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(Received for publication, July 13, 1994; and in revised form, January 16, 1995) From the
Stimulation of resident peritoneal macrophages with S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysSerLys Triggering with bacterial lipopeptides
induced macrophage programmed cell death. In macrophages activated with
lipopeptide, apoptosis was observed even in the absence of nitric oxide
synthesis, therefore indicating the existence of alternative pathways
in the control of programmed cell death in these cells.
The effect of lipopolysaccharide (LPS) ( Activation of M
Figure 1:
Bacterial lipopeptides induce NO
synthesis in M
Figure 2:
Nitrite and nitrate release in M
Since the amount of nitrate released represented 27% of the
nitrite concentration, these results suggested a moderate oxidation of
nitrites in the course of TPP stimulation. Indeed, in addition to NO,
TPP also promoted the release of H
Figure 3:
TPP promotes PDBu-dependent
H
Figure 4:
TPP and LPS antagonize in promoting iNOS
expression. M
Figure 5:
TPP promotes apoptosis in the absence of
NO synthesis. M
Figure 6:
TPP induces apoptosis in arginine-free
medium and promotes iNOS expression. M
Figure 7:
Dose
dependence curve of apoptosis in M
Several bacterial cell wall products, among which are LPS,
lipoproteins, murein, and membrane proteins, share in common the
ability to stimulate various types of immune cells(18) .
However, since the chemical structure of these molecules exhibits great
differences between them, it is conceivable that the signaling pathways
activated after cell triggering with bacterial products might show a
certain degree of specificity. For example, lipopeptides specifically
activate G The results
reported in this work add new information regarding cell activation and
apoptotic death induction of M In rat M An additional difference
between TPP and LPS in M Regarding the mechanism of action of TPP in promoting apoptosis,
several possibilities could be envisaged. The contribution of NO has
been attributed either to a blockage in the energetic metabolism (via
aconitase inhibition) or to a direct alteration in the DNA structure as
result of nucleotide deamination directly due to NO, and both cases
have been discussed previously(9, 17) . In addition to
these mechanisms, there is a possible occurrence in TPP-activated
M Finally, and in keeping with
the view that M
Volume 270,
Number 11,
Issue of March 17, 1995 pp. 6017-6021
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysAlaLys,
two synthetic bacterial lipopeptides, promoted the expression of the
inducible form of nitric oxide synthase, exhibiting a temporal pattern
of nitric oxide release that was delayed with respect to the induction
elicited by bacterial lipopolysaccharide. Treatment of macrophages with
genistein blocked the nitric oxide synthesis triggered by the
lipopeptides or lipopolysaccharide. Simultaneous incubation with
lipopolysaccharide and lipopeptide resulted in an antagonistic effect
on nitric oxide synthase mRNA levels and on nitrite plus nitrate
release to the medium.
)by itself or
in synergism with other cytokines on the induction of NOS in several
tissues is now well
documented(1, 2, 3, 4) . In M
,
IFN-
and TNF-
synergize with LPS in the release of NO which
is involved in the antimicrobial action of activated
M(4) . However, in addition to LPS, other components of
the bacterial cell wall such as membrane lipoproteins are able to
modulate immunological responses in M and B and T lymphocytes,
although their mechanism of action is less
known(5, 6) . Regarding bacterial lipoproteins,
previous work showed that their biological activity can also be
reproduced by synthetic lipopeptides (TPP) that mimic the fatty acid
esterification of these molecules(7) . Indeed, these
lipopeptides have some technical advantages for the use in in vivo experiments since they do not produce necrosis upon injection and
lack toxic and pyrogenic effects(7) . The activation elicited
by these synthetic lipopeptides in M involves an early tyrosine
phosphorylation of substrates and uses an upstream signaling pathway
partially different from that triggered via LPS(8) . usually leads to programmed cell death and the
release of NO by itself has been reported as one of the signals that
mediates apoptosis at least in peritoneal M(9) . However,
it is possible that in addition to this molecule other reactive
intermediates or cytokines produced in the course of M activation
may be involved in the apoptotic process characteristic of activated
cells(9, 10) . Here we show that triggering of
resident peritoneal rat M with TPP promotes NO synthesis with a
kinetics different from that elicited by LPS, and the presence of both
bacterial products results in a significant blockage of NOS expression
and NO release. Moreover, in TPP-activated cells apoptosis may be
obtained in the absence of NO synthesis, suggesting the existence of
alternative pathways in cell death induction by TPP.
Chemicals
Metabolites and biochemicals were from
Sigma. Materials and chemicals for electrophoresis were from Bio-Rad.
TPP and S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysAlaLys (TPP-Ala) were from Boehringer (Mannheim, Germany) and were
tested for endotoxin content that was below 0.1 ng/mg using the Limulus polyphemus test (Sigma). Serum and media were from
Biowhittaker (Walkersville, MD).Preparation of Macrophages
Peritoneal resident
M were prepared from male rats following a previous
protocol(11) . Briefly, after light ether anesthesia the
animals were killed by cervical dislocation and injected
intraperitoneally with 50 ml of sterile RPMI 1640 medium (at 37
°C), and after 10 min of gentle distribution of the medium in the
peritoneal cavity, the ascitic liquid was carefully aspirated to avoid
hemorrhage and kept at 4 °C to prevent the adhesion of the
macrophages to the plastic. After centrifugation at 200 
g for 10 min at 4 °C, the cell pellet was washed once with
ice-cold PBS. Cells were seeded at 1 10
/cm
in RPMI 1640 medium supplemented with 10% of heat inactivated
fetal calf serum. After incubation for 1 h, the non-adherent cells were
removed by extensive washing with ice-cold PBS. Except when otherwise
stated, experiments were carried out in phenol-red free RPMI 1640
medium supplemented with 0.5 mM arginine and 10% of heat
inactivated fetal calf serum. When arginine-free medium was used, the
fetal calf serum was treated for 30 min at 37 °C with 1 unit/ml of
arginase.Determination of NO and
H
NO release was determined
spectrophotometrically by the accumulation of nitrite and nitrate in
the medium (phenol red-free) as follows: 250 µl of culture medium
were transferred to 1.5-ml Eppendorf tubes, and the nitrate was reduced
to nitrite with 0.5 units of nitrate reductase (Boehringer) in the
presence of 50 µM NADPH, 5 µM FAD(12) . The excess of NADPH, which interferes with the
chemical determination of nitrite, was oxidized in the presence of 0.2
mM pyruvate and 1 µg of lactate dehydrogenase. Nitrite was
determined with Griess reagent (11) by adding 1 mM sulfanilic acid and 100 mM HCl (final concentration).
After incubation for 5 min, the tubes were centrifuged and 200 µl
of supernatant were transferred to a 96-well microtiter plate. After a
first reading of the absorbance at 595 nm, 50 µl of
naphthylenediamine (1 mM in the assay) were added. The
reaction was completed after 15 min of incubation, and the absorbance
at 595 nm was compared with a standard of NaNOO. The
HO secretion by cultured macrophages was
measured after triggering with PDBu (13) as follows: cells
attached to coverslips were incubated per triplicate with the indicated
effectors (IFN-, TNF-, and in the presence or absence of TPP)
for 24 h. After two washes with PBS and one with 0.9% NaCl, the
coverslips were placed in a fluorescence cuvette and triggered for 1 h
with 100 ng/ml of PDBu. The release of HO was
measured fluorometrically in the supernatant following the oxidation of
scopoletin at 460 nm and in the presence of horseradish peroxidase that
yields a non-fluorescent product when excited at 350 nm(14) .RNA Extraction and Analysis
Total RNA (3-4
10 cells) was extracted following the guanidinium
thiocyanate method(15) . After electrophoresis in a 0.9%
agarose gel containing 2% formaldehyde, the RNA was transferred to a
Nytran membrane (NY 13-N; Schleicher & Schuell, Germany) with 10
SSC (10 SSC is 1.5 M NaCl, 0.3 M sodium citrate, pH 7.4). The membrane was prehybridized, and the
level of iNOS mRNA was determined using an EcoRI-HindII fragment from the iNOS
cDNA(16) , labeled (45% of efficiency) with
[-
P]dCTP using the Random Primed labeling
kit (Boehringer). The membranes were washed with 0.1 SSC and
0.1% SDS at room temperature for 10 min and twice at 50 °C for 30
min, followed by exposure to x-ray film (Kodak X-OMAT). Quantification
of the films was performed by laser densitometry (Molecular Dynamics),
using the hybridization with a 
-actin probe (0.6-kilobase EcoRI/HindIII fragment isolated from a VC 18 vector)
as an internal standard.Western Blot Analysis of iNOS
M cell layers
(2 10) were rinsed twice with PBS and homogenized
in 500 µl of boiling 250 mM Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, and 2% -mercaptoethanol and after centrifugation in a
microcentrifuge for 5 min, samples were briefly sonicated and
centrifuged. Proteins were size separated in 8% SDS-polyacrylamide gel
electrophoresis. The gels were processed as recommended by the iNOS mAb
supplier (Transduction Laboratories), and after blotting onto a
polyvinylidene difluoride membrane (Millipore) iNOS was revealed
following the ECL technique (Amersham, Bucks, U.K.).Analysis of DNA Fragmentation
Internucleosomal DNA
fragmentation was analyzed by agarose gel electrophoresis. The cell
layers (1-2 10) were washed twice with
ice-cold PBS and filled with 0.5 ml of 20 mM EDTA, 0.5% Triton
X-100, 5 mM Tris-HCl, pH 8.0. After incubation for 15 min at 4
°C, the nuclei were removed by centrifugation at 500 g for 10 min, and the resulting supernatant was centrifuged at
30,000 g for 15 min. The fragmented DNA present in the
soluble fraction was precipitated with 70% ethanol plus 2 mM MgSO, and aliquots were treated for 1 h at 55 °C
with 0.3 mg/ml of proteinase K. After two extractions with
phenol/chloroform, the DNA was resuspended and analyzed in a 2% agarose
gel after staining with 0.5 µg/ml of ethidium bromide.
Alternatively, samples were analyzed using an enzyme-linked
immunosorbent assay cell death kit (Boehringer) in which the
histone-associated DNA fragments of mono- and oligonucleosomes in the
cytosol were detected using a sandwich-enzyme-immunoassay with
anti-histone and anti-DNA-peroxidase antibodies. The quantification of
the relative degree of apoptosis was determined by measuring the
peroxidase activity at 405 nm and subtracting the contribution of cells
at the initial time of culture (endogenous apoptosis). The average A
of samples from control cells was 0.06 O.D.
units.
TPP Promotes the Release of NO and
H
Two synthetic TPP derivatives (TPP
and TPP-Ala) were used to determine the ability of cultured resident
peritoneal MO to release NO to the medium. As Fig. 1A shows, both lipopeptides promoted NO synthesis as reflected by the
presence of nitrate and nitrite in the incubation medium. This M
activation process exhibited a lag period of 6 h followed by a
continuous release up to 48 h. The NO synthesis elicited by the
lipopeptides was delayed when compared to that induced by LPS (Fig. 1A), suggesting that these bacterial products use
partially different pathways in their mechanism of NOS induction.
Additionally, the effect of LPS on NO synthesis resulted quantitatively
higher (35% over the effect of TPP at 24 h) when compared to that
elicited by the lipopeptides. Since both LPS and TPP have been reported
to involve an increased tyrosine phosphorylation following cell
triggering with these bacterial products, we determined the effect of
genistein, a protein tyrosine kinase inhibitor, on the release of NO by
M stimulated with LPS or TPP. As shown in Fig. 1A (bars) the release of nitrate and nitrites was completely
blocked when cells were incubated in the presence of 100 µM of genistein, data that are in agreement with previous
work(8) . To further characterize the inhibitory effect of
genistein, M were stimulated either with TPP or LPS, followed by
the addition of genistein at various times after priming. As Fig. 1B shows, when genistein was added during the 2 h
following M priming, a complete blockage in the NO synthesis was
observed. However, after this initial critical period, genistein
resulted less effective in inhibiting TPP- than LPS-dependent NO
release. The sensitivity to genistein was similar for TPP and LPS (Fig. 1B, inset). Fig. 2shows a very
similar dose-dependent curve for both lipopeptides in nitrite and
nitrate release, exhibiting a half-maximal effect at 0.8 µg/ml
(nitrite concentration). In the following experiments only TPP was
used.
. Cultured resident peritoneal M (3
10
) were incubated with 5 µg/ml of TPP (
), 5
µg/ml of TPP-Ala (
), or 5 µg/ml of LPS (
), and at
the indicated times the concentration of nitrite plus nitrate released
to the medium was measured. Alternatively, cells were incubated for 5
min with 100 µM of genistein prior to stimulation with 5
µg/ml of TPP (solid bars) or 5 µg/ml of LPS (open
bars), and the nitrite and nitrate concentration were measured (panel A). When genistein was added at different times after
stimulation with TPP () or LPS () the NO synthesis was
measured after 24 h of stimulation (panel B). The
dose-dependent curve for the inhibition of NO release by genistein
(added 5 min prior to M triggering) was measured after 24 h of
stimulation with TPP or LPS (inset). Results show the mean
± S.E. of three experiments.
activated with lipopeptides. M were incubated for 36 h with TPP
() or TPP-Ala (), as described in Fig. 1. The
release of nitrate (dotted line) and nitrite (continuous
line) were measured. Results show the mean ± S.E. of three
experiments.
O when
stimulated M cultures were triggered for 1 h with
PDBu(13, 14) . This HO production was near additive to the effect elicited by TNF-
and IFN- (Fig. 3). These results prompted us to study the
role of these cytokines on the TPP-dependent NO release. As Table 1shows, incubation of M with IFN-, TNF-, or
with both molecules inhibited the NO release induced by TPP. Opposite
to this situation, a clear synergism between IFN- and TNF-
was observed in LPS stimulated M, suggesting that TPP and LPS use
different pathways in their mechanism of NOS induction. However, an
unexpected antagonism between TPP and LPS in NO release was observed.
O release in M. Coverslip-attached cells
were stimulated for 24 h with 2 ng/ml of IFN- or TNF- in the
absence (open bars) or presence (dashed bars) of 5
µg/ml of TPP. After this incubation period, the cell layers were
extensively washed and triggered with 100 ng/ml of PDBu for 1 h to
release HO that was determined fluorometrically
by the oxidation of scopoletin in the presence of horseradish
peroxidase. Results show the mean ± S.E. for triplicates from
one of three independent experiments.
TPP and LPS Antagonize in iNOS Expression
The
induction of iNOS mRNA in M stimulated with TPP and LPS was
analyzed to establish the relative levels of message in each condition
and to characterize the inhibitory effect of the simultaneous addition
of both bacterial products at the messenger level. As Fig. 4shows, TPP and LPS increased the iNOS mRNA content (the
4.5 kilobase band) at 4 and 18 h, although to a different extent.
However, when both TPP and LPS were simultaneously added, an important
decrease in the mRNA levels (68% of the LPS-specific response at 18 h)
was observed, in agreement with the inhibition of NO release in these
conditions (Table 1).
were incubated for the indicated periods of time in
the absence (lane 1) or with 5 µg/ml of TPP (lanes 2 and 4) and 1 µg/ml of LPS (lanes 3 and 4), respectively. RNA was extracted and analyzed by Northern
blot using a specific probe for iNOS. After normalization for
-actin content, the relative amount of iNOS mRNA is shown in
arbitrary units (a.u.). The figure shows 1 representative
experiment out of three.
TPP Promotes Apoptosis Through NO-independent
Mechanisms
NO by itself induces apoptosis in cultured M
incubated with NO donors or cytokines that involve NO
synthesis(9, 17) . Because TPP promoted both NO
release and morphological changes associated with apoptosis, we tested
to find out whether this cell death was exclusively due to NO or to
other molecules released in the course of M activation. As Fig. 5A shows, TPP promoted DNA laddering even in the
presence of NOS substrate analogue inhibitors, such as NMA. In this
condition, the amount of nitrite and nitrate released to the medium was
lower than in control unstimulated cells. To determine the effect of NO
on the TPP-induced apoptosis, the nitrite plus nitrate concentration
was measured at the time when the apoptotic process was quantified
following the release to the cytosol of DNA containing nucleosomal
complexes. As Fig. 5, B and C show, in the
presence of NMA the NO synthesis remained unchanged (even lower than in
unstimulated cells); however, a TPP-dependent apoptosis was observed.
Addition of a chemical NO donor such as 3-morpholinosydnonimine (50
µM in the culture medium) to control M incubated
with NMA triggered a rapid apoptotic process, indicating that NMA by
itself did not interfere with the apoptotic response (Fig. 5C). To ascertain that TPP induces apoptosis in
the complete absence of NO synthesis, M were incubated with
arginine-free medium and with serum that was previously treated with
arginase (1 unit/ml), and compared with the response in the presence of
arginine. As Fig. 6shows, in this experimental model apoptosis
prevailed in the absence of NO synthesis (lanes 1 and 2), confirming the existence of a NO-independent pathway
involved in apoptosis induction in cultured rat M. To ensure that
NOS was expressed in response to TPP regardless of the presence of
arginine in the incubation medium, a Western blot of the cytosolic
proteins of these cells was performed using an iNOS antibody. When
cells were simultaneously stimulated with TPP and LPS, apoptosis
prevailed and a decrease in iNOS protein (130 kDa) was evident (lane 5). Taking advantage of this arginine-free model we
determined the degree of apoptosis induced by TPP in the complete
absence of NO synthesis. As Fig. 7shows, in the above mentioned
conditions TPP promoted M apoptosis exhibiting a half-maximal
effect at 0.3 µg/ml, a lower concentration than required to produce
the half-maximal NO synthesis (0.8 µg/ml; Fig. 2).
(2 10) were incubated for 36
h in the absence (lanes 1 and 3) or presence of 5
µg/ml of TPP (lanes 2 and 4) and 1 mM NMA (lanes 3 and 4). The DNA laddering and
nitrite plus nitrate concentration in the medium were assayed (panel A). The time course of NO release in the absence
(, ) or presence of TPP (
, 
) (panel
B), and the relative content in apoptotic cells (panel C)
were determined. Filled symbols correspond to assays in the
presence of 1 mM NMA. To have a positive control of
NO-dependent apoptosis, cells were incubated with 50 µM of
3-morpholinosydnonimine (
), a NO donor (panel C). An A value of 0.06 O.D. units corresponding to
control cells was subtracted from each sample. Results show 1
representative experiment out of three.
were incubated for 36 h in
arginine-free medium (lanes 1 and 2) or in its
presence (lanes 3-5) and were stimulated with 5
µg/ml of TPP (lanes 2 and 4), or TPP plus 1
µg/ml of LPS (lane 5). The extent of apoptosis (open
bars) or NO release (dashed bars) were measured. An A value of 0.05 O.D. units corresponding to
control cells was subtracted from each sample. At the time of sampling,
cells were homogenized and analyzed by Western blot using an iNOS
mAb.
stimulated with TPP in
arginine-free medium. M were incubated for 36 h with the
indicated concentrations of TPP and the extent of apoptosis () and
NO release () were measured. Results show a representative
experiment out of three cell preparations.
proteins in human neutrophils(19) .
Additionally, tyrosine phosphorylation of MAP kinases 1 and 2 has been
reported after TPP activation of murine M from LPS-responsive and
-nonresponsive strains; however, for LPS MAP activation is observed
only in LPS-responsive strains, and the lack of activation in the
LPS-nonresponsive counterparts has been situated at a post-receptor
step but prior to MAP kinase activation(8) . As a result of
M activation with cytokines and bacterial products, these cells
release various secretory molecules (10) exhibiting a high
chemical activity (NO, HO, and
O
). Regarding NO release by TPP-activated
M, it has been shown that tyrosine kinase inhibitors, such as
genistein, effectively cancelled NO production, revealing the necessity
of tyrosine phosphorylation in the pathway that involves NOS
expression, and a similar conclusion applies for LPS(20) .
However, except for a prolonged sensitivity to genistein when added at
various times after LPS triggering, no significant differences have
been observed between LPS and TPP regarding the involvement of protein
tyrosine kinase activation following M activation. by bacterial lipopeptides, which
seem to exhibit some specific characteristics when compared with the
effects elicited by LPS. The release of NO after TPP stimulation is
delayed and quantitatively lower with respect to the response elicited
after LPS challenge. This is opposite to the temporal pattern of MAP
kinase phosphorylation that, at least in murine M, is more
rapidly activated by lipopeptides(8) . Moreover, simultaneous
treatment of the cells with both TPP and LPS results in a blockage of
the response, as reflected by the decrease in iNOS mRNA levels and
protein, and NO release to the medium, which suggests that some signals
are released in the course of the dual stimulation resulting in a
partially antagonistic response. This result was unexpected since
cooperation would be observed in view of the use of some common
transduction pathways (i.e. early protein tyrosine kinase
stimulation and MAP kinase activation). TPP in
addition to NO synthesis promotes the release of
HO, clearly observed after triggering with
phorbol esters, and therefore contributing to cell activation with
various oxygen reactive intermediates; however, the study of the
modulation by IFN- and TNF- of the TPP response revealed that
these cytokines antagonize the production of NO (a behavior opposite to
their cooperative action in LPS stimulated macrophages, refs. 13, 14),
but result additive (and therefore independent) with TPP in the
production of HO. activation concerns the commitment for
apoptosis after stimulation. In the absence of NO synthesis LPS fails
to trigger an effective apoptosis(9, 17) , whereas
under these conditions TPP retains the ability to induce cell death of
activated M, which suggests that an alternative NO-independent
apoptotic pathway is operative in TPP-stimulated cells. Our results
show that when NO is produced, the relative amount of DNA fragmentation
is moderately increased (
30%), suggesting that NO is not the main
apoptotic inducer and that a cooperation exists between pathways that
trigger apoptosis. However, it remains to be determined whether NO may
influence the fraction of the cell population exhibiting DNA cleavage. of an enhanced susceptibility to HO or
other oxidant species released in the course of the activation. An
example of this situation has been reported in retrovirus infected T
cells that exhibit an extreme sensitivity to HO for apoptosis(21) . Indeed, the occurrence of
physiological pathways leading to apoptosis of antigen-presenting
M following CD4
T cell activation has been
proposed to be genetically programmed into the repertoire of M
functions(22, 23) . are programmed for cell death upon
activation(23) , it is possible that the apoptosis observed in
TPP-activated M lies on the release of several reactive oxygen
intermediates (NO, HO,
O) which may trigger the expression of
genes or modulate the activity of transcriptional factors such as NF-kB
or AP-1 that would be responsible for this process(24) .
);
TPP-Ala, S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysAlaLys;
NMA, N
-methyl-L-arginine; M,
macrophage; NOS, nitric oxide synthase; IFN-, interferon ;
TNF-, tumor necrosis factor ; PBS, phosphate-buffered saline;
PDBu, phorbol 12,13-dibutyrate; MAP, mitogen-activated protein.
We thank Dr. Q.-W Xie and Dr. C. F. Nathan for the
iNOS cDNA probe, O. G. Bodelón for technical
assistance and E. Lundin for help in the preparation of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 G. D. Ceneviva, E. Tzeng, D. G. Hoyt, E. Yee, A. Gallagher, J. F. Engelhardt, Y.-M. Kim, T. R. Billiar, S. A. Watkins, and B. R. Pitt Nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells Am J Physiol Lung Cell Mol Physiol, October 1, 1998; 275(4): L717 - L728. [Abstract] [Full Text] [PDF]
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E. Lopez-Collazo, S. Hortelano, A. Rojas, and L. Bosca Triggering of Peritoneal Macrophages with IFN-{alpha}/{beta} Attenuates the Expression of Inducible Nitric Oxide Synthase Through a Decrease in NF-{kappa}B Activation J. Immunol., March 15, 1998; 160(6): 2889 - 2895. [Abstract] [Full Text] [PDF]
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C.-F. Wu, N. H. Bishopric, and R. E. Pratt Atrial Natriuretic Peptide Induces Apoptosis in Neonatal Rat Cardiac Myocytes J. Biol. Chem., June 6, 1997; 272(23): 14860 - 14866. [Abstract] [Full Text] [PDF]
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Y.-M. Kim, M. E. de Vera, S. C. Watkins, and T. R. Billiar Nitric Oxide Protects Cultured Rat Hepatocytes from Tumor Necrosis Factor-alpha -induced Apoptosis by Inducing Heat Shock Protein 70Expression J. Biol. Chem., January 10, 1997; 272(2): 1402 - 1411. [Abstract] [Full Text] [PDF]
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M. J.M. Diaz-Guerra, O. G. Bodelon, M. Velasco, R. Whelan, PeterJ. Parker, and L. Bosca Up-Regulation of Protein Kinase C-epsilon Promotes the Expression of Cytokine-inducible Nitric Oxide Synthase in RAW 264.7Cells J. Biol. Chem., December 13, 1996; 271(50): 32028 - 32033. [Abstract] [Full Text] [PDF]
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M. J.M. Diaz-Guerra, M. Velasco, P. Martin-Sanz, and L. Bosca Evidence for Common Mechanisms in the Transcriptional Control of Type II Nitric Oxide Synthase in Isolated Hepatocytes. REQUIREMENT OF NF-kappa B ACTIVATION AFTER STIMULATION WITH BACTERIAL CELL WALL PRODUCTS AND PHORBOL ESTERS J. Biol. Chem., November 22, 1996; 271(47): 30114 - 30120. [Abstract] [Full Text] [PDF]
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