Molecular mechanisms of antioxidant enzyme expression in lung
during exposure to and recovery from hyperoxia
Linda Biadasz
Clerch1,2,
Donald
Massaro1,3, and
Alla
Berkovich1,2
1 Lung Biology Laboratory, and
Departments of 2 Pediatrics and
3 Medicine, Georgetown
University School of Medicine, Washington, DC 20007-2197
 |
ABSTRACT |
Manganese superoxide dismutase (MnSOD) activity
falls ~50% in lung during 48 h of exposure of adult rats to >95%
O2 (L. B. Clerch and D. Massaro.
J. Clin. Invest. 91: 499-508,
1993). We now show that hyperoxia also decreased MnSOD activity in
lungs of adult baboons, making the phenomenon potentially more
important to humans. In rats, a decrease in lung MnSOD activity during
an initial 48 h of exposure to >95%
O2 and its increase during an immediately subsequent 24 h in air were due to decreases and increases, respectively, in MnSOD specific activity and synthesis rate; the latter
was due to altered translational efficiency. The concentration in the
lung of copper-zinc superoxide dismutase mRNA, catalase mRNA, and
glutathione peroxidase mRNA, unchanged during the initial 48 h of
exposure to O2, rose approximately
twofold during reexposure to O2
after 24 h in air. The demonstration that the fall in MnSOD activity is
translationally and posttranslationally regulated during the initial
exposure to hyperoxia suggests that gene transfer to increase MnSOD
activity in hyperoxic lungs may also require therapy that maintains
translational efficiency and MnSOD specific activity.
translational efficiency; manganese superoxide dismutase; catalase; copper-zinc superoxide dismutase; glutathione peroxidase; gene
expression; baboons; rats
 |
INTRODUCTION |
THE TOXIC EFFECTS of the high concentrations of
inspired O2 often required in the
treatment of respiratory distress syndrome in prematurely born infants
are generally accepted as important risk factors for the development of
bronchopulmonary dysplasia (28).
O2 toxicity also occurs in adults,
but the role it plays in the morbidity and mortality of adults who
receive it as part of the treatment for the respiratory distress
syndrome is less clear than in prematurely born infants (6). That
O2 toxicity may be more prevalent
in adults than is appreciated is suggested by several reports, but
three in particular stand out. Individuals with irreversible brain
damage placed on mechanical ventilation with air for 60-70 h
exhibit a 30% fall in arterial O2
tension; similar mechanical ventilation but with 100%
O2 results in an 80% fall in
arterial O2 tension (1). Exposure
of healthy adults to 100% O2 for
as little as 18 h (12) or to 30-50%
O2 for an average of 45 h (19)
leads to changes in return from bronchoalveolar lavage, indicative of
damage to the alveolar-capillary interface.
The damaging effects O2 has on
cells are caused by intermediates that form as a result of its cellular
metabolism. The production of these intermediates, which include
superoxide,
H2O2,
and the hydroxyl radical (17), increases during exposure to hyperoxia (3, 4, 16). However, cells contain antioxidant enzymes [manganese
superoxide dismutase (MnSOD), copper-zinc superoxide dismutase
(Cu,ZnSOD), catalase, and glutathione peroxidase (GP)] that
protect against these intermediates. MnSOD and Cu,ZnSOD
catalyze the conversion of superoxide to
H2O2;
catalase and GP each convert H2O2
to water (17). Transgenic mice that overexpress Cu,ZnSOD and GP but not
Cu,ZnSOD alone are tolerant to hyperoxia (34). Furthermore, transgenic
mice that overexpress MnSOD in alveolar type II cells and
nonciliated bronchiolar epithelial (Clara) cells at the start of an
exposure to hyperoxia are tolerant to
O2 (36). As interesting and
provocative as these findings are, they do not address the issue of the
basis for tolerance or the lack of tolerance to
O2 as it occurs in nontransgenic
"wild-type" organisms. For example, in otherwise untreated adult
rats exposed to >95% O2, lung
MnSOD activity falls ~50% despite an increase in MnSOD mRNA
concentration, and 70-80% of the rats die within 72 h (9, 15, 32,
33).
The present study was undertaken 1)
to examine the molecular mechanism(s) responsible for the fall in MnSOD
activity that occurs in adult rats during the initial 48 h of exposure
to >95% O2 (9) and, as we now
report, for its increase during a 24-h period in air after 48 h of
hyperoxia; 2) to determine whether the fall in MnSOD activity during exposure to >95%
O2 is peculiar to the adult rat or
whether it occurs in other species, in particular a nonhuman primate,
thereby making it a more important and clinically relevant phenomenon;
and 3) to examine the regulation of
antioxidant enzyme expression during reexposure to
O2 in rats made tolerant to
O2 by a "rest" period in air
(15).
 |
MATERIALS AND METHODS |
Rats.
We used specific pathogen-free adult male Sprague-Dawley rats (Taconic
Farms, Germantown, NY) weighing ~250 g. They were maintained in our
Research Resources Facility on a 12:12-h light-dark cycle and were
allowed food (Rodent Laboratory Chow 5001, Ralston-Purina, St. Louis,
MO) and water ad libitum.
Rats were exposed to >95% O2 at
1 atmosphere or to air from a compressed air generator in identical
3.5-ft3 chambers constructed of
clear plastic. The other conditions of exposure of rats were <0.1%
CO2, 22-25°C, and
40-60% humidity. Exposures were continuous for the times
indicated except for 10-15 min daily when the chambers were opened
for housekeeping purposes. Rats were anesthetized (pentobarbital
sodium, ~80 mg/kg) and then killed by cutting the great vessels of
the abdomen. Lungs were perfused with ice-cold 0.15 M NaCl and either
stored at
80°C or processed immediately for the various
assays. Lungs in which we measured the rates of synthesis of MnSOD and
of general proteins were not perfused but were immediately sliced and
placed in medium at 37°C.
Baboons.
We used lungs from Papio cyanocephalus
anubis, common name olive baboon, and
Papio cyanocephalus
anubis/Papio
cynocephalus, common name olive/yellow
baboon. Baboons were born and raised in the colony at the
Southwest Foundation for Biomedical Research in San Antonio, TX, housed
at the same institution, and fed Purina baboon chow. The baboons were
ventilated with air for 10 ± 0.0 days (mean ± SE,
n = 6) or with 100%
O2 for 7.2 ± 1.4 days
(n = 8). The age of the air-ventilated
baboons was 4.3 ± 0.3 yr and that of the
O2-ventilated baboons was 4.5 ± 0.6 yr. All six air-ventilated baboons were male olive baboons.
Four O2-ventilated baboons were olive baboons, three of which were male; four
O2-ventilated baboons were
olive/yellow baboons, all of which were male. All baboons were killed
by an overdose of pentobarbital sodium. The lungs were not perfused and
were frozen in liquid N2 and
stored at
20°C.
Assays for enzyme activity.
As previously described in detail (9),
27,000-g supernatant fractions of lung
were obtained and used for all enzyme activity assays. Superoxide
dismutase (SOD) activity was determined by two methods: in some
experiments we used the xanthine oxidase ferricytochrome c
assay, which at pH 7.8 distinguishes between Cu,ZnSOD and MnSOD by
differential sensitivity to 1.0 mM sodium cyanide (11); in other
experiments we used the same method with 0.015 M sodium cyanide to
inhibit cytochrome oxidase and diethyldithiocarbamate to distinguish
between Cu,ZnSOD and MnSOD (24). The SOD data in Table
1 were obtained using the first method (11)
and those in Table 2 were obtained using
the second method (24). We do not know whether the use of two different
methods accounts for the large difference in MnSOD activity between
Tables 1 and 2. However, data from others (32, 33) exhibit
substantial differences in MnSOD activity when measured at different
times, even when using the same method; the same is true for Cu,ZnSOD
(32, 33). The same method used to measure MnSOD activity was used for
all lungs from each group of exposures. One unit of SOD activity was the amount that halves the rate of reduction of cytochrome
c. Lung extracts in which catalase and GP activities were
measured were prepared as previously described (9). Catalase activity was measured as the rate of disappearance of
H2O2
at 240 µm (2, 23), and 1 unit of catalase activity decomposes 1 µmol
H2O2/min at 25°C and pH 7.0. GP activity was measured by the
glutathione-oxidized glutathione recycling method (29)
using
H2O2
as substrate and sodium azide to inhibit catalase activity. One unit of
GP activity oxidizes 1 µmol NADPH/min.
Quantitation of MnSOD protein by Western
analysis.
Rat liver MnSOD was purified as previously described (21), and the
antigen was provided to Hazleton Laboratories (Vienna, VA) for antibody
production. The primary antibody used in these studies was rabbit
antiserum against rat MnSOD. To determine the concentration and
specific activity of rat lung MnSOD, we prepared the
27,000-g supernatant fraction of lung
homogenate and dialyzed it overnight and assayed the dialysate for
MnSOD activity. Samples of the dialysate were subjected to
electrophoresis in 8% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gels in the Mini-Protean II cell; transfer
to nitrocellulose was effected in the Mini Trans-Blot cell (Bio-Rad
Laboratories, Hercules, CA). After being blocked with gelatin and
reacted with rabbit anti-rat MnSOD antiserum, the blots were treated
with goat anti-rabbit immunoglobulin G conjugated to horseradish
peroxidase. The horseradish peroxidase band was identified as a purple
color that developed on treatment with 4-chloro-1-naphthol and
H2O2.
The amount of MnSOD protein was estimated by densitometric analysis of
the stained band. Pure MnSOD protein was also assayed to ensure that we
were working in a range within which density was proportional to the amount of MnSOD. Densitometry was performed on a Molecular Dynamics laser densitometer using ImageQuant software (Sunnyvale, CA).
MnSOD and general protein syntheses.
We measured the synthesis of MnSOD and of trichloroacetic
acid-precipitable proteins (general proteins) as previously described in detail (21). Briefly, we incubated 1.0-mm-thick lung slices in 10 ml
of Krebs-Ringer bicarbonate buffer with 5.5 mM glucose, adult rat
plasma concentrations of 19 amino acids (27), and 0.7 mM
L-[3H]phenylalanine.
At this phenylalanine concentration, the specific radioactivity of
tRNA-bound phenylalanine equals that of medium phenylalanine within 15 min of the start of incubation; this allows the use of the medium
specific radioactivity in the calculation of absolute rates of protein
synthesis (8, 21). The flasks were shaken at 120 oscillations/min at
37°C for 4 h under 95% O2-5%
CO2 (21).
At the end of the incubation, lung slices were rinsed in
phosphate-buffered 0.15 M NaCl and homogenized in 10 ml of 2 mM
-mercaptoethanol and 2.5 mM potassium phosphate (pH 8)
(buffer
A) for 3 min using the highest
setting of a Brinkman Instruments Polytron (Westburg, NY). A 200-µl
portion of the homogenate was added to an equal volume of cold 20%
trichloroacetic acid for assay of DNA (30) using calf thymus DNA as a
standard and for extraction of general proteins (21). The remainder of
the homogenate was centrifuged at 100,000 g for 1 h at 4°C. The supernatant
fluid was dialyzed overnight in 4 liters of
buffer
A and then centrifuged at 20,000 g for 20 min at 4°C.
To measure radioactivity incorporated into MnSOD, we added 5 µg of
14C-labeled MnSOD, which had been
labeled by reductive methylation (21), to each
20,000-g supernatant fraction.
Antiserum to MnSOD was added to these fractions and incubated at
30°C for 1 h. Immunoprecipitable material was collected by
centrifugation, denatured, and subjected to SDS-PAGE (21). Tritium
radioactivity migrating with the
14C-MnSOD standard was considered
to be incorporated into MnSOD (21). After electrophoresis, the gels
were sliced and radioactivity was extracted by overnight incubation
with Soluene 350 (Packard Instrument, Meriden, CT) at 56°C;
radioactivity was measured in a scintillation counter. Calculation of
the total disintegrations incorporated per minute was
corrected for recovery of the
14C-MnSOD marker, and the rate of
MnSOD synthesis was calculated (21).
Complementary RNA preparation and solution
hybridization for assay of antioxidant enzyme mRNA.
We obtained the following as gifts: a pGem-blue construct containing
the 3'- end of an
-actin cDNA from Dr. Rudolf K. Werner (Univ.
of Miami, School of Medicine, Miami, FL); a rat liver catalase cDNA,
PMJ 1010, from Dr. Shichi Furuta (Shimshu University School of
Medicine, Nagano, Japan); a rat liver GP cDNA, pGPx 1211, from Dr.
Ambati Reddy (Pennsylvania State Univ., University Park, PA); and a rat
liver MnSOD cDNA from Dr. Ye-Shih Ho (Wayne State Univ., Detroit, MI).
Each cDNA was subcloned into pGem vectors and used to generate sense
and antisense complementary RNAs (cRNAs) as previously described in
detail (7, 8). We generated a rat liver Cu,ZnSOD cDNA (20) and a
galectin-1 cDNA (10). These cDNAs were used to prepare
35S-labeled antisense cRNA probes
and sense cRNA standards as previously described (7-10). The
concentration of mRNA was measured in total nucleic acids isolated from
the lung using the method of Durnam and Palmiter (14). We added
[3H]actin cRNA at the
start of the process to isolate total RNA to enable us to account for
mRNA lost during the extraction (20). The amount of lung mRNA was
corrected for recovery, expressed relative to a standard curve, and
made normal to lung DNA.
Chemical assays.
DNA was assayed by pentose analysis (30) or fluorometrically with a TKO
100 fluorometer (Hoefer Scientific Instruments, San Francisco, CA); the
latter assay is based on the binding of the fluorescent dye Hoechst
3358 to DNA. DNA in all lungs from each set of exposures was measured
by one or the other method. Calf thymus DNA served as the standard for
both methods. Proteins were measured using the Coomassie Plus assay
reagent from Pierce Chemical (Rockford, IL), with bovine serum albumin
as standard.
Data collection and statistical
analysis.
For each parameter measured or calculated from measurements, the values
for individual animals were averaged per experimental group, and the SE
of the group mean was calculated. The significance of the difference
between two groups was determined by an unpaired t-test analysis (31). The significance
of the difference among more than two group means was determined by an
analysis of variance (13, 26) or by a randomized complete-block design
(35).
 |
RESULTS |
Antioxidant enzyme activity in rat lung during exposure to
O2 after a rest period in air and during
reexposure to O2.
As previously reported (9), at the end of 48 h of exposure to >95%
O2, MnSOD activity was
significantly lower in O2-exposed rats than in air-exposed rats, Cu,ZnSOD activity was the same in both
groups, and the activities of catalase and GP were higher in
O2-exposed rats than in
air-exposed rats (Table 1). Twenty-four hours after
O2-exposed rats had been returned
to room air, MnSOD activity was 2.2-fold higher than in rats at the end
of the 48 h of exposure to >95%
O2 and 1.3-fold higher than in
rats not exposed to hyperoxia (Table 1). The rest period did not alter Cu,ZnSOD activity or the effect of
O2 on the activity of catalase or
GP.
Western blot analysis with rabbit antiserum raised against rat liver
MnSOD (21) was used to estimate the amount of MnSOD. Immune antiserum
but not nonimmune antiserum reacted against MnSOD (Fig.
1). To ensure that we worked in a range
within which densitometry units were proportional to the amount of
MnSOD protein, we performed a dose-response assay with increasing
amounts of MnSOD protein (Fig.
2A) and
from those data constructed a standard curve (Fig. 2B) by linear regression analysis.
For assay of lung extracts, we added amounts of extract protein that
allowed us to work in the range within which densitometry units were
proportional to the amount of MnSOD protein. Forty-eight hours of
hyperoxia decreased MnSOD specific activity and concentration
(Table 2). After 24 h in air following 48 h in >95%
O2, both parameters returned to values present in rats exposed only to air (Table 2).

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Fig. 1.
Immunoblot of manganese superoxide dismutase (MnSOD). Samples of lung
from 27,000-g supernatant material
were subjected to SDS-polyacrylamide gel electrophoresis, transferred
to nitrocellulose, and reacted with nonimmune rabbit serum
(lanes 1 and
2) or rabbit anti-rat MnSOD
antiserum (lanes 3 and
4). Lanes
1 and 3 were loaded
with 27,000-g supernatant material
from 1 rat, and lanes 2 and
4 were loaded with material from a
second rat. Each lane contained 50 µg of protein. In
lanes 1 and
2, primary antibody was diluted 1:100
and secondary antibody was diluted 1:3,000. In lanes
3 and 4, primary
antibody was diluted 1:2,500 and secondary antibody was diluted
1:3,000.
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Fig. 2.
A: immunoblot of increasing amounts of
MnSOD protein (lanes 1 and
2, 12.5 ng of MnSOD protein;
lanes 3 and
4, 50 ng of MnSOD protein;
lane 5, 75 ng of MnSOD protein;
lane 6, 100 ng of MnSOD
protein). B: dose-response
relationship between amount of MnSOD protein and densitometry
units determined from immunoblot (A)
using a linear regression analysis.
|
|
Within 24 h of reexposure to >95%
O2, lung MnSOD activity fell to
the level found in air-exposed rats, but by 96 h of reexposure to
O2, it was 2.6-fold higher than in
air-exposed rats. (Fig. 3). Cu,ZnSOD
activity increased by 72 h of reexposure to
O2. The activity of catalase and
GP remained higher in lungs of
O2-exposed rats during each
exposure period (Fig. 3).

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Fig. 3.
Lung antioxidant enzyme activity during reexposure. Rats were placed in
identical chambers and exposed to air or to >95%
O2 for 48 h and then removed from
chambers and allowed to breathe air for 24 h. At end of 24 h in air,
rats were returned to chambers to breathe same concentration of gases
breathed when initially in chamber. Enzyme activities of rats breathing
>95% O2 are expressed relative
to those of rats exposed to air for same period. Number of
O2-exposed rats at each time are
provided at base of each bar. Number of air-only rats varied from 6 to
28/group. Cu,ZnSOD, copper-zinc superoxide dismutase; GP, glutathione
peroxidase.
P < 0.001, P < 0.025, * P < 0.001, # P < 0.005, and
P < 0.001 vs. air only.
|
|
Antioxidant enzyme mRNA concentrations in rat lung during exposure
to O2 after a rest period in air and during
reexposure to O2.
At the end of 48 h in O2, the lung
concentration of MnSOD mRNA was approximately threefold higher in
O2-exposed than in air-exposed rats; this difference was still present after the rest period in air
(Table 3). Forty-eight hours of exposure to
O2 did not affect the
concentration of Cu,ZnSOD mRNA, catalase mRNA, or GP mRNA. At the end
of the 24-h rest period in air, the concentration of Cu,ZnSOD mRNA was
elevated (Table 3). The concentrations of catalase mRNA and GP mRNA
were not altered by the initial exposure to
O2 or by the rest period (Table
3).
By 96 h of reexposure to O2, the
concentration of each mRNA in the
O2-exposed rats was substantially
higher than in the air-exposed rats (Table
4). MnSOD mRNA was elevated 6.4-fold,
whereas the mRNA of each other antioxidant enzyme was elevated
~2.6-fold (Table 4). The increased concentrations of the antioxidant
enzyme mRNAs were not specific; mRNA of galectin-1, an endogenous
-galactoside-binding protein (10) without known antioxidant
activity, was also approximately twofold higher in lungs of rats after
96 h of reexposure to O2 than in
lungs of air-exposed rats (Table 4).
Lung MnSOD synthesis and general protein
synthesis.
The changes in concentration of MnSOD (Table 2) were brought about, at
least in part, by parallel changes in the absolute rate of MnSOD
synthesis (Table 5). The latter occurred as
part of similar changes in general protein synthesis (Table 5).
Studies in baboons.
To determine whether the fall in lung MnSOD activity during exposure to
>95% O2 is peculiar to the
adult rat (Table 1; Ref. 9) or whether it is a more general and hence a
more important phenomenon, we examined lungs from baboons that had been
exposed to 100% O2. Because
primates are more tolerant of hyperoxia than rodents (5, 15, 25),
baboons were exposed longer than rats. In lungs of air-ventilated
baboons, lung MnSOD activity was 69.6 ± 8.3 units/mg DNA
(n = 6); in lungs of baboons
ventilated with 100% O2 for 7.2 days, lung MnSOD activity was 46.9 + 5.4 units/mg DNA
(n = 8, P < 0.05).
 |
DISCUSSION |
The fall in lung MnSOD activity during an initial exposure to
>95% O2 is a posttranscriptional event.
The combination of an elevated concentration of MnSOD mRNA (Table 3), a
diminished rate of MnSOD synthesis (Table 5), and low MnSOD specific
activity (Table 2) indicates that the fall in MnSOD activity that
develops during the initial 48 h of exposure to
O2 is a posttranscriptional event
that has at least two components: impaired translation and
a fall in SOD activity per molecule of MnSOD. Translational regulation
of gene expression may be defined as a change in the number of amino
acids polymerized per unit time per mRNA molecule (22). Our data show
that the rate of MnSOD synthesis was 2.1 pmol · mg
mRNA
1 · h
1
in air-breathing rats, 0.4 pmol · mg
mRNA
1 · h
1
in rats after breathing >95% O2
for 48 h, and 1.1 pmol · mg
mRNA
1 · h
1
in rats after a 24-h rest period in air (calculated from the values in
Tables 3 and 5). Thus for MnSOD there was a 5-fold decrease in
translational efficiency during exposure to >95%
O2 and a 2.8-fold increase toward
that in air-exposed rats during the rest period; that, however, still
left translational efficiency 50% lower than in rats exposed only to
air. The molecular basis for the loss of translational efficiency
remains to be defined; however, the low rate of general protein
synthesis (Table 5) suggests that it involves components of the
protein-synthesizing machinery used to make all proteins.
The low specific activity of MnSOD in lungs from rats exposed to
O2 for 48 h (Table 2) may be
caused, at least in part, by oxidant damage to the protein. This
possibility is supported by the observation that the addition of a
reducing agent to mitochondria-free lung extracts increases MnSOD
activity in extracts of lungs from O2-exposed rats but does not alter
MnSOD activity in extracts from air-exposed rats (9). Because MnSOD
becomes active after it enters mitochondria, the low specific activity
of the enzyme in O2-exposed rats
could also be caused by a defect in transport to or entry into
mitochondria. The increase in the specific activity of MnSOD after the
rest period in air to values present in air-exposed rats (Table 2)
could be due to a more reducing cellular environment after removal from
O2; it could also be caused by the
synthesis of new undamaged enzyme and the degradation of
oxidant-damaged low-specific activity MnSOD molecules at a faster rate
than the rate at which undamaged higher-specific activity MnSOD
molecules are degraded. This difference in degradation is expected
because damaged proteins are "marked" for degradation and hence
are eliminated more rapidly than undamaged molecules (18).
Changes of expression during reexposure to >95%
O2 after the rest period in air.
Our findings raise the possibility that there is a change in the
regulation of antioxidant enzyme expression during reexposure to
O2. Thus, unlike the fall in MnSOD
activity to below that in air-exposed rats, which occurred during the
initial exposure to O2 (Table 1),
MnSOD activity in rats reexposed after a rest period in air remained at
or above the level in air-breathing rats (Fig. 1). Before reexposure to
>95% O2, the greater activity
of catalase and GP in O2-exposed
rats occurred without an increase in the mRNA of either enzyme (Table
3) and therefore reflects posttranscriptional regulation. However,
during reexposure to >95% O2,
the concentrations of catalase mRNA and GP mRNA (Table 4) rose,
indicating regulation, at least in part, at a pretranslational level.
We have not excluded the possibility that the difference in the level
at which catalase and GP gene expression was regulated before and after
the rest period reflects, at least in part, a change in the cellular
composition of the lung. The same considerations apply to Cu,ZnSOD, for
which expression did not change during the initial exposure but which exhibited increased expression mediated pretranslationally, at least in
part, during reexposure to >95%
O2 (Table 3). The resolution of
these issues awaits quantitative, ultrastructural studies of in situ
hybridization.
Implications of our findings.
Our findings indicate that MnSOD, an enzyme that is important for
tolerance to hyperoxia in otherwise unmanipulated rats (9), decreases
during exposure to O2 in rats and
baboons; the latter finding may be particularly relevant as a predictor
of what occurs in humans. Furthermore, the basis for the fall in MnSOD
activity is not due to a failure to increase MnSOD mRNA but is rather
due to impaired translational efficiency and to a posttranslational effect, i.e., a low MnSOD specific activity. These findings imply that
therapies aimed at increasing MnSOD activity during exposure to
O2 by gene transfer may not be
fully successful unless combined with therapy that can increase
translational efficiency and prevent the fall in MnSOD specific
activity.
 |
ACKNOWLEDGEMENTS |
The baboon lung specimens were kindly provided by Dr. Jacqueline
Coalson, Professor of Pathology, Univ. of Texas Health Sciences Center
and the Southwest Foundation for Biomedical Research.
 |
FOOTNOTES |
This work was supported in part by National Heart, Lung, and Blood
Institute Grants HL-47413, HL-20366, HL-48588, and HL-52636 and by a
bequest from the Wiggins Estate.
D. Massaro is Cohen Professor of Pulmonary Research.
Address for reprint requests: D. Massaro, Lung Biology Laboratory,
Georgetown Univ. School of Medicine, 3900 Reservoir Rd., NW,
Washington, DC 20007-2197.
Received 7 June 1996; accepted in final form 24 November 1997.
 |
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