Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase
Plays a Major Role in Preventing Oxidative Injury to Cells*
Masayoshi
Arai
,
Hirotaka
Imai
,
Tomoko
Koumura
,
Madoka
Yoshida
,
Kazuo
Emoto§,
Masato
Umeda§,
Nobuyoshi
Chiba¶, and
Yasuhito
Nakagawa
From the
School of Pharmaceutical Sciences, Kitasato
University, 5-9-1 Shirokane, Minato-ku, Tokyo 108, the
§ Department of Inflammation Research, Tokyo Metropolitan
Institute of Medical Science (Rinshoken), 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113, and the ¶ Japan Energy Corporation,
3-17-35 Niizo-Minami, Toda-shi, Saitama 335, Japan
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ABSTRACT |
Phospholipid hydroperoxide
glutathione peroxidase (PHGPx) is synthesized as a long form
(L-form; 23 kDa) and a short form (S-form; 20 kDa).
The L-form contains a leader sequence that is required for transport to
mitochondria, whereas the S-form lacks the leader sequence. A construct
encoding the leader sequence of PHGPx tagged with green fluorescent
protein was used to transfect RBL-2H3 cells, and the fusion protein was
transported to mitochondria. The L-form of PHGPx was
identified as the mitochondrial form of PHGPx and the S-form as the
non-mitochondrial form of PHGPx since preferential enrichment of
mitochondria for PHGPx was detected in M15 cells that overexpressed the
L-form of PHGPx, whereas no similar enrichment was detected
in L9 cells that overexpressed the S-form. Cell death caused by
mitochondrial injury due to potassium cyanide (KCN) or rotenone
(chemical hypoxia) was considerably suppressed in the M15 cells,
whereas the L9 cells and control RBL-2H3 cells (S1 cells, transfected
with the vector alone) succumbed to the cytotoxic effects of KCN. Flow
cytometric analysis showed that mitochondrial PHGPx suppressed the
generation of hydroperoxide, the loss of mitochondrial membrane
potential, and the loss of plasma membrane integrity that are induced
by KCN. Mitochondrial PHGPx might prevent changes in mitochondrial
functions and cell death by reducing intracellular hydroperoxides.
Mitochondrial PHGPx failed to protect M15 cells from mitochondrial
injury by carbonyl cyanide m-chlorophenylhydrazone, which
directly reduces membrane potential without the generation of
hydroperoxides. M15 cells were more resistant than L9 cells to cell
death caused by direct damage to mitochondria and to extracellular
oxidative stress. L9 cells were more resistant to
tert-butylhydroperoxide than S1 cells, whereas resistance
to t-butylhydroperoxide was even more pronounced in M15
cells than in L9 cells. These results suggest that mitochondria might
be a target for intracellular and extracellular oxidative stress and
that mitochondrial PHGPx, as distinct form non-mitochondrial PHGPx,
might play a primary role in protecting cells from oxidative stress.
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INTRODUCTION |
Mitochondria are a major physiological source of reactive oxygen
species (ROS),1 which can be
generated during mitochondrial respiration (1). Superoxide radicals,
formed by minor side reactions of the mitochondrial electron transport
chain or by an NADH-independent enzyme, can be converted to
H2O2 and to the powerful oxidant, the hydroxyl radical (2). Thus, mitochondria are continually exposed to ROS that
cause peroxidation of membrane lipids, cleavage of mitochondrial DNA,
and impairment of ATP generation, with resultant irreversible damage to
mitochondria. Mitochondrial dysfunction might contribute to the
pathogenesis of various human neurodegenerative disorders, such as
Parkinson's, Alzheimer's, and Huntington's diseases, amyotrophic lateral sclerosis, stroke, epilepsy, aging, and the AIDS dementia complex (3-5). However, ROS don't have exclusively toxic effects; low
levels of ROS generated in mitochondria can act as signaling molecules
under physiological conditions. ROS produced in mitochondria can
activate transcription factors, such as NF
B and AP-1 (6), and can
function as signals in apoptosis that is induced by TNF-
(7),
ceramide (8), and chemical hypoxia (9).
The production of ROS in mitochondria is strictly regulated by
mitochondrial antioxidant enzymes that include phospholipid hydroperoxide glutathione peroxidase (PHGPx), classical glutathione peroxidase (cGPx), and Mn-superoxide dismutase (Mn-SOD). The importance of antioxidant enzymes in mitochondria is indicated by the fact that
knock-out mice without a gene for Mn-SOD suffer from catastrophic effects (10). By contrast, knock-out mice without a gene for cGPx are
quite vigorous. Some of these tissues remain very resistant to
oxidative stress even though GPx is the only antioxidant enzyme that is
known to reduce the H2O2 produced by Mn-SOD in
mitochondria since mitochondria in most mammalian cells lack catalase
activity (11). Two types of GPx, namely cGPx and PHGPx, are located in mitochondria. PHGPx is the only known intracellular antioxidant enzyme
that can directly reduce peroxidized phospholipids (12) and cholesterol
(13) in membranes. Therefore, PHGPx that can reduce
H2O2, rather than cGPx, is thought to
contribute to the enzymatic defenses against oxidative damage to
mitochondria (14). However, the PHGPx in mitochondria has not been
fully characterized.
We previously cloned a cDNA for PHGPx from the rat (15, 16) and
demonstrated that a short 20-kDa (S-form) and a long 23-kDa (L-form)
form of PHGPx were translated from the cDNA, which included two
potential sites for the initiation of translation in vitro (16). We showed that the L-form included a leader sequence
and was selectively imported into the mitochondria of rat liver by an
import system in vitro (16).
Stable transformants of rat basophile leukemia 2H3 (RBL-2H3) cells, in
which the S-form of PHGPx was overexpressed, were resistant to the cell
death caused by a radical initiator or oxidized lipids (17). The S-form
of PHGPx markedly inhibited the production of leukotrienes by
5-lipoxygenase by preventing production of intracellular hydroperoxides
around the nucleus (18).
In the present study, RBL-2H3 cells that overexpressed the
L-form of PHGPx were established and compared with those
that overexpressed the S-form in an attempt to estimate the functional
roles of the two types of PHGPx in protection against intracellular and
extracellular oxidative stress. The L-form of PHGPx was
more effective than the S-form in preventing cell death that was caused
by ROS generated in mitochondria and by exogenously added hydroperoxides.
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EXPERIMENTAL PROCEDURES |
Reagents--
Antibodies against PHGPx and cGPx were prepared as
described previously (17). Monoclonal antibodies against histone H1 and 125I-protein A (2.60-3.70 TBq/g) were purchased from
Cosmobio Co. Ltd. (Tokyo, Japan) and ICN Biochemicals Inc. (Irvine,
CA), respectively. Rhodamine 123 (Rh123),
5,6-carboxy-2',7'-dichlorofluorescein-diacetate (DCFH-DA), and
cis-parinaric acid were obtained from Funakoshi Co. Ltd.
(Tokyo, Japan). Ammonium 7-fluoroben-2-oxa-1,3-diazo-4-sulfonate (SBD-F) and tri-n-butyl phosphine were obtained from Wako
Co. Ltd. (Tokyo, Japan). Propidium iodide (PI) and monoclonal
antibodies against cytochrome oxidase subunit IV was obtained from
Molecular Probes (Leiden, Netherlands). KCN, rotenone, CCCP,
oligomycin, and BSO were purchased from Sigma.
Construction of Plasmids--
A BamHI fragment of
pRPHGPx4 (16) was subcloned into pSR
, as the expression vector, to
construct pSR
-L-form PHGPx that encoded the
L-form of PHGPx (19). S-probe and L-probe were
made from pRPHGPx4 by polymerase chain reactions for construction of S-GFP and L-GFP. The primers for construction of the
S-probe, in which the cDNA encoded the 42 amino acids from the
first residue of the S-form of PHGPx, were
5'-ACATAAGCTTGCTGGCACCATGTGTGCA-3' and
5'-ATTAGGTACCGGCCACGTTGGTGACGAT-3'. The primers for construction of the
L-probe, in which the cDNA encoded the 32 amino acids from the
first residue of the L-form of PHGPx, were
5'-ATTTAAGCTTCCGGCCGCCGAGATGAGC-3' and
5'-ATTAGGTACCGCGGGATGCACACATGGT-3'. The BamHI and
KpnI fragments of S-probe and L-probe were inserted between
the BamHI and KpnI sites of the GFP expression
vector (pCMX-SAP/Y145 F) that had been constructed by Ogawa et
al. (20).
Cell Culture and Transfection--
We used the previously
established control line of cells (S1 cells) and L9 cells that
overexpressed the S-form (non-mitochondrial) of PHGPx (17). M15 cells,
which overexpressed the L-form (mitochondrial) of PHGPx,
were established by the transfection of RBL-2H3 cells with
pSR
-L-form PHGPx and pSV2neo by electroporation, as
described previously (17). A suspension of RBL-2H3 cells (1 × 107 cells/0.25 ml) was transferred to an electroporation
cuvette (0.4-cm gap; Bio-Rad) with a total of 20 µg of linearized
DNA, which consisted of 18 µg of each expression vector and 2 µg of pSV2neo, used to confer resistance to G418 (Geneticin; Life
Technologies, Inc.) (21). A potential difference of 250 V at 500 microfarads was applied at room temperature with a Gene Pulser II
(Bio-Rad), and cell culture was reinitiated after a 10-min recovery
period. Selection for resistance G418 (1 mg/ml) was initiated after
24 h, and cells were subsequently exposed to G418 at 0.5 mg/ml for 2 weeks. Individual G418-resistant colonies were isolated with cloning
cylinders. Levels of expression of PHGPx were determined by
immunoprecipitation with antibodies against PHGPx, and cells that
overexpressed the L-form of PHGPx were isolated. Control cells and cells that overexpressed the L-form or the S-form
of PHGPx were cultured in Dulbecco's modified Eagle's medium that contained 5% fetal calf serum and 0.5 mg/ml G418.
Subcellular Fractionation of Cells--
Cells were labeled with
140 nCi/ml [75Se]sodium selenite (3126 Ci/g; MURR) for
96 h to determine the distribution of PHGPx and cGPx in cells.
Confluent cells in 225-cm2 culture flasks were washed three
times with phosphate-buffered saline (PBS) and harvested by treatment
with trypsin. The cell suspension was centrifuged at 700 × g for 5 min at room temperature and then
[75Se]sodium selenite-labeled cells were fractionated as
described previously (18). The cell pellet was suspended in sucrose
buffer (0.25 M sucrose, 1 mM EDTA, 3 mM imidazole, and 0.1% (v/v) ethanol, with leupeptin,
antipain, chymostatin, and pepstatin A added at a final concentration
of 10 µg/ml each and phenylmethylsulfonyl fluoride at a final
concentration of 100 µg/ml, pH 7.2) and centrifuged at 700 × g for 10 min at 4 °C. Pelleted cells were resuspended in
the same buffer at approximately 1.5 × 107 cells/ml
and homogenized with a Teflon/glass Potter-Elvehjem homogenizer. A
nuclear fraction (pellet) and a postnuclear fraction (supernatant) were
prepared by centrifugation at 700 × g for 10 min. The
nuclear fraction was suspended in 200 µl of the sucrose buffer.
Mitochondrial, microsomal, and cytosolic fractions from the postnuclear
fraction were obtained by differential centrifugation as described by
de Duve et al. (22). Each subcellular fraction was examined
by standard enzymatic assays for activities of cytochrome c
oxidase (a mitochondrial marker), NADPH-cytochrome c
reductase (a microsomal marker), and lactate dehydrogenase (a cytosolic marker) as reported previously (23, 24). The distribution of histone
H1, as a nuclear marker, was determined by immunoblotting with
polyclonal antibodies against histone H1 (25). The purity of each
subcellular fraction of M15 cells was determined according to our
previous paper in which the the purity of each organelle of control
cells and non-mitochondrial PHGPx overexpressing cells including S1 and
L9 cells has been determined (18). The purity of each subfractionation
in M15 cells was the same as those in S1 and L9 cells. Cytochrome
c oxidase was distributed in nuclear (7.9%),
mitochondrial (75.6%), microsomal (3.5%), and cytosolic (12.9%)
fractions of M15 cells. NADPH-cytochrome c reductase
activities were found in nuclear (5.9%), mitochondrial (16.7%),
microsomal (68.3%), and cytosolic (9.1%) fractions of M15 cells.
The nuclear, mitochondrial, and microsomal fractions were solublized in
200 µl of 0.4% Triton X-100 in PBS for 2 h at 4 °C, and each
solution was centrifuged at 100,000 × g for 1 h
at 4 °C. The supernatants were supplemented into 400 µl each of
PBS and subjected to immunoprecipitation with antibodies against PHGPx and cGPx, as described previously (17). Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (12.5% polyacrylamide) under non-reducing conditions. Gels were stained, dried, and subjected to autoradiography. Total levels of PHGPx and cGPx
were calculated from results of scanning densitometry after
autoradiography with a Bio-Imaging Analyzer (BAS2000; Fuji Film, Tokyo).
Activities of PHGPx and cGPx were measured after the fractionation of
cytosol and mitochondria from each cell line (1.5 × 108 cells). Mitochondrial fraction was sonicated and
centrifuged at 10,000 × g for 10 min at 4 °C. The
supernatants obtained from mitochondrial fraction and cytosolic
fraction were used for assays of PHGPx and cGPx activities. PHGPx
activity was determined by using phosphatidylcholine hydroperoxide
(PCOOH) as the substrate according to the previous paper (18). Activity
of cGPx was determined by using hydrogen peroxide as the substrate
(18). The total activity of SOD was measured in terms of the percentage
inhibition of the formation of superoxide by the xanthine-xanthine
oxidase system (26). Mn-SOD activity was measured in the presence of 5 mM KCN, and Cu,Zn-SOD activity was calculated by
subtraction of the activity of Mn-SOD from the total SOD activity.
Distribution of GFP Fusion Proteins in Cultured
Cells--
RBL-2H3 cells were transfected with plasmids that encoded
GFP, L-GFP, or S-GFP by electroporation, as described
above. Transfected cells were cultured on coverslips in 35-mm dishes in
2 ml of Dulbecco's modified Eagle's medium that contained 5% fetal
calf serum at 37 °C in an atmosphere of 5% CO2 in air.
After 24 h, cells were fixed for 20 min on coverslips with 4%
formaldehyde and washed with Hanks' balanced salts solution. The
fluorescence of cells was monitored and photographed with an Axiovert
135M inverted microscope (Carl Zwiss, Germany) equipped with a
Planapochromat 63 × objective and a filter pack appropriate for
GFP fluorescence.
GFP and mitochondria were simultaneously detected in the same cells by
the double staining with GFP fluorescence and a monoclonal antibody of
Cy3-conjugated anti-cytochrome c oxidase subunit IV that was
a specific probe for the mitochondrial staining (27). Fixed cells were
washed with phosphate-buffered saline (PBS) and were blocked with PBS
containing 2% BSA at 25 °C for 30 min. The cells were incubated
with 2 µg/ml mouse anti-cytochrome c oxidase subunit IV
monoclonal antibodies diluted with 2% BSA-PBS at 25 °C for 2 h. Then the cells were washed with PBS and incubated with
Cy3-conjugated goat anti-mouse IgG (Amersham Pharmacia Biotech) diluted
to 10 µg/ml with PBS containing 2% BSA at 25 °C for 1 h.
Fluorescence of GFP and Cy3 in the same cells was monitored and
photographed with an appropriate filter pack.
Cell Viability--
S1, L9, and M15 cells were plated at
0.5 × 105 cells/well in flat-bottomed 96-well culture
plates and cultured for 24 h. Individual transformants were
exposed to indicated doses of KCN, rotenone, CCCP, oligomycin, or
t-BuOOH for appropriate periods. The LDH release assay was
used for the determination of the cell viability, as described
elsewhere (17). In one series of experiments, cells were incubated for
12 h prior to exposure to KCN with 0.5 mM buthionine sulfoxamine (BSO) for depletion of GSH.
Flow Cytometric Analysis--
Changes in the integrity of
plasma membrane and in the mitochondrial membrane potential were
examined by monitoring staining with propidium iodide (PI) and Rh123,
respectively. After treatment with KCN, cells were stained with PI (5 mg/ml) and Rh123 (1 mg/ml) for 10 min. We also used an
oxidation-sensitive fluorescent probe, 5,6-carboxy-2',7'-dichlorofluorescein-diacetate (DCFH-DA), to assess
levels of intracellular peroxides, as follows. Cells were washed with
PBS and incubated with 2.5 µM DCFH-DA in PBS for 15 min.
DCFH-loaded cells were incubated with or without 25 mM KCN for the times indicated. The intensity fluorescence from PI, Rh123, and
dichlorofluorescein (DCF) in cells was analyzed with a flow cytometer
(EPICS® Elite Flow cytometer; Coulter, Hialeah, FL).
Analysis of Cellular Levels of ATP--
Cellular levels of ATP
were determined by the luciferin-luciferase method using a kit from
Sigma (29).
Fluorescence Measurements of Lipid Peroxidation--
Cells
(1 × 106 cells) were loaded with 20 mM
cis-parinaric acid for 1.5 h at 37 °C and then
washed with PBS. The loaded cells were treated with 25 mM
KCN for the times indicated. After incubation, total lipids were
extracted as described by Bligh and Dyer (30). Fluorescence of total
lipids was monitored with a spectrofluorometric detector (RF-550;
Shimazu Co. Ltd., Tokyo) with excitation at 303 nm and emission at 416 nm.
Fluorescence Measurements of GSH in Mitochondria and
Cytosol--
Amounts of GSH in mitochondria and cytosol were measured
according to the previous paper with a slight modification (28). In
brief, S1, L9, and M15 cells (each 2 × 107 cell) were
fractionated into cytosol and mitochondria. Cytosol and mitochondria
dispersed with the sonication were precipitated by the addition of
trichloroacetic acid at a final concentration of 5%. After
centrifugation at 10,000 × g for 10 min, GSH in the supernatant was converted to fluorescent derivative. The reaction was
started by the addition of 0.5 ml of 0.02% ammonium
7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) in 0.25 M borate buffer, pH 10.5, which contained 5 mM EDTA and 1% tri-n-butyl phosphine. The reaction mixture
kept at 60 °C for 30 min, and the reaction was terminated by the
addition of 50 µl of 4 N HCl, and fluorescent thiol
derivatives were separated by reversed-phase high pressure liquid
chromatography (TSK-gel ODS; TOSOH Co. Ltd., Japan). The mobile phase
was 0.1 M citrate buffer, pH 4.0, tetrahydrofuran/acetonitrile (94.8:0.2:5). The fluorescent thiol
derivatives were monitored with emission at 516 nm and excitation at
384 nm during elution at a flow rate of 1.0 ml/min.
Quantitation of Proteins--
Concentrations of protein were
determined with the BCA protein assay reagent (Pierce), with bovine
serum albumin (BSA) as the standard.
Expression of Results--
All data from assays in which the
number of replicates was three or more are expressed as mean
values ± S.D.
 |
RESULTS |
Sorting of Green Fluorescent Protein Tagged with the Leader
Sequence of PHGPx into the Mitochondria of RBL-2H3 Cells--
The
L-form of PHGPx contains a leader sequence, but the S-form does not
(Fig. 1). Chimeric proteins that included
green fluorescent protein (GFP) were expressed in RBL-2H3 cells in
order to determine whether the leader sequence of the L-form could
serve to target GFP to the mitochondria of living cells. One fusion
protein consisted of GFP with the leader sequence of 32 amino acids
from the first residue of L-form (L-GFP) (Fig.
1B). The other was a fusion protein of GFP with 42 amino
acids from the first residue of S-form (S-GFP) (Fig. 1B).
Expression vectors containing cDNA that encoded GFP, L-GFP, or S-GFP were used to transfect RBL-2H3 cells by
electroporation and then the intracellular localization of fluorescence
due to GFP was monitored with a fluorescence microscope 24 h later
(Fig. 2). Fluorescence was diffusely
distributed in cells that expressed S-GFP or GFP (Fig. 2, A
and B). By contrast, discrete regions with strong
fluorescence were observed in cells that expressed L-GFP
(Fig. 2C). GFP and mitochondria in the
L-GFP-transfected cells were simultaneously visualized by
the double staining with GFP fluorescence and a monoclonal antibody of
Cy3-conjugated anti-cytochrome c oxidase subunit IV (Fig. 2,
C and D). The profile of fluorescence due to
L-GFP was identical to that of mitochondrial cytochrome c
oxidase. Efficient import of GFP with the leader sequence of L-form
into mitochondria indicates that the leader sequence at the amino
terminus of mitochondrial PHGPx is the signal for targeting to
mitochondria.

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Fig. 1.
Structure of rat PHGPx and the GFP-PHGPx
fusion proteins. A, the structure of the cDNA
encoding the L-form and the S-form of PHGPx. The cDNA
for rat PHGPx contains two AUG initiation codons. The active site of
PHGPx includes a selenocysteine (Sec) residue encoded by a
UGA termination codon. The selenocysteine insertion sequence region in
the 3'-untranslated region of PHGPx is required for the incorporation
of selenocysteine. B, the structures of GFP-PHGPx fusion
proteins. L-GFP consisted of the leader sequence of PHGPx
(black bar) and GFP (hatched bar). S-GFP
consisted of 42 amino acids from the second initiation site of the
S-form of PHGPx (gray bar) and GFP (hatched
bar).
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Fig. 2.
Targeting of L-GFP to the mitochondria of
RBL-2H3 cells. Expression vectors encoding GFP (A),
S-GFP (B), and L-GFP (C and D) were
used to transfect RBL-2H3 cells. After the incubation for 24 h,
fluorescent images of cells were photographed at higher magnification
under a fluorescence microscope. RBL-2H3 cells that transfected with
expression vector encoding L-GFP were double-stained with
GFP fluorescence (C) and Cy3-conjugated anti-cytochrome
c oxidase monoclonal antibodies, which is a
mitochondrion-specific probe (D). The black bar
has a length of 10 µm.
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Subcellular Localization of PHGPx and cGPx in RBL-2H3 Cells That
Overexpressed the L-form and the S-form of PHGPx--
RBL-2H3 cells
were transfected by electroporation with cDNAs that encoded the
L-form and the S-form of PHGPx (Fig. 1A). Two types of transformant that stably expressed substantial levels of PHGPx
were isolated after appropriate selection. M15 cells strongly expressed
the L-form of PHGPx with the leader sequence and L9 cells expressed the
S-form of PHGPx. The control line of cells (S1) had been transfected
with the expression vector without an insert. The three kinds of
transformants were labeled with [75Se]sodium selenite for
4 days for determination of the amounts of PHGPx and cGPx (Table
I). The total amounts of PHGPx in L9 and
M15 cells were 4 and 3.5 times higher than that in S1 cells, respectively. No significant differences in total respective amounts of
cGPx, Cu,Zn-SOD, and Mn-SOD were detected among L9, M15, and S1
cells.
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Table I
The levels of selenium-labeled PHGPx and cGPx and of Mn-SOD and
Cu,Zn-SOD in S1, L9, and M15 cells
The amounts of PHGPx and cGPx were determined by measurements of total
radioactivities in immunoprecipitated 75Se-labeled proteins.
Levels of SODs were determined from enzymatic activities (units/100
µg of protein). Data are means ± S.D. of triplicate results.
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Fig. 3 shows the subcellular distribution
of 75Se-labeled PHGPx and cGPx in L9, M15, and S1 cells. In
S1 cells, PHGPx was more concentrated in the mitochondria than in the
cytosolic and microsomal fractions (Fig. 3, A and
C). Levels of PHGPx were significantly elevated in the
cytosolic, microsomal, and nuclear fractions in L9 cells. The amount of
PHGPx in the cytosolic fraction from L9 cells was 4 times higher than
that from S1 cells, but the amounts of PHGPx in the mitochondrial
fractions from L9 and S1 cells were similar. The amount of PHGPx
in the mitochondrial fraction from M15 cells was twice that from S1
cells. The overexpression of L-form PHGPx caused the
enhancement of the activity of PHGPx in mitochondria. Specific activity
of PHGPx in mitochondria of M15 cells was 272 ± 12 pmol/min/mg,
whereas specific activities in S1 and L9 were 156 ± 18 and
156 ± 37 pmol/min/mg, respectively. The activities of cytosolic
PHGPx in S1, L9, and M15 cells were 4.0 ± 1.6, 16 ± 1.9, and 8.2 ± 1.7 pmol/min/mg, respectively. These results show that
the leader sequence of the L-form of PHGPx is the targeting
signal for transport to mitochondria, whereas the S-form PHGPx is
widely distributed in various organelles. The L-form of
PHGPx can be considered to be the mitochondrial PHGPx and the S-form to
be the non-mitochondrial PHGPx.

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Fig. 3.
Subcellular localization of PHGPx and cGPx in
PHGPx-overexpressing RBL-2H3 cells. S1 cells (white
bars), L9 cells (gray bars), and M15 cells (black
bars) were incubated with 75Se (sodium selenite; 0.14 mCi/ml) for 4 days. 75Se-labeled cells were fractionated by
differential centrifugation into a nuclear (Nu), a
mitochondrial fraction (Mit), a microsomal fraction
(Mic), and a cytosolic fraction (Cyt).
Distributions of PHGPx and cGPx were determined by immunoprecipitation
with antibodies against PHGPx (A and C) and
against cGPx (B and D). Immunoprecipitates were
analyzed by SDS-polyacrylamide gel electrophoresis (12.5%
acrylamide) with subsequent autoradiography (C and
D). Radioactivity (in relative units) was quantified with a
Bio-Imaging Analyzer.
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In three types of cells, cGPx was localized exclusively in the
cytosolic fraction (Fig. 3, B and D). In S1
cells, the amount of cGPx in the mitochondria was lower than that of
PHGPx. No significant changes of cGPx activities in cytosol and
mitochondria were found by the overexpression of PHGPx (data not shown).
Resistance of Transformant Cells to Oxidative Damage to
Mitochondria--
The sensitivity of mitochondria in M15, L9, and S1
cells to injury was evaluated by exposing cells to KCN, an inhibitor of the respiratory chain (chemical hypoxia) (Fig.
4). The viability of S1 cells and L9
cells decreased rapidly in a time- and dose-dependent manner, and only about 20% of cells remained viable after exposure to
25 mM KCN for 6 h (Fig. 4, A and
B). By contrast, M15 cells were much more resistant to cell
death caused by KCN. The LD50 of KCN for M15 cells was
approximately 30 mM, whereas LD50 for L9 and S1
cells was 20 mM. These results indicated that mitochondrial damage by KCN could be prevented to some extent by the overexpression of PHGPx in mitochondria. However, overexpression of non-mitochondrial PHGPx did not have such a protective effect.

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Fig. 4.
Effects of KCN on the viability of
PHGPx-overexpressing cells. S1 cells (closed circles),
L9 cells (closed triangles), and M15 cells (closed
squares) were exposed to 25 mM KCN for the times
indicated (A). Individual cultures were exposed to the
indicated concentrations of KCN for 6 h (B). Individual
cultures were treated for 12 h with 0.5 mM BSO to
deplete cells of intracellular GSH and were then exposed to the
indicated concentrations of KCN for 6 h. After incubation, cell
viability was determined from the extent of release of LDH. Viability
was expressed as a percentage relative to the total LDH in the cells.
The total LDH in cells was determined after lysis of cells with 0.2%
Triton X-100. Data are the means ± S.D. of results from four
replicates in each case.
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Effects of KCN on the viability of S1, L9, and M15 cells that had been
depleted of GSH were examined to estimate whether or not the resistance
to KCN of M15 cells had resulted from overexpression of mitochondrial
PHGPx activity. Buthionine sulfoximine (BSO), an inhibitor of the
synthesis of glutathione, inhibits the activity of
glutathione-dependent peroxidases, such as cGPx and PHGPx, by lowering the level of glutathione in cytosol and mitochondria of
cells. Amounts of glutathione in cytosol and mitochondria of S1 cells
were 35.6 ± 2.8 and 18.25 ± 1.6 nmol/mg protein,
respectively. No significant changes of glutathione content were
observed in the PHGPx-overexpressing cells. The levels of cellular
glutathione were markedly reduced by the treatment with BSO. Amounts of
glutathione in cytosol and mitochondria of S1 cells reduced to
3.39 ± 2.8 and 2.80 ± 1.32 nmol/mg protein by the treatment
of BSO, respectively. In BSO-treated M15 cells, amounts of glutathione
in cytosol and mitochondria were 3.38 ± 1.33 and 2.75 ± 1.69 nmol/mg protein, respectively. Decrease in the level of
glutathione by BSO was also found in L9 cells at the same extent as
that in M15 cells (data not shown). When M15 cells pretreated with BSO
were exposed to KCN, the cells lost their resistance to KCN toxicity
(Fig. 4C). These results confirm that resistance of M15
cells to KCN is due to the overexpression of PHGPx in mitochondria.
Fig. 5 shows the effects of various
reagents that interfere with mitochondrial function on the viability of
the three lines of transformants (Fig. 5). Rotenone, oligomycin, and
carbonyl cyanide m-chlorophenylhydrazone (CCCP) were used as
an inhibitor of mitochondrial complex I, as an inhibitor of
F0F1-ATPase, and as an uncoupler of oxidative
phosphorylation, respectively. As shown in Fig. 5A, rotenone
had a strong toxic effect on L9 and S1 cells, whereas M15 cells were
more resistant to cell death caused by rotenone. By contrast, the
sensitivity of M15 to mitochondrial injury due to oligomycin or to CCCP
was the same as that of S1 and L9 cells (Fig. 5, B and
C). These results indicate that mitochondrial PHGPx
contributes to the protection of cells from cell death due to
inhibitors of the respiratory chain but not from death due to the
direct effects on membrane potential and reduction on the production of
ATP. Non-mitochondrial PHGPx failed to protect cells from cell death
that was caused by any impairment of mitochondrial function.

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Fig. 5.
Effects of rotenone, oligomycin, and CCCP on
the viability of PHGPx-overexpressing cells. S1 cells
(closed circles), L9 cells (closed triangles),
and M15 cells (closed squares) were exposed to the indicated
concentrations of rotenone (A), oligomycin (B),
and CCCP (C) for 6 h. After treatment, cell viability
was determined as described in the legend to Fig. 4. Data are
means ± S.D. of results from four replicates in each case.
|
|
Inhibition of the Cyanide-induced Generation of Hydroperoxides by
Mitochondrial PHGPx--
The effects of KCN on intracellular levels of
hydroperoxides in the individual transformants were determined by flow
cytometry using the oxidant-sensitive dye 2',7'-dichlorofluorescein
(Fig. 6). In S1 cells, we detected the
rapid generation of hydroperoxides within 30 min (Fig. 6A).
Hydroperoxides were produced in KCN-treated L9 cells to the same extent
and with the same time course as in S1 cells (Fig. 6B).
However, the production of hydroperoxides was considerably suppressed
in KCN-treated M15 cells as compared with KCN-treated S1 and L9 cells
(Fig. 6C).

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Fig. 6.
Flow cytometric analysis of intracellular
hydroperoxides in PHGPx-overexpressing cells exposed to KCN. To
assess levels of intracellular peroxides, flow cytometric analysis was
performed with the fluorescent probe DCFH-DA. S1 cells (A),
L9 cells (B), and M15 cells (C) were preincubated
with DCFH-DA for 5 min at 37 °C and then incubated with 25 mM KCN (black areas) or without KCN (white
areas) for 1 h. The intensity of fluorescence from DCFH of
cells was quantified by flow cytometry and is plotted on a logarithmic
scale, in arbitrary units, against the number of cells.
|
|
Suppression of the KCN-induced Peroxidation of Lipids by
Mitochondrial PHGPx--
We next examined peroxidation of lipids in
KCN-treated transformants. cis-Parinaric acid, which is a
naturally fluorescent polyunsaturated fatty acid, was used as a
sensitive indicator of lipid peroxidation in cells. A reduction in the
intensity of fluorescence of cis-parinaric acid is an
indicator of lipid peroxidation (31). Analysis of the fluorescence of
cis-parinaric acid revealed that the fluorescence decreased
within as little as 1 h after the start of exposure of cells to
KCN. Fluorescence from S1 and L9 cells was reduced to 50% of the
original value after treatment with KCN for 1 h (Fig.
7). By contrast, no reduction in
fluorescence from M15 cells was observed after 1 h, an indication
that lipid peroxidation was suppressed in M15 cells as compared with L9
cells.

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Fig. 7.
Effects of KCN on the peroxidation of lipids
in PHGPx-overexpressing cells. S1 cells (closed
circles), L9 cells (closed triangles), and M15 cells
(closed squares) were incubated with 20 µM
cis-parinaric acid for 90 min at 37 °C. Then the cells
were exposed to 25 mM KCN for the indicated times. After
the incubation with KCN, lipids were extracted, and the fluorescence of
total lipids was quantified with a spectrofluorometric detector. The
intensity of fluorescence from KCN-treated cells is expressed as a
percentage relative to the intensity of fluorescence from control
cells. Data are means ± S.D. of triplicate results.
|
|
Protection from Disruption of Mitochondrial Functions by
Mitochondrial PHGPx--
To assess changes in mitochondrial membrane
potential (
) and in the integrity of plasma membranes in
transformant cells, we performed flow cytometric analysis after double
staining with rhodamine 123 (Rh123) and propidium iodide (PI) (Fig.
8). Rh123, a lipophilic cation, is
selectively taken up by mitochondria, and uptake is directly
proportional to mitochondrial 
. PI is imported into cells and
binds to cellular DNA when the integrity of plasma membranes is lost.
Cells that had not been treated with KCN were predominantly located in
the PI-negative and high 
field (PI(
)-
high) (Fig. 8,
A, D, and G). New subsets of S1 cells
appeared after treatment of S1 cells with KCN for 2 h; a large
number of subsets was located in the PI(
)-
low field and a
smaller subset was present in the PI(+)-
low field (Fig.
8B). Most S1 cells lost membrane integrity upon exposure to
KCN for 4 h and moved to the PI(+)-
low field (Fig.
8C). Changes of the distribution of L9 cells upon treatment
with KCN were expected to be identical to those of S1 cells. Indeed, L9
cells also shifted from the PI(
)-
low field to the
PI(+)-
low field upon exposure to KCN (Fig. 8, E and
F). By contrast, M15 cells retained their mitochondrial
membrane potential and remained in the PI(
)-
high field after
the exposure to KCN for 2 h (Fig. 8H). Many M15 cells retained their mitochondrial membrane potential even after long term
treatment with KCN (4 h) (Fig. 8I). These observations
indicate that mitochondrial PHGPx protects cells by preventing loss of membrane potential and loss of membrane integrity, whereas
non-mitochondrial PHGPx can't protect cells from the effects of
KCN.

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Fig. 8.
Flow cytometric analysis of changes in
mitochondrial membrane potential and in the integrity of plasma
membranes of PHGPx-overexpressing cells exposed to KCN. S1 cells
(A-C), L9 cells (D-F), and M15 cells
(G-I) were double-stained with Rh123 and PI after the
incubation with 25 mM KCN for 0 h (A,
D, and G), 2 h (B, E,
and H) and 4 h (C, F, and
I). The intensity of fluorescence from PI was plotted
against that from Rh123. Similar results were obtained in three
independent experiments.
|
|
Rapid reductions in levels of cellular ATP were observed in S1 and L9
cells after 2 h of exposure to KCN, with further losses within the
next 2 h (Table II). The level of
ATP in M15 cells after 3 h of treatment with KCN was clearly much
higher than in S1 and L9 cells. The decreases in levels of ATP
corresponded closely to the loss of mitochondrial membrane potential in
transformant cells upon treatment with KCN.
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Table II
Effects of KCN on the intracellular levels of ATP in S1, L9, and M15
cells
S1, L9, and M15 cells were treated with 25 mM KCN for the
times indicated. Amounts of ATP (nmol/mg protein) were determined by
the luciferin-luciferase method. Values given are means ± S.D. of
results from triplicate experiments.
|
|
Resistance of Transformant Cells to Extracellular Oxidative Damage
by t--
BuOOH
Each line of transformants exhibited different
sensitivity to extracellular oxidative damage by t-BuOOH
(Fig. 9). Numbers of surviving S1 cells
decreased rapidly and in a dose-dependent manner. L9 cells
were more resistant to the cytotoxic effects of t-BuOOH than
S1 cells, and M15 cells were even more resistant. In M15 cells,
half-maximal killing with t-BuOOH occurred at less than 50 µM of t-BuOOH. In contrast, the
LD50 of L9 cells was accounted for 32 µM,
although the total expressions of PHGPx in whole cells were the
same between M15 and L9 cells. Thus, overexpression of mitochondrial
PHGPx was more effective in protecting cells from extracellular
oxidative injury than that of non-mitochondrial PHGPx.

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Fig. 9.
Effects of t-BuOOH on the
viability of PHGPx-overexpressing cells. S1 cells (closed
circles), L9 cells (closed triangles), and M15 cells
(closed squares) were exposed to the indicated
concentrations of t-BuOOH for 2 h. Then viability was
determined as described in the legend to Fig. 4. Data are means ± S.D. of the results from four replicates in each case.
|
|
Changes in Mitochondrial Functions Induced by t-BuOOH--
Changes
in mitochondrial functions induced by t-BuOOH were estimated
by flow cytometric analysis (Fig. 10).
Three subsets of S1 cells appeared after exposure to t-BuOOH
for 1 h as follows: cells in the PI(
)-
high field; cells in
the PI(±)-
moderate field; and cells in the PI(+)-
low
field. Thus, S1 cells lost mitochondrial membrane potential and also
plasma membrane integrity, and they were located predominantly in the
PI(+)-
low field after treatment with t-BuOOH (Fig.
10A). Moderate decomposition of plasma membranes and the
moderate loss of mitochondrial potential were observed in L9 cells.
More L9 cells remained in the PI(±)-
moderate field than S1 cells
(Fig. 10B). The membrane potential of M15 cells was
retained, and cells with a low membrane potential were fewer than in
the case of L9 cells (Fig. 10C). The population of M15 cells
located in the PI(±)-
moderate field was also smaller than in the
case of L9 and S1 cells.

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Fig. 10.
Flow cytometric analysis of changes in
mitochondrial membrane potential and the integrity of plasma membranes
of PHGPx-overexpressing cells exposed to t-BuOOH.
S1 cells (A), L9 cells (B), and M15 cells
(C) were double-stained with Rh123 and PI after incubation
with 50 µM t-BuOOH for 60 min. The intensity
of fluorescence from PI was plotted against that from Rh123. Similar
results were obtained in three independent experiments.
|
|
 |
DISCUSSION |
Some proteins in mitochondria are initially synthesized in the
cytoplasm as larger precursors, and then they are imported into the
mitochondria where proteolytic cleavage yields the mature forms. The
leader sequence of the 23-kDa precursor to PHGPx is the signal for
import of this protein into the mitochondria by using in
vitro import system (16). In the present study, we examined the
transport of PHGPx into the mitochondria of living cells. Green
fluorescent protein (GFP) has proved useful as a probe in an attempt to
visualize the translocation of proteins in living cells. However, few
studies have used GFP to study the transport of proteins into
mitochondria in cells. Yano et al. (32) successfully
visualized the translocation of a chimeric protein that consisted of
GFP and the presequence of ornithine transcarbamylase among the
organelles of COS 7 cells. We constructed a plasmid that encoded GFP
protein linked to the leader sequence of PHGPx (L-GFP) to
investigate the role of the leader sequence. L-GFP was
correctly targeted to the mitochondria, whereas S-GFP, which lacked the
leader sequence, was not detected in mitochondria (Fig. 2). Thus, the
leader sequence of 23-kDa PHGPx (L-form) appears to be
required not only as an import signal but also as a targeting signal.
There are four known isozymes of glutathione peroxidases as follows:
cytosolic GPx (cGPx), PHGPx, plasma GPx, and gastrointestinal GPx (33).
PHGPx is unique among these isozymes in having a leader sequence for
transport to mitochondria. cGPx is located in mitochondria but is not
translated with a signal peptide.
The leader sequence of PHGPx is located between two different sites
for initiation of translation of the cDNA for PHGPx. Pushpa-Rekha et al. (34) demonstrated that the gene for PHGPx has
alternative transcription sites and that these sites result in two
populations of mRNAs for PHGPx. They found that one mRNA had an
upstream AUG codon that was primarily utilized in rat testis and was
translated to yield the mitochondrial PHGPx (L-form). The
other mRNA lacked the upstream AUG codon, and it encoded
non-mitochondrial PHGPx (S-form), which was synthesized predominantly
in somatic cells. In RBL-2H3 cells, the upstream initiation site
appears to be used predominantly, with resultant transcription of
mRNA for mitochondrial PHGPx, since the amount of PHGPx in
mitochondria was higher than that in other organelles (Fig. 3). The
mechanism for regulation of transcription from the two initiation sites
in a single gene for PHGPx remains to be resolved.
A considerable amount of PHGPx was found in mitochondria when RBL-2H3
cells were transfected with a plasmid encoding the L-form of PHGPx (M15 cells). Levels of PHGPx were also elevated in cytosolic, microsomal, and nuclear fractions of M15 cells as compared with S1
cells (control transfected cells). Two possibilities can be considered
to explain the increased amounts of PHGPx in organelles other than
mitochondria in M15 cells. One possibility is that mRNA for the
S-form of PHGPx is transcribed from the downstream initiation site, as
well as from the upstream site, when the L-form of PHGPx is
overexpressed. Alternatively, the signal peptide of PHGPx might be
partially removed by proteolysis before import into mitochondria can be
completed. The latter possibility can be rejected since the fusion
protein that consisted of GFP with the signal peptide was efficiently
transported into mitochondria (Fig. 2).
The overexpression of the mitochondrial type of PHGPx protected RBL-2H3
cells from cell death due to mitochondrial oxidative stress that
resulted from exposure of cells to KCN and rotenone (chemical hypoxia).
Neither S1 nor L9 cells were resistant to the cytotoxicity of KCN.
Resistance of M15 cells was eliminated when the activity of PHGPx in
these cells was inhibited by depletion of cytosolic and mitochondrial
glutathione with buthionine sulfoximine (BSO). Thus, overexpression of
mitochondrial PHGPx clearly contributed to protection from
mitochondrial damage.
We next investigated how mitochondrial PHGPx interferes with the
toxicity of KCN, an inhibitor of complex IV of the mitochondrial respiratory chain. KCN causes various types of damage to mammalian cells, inducing the rapid generation of ROS (35), the reduction of
mitochondrial 
(36), a decrease in cellular levels of ATP (36),
and lipid peroxidation (37), for example. These phenomena developed in
KCN-treated RBL-2H3 cells in a time-dependent manner. Flow
cytometric analysis revealed that KCN induced the rapid generation of
hydroperoxides in S1 cells within 30 min (Fig. 6A). Lipid
peroxidation was induced within 1 h (Fig. 7). After the production
of hydroperoxides, loss of mitochondrial membrane potential and a
reduction in levels of intracellular ATP were observed from 2 to 4 h (Fig. 8 and Table II). Such mitochondrial injury induced the loss of
plasma membrane integrity (Fig. 8) and, finally, cell death at 8 h
(Fig. 4). The overexpression of mitochondrial PHGPx prevented cell
death caused by KCN. Mitochondrial PHGPx hindered the generation of
hydroperoxides, which was an early feature of cell damage.
Mitochondrial PHGPx failed to prevent cell death in response to CCCP or
oligomycin, both of which directly reduced the membrane potential and
the level of cellular ATP without the production of hydroperoxides. Thus, mitochondrial PHGPx appeared to maintain the functions of mitochondria by reduction of intracellular hydroperoxides generated as
a result of damage to the mitochondrial respiratory machinery.
PHGPx in mitochondria effectively reduced the
H2O2 generated in mitochondria that had been
damaged by exposure to KCN since the extent of lipid peroxidation
caused by H2O2 was significantly reduced in
KCN-treated M15 cells. In general, PHGPx reduces
H2O2 less effectively than cGPx. The present
results suggest that PHGPx in mitochondria might limit local increases
in concentrations of H2O2. By contrast, PHGPx
in the cytosol might reduce H2O2 that is
dispersed in the cytosol much less efficiently.
M15 and L9 cells exhibited different levels of sensitivity to
extracellular oxidative damage (Fig. 9). M15 cells were more resistant
to the cytotoxic effects of t-BuOOH than L9 cells even though the total activity of intracellular PHGPx was similar in both
lines of transformed cells. These results suggest that mitochondria might be a primary target for t-BuOOH. Mitochondrial PHGPx
prevents cell death by protecting the mitochondrial machinery from
t-BuOOH (Fig. 10). Mitochondrial PHGPx not only prevents
direct injury of mitochondria by KCN, but it also reduces the
cytotoxicity of exogenously added t-BuOOH by protecting
mitochondrial functions. Levels of ROS in mitochondria increase
substantially in pathological situations, such as ischemia reperfusion
(38). Several factors have been reported that protect mitochondria
against oxidative damage. Mitochondrial SOD (Mn-SOD) is induced by
TNF-
, which initiates cell death through the elevation of levels of
ROS in mitochondria (39). Induced Mn-SOD might protect cells from the toxic effects of TNF-
(40). Heat shock proteins are also induced to
protect cells from stresses that include ROS. Overexpression of heat
shock protein 70 results in resistance to the cytotoxicity of TNF-
.
Polla et al. (41) demonstrated that heat shock protein 70 prevented changes in mitochondrial membrane potential by
H2O2, and they suggested that mitochondria
might be selective targets for protective effects against the oxidative
injury. Bcl-2, which is an anti-apoptosis protein, is located primarily
on the outer membrane of mitochondria. Bcl-2 prevents cells from
undergoing necrosis in response to inhibitors of the respiratory chain
(chemical hypoxia) such as rotenone, antimycin A, and KCN (36) and from apoptosis in response to a variety of other stimuli (42, 43). Bcl-2
prevents cell death by inhibiting the loss of mitochondrial membrane
potential (36) and by inhibiting the release of cytochrome c, an activator of caspases (44, 45), from the mitochondria to the cytosol. Exogenous H2O2 is a potent
inducer of the liberation of cytochrome c from damaged
mitochondria, and Bcl-2 effectively prevents the toxic effects of
exogenous H2O2 that lead to cell death (43).
These earlier results suggest that the generation of ROS in the
mitochondria might be a key step in the initiation of cell death and
that mitochondria are well placed to be sensors of oxidation damage to
cells (46). PHGPx is localized predominantly at contact sites between
the outer and inner membranes of mitochondria in the rat testes (47).
The reduction of levels of ROS in mitochondria by PHGPx might also be
associated with the defenses of the cell against apoptosis that is
mediated by damages to mitochondria.
Recent reports indicate that ROS play an important role as mediators or
modulators in cellular signaling pathways, such as the Ras signaling
pathway (48), the mitogen-activated protein kinase pathway (49), and
the activation of nuclear factor
B (NF
B) (50). ROS generated in
mitochondria might be responsible for the activation of NF
B (51) and
of Sterile 20 (Ste 20), which is likely to be an
oxidant-stress-responsive kinase-1 (SOK-1) (52). Brigelius-Flohe
et al. (53) found that the activation of NF
B by
interleukin-1 was inhibited in ECV304 cells that overexpressed PHGPx.
The present study also suggests that mitochondrial PHGPx might
participate in the regulation of signal transduction pathways that are
triggered by ROS in mitochondria.
 |
ACKNOWLEDGEMENTS |
We thank Akiko Toudo, Aya Teshirogi, and
Atsuko Saito for their expert technical assistance. We also thank
Dr. Kazuhiko Umesono and Dr. Junichiro Inoue for the kind gift of the
GFP expression vector, and we thank members of the Kitasato Institute
for help in the flow cytometric analysis.
 |
FOOTNOTES |
*
This work was supported in part by Special Coordination
Funds for Promoting Science and Technology, and by Grants-in-aid
10672052 and 10780389 from the Ministry of Education, Science and
Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: School of
Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane,
Minato-ku, Tokyo 108, Japan. Fax: 81-3-3444-4943; E-mail:
nakagaway{at}pharm.kitasato-u.ac.jp.
 |
ABBREVIATIONS |
The abbreviations used are:
ROS, reactive oxygen
species;
BSO, buthionine sulfoximine;
CCCP, carbonyl cyanide
m-chlorophenylhydrazone;
cGPx, cytosolic glutathione
peroxidase;
DCFH-DA, 5,6-carboxy-2',7'-dichlorofluorescein diacetate;
GFP, green fluorescent protein;
LDH, lactate dehydrogenase;
PBS, phosphate-buffered saline;
PHGPx, phospholipid hydroperoxide
glutathione peroxidase;
PI, propidium iodide;
RBL, rat basophile
leukemia cells;
Rh123, rhodamine 123;
t-BuOOH, tert-butylhydroperoxide;
TNF-
, tumor necrosis factor-
;
SOD, superoxide dismutase;
BSA, bovine serum albumin.
 |
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