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* Departamento de Fisiología,
Departamento de Patología, and
Departamento de Bioquímica y Biología Molecular, Universidad de Valencia, 46010 Valencia, Spain
2Correspondence: Departamento de Fisiología, Facultad de Medicina, Avenida Blasco Ibañez 17, 46010 Valencia, Spain. E-mail Federico.V.Pallardo{at}uv.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: oxidative stress • mtDNA • mammary gland • glutathione
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although apoptosis is a well-defined morphological process, the
biochemical mechanisms involved remain under investigation. It is well
known that cellular redox status modulates various aspects of cellular
function. Kane et al. (1)
have suggested that proto-oncogene Bcl-2, an
inhibitor of apoptosis, exerts its action by reducing the production of
reactive oxygen intermediates (ROI),3
thus working as an antioxidant in neurons. Recent reports have
emphasized the role of oxidative stress and nuclear DNA damage in
apoptosis (2,
3)
. In addition, antioxidants have been shown to protect
against apoptosis in different experimental models. However, normal
apoptosis occurs in very low oxygen environments (4)
. Although Zamzami
et al. have shown that apoptosis is closely related to mitochondrial
impairment (5)
, the possible effect of apoptosis on mitochondrial DNA
(mtDNA) is not known. Apoptosis is a highly complex biochemical process
that may differ according to the model; therefore, we used more than
one model in our studies. We chose an in vitro model
(cultured fibroblasts deprived of growth factors) and an in
vivo model (the mammary gland at the peak of lactation).
Van den Dobbelsteen et al. (3)
reported that apoptosis in JURKAT
cells causes an efflux, but not an oxidation, of cellular glutathione.
We have developed a method to determine oxidized glutathione (GSSG)
minimizing oxidation of reduced glutathione (GSH) to less than 0.5%,
and we have validated it in various physiological and pathological
models (6,
7)
. Using this method, we show here that glutathione is
indeed oxidized in apoptosis. Thus, the main goal of this study was to
find the origin of the oxidative stress generated during the apoptotic
process:
We report here that the following events take place during the apoptotic process: mtDNA oxidation, increased mitochondrial peroxide production and cytosolic peroxide levels, early oxidation of the mitochondrial and cytosolic GSSG/GSH couple, and decreased mitochondrial membrane potential. Thus, the emerging picture of our experiments is that oxidative stress is an early event in apoptosis and that mitochondria contribute significantly to such stress by increasing the generation of peroxides.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
3Y1 fibroblasts were cultured in Dulbecco's modified Eagle's
medium supplemented with 10% FCS, antibiotics (25 U/ml penicillin and
25 µg/ml streptomycin), and amphotericin B (0.3 µg/ml) in 5%
CO2 in air at 37°C in 75
cm2 flasks. Experiments were performed 4, 8, 24,
and 48 h after FCS removal. In experiments under anaerobic
conditions, media, reagents, and equipment were maintained in the
anaerobic incubator for 24 h before use. The oxygen concentration
in the anaerobic atmosphere was 2 to 8 p.p.m. In experiments using
glutathione monoethylester, fibroblasts were cultured with and without
glutathione monoethylester (0.2 mM) for 48 h.
Lactating mammary gland
Female Wistar rats had free access to water, were fed ad
libitum, and kept on a 12 h dark/12 h light cycle. The
lactating mammary gland is a well-known model of apoptosis for in
vivo studies (8)
. Rats were anesthetized with pentobarbital sodium
(50 mg/kg body wt i.p.). Mammary glands were removed at the peak of
lactation (days 14–15) from rats whose pups were removed from the
mother 6, 12, 24, and 48 h before performing the experiment (days
12–13 of lactation). Figure 1
A shows a typical ladder-type DNA pattern of nuclear
apoptosis from the weaned lactating mammary gland 24 h after
weaning. Apoptosis was also assessed in the mammary gland by the
standard kit for in situ quantification of apoptosis:
`ApopDETEK' (ENZO). This is a method for detection of the early
stages of the chromosome breakdown, based on the incorporation of
BIO-16-dUTP by a terminal deoxynucleotide transferase on the 3'-OH
termini produced by the endonucleolytic activity associated with the
apoptotic process.
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Lactating mammary gland blood samples
The release of GSH and GSSG in blood by the mammary gland was
determined in blood collected from the pudic-epigastric vein of control
lactating rats and in animals 12 h after weaning. After blood
sampling, the mammary glands were removed. Blood samples were treated
with 6% perchloric acid containing 1 mM EDTA to determine GSH or with
6% perchloric acid containing 20 mM N-ethylmaleimide and 1 mM EDTA to
determine GSSG. Then samples were centrifuged for 10 min at 3500 rpm,
and the acidic supernatants were neutralized and used to determine
glutathione, as described previously (6,
7)
.
Isolation of mitochondria
Mammary glands were quickly removed from the animals. Isolation
of mitochondria was performed using the procedure described by Rickwood
et al. (9)
.
Flow cytometry
Flow cytometry was performed using an EPICS PROFILE II flow
cytometer (Coulter Electronics, Hialeah, Fla.). Fluorochromes were
excited with an argon laser tuned at 488 nm. Forward-angle light
scatter and side-angle light scatter (90°) were measured and
fluorescence was detected through a 488 nm blocking filter, a 550 nm
long pass dichroic, a 525 nm band pass, or a 575 nm long pass; 10,000
individual cells or 10,000 mitochondria were counted for each sample.
To determine the percentage of apoptotic cells, ~106
cells/sample were washed with ice-cold phosphate-buffered saline (PBS)
and fixed in 95% ethanol. Cells were then resuspended in 1 mg/ml RNase
for 30 min at 37°C and stained with 0.05 mg/ml of PI for 24 h
Cells were excited at 488 nm, and the emission was detected through a
630/22 nm band pass filter. 10,000 cells were analyzed for each sample.
Cell cycle analysis was performed using software E 11/94 version 4.0
EPICS. Cells were considered to be in apoptosis if they exhibited
sub-G1 DNA fluorescence and the same forward
angle light scatter as G1 cells. Cellular debris
was gated out using an electronic threshold. Figure 1B
shows
a characteristic histogram of control and apoptotic fibroblasts after
24 h of culture using PI as a fluorescent probe.
Mitochondrial membrane potential from fibroblasts was measured using
rhodamine 123 (RH123). Approximately 500,000 cells were resuspended in
1 ml of culture medium, 2 µl RH123 (10 µg/ml) was added, and
fluorescence emission was measured at 525 ± 5 nm (11)
.
Intracellular peroxide levels were measured with two different
fluorochromes: 2',7'-dichlorofluorescin diacetate (DCFH) (12)
and
dihydroethidium (DHE) (12)
. DCFH was used to determine peroxide levels
in cells according to the method of Rothe and Valet (12)
. One
milliliter of cell suspension (4–4.5 x 105 cells) and 5 µl
DCFH (1 mM) were used. In studies with DHE, fibroblasts (4.0–4.5 x 105 cells) were resuspended in 1 ml of PBS and incubated
with DHE 5 µl (1 mM) for 15 min at 37°C in the dark (12)
.
To measure peroxide production in isolated mitochondria from the lactating mammary gland, we used dihydrorhodamine 123 (DHRH 123) as a fluorescent probe (DHRH 123 is oxidized to RH123 as a function of peroxide formation).
DNA electrophoresis
The protocol described by Gong et al. (13)
was used to detect
digested DNA from apoptotic mammary gland tissue. To obtain each DNA
sample, 0.5 g of tissue was collected.
Glutathione determination
GSH was measured using the glutathione-S-transferase assay (14)
.
Fibroblast samples were harvested, washed, and resuspended in TCA 15%
EDTA 1 mM. Samples from the lactating mammary gland were obtained by
quick homogenization in 6% (w/v) PCA containing 1 mM EDTA.
Determination of GSSG was carried out by a high-performance liquid
chromatography method with UV-V detection that we recently developed to
measure GSSG in the presence of a large excess of GSH (6,
7)
. The
essence of this method consists of minimizing GSH oxidation, which
otherwise results in large increase in GSSG (6,
7)
.
Purification of mitochondrial DNA
The method of Latorre et al. (15)
was used. The purity of mtDNA
was determined spectrophotometrically by measuring the absorbance ratio
at 260/280 nm. The value found was 1.7–1.8, which is in accordance
with previous studies (16)
. The mtDNA preparation was free of any
detectable nuclear DNA, as tested by agarose gel electrophoresis and
staining with ethidium bromide.
Digestion of mtDNA to nucleosides
After isolation, mtDNA was digested to nucleosides (17,
18)
using nuclease P1, then alkaline phosphatase was added. To liberate the
nucleosides from phosphate residues, the resultant solution was
incubated at 37°C for 60 min. An 8-oxo-7,8-dihydro-2'-deoxyguanosine
(oxo8dG) standard was synthesized and purified in our laboratory as
described in ref 19
.
Measurement of oxo8dG
The oxo8dG present in DNA hydrolysates was separated
isocratically on a 25 x 0.46 cm ODS-2 Spherisorb column, with a
mobile phase of 1% methanol in 50 mM potassium phosphate buffer, pH
5.5, pumped at a flow rate of 1 ml/min. Electrochemical detection of
oxo8dG was performed on an ESA Coulochem II (Bedford, Mass.) model 5200
equipped with a 5011 analytical cell and a 5021 guard cell. The
settings for the dual coulometric detector were +0.15 for detector 1
and +0.45 for detector 2.
Statistics
Results are expressed as mean ± SD.
Statistical analyses were performed by the least-significant difference
test which consists of two steps. First, an analysis of variance was
performed. The null hypothesis was accepted for all numbers in which F
was nonsignificant at the level of P<0.05. Second, the sets
of data in which F was significant were examined by the modified
t test, using P<0.05 as the critical limit.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that induction of apoptosis by FCS deprivation in fibroblasts
increases over different periods of time. The percentage of apoptotic
fibroblasts is significantly higher at 24 h of calf serum
deprivation, but not before this time, and remains high during the next
24 h of cell culture. The percentage of apoptosis in the lactating
mammary gland determined morphologically was 4% in control tissues (at
the peak of lactation) and 16%, 38%, 42%, and 24% at 6, 12, 24, and
48 h after weaning, respectively (the morphological experiment was
repeated twice with similar results).
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Glutathione oxidation is an early event in apoptosis
Cellular GSSG/GSH ratio increases 8 h after culture in
serum-deprived medium, i.e., clearly before the occurrence of
apoptosis, as shown in Fig. 3
. At 24 h of serum deprivation GSSG level remains high, GSH level
decreases, and a clear correlation exists between glutathione oxidation
and an increase in the percentage of apoptosis in fibroblasts (see
Fig. 4
A, B).
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Glutathione oxidation in the regressing mammary gland
Apoptosis also causes an increase in GSSG levels concurrent with a
decrease in GSH levels in the mammary gland 24 h after weaning
both in whole tissue (Fig. 5
A, B) and in mitochondria from the regressing mammary gland
(Fig. 6
). We also measured the release of GSH and GSSG in blood from the
mammary gland and have found that GSSG, but not GSH release, increases
significantly 12 h after weaning. In blood collected from the
pudic-epigastric vein, GSSG levels increased from 0.05 ± 0.03
µmol/ml (n=5) in controls to 0.11 ± 0.06 µmol/ml
(n=5) 12 h after weaning (P<0.05). These
results demonstrate that the loss of GSH in the regressing mammary
gland is accompanied by an increase of GSSG in the tissue that is
excreted from the gland and washed out by blood.
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Damage to mtDNA in apoptosis
We recently found that age-associated oxidation of glutathione
directly correlates with oxidative damage to mtDNA (20)
. Thus,
oxidation of glutathione associated with apoptosis led us to examine
whether mtDNA is affected in the apoptotic mammary gland. We found that
apoptosis causes an oxidation of mtDNA in the mammary gland (Fig. 7
). The mammary gland at the peak of lactation had a content of the
oxidatively modified DNA base, oxo8dG of 0.068 ± 0.044
(n=6) pmol/µg DNA, whereas the mammary gland in which
apoptosis had been induced by removing the pups had an oxo8dG
concentration of 0.200 ± 0.069 (n=4) pmol/µg DNA
(P<0.05). Thus, the apoptotic tissue had a content of
oxidatively modified DNA bases that is more than 300% of the control.
We could not study oxidation of mitochondrial DNA in fibroblasts in
culture because the amount of tissue required for this measurement made
it impractical to measure oxidation of mtDNA in these cells.
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Mitochondria from apoptotic mammary glands produce more
peroxides than those from controls
In an attempt to determine the possible reason for
oxidative stress associated with apoptosis, we measured peroxide
production by mitochondria from control and apoptotic mammary glands.
Figure 8
shows peroxide production by mitochondria isolated from the mammary
gland. Peroxide production from mitochondria isolated from apoptotic
glands is 300% higher than in controls.
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Increased peroxide levels in apoptotic fibroblasts
A moderate increase in peroxide levels may be expected as a result
of increased peroxide production. We measured such levels using two
different fluorochromes (as described in Materials and Methods) and
found that the peroxide content of apoptotic fibroblasts is
significantly higher than in controls (see Fig. 9
).
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Apoptosis decreases mitochondrial membrane potential
The role of mitochondria in apoptosis was reviewed in ref 21
. We
tested the effect of apoptosis on the major mitochondrial function:
oxidative phosphorylation coupled with respiration. This can be tested
by determining mitochondrial membrane potential (22)
. We found that
mitochondria from apoptotic fibroblasts have a lower membrane potential
than controls (Fig. 9)
. This is in keeping with previous reports in
which mitochondrial damage in apoptosis was observed (for a review, see
ref 21
).
Glutathione monoethyl ester delays apoptosis
Work from Anderson and Meister (23)
showed that glutathione
monoethyl ester enters cells where it is converted to free glutathione,
thus raising intracellular GSH levels. We found that fibroblasts
incubated with glutathione monoethyl ester have a lower index of
apoptosis than controls (see Fig. 10
). Glutathione ester produces a transient decrease in apoptosis after
FCS removal. In fact, 48 h after FCS removal the index of
apoptosis rises again (results not shown).
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Glutathione is not oxidized under anaerobic conditions
Fibroblasts were incubated under anaerobic conditions in
order to find whether the glutathione oxidation process takes place in
apoptosis under anaerobic conditions, when reactive oxygen species
(ROS) production is decreased. The results presented in Fig. 11
show that under anaerobic conditions,
the percentage of apoptosis decreases, GSH is not oxidized, and there
are no significant changes in peroxide levels or in the mitochondrial
membrane potential (results not shown). These results contrast with the
clear increase in GSSG and peroxides levels and the decrease in
mitochondrial membrane potential under physiological aerobic
conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. On the other hand, apoptosis can
occur at very low oxygen tension, when oxygen radicals are unlikely to
occur. The proto-oncogene product Bcl-2 can inhibit apoptosis in the
absence of reactive oxygen products (4)
.
The glutathione redox pair is an index of oxidative stress (24)
. The
GSH/GSSG ratio is at equilibrium with thiol and disulfides in proteins,
and has been proposed as an important regulator of enzyme activities
(25)
.
Reports by Van Dobbelsteen et al. (3)
and Ghibelli et al. (26)
have
proposed that glutathione depletion in apoptosis is due to an increased
efflux of the reduced form and that such loss is therefore
nonoxidative, i.e., not due to oxidative stress. In this paper we
report that although GSH extrusion is the major source of GSH loss (3,
26)
, apoptosis causes an oxidation of glutathione (see Figs. 3
and 4
)
in aerobic conditions. This oxidation of glutathione is an index of
oxidative stress, which occurs both in the cytosol and in the
mitochondria. Glutathione oxidation is an early event in the apoptotic
process and may be a metabolic signal or at least an early warning of
apoptosis.
The results presented in Fig. 11
show that apoptosis takes place in
anaerobiosis, although to a lesser extent. Under anaerobic conditions,
however, GSSG, cellular peroxide levels and mitochondrial membrane
potential did not change. These results contrast with the clear
increase in GSSG and peroxide levels and the decrease in mitochondrial
membrane potential shown in those apoptotic fibroblasts cultured in
aerobic, physiological conditions. These new results demonstrate that,
as it is known, apoptosis can follow two possible pathways:
1) a free radical-dependent pathway and 2) a free
radical-independent pathway. It appears that regulation of cytochrome
c release by mitochondria is a key factor in the induction
of apoptosis (29)
. The existence of a mitochondrial permeability
transition channel (MPT) that is independent of free radicals would
explain the results obtained in anaerobiosis and in aerobiosis.
We show here that glutathione oxidation precedes nuclear DNA
degradation and that mitochondrial oxidative stress plays a key role in
the induction of the apoptotic machinery under aerobic conditions. Yang
and Cortopassi (29)
reported recently that oxidative stress can
regulate cytochrome c release (a common final activator of
apoptosis) from mitochondria by the MPT. They identify the MPT as
intracellular sensors of oxidants and other toxins. Our results
coincide with those found by Yang and Cortopassi (29)
and suggest that
oxidative stress, at least under aerobic conditions, is a cause and not
a consequence of the apoptotic process. Polyak et al. (2)
showed that
p53 transcriptionally induces redox-controlling genes, resulting in the
production of ROS, which in turn leads to oxidative damage to
mitochondria and apoptosis.
Apoptosis in fibroblasts and the mammary gland
To study the role of oxidative stress in apoptosis, we used
two models: serum deprived fibroblasts and the mammary gland of the
lactating rat deprived of pups. The reason for using two different
models is twofold. First, because the mechanisms of apoptosis may be
different depending on the model used. Second, we wanted to test the
involvement of damage to mtDNA in apoptosis. Damage of mtDNA occurs in
aging and is related to oxidation of glutathione (20)
. For technical
reasons, isolation of mtDNA cannot be achieved with fibroblasts in
culture (large amounts of cells are required). The lactating mammary
gland was a suitable material and, as shown in Results, mtDNA of
apoptotic tissue had higher oxidative damage than controls.
Peroxide production by mitochondria in apoptosis
Many papers have dealt recently with the occurrence of oxidative
stress in apoptosis. We have shown here that glutathione oxidation (not
merely depletion) and mtDNA oxidative damage take place in apoptosis,
but we wanted to investigate the reason for this oxidative stress.
An interesting paper by Zamzami et al. (5)
stressed the importance of
the mitochondrial control of nuclear apoptosis. Consequently, we
decided to test whether mitochondrial free radical production could be
involved in the oxidative stress associated with the apoptosis we had
detected. Free radical production can be tested by flow cytometry.
Using this method, we have recently found that mitochondria from old
animals produce more peroxide that those from young ones (30)
.
We have found that apoptotic cells have higher peroxide levels than
controls and that this is due, at least in part, to an increased
peroxide production by mitochondria (see Figs. 3
and 4
). Recent results
by Cai and Jones (31)
have shown that superoxide may be generated in
apoptosis due to cytochrome c release from mitochondria.
To summarize the relevant facts reported in this paper: 1) Glutathione oxidation takes place in fibroblasts in culture and in the lactating mammary in vivo during the apoptotic process; 2) glutathione oxidation precedes fragmentation of the nuclear DNA; 3) apoptosis in fibroblasts can be delayed when high glutathione levels are maintained by treatment with glutathione monoethyl ester; 4) mitochondria from apoptotic tissues show oxidative stress evidenced by an increase in GSSG and oxidative damage to mtDNA; and 5) apoptotic cells have a higher peroxide level than controls, which can be explained at least in part by increased mitochondrial peroxide production. The emerging picture of our experiments is that oxidative stress is an early event in apoptosis. Mitochondrial DNA is damaged and mitochondria contribute significantly to the oxidative stress associated with apoptosis by increasing the generation of peroxides.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations: DCFH, 2',7'-dichlorofluorescin diacetate;
DHE, dihydroethidium; DHRH 123, dihydrorhodamine 123; FCS, fetal calf
serum; GSH, glutathione; GSSG, oxidized glutathione; MPT, mitochondrial
permeability transition channel; mtDNA, mitochondrial DNA; oxo8dG,
8-oxo-7,8-dihydro-2'-deoxyguanosine; PBS, phosphate-buffered saline;
PI, propidium iodide; RH123, rhodamine 123; ROI, reactive oxygen
intermediates; ROS, reactive oxygen species.
Received for publication July 1, 1997.
Revision received January 18, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), apoptosis (•). Each point represents an
independent experiment performed 0 h after weaning (control) or
48 h after weaning (apoptosis).
