumably due to their rapid lung clearance and slow cellular uptake.
These results suggest that to be biologically active in vivo, the ON
must be delivered by appropriate means to enhance their residence time
and cellular uptake characteristics.
In summary, we demonstrated that the 3 (
510) site of TNF-
gene
promoter was required for maximum LPS inducibility in macrophage RAW
cells. Mutation of this site caused a major reduction in LPS inducibility of the TNF-
gene. EMSA studies showed that ON carrying the
3 sequence were able to compete for NF-
B binding. Supershift assays revealed that the NF-
B complexes were composed of the p65/p50
heterodimer and the p50/p50 homodimer. The
3 ON was effective in
inhibiting LPS-induced TNF-
gene expression and neutrophil infiltration in a murine lung inflammation model. These findings have a
direct implication on the therapeutic utilization of this compound in
inflammatory and immune diseases. A similar gene inhibition approach
may be employed to aid the study of other gene functions and their
roles in disease pathogenesis.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-62959.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Rojanasakul, Dept. of Basic Pharmaceutical Sciences, West Virginia Univ. Health Sciences Center, PO Box 9530, Morgantown, WV 26506.
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.
First published October 11, 2002;10.1152/ajplung.00134.2002
Received 6 May 2002; accepted in final form 10 September 2002.
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