1 Department of Pediatrics, The Evanston Hospital, Northwestern University Medical School, Evanston, Illinois 60201; 2 Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota 55455; 3 Genzyme Corporation, Framingham, Massachusetts 01701-9322; and 4 Department of Anesthesiology and Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35233
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ABSTRACT |
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Nitric oxide (· NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous · NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK1) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK1-WTCFTR). Exposure of these cells to 125 µM DETA NONOate at 37°C for 24 h (a chemical · NO donor) diminished Zn2+-induced and uninduced CFTR protein levels by 43.3 ± 5.1 and 34.4 ± 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of · NO, were added to the medium. Exposure to · NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK1-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible · NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM NG-monomethyl-L-arginine in the medium. Pretreatment of LLC-PK1-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of · NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that · NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.
peroxynitrite; guanosine 3',5'-cyclic monophosphate; cystic fibrosis; gene transfer; inflammation; nitric oxide synthase; DETA NONOate; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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THE STRATEGY OF GENE therapy for the treatment of
recessive hereditary diseases involves substitution of the defective
gene product with a heterologously expressed wild-type protein. Gene therapy has been proposed for the treatment of cystic fibrosis (CF) and
a number of other diseases. CF is caused by mutations of cystic
fibrosis transmembrane conductance regulator (CFTR), a cAMP-stimulated
Cl channel that is
predominantly localized to the apical plasma membranes of epithelial
cells. Substitution of the mutant CFTR with heterologously expressed
wild-type gene has been accomplished in tissue cultures of CF
epithelial cells and has resulted in correction of the defective
Cl
transport in these cells
(17, 33).
However, several problems have been encountered with gene transfer in vivo. Synthetic DNA constructs have to be delivered to the target cells, enter the cells, and translocate in the nucleus, and the transgenes have to be expressed at a sufficiently high level for functional correction of the missing or defective protein. In the case of gene therapy for CF, delivery of gene constructs to the targets (airway epithelial cells) is hampered by airway obstruction; in addition, airway epithelial cells are notoriously difficult to transfect. Considerable effort has been focused on bypassing these obstacles. However, even after efficient delivery and transfection, the longevity of transgene expression in target cells is limited by a variety of mechanisms (18). Understanding the mechanisms that may limit the longevity of heterologous gene expression in epithelial cells will be critical for the development of clinically useful CF gene therapy.
Current methods of gene transfer in vivo achieve only short-lived, transient gene expression. Immune reaction against transfected cells and/or against the gene delivery vehicles is one of the main mechanisms that limits the longevity and level of transgene expression. An immune response mediated by cytotoxic T and T helper cells destroys transduced cells after gene transfer using adenoviral vectors with an intact E4 region in immunocompetent mice (6, 18, 42). On the other hand, transgene expression declines even in immunodeficient nude mice after gene transfer using adenoviral vectors with truncated E4 regions (18). Transgene expression is also transient after in vivo gene transfer using cationic lipids or naked DNA (12, 40). Furthermore, loss of transgene expression has been observed in vitro in the absence of immune cells (22). The variables contributing to the loss of heterologous gene expression in the absence of a clear-cut immune response are poorly understood. Emerging evidence indicates that nitric oxide (· NO) can modify gene expression by either cGMP-dependent (8, 31) or cGMP-independent mechanisms (21, 29) and may modify gene transfer by regulating the expression of transgenes. · NO produced under basal conditions by lung cells, including human airway and bronchial cells (32), serves as a key signaling molecule in diverse physiological processes (27). In addition, exposure to inflammatory mediators such as proinflammatory cytokines (14), lipopolysaccharide (35, 39), and viruses (1, 9) induces endothelial, epithelial, and inflammatory cells to generate large amounts of · NO, mainly via the inducible isoform of nitric oxide synthase (iNOS). Furthermore, · NO production is increased in most human inflammatory lung diseases in which gene therapy is advocated, including CF (3, 4).
Previously, we infected murine fibroblast cells that express the human
iNOS (NIH/3T3-iNOS) but lacked tetrahydrobiopterin (BH4), a cofactor necessary for · NO production
(38), with replication-deficient adenovirus (E1-deleted) vectors
containing reporter genes. Our results indicate that addition of
sepiapterin before infection upregulated endogenous · NO
production and decreased both luciferase and -galactosidase protein
expression in NIH/3T3-iNOS to ~60% of their control values.
Furthermore, this decrease was prevented by coincubating NIH/3T3-iNOS
cells with
NG-monomethyl-L-arginine
(L-NMMA; 1 mM), which decreased · NO to baseline
levels (15). The present study was designed to determine whether
exogenous · NO produced either by · NO donors or by
NIH/3T3-iNOS cells affects CFTR transgene expression in epithelial
cells and whether the · NO effects on CFTR transgene
expression are mediated by cGMP-dependent or -independent mechanisms.
Our results indicate that heterologous expression of human wild-type
CFTR in the porcine kidney LLC-PK1
cells (LLC-PK1-WTCFTR) is impaired
by physiological (nanomolar) levels of · NO, likely to be
present in inflamed tissues. The noted effects of · NO on
CFTR expression were not mediated by changes in cGMP levels and were
not due to cytotoxicity or increased apoptosis.
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MATERIALS AND METHODS |
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Chemicals. Tissue culture media and
defined FBS were purchased from GIBCO BRL (Gaithersburg, MD) and from
HyClone (Logan, UT), respectively. Acrylamide, bis-acrylamide, ammonium
persulfate, N,N,N',N'-tetramethylethylenediamine,
urea, and -mercaptoethanol were from Bio-Rad Laboratories (Richmond,
CA). Protein G immobilized on agarose beads was from
Boehringer-Mannheim (Indianapolis, IN). 14C-labeled SDS-PAGE
molecular-weight markers and
[
-32P]ATP were from
Amersham (Arlington Heights, IL). The monoclonal antibody used for CFTR
immunoprecipitation was from Genzyme (mAb 24-1).
2,2'-(Hydroxynitrosohydrazo)bis-ethanamine (DETA) NONOate (28) was from Cayman Biochemicals and was dissolved as a 100 mM stock
in 0.1 N NaOH. All other chemicals were from Sigma (St. Louis, MO).
Isolation of human red blood cells. Human red blood cells (RBCs) were freshly isolated for each experiment from heparinized blood provided by one of the authors. Blood was diluted 1:3 with DMEM and was passed through Ficoll by centrifuging at 2,000 g for 10 min. RBCs were removed from the bottom layer, washed two times in PBS, and resuspended in DMEM at 50% vol/vol.
Tissue culture and · NO generation. NIH/3T3-iNOS cells were a generous gift from Dr. Edith Tzeng (Department of Surgery, University of Pittsburgh, Pittsburgh, PA). NIH/3T3-iNOS cells were generated by transfecting NIH/3T3 cells with a retroviral vector with recombinant human iNOS cDNA and neomycin-resistant gene inserts, followed by selection of stably expressing clones in neomycin (38). LLC-PK1-WTCFTR were generated by a similar strategy utilizing the bovine papilloma virus-based pBPV-CFTR vector construct and selection with neomycin (24). In this cell line, the expression of human wild-type CFTR is under the control of a heavy metal-sensitive metallothionein promoter. Both LLC-PK1-WTCFTR and NIH/3T3-iNOS cells were maintained in DMEM supplemented with 10% FBS, 400 µg/ml geneticin, and 2% penicillin-streptomycin. Experiments were performed after at least 3 days in culture at 75-90% confluence. CFTR expression in LLC-PK1-WTCFTR cells was induced by including 50 µM ZnSO4 in the culture medium.
In the first series of experiments, LLC-PK1-WTCFTR cells, seeded in 12-well multiwell dishes in DMEM without FBS, were exposed to · NO by adding 125 mM DETA NONOate to the medium for 24 h. In additional experiments, LLC-PK1-WTCFTR cells were cocultured with NIH/3T3-iNOS cells in a 2:1 ratio for 24 or 48 h. In some studies, 105 freshly isolated RBCs were included in each well of a 12-well dish to scavenge · NO.
Measurements of nitrite and · NO. Nitrite levels were determined in the cell culture medium by the Greiss assay, with sodium nitrite as the standard. Before the measurements, nitrate was reduced to nitrite using Escherichia coli nitrate reductase as previously described (15). Evolution of · NO in the medium (pH 5.0-7.4, 37°C) by DETA NONOate was measured with an ISO-NO electrochemical probe (World Precision Instruments, Sarasota, FL) connected to an IBM-compatible computer via an analog-to-digital converter. Concentrations of · NO were derived by comparing the signal against the one obtained from a · NO-saturated water solution (1.94 mM · NO).
Measurements of lactate dehydrogenase release and apoptosis. Lactate dehydrogenase (LDH) levels in the media and in cell lysates were measured spectrophotometrically as the rate of NADH consumption in the presence of pyruvate. LDH release was expressed as the percentage of total cellular LDH released. Apoptosis of LLC-PK1-WTCFTR was assayed using fluorescence (FITC; excitation: 494 nm; emission: 518 nm) TUNEL staining of fragmented DNA with a kit (Promega) according to the manufacturer's instructions. The total number of nuclei was assessed by counterstaining LLC-PK1-WTCFTR cells with Hoechst 33258 DNA stain (excitation: 352 nm; emission: 461 nm). Fluorescence microslides were viewed with a fluorescence microscope, and the number of apoptotic nuclei (FITC) and the total number of nuclei (Hoechst) were counted for five fields of view of each preparation under a ×20 objective.
Immunoprecipitation of CFTR.
LLC-PK1-WTCFTR cells exposed to
· NO for 24 h as described above were lysed for 30 min at
4°C in lysis buffer [1% Nonidet P-40, 150 mM NaCl, 1 mM
EDTA, 20 mM HEPES (pH 7.4), 1 µg/ml aprotinin, 1 µg/ml
leupeptin, and 1 µg/ml 4-(2-aminoethyl)benzenesulfonyl
fluoride]. Lysates were centrifuged at 16,000 g for 5 min, and the supernatants were
removed and mixed with 1/4 volume of 5×
radioimmunoprecipitation assay (RIPA) buffer [250 mM Tris (pH
7.5), 120 mM sodium deoxycholate, 650 mM NaCl, 5% Triton X-100
(vol/vol), and 0.5% SDS] and 0.8 µg of monoclonal -CFTR
(COOH terminus-specific) antibody or nonimmune mouse IgG. After 90 min
of incubation at 4°C, antigen-antibody complexes were collected by
capture on 5 µl of protein G-conjugated agarose beads
(Boehringer-Mannheim) for 90 min at 4°C. Precipitates were washed
two times in 1× RIPA and then two times in kinase buffer [50 mM Tris · HCl (pH 7.5), 10 mM
MgCl2, and 0.01% BSA] and
were subjected to phosphorylation for 30 min at 30°C in kinase
buffer using the purified catalytic subunit of the cAMP-dependent
protein kinase (15 U/reaction; Promega) and
[
-32P]ATP (6,000 Ci/mmol; 10 µCi/reaction; NEN, Boston, MA). Subsequently, samples
were washed two times in 1× RIPA and were separated by SDS-PAGE.
The intensity of different bands was quantified by phosphorimaging using the IPLab Spectrum software. Band intensities were calculated as
the sum of pixel intensities above background within the area corresponding to band C (i.e., the fully glycosylated 170- to 190-kDa
band of CFTR). CFTR expression in each sample is expressed relative to
the mean CFTR band intensity of duplicate or triplicate samples of
untreated controls.
Data analysis. In general, data are shown as means ± SE unless otherwise noted. Experiments were performed with duplicate or triplicate samples. The mean value of untreated controls was considered 100%, and all individual samples, including controls, were normalized to this value. Paired or unpaired Student's t-test was used for statistical analysis between two group means. Statistical differences among multiple group means were determined using one-way ANOVA and the Bonferroni modification of the t-test. Statistical significance was considered at P < 0.05.
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RESULTS |
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LLC-PK1-WTCFTR cells.
LLC-PK1-WTCFTR cells were
generated to study CFTR expression and function in the context of
polarized epithelial cells (24). LLC-PK1-WTCFTR cells form
polarized monolayers and, unlike the parental
LLC-PK1 line, transport
Cl in a vectorial and
cAMP-dependent manner (2). To relate their levels of CFTR expression to
a cell line with endogenous CFTR expression, we performed parallel CFTR
immunoprecipitation experiments using equal amounts of total cellular
protein from cell lysates of
LLC-PK1-WTCFTR and HT-29-CL19A
colonic adenocarcinoma cells. Figure 1
depicts results from a representative experiment, indicating that in
the absence of Zn2+,
LLC-PK1-WTCFTR cells express
slightly less CFTR than HT-29-CL19A cells. After stimulation of the
metallothionein promoter with 50 µM
Zn2+, CFTR expression in
LLC-PK1-WTCFTR cells increased
manyfold, surpassing the expression levels in HT-29-CL19A cells.
Immunoprecipitation performed with nonimmune IgG (Fig. 1,
lane 2) verified the lack of
nonspecific protein bands at and near the region of CFTR.
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Quantification of · NO release by DETA NONOate.
Because our experimental strategy relied heavily on the use of DETA
NONOate, we sought to determine whether the · NO
concentration released from this chemical · NO donor
corresponded to · NO concentrations that may be encountered
in vivo. Addition of 125 µM DETA NONOate at pH 7.4 to the medium
generated maximum steady-state · NO concentration ([NO]max)
values that were near the detection limit of the ISO-NO electrode
(<100 nM; Fig.
2A).
In contrast, 125 µM DETA NONOate generated significantly higher
peak [NO]max values at
lower pH values (5.5-7.0; Fig.
2A). To verify our measurement of
[NO]max at pH 7.4, the
predicted [NO]max
value at pH 7.4 (87 nM) was extrapolated from a curve fitted to the
more reliable [NO]max
measurements at pH values between 5.5 and 7.0 (Fig.
2B). These results indicate that
peak · NO concentrations used in this study were
<100 nM, comparable to · NO concentrations in inflamed
tissues in vivo (34, 41).
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Exposure of LLC-PK1-WTCFTR cells to
· NO caused no significant cytotoxic or proapoptotic effect.
Exposure of LLC-PK1-WTCFTR cells
to 50 µM Zn2+ increased LDH
release significantly; however, LDH levels in the medium were still small (<4% of total cellular LDH; Table
2). Treatment with 125 µM DETA NONOate
did not significantly increase LDH release above values noted in the
presence of Zn2+ alone
(Zn2+ and
Zn2+ + 125 µM DETA NONOate;
P = 0.42). In addition, exposure of
LLC-PK1-WTCFTR cells to 125 µM
DETA NONOate did not increase the number of apoptotic cells (data not
shown). These data indicate that none of our treatment conditions
resulted in widespread cytotoxicity; thus the inhibition of CFTR
expression by · NO was not a consequence of cytotoxicity.
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DISCUSSION |
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Gene therapy is based on the principle that a gene construct substituting for a missing or defective endogenous gene can be delivered and heterologously expressed in cells using vector- or virus-based delivery/expression systems. Gene therapy has been proposed and tested in preclinical and clinical trials for a broad range of inherited and acquired disorders including CF, hemophilia A and B, Duchenne's muscular dystrophy, Parkinson's disease, neoplasias, and hematopoietic disorders. To achieve a therapeutic effect, persistent expression of the heterologous gene product is desirable for most applications. Furthermore, transgene expression has to be maintained at a sufficiently high level for the functional substitution of the missing or defective endogenous gene product. To develop efficient gene therapy for treatment of genetic diseases, host mechanisms antagonizing heterologous gene expression have to be identified to create effective strategies to circumvent these limitations.
· NO has been shown to inhibit the expression of heterologous reporter genes in tissue culture and after gene delivery to rodent airways (15). The present study was designed to directly test whether the heterologous expression of CFTR is inhibited by · NO in polarized epithelial cells and to provide some insight into putative mechanism(s) by which · NO inhibits CFTR expression. We have chosen a stable expression system for our studies that provides sufficiently high levels of expression for the reproducible detection of CFTR protein from 10,000 to 50,000 polarized epithelial cells grown on permeable filters. The results indicate that very small concentrations of · NO produced either by a chemical · NO donor (DETA NONOate) or by NIH/3T3-iNOS cells inhibit heterologous CFTR expression. Furthermore, the fact that inhibition of CFTR expression was reversed when · NO concentrations were reduced to control levels by the use of · NO scavengers (RBCs) or inhibitors of · NO production (L-NMMA) strongly suggests that the noted effects were due to the actions of · NO per se.
The chemical · NO donor DETA NONOate has been known to
release · NO at a slow rate (half-time 56 h), without the
generation of superoxide. We estimated that the peak concentration of
· NO under our experimental conditions was between 50 and 100 nM, a range of concentrations comparable to steady-state tissue
concentrations likely to be found in inflamed tissues (34, 41). At
these low · NO concentrations, nitrogen dioxide production
from the reaction of · NO with oxygen will be very low.
NIH/3T3-iNOS cells are deficient in BH4 synthesis and when
cultured alone do not produce · NO without addition of
sepiapterin, i.e., a precursor of BH4 (38). On coculture
with LLC-PK1-WTCFTR cells,
NIH/3T3-iNOS cells produced NO in the absence of sepiapterin. We
attribute this phenomenon to the release of BH4 or one of
its precursors by LLC-PK1-WTCFTR
cells in the media (see RESULTS). The rate of nitrite
accumulation in the tissue culture media after 24 h of treatment with
125 µM DETA NONOate (119 µM/24 h) and after coculture of
LLC-PK1-WTCFTR and NIH/3T3-iNOS
cells (20 µM · 106
cells
1 · 24 h
1) was similar to the
rate of nitrite production by murine peritoneal macrophages (60 µM · 106
cells
1 · 24 h
1; see Ref. 20).
Although our studies did not provide a definitive answer regarding the exact mechanism by which · NO inhibits heterologous CFTR expression in LLC-PK1-WTCFTR cells, we have excluded the role of cGMP in the regulation and the role of cytotoxic or proapoptotic effects of · NO on LLC-PK1-WTCFTR cells. The presence of ODQ, a specific and potent inhibitor of guanylyl cyclase (36), had no effect on inhibition of CFTR expression by · NO. Additionally, treatment of LLC-PK1-WTCFTR cells for 24 h with 8-bromo-cGMP, a cell-permeant analog of cGMP, did not change CFTR expression levels. These data indicate that · NO inhibits CFTR expression via a cGMP-independent mechanism. Furthermore, our results indicate that the observed effects were not due to · NO-induced cell injury or apoptosis.
The vector construct providing stable CFTR expression in
LLC-PK1-WTCFTR cells is based on
the bovine papilloma virus genome. Bovine papilloma virus-based vectors
have been known to replicate episomally (30). One possible explanation
for the inhibitory effect on CFTR transgene expression is related to
the stability of episomal DNA. This possibility is supported by the
observation that airway epithelial cells transduced with an episomally
replicating CFTR cDNA construct have lost the episomal vector in the
absence of selection in hygromycin (22). Similarly,
LLC-PK1-WTCFTR cells have to be
maintained under continuous selection in G418 to maintain CFTR
expression (unpublished observations). A facilitating effect of
· NO on the naturally occurring elimination of episomal DNA could explain the inhibitory effect of · NO on heterologous
CFTR expression in our model system. Transcription from viral vectors typically requires the contribution of transcription factors from the
host cells (7, 11, 23) and from the viral genome (30). Reactive
oxygen-nitrogen intermediates such as peroxynitrite are known to modify
proteins and affect their function either by nitrating or oxidizing key
amino acids (13) or by S-nitrosylation of critical thiol groups (25).
Via such mechanisms, · NO can inhibit the function of
transcription factors such as nuclear factor-B (25, 37). Whether the
observed inhibition of heterologous CFTR expression and reporter
molecule expression occurs at the level of episomal plasmid replication
and/or transcriptional regulation and whether its mechanism involves
the · NO-dependent inhibition of transcriptional factors are
yet to be determined.
Although the continuous improvement of pharmacological approaches steadily improves the outlook for CF patients, gene therapy is regarded by many as the ultimate solution to not only treating but also curing CF patients. Current experimental gene therapy approaches provide only short-lived transient gene expression. Our current study identifies inflammatory · NO production as a host reaction that might be partially responsible for the low level and transient nature of transgene expression in CF gene therapy. The relative quantity of · NO production in the CF lung is poorly characterized, especially in the context of gene therapy where the airway surfaces are exposed to large amounts of synthetic materials or viral DNA, which may trigger · NO production. Therefore, it is yet to be determined whether sufficient · NO production exists in distal CF airways in vivo after gene delivery that could limit the expression of the CFTR transgene. Recent studies indicate the absence of iNOS in the epithelium of CF airways (19, 26). Furthermore, there is considerable controversy regarding the levels of exhaled · NO in patients with CF. A recent study clearly demonstrated the presence of increased levels of nitrite in breath condensates of patients with CF (16). This finding indicates that, in patients with CF, exhaled · NO may not reflect the total amount of · NO produced by airway cells, perhaps due to the difficulty of getting through the thick airway secretions. Additionally, inflammatory cells of patients with CF have normal levels of iNOS and thus may contribute considerable levels of · NO in the close vicinity of airway cells (26).
In summary, our data indicate that · NO inhibits CFTR transgene expression in epithelial cells via a mechanism that is not mediated by cGMP. The exact role and quantity of inflammatory · NO in CF is controversial; however, there is sufficient evidence indicating the potential presence of large amounts of · NO in the CF lung. Considering the possibility that viral vectors or chemical irritants might exaggerate · NO production during and subsequent to gene transfer, inflammatory · NO production might be a major obstacle for successful CF gene therapy. A better understanding of the mechanism by which · NO inhibits CFTR expression might aid in designing strategies for more efficient therapeutic gene transfer.
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ACKNOWLEDGEMENTS |
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We thank Kevin L. Kirk and Eric J. Sorscher for allowing access to their laboratory resources in support of this project and for their many helpful discussions. Ping Hu provided valuable technical assistance for the determination of · NO2 and · NO3.
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FOOTNOTES |
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This work was supported by the Jessica Jacobi Golder Endowment (T. Jilling), National Heart, Lung, and Blood Institute Grants HL-31197 and HL-51173, and Office of Naval Research Grant N00014-97-1-0309 (S. Matalon).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 19th St., THT 940, Birmingham, AL 35233 (E-mail: sadis.matalon{at}ccc.uab.edu).
Received 2 April 1998; accepted in final form 5 March 1999.
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