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*
Departments of Surgery and
Anesthesiology, Georgetown University Medical Center, Washington, D.C. 20007
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, using a model of endotoxin-mediated iNOS expression in ANA-1 murine macrophages, we demonstrate that NO inhibits total protein synthesis and cell proliferation without alteration in global gene transcription. However, induction of iNOS and inhibition of total protein synthesis in this cell line are associated with a specific and reproducible cleavage pattern in 28S and 18S rRNA that is both NO- and time-dependent. We examined enzymatic function of the 60S ribosome, which is largely composed of intact 28S and 18S rRNA. Levels of 60S ribosome and 60S ribosome-associated peptidyl transferase activity, an essential part of peptide chain elongation are both significantly depressed in the settings of both LPS-mediated endogenous NO synthesis and exposure to an exogenous NO donor, S-nitroso-N-acetylpenicillamine (SNAP). We conclude that NO-mediated inhibition of total protein synthesis is the result of decreased 60S ribosome-associated peptidyl transferase activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ANA-1 murine macrophages, a gift from Dr. George Cox (Uniformed Services University of the Health Sciences, Bethesda, MD), were maintained in DMEM with 5% heat-inactivated FCS. LPS (Escherichia coli serotype 0111:B4; 0–10,000 ng/ml) was added to the medium to induce NO synthesis. In selected instances, the competitive substrate inhibitor of NOS, NG-nitro-L-arginine methyl ester (L-NAME; 4 mM), alone or together with LPS and the NO donors, SNAP or S-nitroso-N-acetylcysteine were added. Cells were harvested after incubation for 2–20 h at 37°C in 95% O2/5% CO2. To determine cell viability, cells were washed with ice-cold PBS three times, stained with 0.2% trypan blue solution (w/v) for 10 min, and viable cells were counted under the light microscope. Cell viability was routinely >95% under all treatment conditions.
Determination of NO synthesis
NO release from cells in culture was quantified by measurement of the NO metabolite, nitrite, using the technique of Snell and Snell (6). To reduce nitrate to nitrite, 200 µl conditioned media was incubated in the presence of 1.0 U nitrate reductase, 50 µM NADPH, and 5 µM flavin adenine dinucleotide. Sulfanilamide (1%) in 0.5N hydrochloric acid (50% v/v) was then added. After a 5-min incubation at room temperature, an equal volume of 0.02% N-(1-(naphthyl))ethylenediamine was added; following incubation at room temperature for 10 min, absorbance at 570 nm was compared with that of a NaNO2 standard.
RNA and DNA extraction and Northern blot analysis
Total RNA was purified from cultured ANA-1 macrophages with the use of TRIzol Reagent (Life Technologies, Rockville, MD). The purified total RNA (5 µg) was subjected to electrophoresis on a 1% (w/v) agarose gel for 3 h at 80 v. The RNA was blotted onto Nytran nylon transfer membrane (Schleicher & Schuell, Keene, NH) with 10x SSC (1.5 M NaCl/0.15 M sodium citrate) prepared with diethyl pyrocarbonate-treated water, then cross-linked to the membrane by UV radiation. cDNA probes complementary to murine 28S and 18S rRNA were prepared by PCR amplification with primers based on their cDNA sequence and randomly labeled with [32P]dCTP. Hybridization of the labeled probe was performed in hybridization solution (50% formatted, 5x SSC, 5 mM EDTA, 20 mM sodium phosphate buffer (pH 7), 1% SDS, 200 µg/ml salmon sperm DNA, and 5x Denhardt’s solution) at 42°C overnight with 3 h of prehybridization at 42°C. The blots were washed briefly with 2x SSC containing 0.1% SDS at room temperature, once for 15 min with 0.5x SSC containing 0.1% SDS at 42°C, and once for 15 min with 0.2x SSC containing 0.1% SDS at 50°C and 65°C. Autoradiographs were exposed at room temperature. Genomic DNA was purified from cultured ANA-1 macrophages with the DNAzol Reagent (Life Technologies). The purified genomic DNA were subjected to electrophoresis on a 0.6% (w/v) agarose gel for 3 h at 100 V.
Determination of protein synthesis
Cells were washed with methionine-free DMEM three times and incubated for 20 min in short-term medium (methionine-free DMEM containing 5% dialyzed FCS) in a humidified 37°C, 5% CO2 incubator to deplete intracellular pools of methionine. Medium was then replaced with fresh short-term labeling medium containing 10 µCi/ml [35S]methionine. After 15-, 30-, 60-, 120-, and 240-min labeling, cells were washed with PBS twice and lysed in TE buffer supplied with 1% SDS. The cell lysis was then passed through a 21 gauge needle several times. A total of 50 µl labeled cell lysis was added to 0.5 ml BSA (0.1 mg/ml) on ice and an equal amount (0.5 ml) of ice-cold 20% trichloroacetic acid was added. The solution was vortexed vigorously and incubated for 30 min on ice. The precipitate was collected on filter papers and was washed once with ice-cold 10% TCA and twice with 95–100% ethanol. When dry, the filter papers were transferred to a scintillation vial containing 4 ml scintillation fluid. Radioactivity was then measured.
Cell proliferation assays
ANA-1 macrophages were labeled with 5 µCi/ml [3H]thymidine for 20 h in the presence or absence of LPS. The plates were treated with 0.5 ml/well ice-cold 10% trichloroacetic acid and kept at 4°C overnight. The plates were washed three times with distilled water and dried at room temperature. Cells were collected with 0.5 ml/well 0.33 mM NaOH and 300 µ1 solution was transferred into a scintillation vial containing scintillation fluid. [3H]Thymidine incorporation into DNA was measured using a scintillation counter.
Nuclear run-on analysis
A total of 100 µl ANA-1 macrophage nuclei was incubated for 5
min at 30°C with 150 µCi of [32P]rUTP (800
Ci/mmol) in 100 µl 10 mM Tris HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl,
and 5.0 mM (each) ATP, CTP, and GTP. Labeled RNA was isolated by the
acid-guanidinium thiocyanate method. Before ethanol precipitation,
labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution
was neutralized by the addition of HEPES (acid free) to a final
concentration of 0.24 M. After ethanol precipitation, the RNA pellet
was re-suspended in 10 mM N-tris(hydroxymethyl)methyl-2
minoethanesulfonic acid (pH 7.4), 0.2% SDS, and 10 mM EDTA. Target DNA
for cyclophillin, ß-actin, and GAPDH was spotted onto nylon membranes
with a slot blot apparatus. 
-Bacteriophage DNA served as negative
controls. Hybridization was performed at 42°C for 48 h with
5 x 106 cpm of labeled RNA in hybridization
buffer (50% formamide, 4x SSC, 0.1% SDS, 5x Denhardt’s solution,
0.1 M sodium phosphate (pH 7.2), and 10 µg/ml salmon sperm DNA).
After hybridization, the membranes were washed twice at room
temperature in 2x SSC and 0.1% SDS, and three times at 56°C in
0.1x SSC and 0.1% SDS. The membranes were then exposed to x-ray film
and scanned.
Cellular ribosome profile
ANA-1 murine macrophages were isolated in ice-cold PBS. The cells were pelleted at maximum speed in a microcentrifuge and resuspended in hypotonic buffer (40 mM Tris-HCl (pH 7.4), 20 mM KCl, 3 mM MgCl2, 20 mM sodium fluoride, and 150 mM sucrose) and kept on ice for an additional 10 min. Following cell lysis in 1% Triton X-100 and 1% deoxycholate, the preparation was vortexed and centrifuged for 1 min in a microcentrifuge. The supernatant formed the total ribosomal fraction and a portion was used for peptidyl transferase activity. The remaining supernatant was layered onto a linear 10–45% sucrose gradient containing 25 mM Tris-HCl, 80 mM KCl, 4 mM MgCl2, and 20 mM sodium fluoride. Following centrifugation for 4 h in a Beckman (Fullerton, CA) SW-41 rotor, ribosomal profiles were monitored by continuously measuring A280.
Ribosome-associated peptidyl transferase activity
Peptidyl transferase activity was measured in ANA-1 macrophage total ribosomal fractions using a peptidyl transferase reaction with full-length formyl-[3H]Met RNA as a donor substrate (7). Typically, cell fractions were incubated with 2.5 pmol formyl-[3H]Met RNA in 40 µl buffer containing 20 mM Tris-HCl (pH 8.0), 400 mM KCl, 20 mM MgCl2, and 1 mM puromycin. Reactions were initiated by adding 20 µl cold methanol and incubated for 20 min at 0°C. To terminate the reaction, 10 µl 4 M KOH was added and incubated for an additional 20 min at 37°C. After the addition of 200 µl 1 M KH2PO4, the reaction mixture was extracted with 1 ml ethyl acetate. A portion of the extract was mixed with scintillation mixture and counted. Peptidyl transferase activity is expressed as fmol/mg protein/min.
Statistical analysis
Data are presented as mean ± SEM of three or four experiments each performed in triplicate. Statistical analysis was performed using the Student t test or rank sum test, as appropriate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following stimulation with 10–10,000 ng/ml LPS for a period of
20 h, ANA-1 murine macrophage production of NO was determined by
measuring levels of nitrite and nitrate in the culture media (Fig. 1
). The dose response was plotted in a
semilogarithmic fashion. In the absence of LPS, the nitrite level in
the media was 9.9 ± 1.0 nmol/mg protein. NO production increased
in a significant dose-related fashion at LPS concentrations of 10, 100,
1,000, and 10,000 ng/ml (p < 0.01 vs
control for LPS = 100, 1,000, and 10,000 ng/ml). The competitive
substrate inhibitor L-NAME (4 mM) was added. In
the presence of L-NAME alone, nitrite production
was not significantly altered from that of control cells (10.1 ±
1.1 nmol/mg). In cells treated with LPS + L-NAME,
NO production was ablated at all concentrations of LPS (10–10,000
ng/ml) and did not significantly differ from that of control cells. In
addition, trypan blue exclusion and media levels of lactate
dehydrogenase were determined as measures of cell viability. Under all
treatment conditions, cell viability was routinely >95%, and LDH
levels did not differ among the various treatment groups. These data
indicate that LPS-mediated NO production is dose-dependent and does not
alter cell viability. Subsequent studies use 100 ng/ml LPS and 4 mM
L-NAME, unless stated otherwise.
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Following a 20-h incubation, total protein synthesis was
determined by [35S]methionine incorporation in
control, LPS-, LPS + L-NAME-, and SNAP 400 µM-treated
macrophages (Fig. 2A). In all
instances, there was a significant linear, time-dependent increase in
protein synthesis. In LPS-treated cells, total protein synthesis was
consistently decreased by 40–70% at all time points when compared
with those of control and LPS + L-NAME
(p < 0.05 vs control and LPS +
L-NAME). The greatest incremental difference in
protein synthesis was present following a 15-min pulse. Protein
synthesis in control and LPS + L-NAME cells was
not significantly different. In SNAP-treated cells, total protein
synthesis was significantly decreased at all time points, in comparison
to control and LPS + L-NAME and also, when
compared with LPS alone (p < 0.05 vs control,
LPS + L-NAME, and LPS). When cells were incubated
in the presence of LPS + L-NAME + SNAP, the
protein synthesis curve was not statistically different from that
of SNAP alone (data not shown). These data suggest that NO, whether
from an exogenous or endogenous source, is associated with significant
depression of total protein synthesis in ANA-1 murine macrophages.
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B). There was no
difference in protein synthesis following 8 h of LPS stimulation.
However, at LPS incubation times greater than 12 h, protein
synthesis progressively decreases in a significant fashion
(p < 0.05 vs control and LPS +
L-NAME for 16 and 20 h). The time dependence
of total NO synthesis was also determined and plotted in Fig. 2B. NO production increases dramatically following 12 h
of LPS stimulation and reaches a near maxium after
20 h of LPS stimulation. The time course of this increase in NO is
associated with a parallel decline in total protein synthesis. When
[35S]methionine incorporation is plotted vs
nitrite from LPS stimulated cells, an inverse linear relationship
(r2 = 0.70) is present (Fig. 2C). This suggests that NO production is inversely
associated with decreased protein synthesis in LPS stimulated
macrophages.
ANA-1 cell proliferation in the setting of LPS-induced NO synthesis was
determined by the incorporation of
[3H]thymidine (Fig. 3). LPS-mediated NO synthesis was
associated with a significant 50% decrease in tritiated thymidine
incorporation following a 20-h incubation in the presence of LPS
(p < 0.05 vs control and LPS +
L-NAME).
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LPS-mediated NO production and gene transcription
To determine the role of NO production in ANA-1 macrophage gene
transcription, nuclear run-on analysis was performed using ß-actin,
GAPDH, and cyclophillin as target "housekeeping" genes (Fig. 4). Following 20 h of LPS
incubation, there was no difference in gene transcription for any of
the target genes tested. Transcription was not significantly
different among control, LPS-, L-NAME-, and LPS +
L-NAME-treated cells. These results indicate that
inhibition of total protein synthesis is not the result of decreased
global gene transcription in LPS-treated ANA-1 murine macrophages.
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The pattern of total RNA expression was then examined in this
experimental model (Fig. 5A).
In control cells, the typical electrophoretic pattern of 28S and 18S
rRNA expression was found. In contrast, in the presence of 50 and 100
ng/ml LPS, a distinctive cleavage pattern of 18S and 28S rRNA was
found. This pattern is associated with decreased levels of 18S and 28S
rRNA, as determined by intensity of the bands on gel electrophoresis.
Inhibition of NO production by the addition of
L-NAME resulted in restoration of the normal rRNA
pattern. The electrophoretic pattern of genomic DNA expression in LPS-
and LPS + L-NAME-treated cells did not differ and
specifically did not exhibit an apoptotic pattern (Fig. 5B).
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A). Again, a specific
cleavage pattern of rRNA was exhibited in all cells treated with LPS.
In contrast, the typical rRNA pattern was found in control,
L-NAME-, and LPS +
L-NAME-treated cells. A transition in the
intensity of RNA degradation was noted between LPS concentrations of
50–200 ng/ml. The experiments were then repeated using LPS
concentrations of 50, 100, 150, 200, and 250 ng/ml to better
characterize the transition (Fig. 6B). No distinct
transition corresponding to a specific LPS concentration was evident
from these experiments.
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, B and
C).
To determine the identity of these various RNA bands, Northern blot
analysis was performed (Fig. 7). A
full-length oligonucleotide probe was constructed that was
complementary to murine 18S rRNA (1869 bp). Due to the size of 28S
rRNA, three separate cDNA probes were made as follows: 28S-1 (nt
0–1500), 28S-2 (nt 1559–3268), and 28S-3 (nt 3275–4712).
Based upon the results of the Northern blot analysis, 18S rRNA is
cleaved into three fragments of 
1.0, 0.8, and 0.6 kb. In contrast,
28S rRNA is cleaved into four fragments. One binds 28S-1 (size 3.3
kb); another (size 2.6 kb) binds 28S-2, and a final two are
complementary to 28S-3 (sizes 1.5 and 1.0 kb). These data
indicate that NO, from both endogenous and exogenous sources, induces a
specific time-dependent cleavage pattern in rRNA resulting in decreased
levels of 18S and 28S rRNA expression.
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ANA-1 macrophages were exposed to 100 ng/ml LPS for 20 h. Unstimulated control and LPS + L-NAME-treated cells served as comparison groups. In cells exposed to LPS, there was a marked 4-fold decrease in 60S ribosomal A280 (0.025 ± 0.012) compared with that of control (0.102 ± 0.14; p < 0.05 vs LPS) and LPS + L-NAME (0.115 ± 0.18; p < 0.05 vs LPS) cells, indicating a decrease in quantity of 60S ribosomes. In addition, predictably, the A280 of the 80S ribosomal particle composed of 60S and 40S ribosomes was also decreased in LPS-treated cells (0.012 ± 0.009) compared with that of control (0.265 ± 0.019; p < 0.05 vs LPS) and LPS + L-NAME-treated (0.281 ± 0.023; p < 0.05 vs LPS) cells. These data suggest that LPS-mediated NO synthesis is associated with a decrease in relative quantities of 60S and 80S ribosomal particles, which are requisite for protein synthesis.
Ribosome-associated peptidyl transferase activity
Cleavage of 28S and 18S rRNA may alter 60S ribosomal peptidyl
transferase activity and inhibit total protein synthesis. Peptidyl
transferase activity from ANA-1 total ribosomal fractions was
determined using LPS concentrations of 0, 1, 5, 10, 25, 50, 100, and
1000 ng/ml at time points varying from 0 to 20 h (Fig. 8A). In certain instances, 4
mM L-NAME was also added. In unstimulated control
cells, peptidyl transferase activity was maintained throughout the
study period. LPS concentrations of 1, 5, 10, and 25 ng/ml were
associated with a progressive, concentration- and time-dependent
decrease in peptidyl transferase activity beginning at 12 h of
incubation. In contrast, LPS treatment at concentrations of 50, 100,
and 1000 ng/ml was associated with maximally depressed enzyme activity
beginning at 12 h with a subsequent decline thereafter at 16 and
20 h of incubation. At LPS concentrations of 50, 100, and 1000
ng/ml, peptidyl transferase activity was 2-fold less than that of
controls at 20 h of incubation. Ablation of NO synthesis by
addition of 4 mM L-NAME restored peptidyl
transferase activity to control levels at all time points studied. The
addition of an exogenous source of NO in the form of SNAP significantly
depressed enzyme activity at all time points compared with both control
and LPS cells. L-NAME alone did not alter
peptidyl transferase activity in comparison to those of control cells
(data not shown). To further examine the threshold characteristic of
these data, the IC50 of peptidyl transferase activity was
plotted as a function of LPS concentration (Fig. 8B). These
data suggest that NO, from either exogenous or endogenous sources,
inhibits ribosome fraction-associated peptidyl transferase
activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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12 h of
exposure to NO and reaches its maximum following 20 h of exposure.
In addition, 28S and 18S rRNA cleavage occur at all concentrations of
LPS examined during a standard 20-h incubation, suggesting that rRNA
cleavage is both NO- and time-dependent. Transcription, as determined
by nuclear run-on analysis of the standard "housekeeping" genes,
ß-actin, GAPDH, and cyclophillin, is not altered by LPS-mediated NO
production. Northern blot analysis demonstrated the presence of two
cleavage sites in both 18S and 28S rRNA. The time course and NO
concentration dependence of decreased total protein synthesis
correlates with appearance of this distinctive rRNA cleavage pattern.
We hypothesized that NO-mediated inhibition of cellular protein
synthesis may be the result of fragmentation of the rRNA components
essential to 60S ribosome-mediated protein elongation. Subsequent
studies found that levels of 60S ribosome and ribosomal peptidyl
transferase activity were significantly decreased in ANA-1 murine
macrophages exposed to endogenous and exogenous sources of NO. We
conclude that NO-mediated inhibition of total protein synthesis is the
result of depressed 60S ribosome-associated peptidyl transferase
activity. Eukaryotic protein synthesis is catalyzed by ribosomes, which are large complexes of proteins and rRNA (8). The smaller 40S rRNA subunit binds both mRNA and tRNA, while the larger 60S rRNA subunit catalyzes peptide bond formation. Together the 60S and 40S ribosomes compromise the larger functional unit of the 80S ribosome. The process of protein translation centers upon binding of an aminoacyl-tRNA molecule to the vacant A site on the 40S ribosomal subunit. The carboxyl end of the polypeptide chain is uncoupled from the tRNA molecule linked to the growing end of the polypeptide chain at the adjacent P site; a peptide bond is formed to the amino acid linked to the tRNA molecule in the A site. This central reaction, termed peptidyl transferase, requires an intact 28S rRNA in combination with four ribosomal proteins in the 60S ribosome. Work based upon the prokaryotic model indicates that the 28S rRNA equivalent is a critical component of the basic catalytic unit (9, 10). The effect of NO on peptidyl transferase activity has not been previously examined. However, a single conserved histidine residue associated with the catalytic site of peptidyl transferase is sensitive to oxidative stress induced by singlet oxygen generation (8). Although our data suggests that NO induces a specific pattern of rRNA degradation, the sensitivity of the active site of peptidyl transferase to oxidative stress suggests another possible mechanism by which NO may modulate peptidyl transferase function and ultimately, total protein synthesis.
In our model, we hypothesize that NO mediates a specific pattern of 18S
and 28S rRNA cleavage. As a result of depletion of intact components of
the 60S ribosome, peptidyl transferase activity is decreased, total
protein synthesis is depressed, and inhibition of cell proliferation
ensues. Alternatively, it is possible that these RNA bands are the
result of altered maturation or cleavage of rRNA precursors rather than
or in addition to that of the mature rRNA forms. NO-mediated inhibition
of protein synthesis has been previously observed in multiple
experimental models using hepatocytes and macrophages. However, the
underlying mechanism has not been not extensively studied. Recently,
Kim et al. (5) examined NO-mediated inhibition of total
protein synthesis in RAW 264.7 murine macrophages. They found that
exposure to NO was associated with phosphorylation of the protein
synthesis initiation factor, eIF-2
. 80S ribosome formation was
inhibited following a 14-h incubation with LPS. They postulated that NO
impairs protein translation by phosphorylation of the subunit of
eIF-2 and inhibits binding of the initiator methionyl-tRNA to 40S
rRNA. These investigators did not specifically address the role of NO
in rRNA processing.
In a similar fashion, our results also indicate diminution in
quantities of both 80S and 60S ribosome, which are essential structural
scaffolds upon which translation occurs. In addition, 60S ribosomal
peptidyl transferase activity paralleled the time course of 28S and 18S
rRNA degradation. We did not specifically address eIF-2 activity.
However, NO may certainly act at multiple points in the protein
synthetic pathway, including eIF-2 phosphorylation and inhibition of
60S ribosome formation with consequent ablation of peptidyl transferase
activity.
In this experimental model, NO mediates a specific cleavage pattern in
rRNA that is not paralleled by apoptosis. The effect of NO on rRNA
metabolism has not been previously addressed. However, work in the
early 1980’s by Varesio and colleagues (11, 12, 13, 14) examined
murine macrophages activated by individual exposure to IFN-,
IFN-
, and LPS. Similar to that found in our study, they demonstrated
a specific cleavage pattern in 28S rRNA with decreased accumulation of
28S rRNA and associated ribosomal particles. This pattern was
associated with a cytotoxic functional profile rather than a
suppressive profile. Given that this body of work predates the
discovery of NO, the authors did not specifically address the presence
or absence of NO. However, the similarities with the results from our
study are compelling. Varesio et al. (12) may have been
the first to identify a specific rRNA cleavage pattern that is mediated
by cytokine- or LPS-mediated NO synthesis in macrophages. The specific
sites of rRNA cleavage in 18S and 28S rRNA are the subject of ongoing
investigation in our lab. It is unclear whether the cleavage site is
specific for a defined nucleotide sequence or a rRNA tertiary
structural motif. In addition, the mechanism may reside in enzymatic
endonuclease or ribozyme activity. Finally, it will be important to
determine the signaling pathway for the induction of this cleavage
pattern.
The functional correlate of NO-mediated inhibition of total protein synthesis in the setting of LPS stimulation is unknown. Is it a cytotoxic or cytoprotective response? It has been hypothesized that inhibition of protein synthesis and mitochondrial respiration may serve as a stress response (5). The purpose may be to transiently shut-down nonessential cellular functions and conserve cellular resources in the presence of proinflammatory cytokines and/or endotoxemia. However, when chronically maintained, this process may lead to ultimate loss of cell viability. In our study, there were no discernible differences in cell viability based upon trypan blue exclusion or LDH release.
These considerations notwithstanding, we have demonstrated that LPS-mediated NO production is associated with decreased total protein synthesis without change in global gene transcription programming. This process is paralleled by a specific, reproducible, NO- and time-dependent cleavage pattern in rRNA constituents of the 60S ribosome, 28S and 18S rRNA. This results in decreased expression of both the 60S ribosome and the complete 80S ribosome, both of which are essential for protein synthesis, and significant inhibition of 60S ribosome-associated peptidyl transferase. The time course of decreased peptidyl transferase activity closely parallels that noted with cleavage of 28S and 18S rRNA. In this model of LPS-treated ANA-1 murine macrophages, we conclude that one potential mechanism underlying the inhibition of total protein synthesis is NO-mediated degradation of constituent rRNA components of the 60S ribosome and decreased 60S-associated peptidyl transferase activity, an essential central step in the protein synthetic pathway.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul C. Kuo, Georgetown University Medical Center, Department of Surgery, 4 PHC, 3800 Reservoir Rd, N.W., Washington, D.C. 20007.
3 Abbreviations used in this paper: iNOS, inducible NO synthase; SNAP, S-nitroso-N-acetylpenicillamine; L-NAME, NG-nitro-L-arginine methyl ester.
Received for publication February 8, 2000. Accepted for publication July 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Mol. Med. 4:179.[Medline]
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