From the Department of Health Chemistry and
** Laboratory of Biomedical Genetics, Graduate School of
Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
and ¶ Banyu Tsukuba Research Institute (Merck), Tsukuba,
Ibaraki 300-0026, Japan
Received for publication, November 20, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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Intracellular type I platelet activating
factor-acetylhydrolase is a phospholipase that consists of a dimer of
two homologous catalytic subunits Platelet-activating factor
(PAF)1 is a potent signaling
phospholipid involved in diverse physiological events, such as
inflammation and anaphylaxis (1). In addition, PAF has been implicated
in the central nervous system (2, 3) and the reproductive system (4,
5). PAF is hydrolyzed to an inactive metabolite by a specific enzyme
called PAF-acetylhydrolase (PAF-AH). At least three types of PAF-AH
exist in mammals, namely the intracellular types I and II (6, 7) and a
plasma type (8). Intracellular type I PAF-AH (PAF-AH (I)) is an
oligomeric complex. It contains a dimer of two homologous catalytic
subunits, The Generation of Antibodies--
Mouse monoclonal antibodies against Histological and Immunohistochemical Analyses--
Testes were
dissected and fixed overnight in Bouin's fixative at 4 °C. Paraffin
sections (5 µm) were prepared and stained with Periodic Acid-Schiff
(PAS) and hematoxylin. For immunohistochemistry, mice under anesthesia
were perfused with phosphate-buffered saline (PBS) and then with 4%
paraformaldehyde in PBS. Testes were dissected and refixed overnight in
4% paraformaldehyde at 4 °C. Paraffin sections (5 µm) were boiled
in a microwave oven in 10 mM sodium citrate buffer (pH 6.0)
for antigen retrieval. Subsequent immunodetection was performed using a
Vector M.O.M. immunodetection kit (Vector Laboratories) for Western Blot Analysis--
Tissues were homogenized in quadruple
volumes (w/v) of SET buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 250 mM sucrose) with protease
inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mg/ml pepstatin, 10 mg/ml leupeptin, 10 mg/ml aprotinin) and phosphatase inhibitors (50 mM NaF, 10 mM
Na3PO4). After centrifugation at 1,000 × g at 4 °C, the supernatants were used as the total
protein lysates. The protein concentrations of samples were determined by the BCA assay (PIERCE). Each total protein lysate (50 µg/lane) was
separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes or nitrocellulose membranes. The membranes were blocked with
5% (w/v) skim milk (Wako) in TTBS buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% (w/v) Tween 20) and incubated
with antibodies in TTBS. Chemiluminescence (ECL kit, Amersham
Biosciences) was used for analyzing levels of protein according
to the manufacturer's instructions.
Northern Blot Analysis--
Total RNA was extracted from mouse
tissues using Isogen (Nippongene). Total RNA (10 µg/lane) was
separated by 1% agarose-formaldehyde gel electrophoresis and
transferred to Hybond-N membranes (Amersham Biosciences) in 20× SSC.
The membranes were hybridized in Rapid-hyb buffer (Amersham
Biosciences) at 65 °C and washed with 0.5× SSC, 0.1% SDS at
65 °C. Probes for We established monoclonal antibodies against 1 and
2 as well as LIS1,
a product of the causative gene for type I lissencephaly. LIS1 plays an
important role in neuronal migration during brain development, but the
in vivo function of the catalytic subunits remains unclear.
In this study, we generated
1- and
a2-deficient mice by targeted disruption.
1
/
mice are indistinguishable from
wild-type mice, whereas
2
/
male mice
show a significant reduction in testis size. Double-mutant male mice
are sterile because of severe impairment of spermatogenesis. Histological examination revealed marked degeneration at the
spermatocyte stage and an increase of apoptotic cells in the
seminiferous tubules. The catalytic subunits are expressed at high
levels in testis as well as brain in mice. In wild-type mice,
2 is
expressed in all seminiferous tubule cell types, whereas
1 is
expressed only in the spermatogonia. This expression pattern parallels
the finding that deletion of both subunits induces a marked loss of
germ cells at an early spermatogenic stage. We also found that
the LIS1 protein levels, but not the mRNA levels, were
significantly reduced in
2
/
and
double-mutant mice, suggesting that the catalytic subunits, especially
2, are a determinant of LIS1 expression level.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
2, and a non-catalytic
subunit (6, 9-11).
Interestingly, the
subunit was later found to be identical to LIS1,
the product of the causative gene for type I lissencephaly (10, 12).
Type I lissencephaly is a genetic brain malformation showing a smooth
cerebral surface without gyri, caused by abnormal neuronal migration at
early developmental stages. Mice homozygous for the Lis1
null mutation die early in embryogenesis soon after implantation (13).
Heterozygous and compound heterozygous mice have expression
level-dependent defects in neuronal migration (13). A
series of recent studies has suggested that LIS1 interacts not only
with PAF-AH (I) catalytic subunits but also with a number of proteins,
including tubulin (14), cytoplasmic dynein (15, 16), and NUDE
(17-20). Through interaction with these proteins, LIS1 plays important
roles in microtubule-associated cellular functions such as mitotic cell
division, chromosomal segregation, and neuronal migration. In contrast,
the biological role of the catalytic subunits of PAF-AH (I) remains a
complete enigma. Nothwang et al. (21) have described a case
of functional hemizygosity of
1, possibly responsible for the
resulting mental retardation, ataxia, and brain atrophy in this
patient. Furthermore, Lecointe et al. (22) have proposed
that deregulation of transcription of the human
2 gene is
associated with the development of a certain lymphoma. Therefore, the
PAF-AH (I) catalytic subunit is also likely to play an important role
in some pathological conditions.
1 and
2 catalytic subunits belong to a novel serine esterase
family (9). These subunits, which show ~60% amino acid homology with
each other, form homodimers and a heterodimer. Ho et al.
(23) have reported the x-ray crystal structure of the
1 homodimer.
The folding is unique among known lipases and phospholipases. The
structure unexpectedly resembles those of the G-protein family such as
p21ras and G
. To elucidate the in vivo
function of the catalytic subunits of PAF-AH (I), namely
1 and
2,
we generated mice lacking either one or both of these two proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,
2 Mutant Mice--
1,
2 genomic clones were isolated from a mouse 129/SvJ
genomic library in the Lambda FIXII vector (Stratagene). Targeting vectors were constructed for replacing part of exon 2 and 3 of the
1 and
2 genes, which include a translation
initiation site and catalytic motif (GXSXV) with
a PGKneobpA cassette (24). A PGKDTA (diphtheria toxin A fragment) cassette was inserted at the 3'-end of the short arm for
negative selection. The targeting vectors were linearized and
electroporated into ES cell line RW4 (Genome Systems), which was
cultured on neomycin-resistant mouse embryonic fibroblasts.
G418-resistant colonies were screened for homologous recombinants by
PCR. Candidates of homologous recombinants were verified by Southern
analysis using fragments at the 3'-ends of the genes, external to the
targeting vectors as probes. Chimeric mice were generated by injection
of the ES cells into C57BL/6N blastocysts, followed by transfers to
foster mothers, and backcrossed to C57BL/6N mice. Genotypes were
determined by PCR and/or Southern analysis of the tail DNA samples.
1 and
2 were established as follows.
1
/
and
2
/
female mice were immunized with each
purified recombinant rat protein with Freund's complete adjuvant
(DIFCO), followed by six boosters at 2-week intervals with 20 µg of
protein and established monoclonal antibody, producing hybridoma cell
lines as previously described (11). Monoclonal antibody against LIS1
(clone 338, a kind gift from Dr. O. Reiner, Weizmann Institute,
Rehovot, Israel) and
-tubulin (clone DM1A, Sigma) were used for a
Western blot analysis. Polyclonal antibody against LIS1 (N-19, Santa
Cruz Biotechnology) was used for an immunohistochemical analysis.
1 and
2 and Vectastain ABC kit (Vector Laboratories) for LIS1.
Immunostaining was visualized using diaminobenzidine and
counterstained with hematoxylin. For detection of apoptotic germ cells,
Bouin's-fixed, paraffin-embedded testis sections were subjected to
TUNEL staining using an in situ cell death detection kit,
POD (Roche Molecular Biochemicals) according to the manufacturer's instructions.
1,
2, and LIS1 were obtained by RT-PCR from
mouse RNA and labeled by 32P[dCTP] (Amersham Biosciences)
using the Rediprime II DNA labeling system (Amersham Biosciences).
Membranes were stripped and rehybridized with human
glyceraldehyde-3-phosphate dehydrogenase cDNA probe (Clontech) to ensure equal loading.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
2 by
immunizing the respective knockout mice. A Western blot analysis of
adult mouse tissue shows that
1,
2, and LIS1 were most abundantly expressed in brain and testis (Fig. 1).
Expression of
2 and LIS1 were observed in other tissues as well,
whereas
1 expression was restricted to embryonic brain and adult
testis (Fig. 1). The expression levels of
2 and LIS1 were observed
to be essentially proportional to that of
-tubulin, a component of
microtubules. Because LIS1 plays an important role in microtubule
dynamics (14, 16), it can be postulated that both
subunits are also
involved in this process.
View larger version (70K):
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Fig. 1.
Expression pattern of PAF-AH (I) subunits in
mice. Expression of 1,
2, LIS1, and
-tubulin in various
mouse tissues was examined by Western blotting using specific
antibodies.
Immunohistochemical staining of adult mouse testes revealed that 2
and LIS1 immunoreactivity was present in all seminiferous tubule cell
types (Fig. 2, D and
G). Intense staining of
2 and LIS1 was observed in
meiotically dividing spermatocytes and elongating spermatids. In
contrast,
1 staining was restricted to the cells lining the basal
compartment of seminiferous tubules (Fig. 2A). Magnification
revealed that
1 was specifically localized in spermatogonia cytoplasm (Fig. 2B, arrow), whereas
2 and LIS1
were expressed in the cytoplasm of all types of spermatogenic cells and
Sertoli cells (Fig. 2, E and H), suggesting that
1 is involved specifically in proliferation and/or differentiation
of spermatogonia. LIS1 was also localized at meiotic spindles of
spermatocytes (Fig. 2H, arrowhead) and manchettes
of elongating spermatids (Fig. 2H, arrow), both
of which are specific microtubule structures. No staining of
1 or
2 was detected in the seminiferous tubules of null mutant mice (Fig.
2, C and F).
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We used homologous recombination in embryonic stem cells to generate
mice lacking the 1 (Pafah1b3) and
a2 (Pafah1b2) genes. Parts of exon 2 and exon 3 of each gene, including the translation initiation site and the
catalytic serine residue, were replaced with a neomycin-resistance gene
(Fig. 3A). Targeted embryonic stem cell clones and subsequent germ line transmissions were detected by PCR and/or Southern blot analysis (Fig. 3B). Both
1
/
and
2
/
mice were born with the expected Mendelian frequencies, viable and
apparently indistinguishable from their wild-type littermates. Western
blot analysis of the brain and the testis homogenates showed no
immunoreactive bands in either
1
/
or
2
/
mice (Fig. 6A).
1
/
/
2
/
mice
were also viable and apparently indistinguishable from wild-type mice.
However,
1
/
/
2
/
males
were found to be infertile, whereas female fertility was not affected.
Testes weights of 5-week-old
1
/
/
2
/
mice
were significantly (~35%) smaller than those of wild-type mice
(Table I). Testes weights were not
noticeably reduced in
1
/
mice, whereas
they were reduced to 60% in
2
/
mice
(Table I). There was no significant difference in body weight among any
of the genotypic combinations (Table I).
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The histology of mutant testes was examined at age 5 weeks, the time
when the first wave of murine spermatogenesis is completed. The
seminiferous tubules of
1
/
/
2
/
mice
showed a 50% reduction in diameter, and spermatogenic cells were
dramatically decreased (Fig.
4F) when compared with
wild-type mice (Fig. 4A). Spermatocytes beyond the pachytene
stage and round spermatids were significantly reduced in number.
Elongated spermatids were rare, and the few remaining spermatids had
deformed nuclei. Some germ cells appeared to be detached from the
Sertoli cells. No spermatozoa were observed in the epididymis (data not
shown). In older mice, early germ cell stages were more severely
affected, leading to increased depletion of spermatocytes and
spermatogonia (data not shown). In TUNEL assays, apoptotic cells were
rare in wild-type testes as previously reported (25) (Fig.
5A), whereas
1
/
/
2
/
testes had a significantly larger number of apoptotic cells (Fig. 5B). The cells undergoing apoptosis in
1
/
/
2
/
testes were predominantly spermatocytes. These results indicate that in
the absence of the catalytic subunits of PAF-AH (I), the differentiation of prehaploid stages of spermatogenesis fails, leading
to induction of programmed cell death in the germ cell compartment.
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Histologically, 1
/
mice testes showed no
apparent impairment of spermatogenesis (Fig. 4B). On the
other hand,
2
/
mice testes showed
significant weight reduction and varying germ cell impairment (Fig.
4C and Table I). When comparing
1
/
/
2+/
mice
testes with
1+/
/
2
/
mice
testes from both the histological and weight points of view, impairment
was more severe when both
2 alleles were missing (Fig. 4,
D and E, and Table I). These results
indicate that
2 plays a more important role in spermatogenesis than
1 and that missing
1 alleles can in part be compensated by the
presence of
2. However, it is evident that
1 also plays a role in
male fertility based on the observation that the absence of both
2
alleles can be partly compensated by the presence of
1 alleles (Fig.
4, C, E, and F, and Table I). The
1 protein level was reduced to about 20% of the normal level in
2
/
mice (Fig. 3C) even though
there was no reduction of the mRNA level (data not shown), whereas
the
2 level was not changed in
1
/
mice.
Because a large portion of LIS1 forms complexes with 1 and/or
2
in the cytosolic fraction, we examined the LIS1 protein levels in
1
and/or
2 mutant adult mice. In
2
/
mice, LIS1 levels in both brain and testis were reduced to ~30% compared with wild-type mice (Fig.
6A). In contrast, no reduction of LIS1 was observed in
1
/
mice (Fig.
6A). In
1
/
/
2
/
mice,
as in
2
/
mice, LIS1 levels were reduced
to about 20% of the levels in the wild type mice. Lis1
mRNA expression in
2
/
mice was either
the same as or slightly higher than the expression in wild type mice
(Fig. 6B). On the other hand, LIS1 expression in E14.5
(embryonic day 14.5) brain of each mutant mouse was not significantly
less than that in wild-type mice (Fig. 6C).
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DISCUSSION |
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In this study, we demonstrated that the catalytic subunits of
intracellular PAF-AH (I) are involved in murine spermatogenesis. Our
study gives new insights into the in vivo function of PAF-AH (I). We showed by Western blotting that in mice the catalytic subunits
1 and
2 as well as LIS1 are present at high levels in both brain
and testis. Interestingly, it was noted that PAF-AH (I) subunits
exhibit expression levels proportional to those of
-tubulin, a major
component of intracellular microtubules. The above findings and the
fact that LIS1 is a microtubule-associated protein lead us to speculate
that the catalytic subunits are also involved to a major extent in
microtubule dynamics. Microtubule structures undergo dramatic
rearrangements in the process of spermatogenesis. Processes involving
microtubule rearranging include mitotic division of spermatogonia,
meiotic division of spermatocytes, manchette formation, and flagellar
axoneme assembly in spermatids. The most severe degeneration occurs in
primary spermatocytes of
1
/
/
2
/
mice,
but degeneration also occurs in meiotically dividing spermatocytes (increased apoptosis) and in elongating spermatids (abnormal nuclear morphogenesis). Therefore, it can be stated that PAF-AH (I) catalytic subunits are involved in the several processes of spermatogenesis and
not just in a specific stage of spermatogenesis.
Although the exact molecular mechanism and function of the PAF-AH (I)
catalytic subunits in spermatogenesis are unclear, we found that
depletion of both catalytic subunits leads to a major decrease in LIS1
protein levels, suggesting that the catalytic subunits are associated
with LIS1. It is most likely that LIS1 protein levels are
post-transcriptionally influenced by the catalytic subunits, because
LIS1 mRNA levels are not altered in
2
/
mice. Because LIS1 levels are crucial
for cortical brain development (13), we speculate that LIS1 is involved
in microtubule organization of spermatogenesis and that reduced LIS1
protein levels are responsible for the testicular defects occurring in
1
/
/
2
/
mice.
However, our studies also revealed that the more severe testicular
degeneration in
1
/
/
2
/
mice
than in
2
/
mice cannot be explained
solely by the amount of reduction in the level of LIS1, because the
reduction of LIS1 was not very different between
2
/
and
1
/
/
2
/
mice.
Given that PAF-AH (I) closely resembles trimeric G-proteins (23), the
catalytic subunits may mediate a novel intracellular signaling to LIS1
in mammals, and depletion of this signaling pathway may result in
severe impairment in spermatogenesis.
Because PAF-AH (I) catalytic subunits are predominantly expressed in
brain as well as in testis and because haplo-insufficiency of LIS1
leads to severe brain malformation in both humans and mice (13), we
expected that mice lacking the catalytic subunits would exhibit brain
abnormalities. However, Nissl staining of adult
1
/
/
2
/
mice
brain showed no obvious abnormalities in lamination of neurons in the
cerebral cortex, hippocampus, or cerebellum (data not shown). To our
surprise, in E14.5 brain of
1
/
/
2
/
mice,
there was no significant reduction of LIS1 protein levels, suggesting
that there is a mechanism to maintain LIS1 protein levels and the
function of catalytic subunits in brain.
In 2
/
mice, both
1 and LIS1 protein
levels were significantly reduced compared with wild-type mice, whereas
in
1
/
mice both
2 and LIS1 levels were
not changed markedly. In preliminary experiments, supernatants of mice
testis or brain homogenates were subjected to DEAE-Sepharose ion
exchange column chromatography. In the case of the
1
/
homogenates,
2 eluted in the same
fraction as LIS1, whereas in the case of the
2
/
homogenates
1 and LIS1 eluted at
the different positions.2
These results suggest that
2, probably the
2 homodimer, has a
strong affinity for LIS1 and that the
1 homodimer has a weak or
negligible affinity for LIS1 in vivo. Considering the fact that
1 mRNA levels are not changed in
2
/
mice (data not shown), the present
results also suggest that the
1 protein is not stably expressed in
the absence of
2 in vivo. These observations are
consistent with our previous report (26) that
1/
2 heterodimers
and
2 homodimers are the major PAF-AH (I) catalytic units present
in vivo.
Immunostaining studies revealed that 2 is expressed in all
spermatogenic cells, whereas
1 is expressed only in spermatogonia. This expression pattern parallels the finding that deletion of both
subunits induces a marked loss of germ cells, even at an early
spermatogenic stage. We have previously shown that
1 is specifically
expressed in migrating neurons in the embryonic and postnatal stages,
whereas the
2 expression level is almost constant from the fetal
stages through adulthood (26, 27). As a result, the catalytic subunits
change from the
1/
2 heterodimer to the
2/
2 homodimer in
neurons during brain development. It is likely that the same type of
alteration in the catalytic dimer occurs during differentiation from
spermatogonia to spermatocytes. Interestingly, it has been shown that
undifferentiated spermatogonia move to specific sites within the
seminiferous tubule and spread their progeny laterally along the base
of the tubule (28). Transplantation experiments demonstrated that
spermatogonia are capable of moving along the length of the
seminiferous tubule at a rate of more than 50 µm/day (29, 30).
Although the biological significance of the change in the catalytic
subunit combination is not known, it is interesting to speculate
that
1 mediates a common signaling pathway in migrating neurons and spermatogonia.
The cellular function of the enzyme activity and the physiological substrate of this enzyme are largely unknown. PAF has been detected in sperm from several mammalian species and has been shown to affect sperm motility and fertility (4). High-fertility spermatozoa, for example, have a substantially greater PAF content than low-fertility spermatozoa (31, 32). Exogenously added PAF increases the motility of human spermatozoa (33). Our study gives hints at the possibility that PAF is not only involved in spermatozoal maturation and penetration but is also involved in spermatogenesis itself. On the other hand, PAF-AH (I) shows striking substrate specificity for an acetyl group but hydrolyzes other types of acetyl-containing esters in vitro (34). Studies on the tertiary structure of the catalytic dimer suggest that the substrate of this enzyme is not necessarily a lipophilic substance (23). Because PAF-AH (I) shows similarities to trimeric G-proteins (23), the PAF-AH (I)-mediated novel intracellular signaling is likely operating in mammals, with PAF or a related substance as a GTP-like switch.
When measuring cytosolic PAF-AH activity of
1
/
/
2
/
mice
in brain and testis, enzymatic reduction to ~65% of wild-type mice was seen in both tissues (data not shown). This phenomenon is likely
because type I PAF-AH is the only affected subtype, whereas enzymatic
activities of type II PAF-AH and further not yet identified PAF-AH
subtypes are probably responsible for the remaining activity.
In conclusion, we found that the depletion of the PAF-AH (I) catalytic
subunits induces reduction of LIS1 protein on the cellular level and
severe testicular malformation on the phenotypic level. The next
question to be considered is whether the catalytic activity of PAF-AH
(I) is required for LIS1 protein stability and spermatogenesis. To
answer these questions, we are planning to insert the catalytically inactive 1 and
2 subunit genes into our double-knockout mouse model.
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ACKNOWLEDGEMENTS |
---|
We thank O. Reiner for the LIS1 antibody and S. Ishikawa and staff for animal care.
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FOOTNOTES |
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* This work was supported by the Joint Research Fund between the University of Tokyo and Banyu Pharmaceutical Co.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.
§ Present address: Division of Molecular Neurobiology, Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.
Present address: Dept. of Pharmacology, Graduate School of
Medicine, Kyoto University, Kyoto 606-8501, Japan.
Present address: Faculty of Pharmaceutical Sciences, Teikyo
University, Kanagawa 199-0195, Japan.
§§ To whom correspondence should be addressed: Dept. of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4720; Fax: 81-3-3818-3173; E-mail: harai@mol.f.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M211836200
2 H. Koizumi, N. Yamaguchi, J. Aoki, K. Inoue, and H. Arai, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PAF, platelet-activating factor; PAF-AH, platelet-activating factor acetylhydrolase; TUNEL, TdT-mediated dUTP nick-end labeling.
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REFERENCES |
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