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* Institute of Pharmaceutical Chemistry and Clinics of Anaesthesiology and Intensive Care Medicine, Jena, Germany,
Department of Anatomy and Cell Biology III, and the
Institute of Pathochemistry and Neurochemistry, University of Heidelberg, 69120 Heidelberg, Germany
1Correspondence: Clinics of Anaesthesiology and Intensive Care Medicine, University of Jena, 07740 Jena, Germany. E-mail: hans-peter.deigner{at}med.uni-jena.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
fibroblasts initiated by oxidized low density lipoproteins (minimally
modified LDL, mmLDL) is associated with an increase in acid SMase
(aSMase, E.C. 3.1.4.12) expression and ceramide concentration.
Application of a novel, potent, and specific inhibitor of aSMase
expression (NB6) diminished the effects of mmLDL and
C6-ceramide treatment by inhibiting transcription via Sp1
and AP-2. Moreover, apoptosis was abolished after mmLDL and
C6-ceramide treatment of hereditary aSMase-deficient
fibroblasts (from Niemann-Pick patients). We suggest that in
mmLDL-initiated apoptosis 1) enhanced ceramide
generation via aSMase appears to be required as well as
2) a positive feedback control of aSMase expression by
the increase in intracellular ceramide concentration.—Deigner, H.-P.,
Claus, R., Bonaterra, G. A., Gehrke, C., Bibak, N., Blaess, M.,
Cantz, M., Metz, J., Kinscherf, R. Ceramide induces aSMase expression:
implications for oxLDL-induced apoptosis.
Key Words: acid sphingomyelinase • macrophages • Niemann-Pick • programmed cell death • C6-ceramide
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and to function as a common lipid mediator in
programmed cell death generated in cells by signaling through
CD95-Fas/Apo1 (2)
, tumor necrosis factor 
(3)
, ionizing radiation (4)
, and
chemotherapeutic agents (5)
.
Oxidized lipoproteins, modified by either transition metal- or by
cell-mediated oxidation, undergo increased uptake by M
(6)
, leading to cell damage. Several cytotoxic effects
have been characterized such as the accumulation of cholesterol esters
and modified cholesterol, leading to the formation of foam cells,
lysosomal destabilization (7)
, and, finally, apoptosis and
postapoptotic necrosis (8
9
10)
. Stimulation of both acid
sphingomyelinase (aSMase) and neutral SMase (nSMase) was observed using
sphingomyelin substrate from endogenous and exogenous sources (9
, 11)
. Several recent reports implicate ceramide in modified low
density lipoprotein (mLDL) -induced apoptosis as a product of
sphingomyelin hydrolysis by aSMase/nSMase (9
10
11
12
13
14)
. The
exposure of human M to mLDL, containing a considerable amount of
sphingomyelin, has been shown to cause an increase in the concentration
of cell-associated ceramide (10)
. Langmann and colleagues
have suggested that the activity of aSMase is mainly determined by its
expression (15)
.
This study was carried out to analyze the role of aSMase and ceramide
in minimally modified LDL (mmLDL) -initiated apoptosis in human M
and fibroblasts. In addition, NB6, a novel inhibitor of aSMase
expression developed by our group, and hereditary aSMase-deficient
Niemann-Pick fibroblasts were used.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and fibroblasts
In vitro conditions
M or fibroblasts were cultured in RPMI 1640 medium
supplemented with fetal calf serum (FCS), glutamine, and
penicillin/streptomycin. Nonadherent cells were removed by washing with
supplemented medium. Cells were cultured with lipoprotein-deficient
serum (prepared by ultracentrifugation of FCS, 
>1.21)
(16)
for 24 h prior to experimentation.
LDL was isolated according to Havel et al. (16)
or Himber
et al. (17)
; concentrations refer to protein
concentrations (apoB, 500 kDa) determined by the modified Lowry method
(18)
. LDL was pooled from three donors for each
preparation, dialyzed against Tris (pH 7.4, 20 mM, NaCl 150 mM, 0.1 mM
EDTA), sterilized by filtration through a 0.2 µm membrane filter, and
stored at 4°C under argon (up to 2 wk). mmLDL was obtained by
Fe-catalyzed oxidation as described by Watson et al. (19)
.
After incubation with 20 µM Fe2+ at 20°C for
48 h, the peroxide content as determined according to El-Saadani
et al. (20)
typically increased from 30 nmol/mg to
800-1000 nmol/mg; electrophoretic mobility was only marginally enhanced
by this procedure.
M or fibroblasts were exposed to mmLDL (27 µg/ml),
C6-ceramide (10 µM), or
H2O2 (30 µM) and
apoptotic cells were identified by YOPRO-1 staining (21)
and/or by the TUNEL technique (Quantum Appligene, Heidelberg, Germany).
The percentage of apoptotic cells was counted using a microscope fitted
with mercury light source and fluorescence assembly in combination with
a computer-assisted morphometry system developed by our group
(22)
.
ß-Hexosaminidase and ß-glucosidase activities were determined as
described previously (23)
.
Preparation of the inhibitor of aSMase expression, NB6
((3-carbazol-9-yl-propyl)-[2-(3,4-dimethoxy-phenyl)-ethyl)-methyl-amine),
will be reported elsewhere. Cytotoxicity was determined by measurement
of lactate dehydrogenase (LDH) released from the cytosol of damaged
human M or fibroblasts into the supernatant after treatment with
NB6. Cells were incubated in 96-well plates in phenol red-free RPMI
(for 18 h, 37°C; 5% CO2 at concentrations
up to 10 µM) according to the manufacturer’s instructions
(Cytotoxicity Detection Kit-LDH, Roche Molecular Biochemicals,
Mannheim, Germany) using an ELISA plate reader (Bio-Rad 450) at 490 nm.
Reverse transcription-polymerase chain reaction (RT-PCR) and
Western blotting
aSMase expression was investigated by Western blotting
(24
, 25)
using polyclonal antibodies (kindly provided by
K. Sandhoff and K. Ferlinz, Bonn, Germany) and by RT-PCR. For
amplification of aSMase, primers were used under the following
conditions: 5'-CAG GGT TCC TGG CTG GGC AGC A-3' (forward) and 5'-GGT
CCT GGA CC ATG AGA CCT AC-3', 94°C, 60°C, 72°C, 1 min each, 40
cycles.
Analysis of the sequence of the purified PCR product was performed by Dr. H. Delius (Deutsches Krebsforschungszentrum, Heidelberg, Germany).
Determination of SMase activity and of ceramide concentration in cell
lysates was performed as described previously (11)
.
Quantification of ceramide concentration was assayed by the DAG method
under appropriate conditions (26
, 27)
.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared by the mini-extraction procedure
as described earlier with minor modifications (28)
: after
washing with phosphate-buffered saline, (stimulated) M were
centrifuged for 15 s at 900 g at 4°C in a Hermle Z160
centrifuge (Wehingen, Germany). Then pellets were resuspended in 50
µl of buffer A (HEPES/KOH [20 mM], 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, pH
7.6), incubated on ice for 10 min, and centrifuged for 10 min at 3000
g. The supernatants were discarded and the pelleted nuclei
were resuspended in 50 µl of buffer C (HEPES/KOH [20 mM], 0.42 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 15% glycerol,
0.5 mM DTT, 0.5 mM PMSF, 0.2% Nonidet P-40) and incubated on ice for
15 min. After centrifugation (5 min, 14,000 g, 4°C) the
supernatants containing nuclear proteins were harvested, protein
concentrations determined by the Bradford method, and samples stored at
-80°C for later use in the binding assays. EMSA was performed as
described by Suzuki et al. (28)
: reaction mixtures (final
volume 16 µl) containing 8 µg of nuclear extracts, 0.5 µg
poly(dI-dC) (Gibco-BRL, Karlsruhe, Germany), and 60,000 cpm
[32P]-labeled probe in binding buffer were
incubated at 30°C for 30 min. Samples were then analyzed by native
5% polyacrylamide gels and imaged by autoradiography (48 h).
Double-stranded oligonucleotide containing consensus binding sides for
SP-1, AP-2, and oct-1 (MWG-Biotech, Ebersberg, Germany) were labeled
with -[32P]-dCTP (Amersham Pharmacia,
Freiburg, Germany) using Klenow fragment (GibcoBRL) and purified by
centrifugation (735 g, 2 min) in MicroSpinTM G-25 columns
(Amersham/Pharmacia). The following oligo sequences were used: Sp-1:
5'-TGG AAC CGG GCG GGC GGG CTA CCG GGC GGG CT-3'; 5'-TGG AAG CCC GCC
CGG TAG CCC GCC CGC CCG GT-3'. AP-2: 5'-TGG ATC GAA CTG ACC GCC CGC
GGC-3'; 5'-AAG GGC CGC GGG CGG TCA GTT GGA-3'. oct-1: 5'-TGG ATG TCG
AAT GCA AAT CAC TAG AA-3'; 5'-TGG ATT CTA GTG ATT TGC ATT CGT CA-3'.
Statistical analyses
Results are presented as means + SE. Statistical
procedures were performed by the Mann Whitney U-Wilcoxon
Rank Sum W Test or the impaired Student’s t test
using the SIMSTAT program (Provalis Research, Montreal Canada). A
P value of 0.05 or less was chosen for statistical
significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, we investigated the expression of this
enzyme. Using PCR analysis, we show that mmLDL treatment raised the
level of aSMase-mRNA in M (Fig. 1
). Sequence analysis the 687 bp product confirmed the amplification of
the respective aSMase fragment. In parallel with stimulation of aSMase
expression, a significant induction of apoptosis was found [0 µg/ml
mmLDL, 7±2.1% (apoptotic cells); 27 µg/ml, 20.7±2.7%; 54 µg/ml,
29.3±3.8%; 100 µg/ml, 37±4%; data are means ±
SE of three experiments].
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There is convincing evidence that mLDL-mediated apoptosis involves
generation of ceramide (10
, 11)
. It has not been studied
whether ceramide itself stimulates the production of the
ceramide-delivering enzyme, aSMase. Here we show for the first time
that not only mmLDL (see Fig. 1
), but also ceramide alone, induced
aSMase expression (see Fig. 3
). The latter also occurred after
H2O2 (30 µM; not shown)
treatment. To provide further evidence for the role of aSMase in mmLDL
or ceramide initiated cell death, we performed inhibition experiments.
We selected one of several compounds capable of inhibiting aSMase
expression and activity in pretreated M: NB6 (Fig. 2
). This agent did not show cytotoxic effects in M or fibroblasts at
concentrations of up to 10 µM (18 h treatment) as determined by LDH
release; the synthesis and analytical data of NB6 will be reported in
more detail elsewhere. We found that NB6 pretreatment of M abolished
ceramide-stimulated aSMase expression, induction of apoptosis
(Fig. 3
), and inhibited the enhancement of enzymatic activity
[IC50=4 µM (mean of 4 experiments)] in
lysates of pretreated cells (30 min) stimulated with 27 µg/ml mmLDL
(4 h).
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To confirm a critical role of lysosomal enzymes in mmLDL-induced
apoptosis, we investigated two other lysosomal enzymes as well as the
effect of modulating the intralysosomal pH. We found that activities of
ß-hexosaminidase and ß-glucosidase were not significantly different
after mmLDL treatment. Untreated cells revealed ß-hexosaminidase and
ß-glucosidase activities of 125.2 ± 7.4 and 0.205 ±
0.025; in mmLDL-treated cells we observed 145.2 ± 10.1 and
0.185 ± 0.005 activities of both enzymes, respectively.
Furthermore, we raised the intralysosomal pH in fibroblasts by
pretreatment (18 h) with the lysosomotropic agent ammonium chloride (9
mM) inhibiting lysosomal function (29)
. This treatment
impaired the apoptogenic potential of mmLDL, i.e., no significant
increase in the number of apoptotic cells could be observed (three
experiments) upon addition of 54 µg/ml mmLDL.
To further analyze the effect of aSMase on apoptosis initiated by
mmLDL, we exposed aSMase-deficient human fibroblasts to mmLDL and
C6-ceramide. Since significant differences were
observed in mmLDL-treated normal human fibroblasts after 16 h
(Fig. 4A
, inset), this time was chosen. In contrast to the normal
fibroblasts, where mmLDL and C6-ceramide
significantly induced apoptosis (Fig. 4A
), programmed cell
death was abolished in Niemann-Pick fibroblasts (Fig. 4B
).
H2O2 treatment, however,
induced apoptosis in Niemann-Pick fibroblasts similar to the control.
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Comparable to M, mmLDL increased the level of aSMase-mRNA in
fibroblasts in a concentration-dependent manner (Fig. 5
), whereas NB6 inhibited the ceramide- and mmLDL-mediated induction of
aSMase expression (Figs. 6A
, B
). Pretreatment of fibroblasts with NB6 (10 µM)
prevented the rise of aSMase activity [pmol/(h x mg)]: control:
910 ± 58, mmLDL: 1597 ± 88, ceramide: 1466 ± 111, and
H2O2: 2154 ± 164 vs.
control + NB6: 780 ± 69; mmLDL + NB6: 712 ± 118, ceramide +
NB6: 843 ± 123, and
H2O2 + NB6: 2213 ±
241. NB6 pretreatment inhibited not only an increase in aSMase activity
after stimulation, but also reduced its basal activity; no direct
effect on acid/neutral SMase activity was found in cell lysates. To
study potential changes in the transcriptional level, we investigated
the effects of mmLDL, ceramide and hydrogen peroxide on Sp1 and AP-2
activities and their modulation by NB6. mmLDL, but ceramide in
particular, enhanced the binding capacity of both transcription factors
Sp1 and AP-2 in the nuclear extracts of fibroblasts (Fig. 7
). Pretreatment of these cells with NB6 reduced the mmLDL- or
ceramide-mediated increase in Sp1 and AP-2 affinity (Fig. 7)
. A minor
increase in transcription factor activity was observed in response to
the plain compound NB6. Specificity of binding was confirmed by the
effective competition of a 40-fold molar excess of unlabeled probe.
Furthermore, NB6 inhibited the mmLDL- and
C6-ceramide-induced apoptosis in control
fibroblasts similar to aSMase-deficient Niemann-Pick fibroblasts
(Fig. 8
). As depicted in Fig. 9
, the cell-associated ceramide level, which roughly corresponded to the
respective aSMase expression, increased after mmLDL, ceramide or
H2O2 treatment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10
11
12
13
14)
. Our results now demonstrate that initiation of
apoptosis by treatment of M with mmLDL leads to an increase in both
the expression of aSMase and the intracellular concentration of
ceramide. Inhibition of aSMase by NB6, a novel aSMase transcription
inhibitor synthesized in our lab, abolished the effects of mmLDL
treatment. In aSMase-deficient (Niemann-Pick) fibroblasts, mmLDL
treatment did not initiate apoptosis.
mmLDL is known to be internalized mainly via the LDL receptor pathway
when exposed to M (30)
. The resulting endocytotic
vesicles do not necessarily fuse with lysosomes because signaling via
aSMase was demonstrated not to depend on ingestion and processing of
mLDL in these organelles (31)
. mmLDL was shown to damage
the lysosomal membranes of M (7)
, suggesting a leakage
of ceramide into the cytosol under these conditions. Although exchange
of ceramide over intact membrane bilayers requires days and thus likely
is too slow to participate in signaling, a trans-bilayer
transport (flip-flop) is expected to occur much faster
(32)
. Several possibilities have been proposed as to how
ceramide may interact with its targets: 1) potential target
molecules might diffuse to the site of ceramide generation,
2) ceramide might act by changing the physical properties of
the membrane where it has been synthesized, and 3) ceramide
could be delivered by lipid transfer proteins (33)
.
Furthermore, endosomal/lysosomal cathepsin D, an enzyme involved in
mediation of apoptosis (34)
by initiating a proteolytic
cascade, has recently been identified as likely to convey downstream
signaling of aSMase by ceramide (35)
. The release of
cathepsin D into the cytosol was also found after treatment of M
with mLDL (7)
, i.e., under conditions where an eventual
increase in cellular ceramide might be assumed. The key role of aSMase
and ceramide in cell death is further supported by 1) the
fact that a raise of intralysosomal pH by ammonium chloride diminishes
mmLDL-initiated apoptosis (this report) and 2) data of
Lepple-Wienhus et al. (36)
, who found that effects on
Ca2+ influx after CD95/CD95L interactions could
be mimicked by ceramide and sphingosine generated by aSMase.
Furthermore, Farina et al. have most recently shown that ceramide
accumulation due to a ceramidase deficiency (Farber disease) induces
abundant morphological changes associated with apoptosis in colonocytes
(37)
. Overexpression of another ceramide-regulating enzyme
in lysosomes, acid ceramidase, was consistently shown to protect from
apoptosis (38)
, and unpublished experiments from our
laboratories demonstrate a decreased sensitivity of ceramidase
overexpressing fibroblasts (kindly provided by D. Adam, Kiel, Germany)
toward apoptosis, induced by mLDL.
To further analyze the prominent role of aSMase and subsequent ceramide
generation in the initiation of the death program after mmLDL
treatment, we tested NB6, a novel inhibitor of aSMase transcription,
and Niemann-Pick fibroblasts, which differ from fibroblasts by the
lack of aSMase activity. NB6 inhibited mmLDL or ceramide-induced aSMase
expression, stimulation of ceramide production, and consequently
reduced programmed cell death in M and fibroblasts. In
Niemann-Pick fibroblasts, mmLDL and ceramide (used in
concentrations like in control fibroblasts) failed to induce apoptosis.
These data provide evidence for a similar antiapoptogenic mechanism in
NB6-treated or Niemann-Pick fibroblasts during mmLDL/ceramide-mediated
apoptosis, involving inhibition of aSMase transcription and, to some
extent, ceramide formation.
Here we show for the first time that mmLDL and ceramide are both able
to stimulate aSMase expression. The recent findings of Langmann and
colleagues indicate cooperative regulation of aSMase by redox-sensitive
transcription factors AP-2 and Sp1 (15
, 39
, 40)
. Our data
demonstrate an enhancement of transcription factor activities in
response to mmLDL and C6-ceramide treatment. In
particular, Sp1 binding activity is markedly increased after exposure
to C6-ceramide and is reducible by NB6
pretreatment. Hence, this agent inhibits mmLDL/ceramide-induced
expression of aSMase by yet unknown mechanisms. Furthermore,
antiapoptotic effects may include the modulation of other factors such
as stress-inducible manganese superoxide dismutase (10)
,
known to be controlled by Sp1 (41)
.
The addition of mmLDL as well as plain ceramide further stimulate
aSMase-mediated ceramide production and enzyme expression, thus
suggesting an auto-feedback mechanism being interruptible by NB6
(Fig. 10
). This hypothesis is supported by the fact that exogenous ceramide at
concentrations applied to fibroblasts did not result in
increased apoptosis in Niemann-Pick fibroblasts. Treatment of
fibroblasts with the inhibitor of aSMase transcription, NB6, prior to
mmLDL or ceramide exposure also neutralized programmed cell death
induction. These results could be explained by the absence of aSMase
stimulation via a positive auto-regulative loop, thus preventing the
generation of the lipid mediator at sites critical to apoptogenic
signaling. Comparable to our findings with aSMase, Jaffrézou
et al. (42)
have suggested a positive feedback control of
nSMase activity by ceramide in M, but no data about enzyme
expression have been provided.
|
Addition of ceramide to fibroblasts raises ceramide concentrations comparable to those obtained after exposure to mmLDL. However, a marginal reduction of the ceramide-induced increase to a level similar to that found after mmLDL addition is associated with a decrease in apoptosis. These observations indicate that the ceramide concentration per se is not critical to apoptosis induction, but rather generation in the lysosomal compartment. The importance of the total amount of cell associated ceramide, however, should not be overestimated as further distribution and localization in subcellular compartments have not been addressed in this study. Thus, exogenous C6-ceramide may not completely mimic ceramide species generated from endogenous sphingomyelin on stimulation. However, there is no experimental option as long-chain analogs added to the medium do not enter the cell or distribute intracellularly. In the context of this study, apoptosis can be induced by passing the aSMase deficiency with higher concentrations of exogenous C6-ceramide (50 µM) (not shown).
Our results agree with previous findings with Niemann-Pick
patients, whose cells did not respond to ionizing radiation with
ceramide formation and apoptosis (43)
. Furthermore,
irradiated endothelial cells of aSMase knockout mice revealed a 70%
reduction of apoptosis (44)
. Similarly, the induction of
oxidative stress by photodynamic therapy was shown to be absent in
Niemann-Pick lymphoblasts (36
, 45
, 46)
. The combination of
both exogenous SMase and photodynamic therapy was required to induce
oxidative stress, whereas the addition of the enzyme alone failed to
induce apoptosis, despite of an intracellular increase in ceramide.
Evidence for stress as an integral element of
mLDL/ceramide/aSMase-induced cell death is also supported by our recent
observations that mLDL- and ceramide-mediated apoptosis is associated
with an increase in manganese superoxide dismutase expression/activity
and a decrease in intracellular glutathione levels in M
(10)
. Vice versa, stress-induced apoptosis apparently
requires the generation of ceramide in U937 cells (47)
.
The latter study indicates that exogenous addition of hydrogen peroxide
increases ceramide levels. Stress-induced apoptosis in turn requires
the generation of ceramide in U937 cells (47)
. However,
the addition of hydrogen peroxide also induced apoptosis in
Niemann-Pick fibroblasts, which may be due to additional or alternative
pathways. One possible mechanism might be the direct and
concentration-dependent damage of mitochondrial DNA, which was more
extensive and persisted longer than nuclear DNA damage in human cells
during oxidative stress (48)
.
Until now there were no data available to clarify whether aSMase
deficiency (Niemann-Pick patients) inhibits atherosclerosis.
Nevertheless, reduction of aSMase activity could be a principle to
control mmLDL-mediated cell death. This concept may have further
implications for various states of human diseases involving
ceramide-dependent signaling (49
50
51)
. In atherogenesis,
for instance, apoptosis may be detrimental, since it could lead to
plaque rupture and thrombosis (52)
.
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ACKNOWLEDGMENTS |
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Received for publication April 23, 2000.
Revision received July 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05 vs.
control or #P 
