1 Institut National de la
Santé et de la Recherche Médicale, The distributions
of the mRNAs for estrogen receptors (ER
human myometria
SMOOTH MUSCLE CELLS retain remarkable plasticity during
normal and pathological development, as their phenotype can change rapidly and reversibly in response to changes in their hormonal environment. Pregnancy and leiomyoma, a monoclonal benign myometrial tumor, both lead to major morphological and biochemical modifications in the myometrium, illustrating the difference between the normal and
pathological development of this tissue. There is compelling evidence
that steroid hormones, especially estrogens, have a central role in
modulating the growth, differentiation, and functions of target tissues
like the myometrium (10). These hormones are produced in large amounts
by the placenta during pregnancy, and their pleiotropic actions ensure
optimal myometrial responses for a successful pregnancy outcome.
Leiomyoma is another estrogen-sensitive form of myometrial growth. This
disorder is rare before puberty but is the most frequent benign uterine
smooth muscle cell tumor in women of reproductive age. Leiomyoma can
also rapidly increase in size during pregnancy, leading to fetal
wastage, whereas they regress after menopause, ovariectomy, or
gonadotropin-releasing hormone agonist therapy (32).
Classically steroid hormones act via specific intracellular receptor
proteins (7, 20). There is no real consensus about the myometrial
steroid receptor status during human gestation. Some have reported the
presence of estrogen and/or progesterone receptors in the myometria of
pregnant women at the end of pregnancy while others have not found them
(29, 18, 31). There are also conflicting data on the relative abundance
of steroid receptors in leiomyoma, with reports of overproduction or no
change compared with normal tissue (4, 24, 33). It is thus important to know how the estrogen-receptor (ER) status changes during the normal
and physiopathological growth of the human myometrium. This is
particularly significant since several studies have shown that both
leiomyoma and myometrium of pregnant women, especially at the end of
pregnancy, show similar overexpression of a number of genes regulated
by steroid hormones, such as the genes coding for connexin-43,
insulin-like growth factors, epidermal growth factors,
c-myc, and collagen. This
had led certain authors to consider leiomyoma as a
"pseudopregnant" myometrium (2).
Steroid hormone receptors are nuclear receptors that continuously
shuttle between the nucleus and the cytoplasm (16). They are
hormone-activated transcription factors that regulate the expression of
specific genes by binding to steroid-responsive elements. Until
recently, it was generally accepted that there was only one ER gene
coding for the classical ER (ER Knowledge of the distributions of ER Tissues. Biopsies of myometria from
pregnant women were obtained from patients
(n = 8) with normal pregnancy who were
delivered by elective cesarean section. The cesarean section was done
for previously diagnosed cephalopelvic disproportion before the onset of labor between the 38th and 40th wk of amenorrhea. This procedure was
approved by the Consultative Committee of Persons Involved in
Biomedical Research of Paris-Cochin, and all subjects gave their
informed written consent.
Normal and pathological (leiomyoma) nonpregnant myometrial samples were
obtained from cyclic women (n = 10)
aged 39-51 yr, undergoing hysterectomy for benign gynecological
indications. Tissue samples were excised from normal muscle in areas
free of macroscopically visible abnormalities
(n = 10). Samples of intramural leiomyoma (n = 5) were collected as
previously described (5), and the uterus was examined by a pathologist
to exclude adenomyosis or malignant changes. All of the leiomyoma were
similar in size (2-cm diameter). None of the patients had been on
hormonal medication for at least 3 mo before hospitalization. Surgery
was scheduled during the luteal or follicular phases of the menstrual
cycle. The stage of the menstrual cycle was estimated histologically by
dating the endometrium (5). The biopsies were immediately frozen in
liquid nitrogen and were stored at RNA preparation and RT. All reagents
used for RNA isolation were molecular reagents from Sigma Chemical (St.
Louis, MO). Total RNA was isolated from myometria of pregnant
(n = 5) or nonpregnant (n = 5) women and leiomyoma
(n = 5) using an acid
guanidinium-phenol-chloroform procedure (9). RT was performed using
random hexanucleotides (20 µM) as primers on 4 µg of total RNA plus
200 U Moloney murine leukemia virus RT in a final volume of 25 µl at
39°C for 60 min, as in the manufacter's specifications (Life
Technologies). The cDNA products were stored at PCR. The primers used for human ER The PCR primers used to amplify human ER An aliquot (15 µl) of each PCR product was separated by
electrophoresis (3% Nusieve agarose gel in Tris-borate-EDTA buffer containing 0.01% ethidium bromide). A molecular weight
standard (lambda 123-bp DNA ladder; Life Technologies) was used to
confirm the predicted PCR product sizes. Lack of genomic DNA
contamination was checked in all experiments by conducting a control
reaction containing mRNA without RT. Normalization of mRNA amounts in
all samples studied was checked by amplifying an endogenous gene, human
The intensities of the specific bands for ER Preparation of protein fractions. Each
frozen sample of myometrium was thawed on ice and homogenized in
ice-cold 12 mM Tris, 1.5 mM EDTA, 10% glycerol, and 2 mM
Na2MO4
(pH 7.4) using a Polytron homogenizer and a tissue-to-buffer ratio of
1:10 (wt/vol). The homogenate was centrifuged at 1,000 g for 10 min, and the supernatant was
transferred to an ultracentrifuge tube and centrifuged at 105,000 g for 60 min. The resulting cytosol
was used in receptor-binding assays, as this procedure extracted a
large percentage of the nuclear untransformed receptors (14, 22). All
cytosol fractions were kept at Binding analysis of ER. Before the
binding assay, the cytosol samples were treated with a dextran-coated
charcoal pellet (10% NoritA and 0.1% dextran T70) for 1 h at 4°C
to remove endogenous steroids. Aliquots of charcoal-treated cytosol
(500 µg proteins) were then incubated (16 h at 4°C) with
increasing concentrations (0.25-12 nM) of highly tritiated
17 The binding parameters, affinity constant
(Ka = 109
M Statistical analysis. The
nonparametric Mann-Whitney test for unpaired samples was used to
compare the levels of ER The Mann-Whitney test for unpaired samples was also used to compare the
kinetic parameters in the myometria of pregnant and nonpregnant women
and in leiomyoma. All results are expressed as means ± SE.
The Statview statistical package for Mackintosh (Abacus Concepts,
Berkeley, CA) was used in these analyses.
P values of <0.05. were considered significant.
ER
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and ER
) and their binding
properties in myometria of pregnant and nonpregnant women and in
leiomyoma were studied. RT-PCR analysis indicated that the term
pregnancy myometria had little ER
mRNA, whereas the amounts of ER
mRNAs in pregnant or nonpregnant myometria appeared to be similar. Both
ER
and ER
mRNA were greater in certain leiomyoma than in normal
nonpregnant myometria. The binding kinetics revealed that two specific
binding sites (with high or low affinity) for 17
-estradiol were
present in the nonpregnant myometrium. Only the low-affinity binding
sites were detectable in late-pregnancy myometria and in leiomyoma, and
their capacities were increased two- to threefold
(P < 0.001) in leiomyoma. The pregnancy- and leiomyoma-related changes in myometrial ER status, especially the low concentration of ER
mRNA and the lack of
high-affinity ER in pregnant women, plus the increased ER
and ER
mRNAs and the increased low-affinity ER in some leiomyoma, suggest that the redistribution of ER subtypes is associated with the pathological and/or normal growth of the myometrium.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
), which bound estrogen with high
affinity. The description of a new gene encoding a second type of ER,
termed ER
(13), has prompted reevaluation of the estrogen signaling
system. This new full-length ER
cDNA was cloned from the human ovary
and testis (23, 27). The chromosomal locations of the ER
and ER
genes indicate that there are two independent ER genes in humans (13).
Both of these forms of ER have been found in steroid-sensitive tissues
(13, 19, 22, 23, 27), showing the complexity of the tissue response to
estrogen. This also increases the likelihood that any selective effect
of estrogen could be due to the differential expression of these two ER
genes, depending on the physiological and/or physiopathological state
of the target tissues.
and ER
in the human
myometrium is needed for studies on the respective function and importance of these ER subtypes in normal and abnormal growth. We have
therefore compared the pattern of ER (ER
and ER
) expression in
the myometria of pregnant and nonpregnant women and in leiomyoma. We
first analyzed the distributions of the ER
and ER
mRNAs using RT-PCR and then determined the concentrations and affinity constants of
the ER in these three tissues by ligand binding. Our results indicate
that both ER
and ER
mRNAs are present in the myometria of
nonpregnant and pregnant women at term and in leiomyoma, but the
amounts of these mRNAs and the estrogen-binding properties change
according to the physiological and physiopathological growth of the myometrium.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80°C.
20°C until
required for PCR.
cDNA were as follows: ER
upper (sense) 5'-CAG GGG TGA AGT GGG
GTC TGC TG-3' (priming site in exon 4, nucleotides 1060-1082
as numbered in Ref. 15) and ER
lower (antisense) 5'-ATG CGG
AAC CGA GAT GAT GTA GC-3' (priming site in exon 6, nucleotides
1520-1542; Genosys Biotechnologies). Briefly, 2.5 µl of cDNA
reaction mixture were used for amplification in the presence of 0.1 mM
each dNTP, with 0.2 µM primers, 1 mM MgCl2, and 2.5 U
Taq polymerase (Life Technologies,
France) in 25 µl PCR buffer. The amplification profile consisted of
22 cycles with denaturation at 94°C for 1 min, annealing at
66°C for 1 min, and extension at 72°C for 1 min, with a final
extension at 72°C for 10 min.
cDNA were as follows: ER
upper (sense) 5'-TGC TTT GGT TTG GGT GAT TGC-3'
(nucleotides 1164-1184 as numbered in Ref. 27) and ER
lower
(antisense) 5'-TTT GCT TTT ACT GTC CTC TGC-3' (nucleotides
1402-1422) with 0.1 mM of each dNTP, 0.4 µM primers, 1 mM
MgCl2, and 2.5 U
Taq polymerase (Life Technologies) in
25 µl PCR buffer. The amplification profile was 30 cycles with
denaturation at 94°C for 1 min, annealing at 58°C for 1 min,
and extension at 72°C for 1 min, with a final extension at 72°C
for 10 min.
2-microglobulin cDNA, using an
additional pair of primers (17). The PCR products were also checked by
Southern blot analysis with specific internal oligonucleotides for each
sequence, 5'-TAGAGCGTTTGATCATGAGCGGG-3' for ER
cDNA and
5'-CAGGAGCATCAGGAGGT-3' for ER
cDNA. Hybridization was
performed with the specific probes labeled with
fluorescein-11-desoxy-UTP using an enhanced chemiluminescence 3'
oligolabeling and detection kit (Amersham) according to the
manufacturer's instructions.
, ER
, and
2-microglobulin mRNAs were
analyzed densitometrically (Gel Scan DU Series 600 spectrophotometer;
Beckman Instruments). Results were expressed as relative levels of
specific mRNAs normalized to
2-microglobulin mRNA in each
amplified sample.
80°C. The protein
concentration in each cytosol was determined by the dye-binding assay
(Bio-Rad, Richmond, CA) with BSA as the standard.
-[2,4,6,7-3H]estradiol
(85-110 Ci/mmol; Amersham) or labeled estradiol plus a 500-fold
molar excess of unlabeled diethylstilbestrol. The final volume of the
incubation mixture was 0.5 ml. A 10-µl aliquot was then taken to
measure total radioactivity. Bound and free hormone fractions were
separated by adding an equal volume of a suspension of 1% charcoal and
0.1% dextran (wt/vol) for 10 min at 0°C and centrifugation at
1,500 g for 10 min. Aliquots of
supernatant were counted in 4 ml of Ultima-gold XR scintillation fluid
(Packard) in a Beckman LS 6000-IC scintillation counter.
1), and binding capacity
(equivalent to total number of binding sites: fmol/mg protein) were
estimated by Scatchard graphical analysis after subtraction of the
nonspecific binding. When more than one type of binding site was
detected (biphasic curves), the Rosenthal graphical correction (34) was
applied to determine the binding parameters of the first class of
binding sites.
and ER
mRNAs in the myometria of
pregnant and nonpregnant women. The Wilcoxon signed-rank test for
paired samples was used to determine significant difference in the
levels of ER
and ER
mRNAs between the leiomyoma and nonpregnant myometria.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and ER
mRNAs in the myometria of pregnant and
nonpregnant women and in leiomyoma. To determine the
distribution of ER
and ER
mRNAs in the myometria of pregnant and
nonpregnant women and in leiomyoma, total RNAs from each tissue were
reverse transcribed. The resulting cDNAs were amplified by PCR using a
set of primers specific for each ER cDNA sequence. As illustrated in
Fig. 1, electrophoresis on agarose gel
revealed PCR products of the predicted sizes 483 bp for ER
and 259 bp for ER
in all tissues. The successful normalization of RNA
amounts during the RT step was verified by amplification of a fragment
of the reference standard,
2-microglobulin cDNA, in all of
the samples analyzed (Fig. 1).
View larger version (35K):
[in a new window]
Fig. 1.
Estrogen receptor (ER) (A), ER
(B), and
2-microglobulin (C)
mRNAs in the myometria of pregnant and nonpregnant women and in
leiomyoma, analyzed by RT-PCR. Typical ethidium bromide-stained gels
representative of each mRNA subtype (ER
, ER
, and
2-microglobulin) are shown.
Same samples of pregnant and nonpregnant myometria and leiomyoma were
used for each amplification. RT was included (+) or omitted (
).
2-Microglobulin cDNA was used
as an endogenous reference gene; equivalent band intensities were found
in all samples studied.
The apparent amounts of ER and ER
mRNAs in the three tissues were
compared after Southern blotting and hybridization with oligonucleotide
probes specific for each ER subtype (Fig.
2A). Densitometric analysis of the signals indicated wide variations in the
ER
and ER
(Fig. 2B) mRNA
levels in normal myometrial samples from different nonpregnant patients
without any evident correlation with the stage of the menstrual cycle.
A comparison of the signals in the myometrium and the leiomyoma from
the same patient indicated that these signals were increased (3- to
8-fold) for ER
mRNA in three leiomyoma (Fig.
2B, lanes 2-3,
11-12, and 14-15), whereas the signals for
the two other patients were more similar (Fig.
2B, lanes
5-6 and 8-9;
P < 0.06, leiomyoma vs. nonpregnant myometria). The weakest signals for ER
mRNA were from the pregnant myometria (P < 0.01, pregnant vs.
nonpregnant groups).
|
The signals for ER transcripts also appeared to be greater (1.5- to
4-fold) in leiomyoma (Fig. 2B,
lanes 2-3, 5-6, 8-9,
and 14-15) than in nonpregnant
myometria, except for one case (Fig. 2B, lanes
11-12; P < 0.06, leimyoma vs. nonpregnant myometria). Homogenous amounts of ER
mRNA were found in pregnant myometria except for in one woman (Fig.
2B, lane
1). Globally, the intensities for ER
mRNA in
pregnant and nonpregnant myometria were similar.
Binding properties of ER in the myometria of pregnant
and nonpregnant women and in leiomyoma. The binding
patterns of untransformed ER isolated from myometrial cytosol of
pregnant or nonpregnant women and from leiomyoma are shown in Fig.
3. Table 1
summarizes the binding kinetic values with the
Ka and number of
binding sites (n) determined after
graphical Scatchard analysis. The specific binding of
17-[2,4,6,7-3H]estradiol
to the myometrial receptors in nonpregnant women followed a biphasic
saturation curve (Fig. 3) with a curvilinear Scatchard plot, indicating
two binding components: one class of binding sites (I) with high
affinity
[Ka(I) = 4.8 ± 0.6 × 109
M
1] and low capacity
[n(I) = 10.2 ± 1.6 fmol/mg
protein] and a second class (II) with a lower affinity
[Ka(II) = 0.3 ± 0.1 × 109
M
1] and a greater
capacity [n(II) = 122 ± 15 fmol/mg protein]. The stage of the menstrual cycle had no evident
influence on these binding parameters under our experimental
conditions. By contrast, linear transformation of saturation data
revealed a single population of 17
-estradiol-binding sites in the
myometria of pregnant women. High-affinity binding sites, typical of
type I, were not detected in pregnant myometria (Fig. 3). Only the
low-affinity binding sites
(Ka = 0.2 ± 0.1 × 109
M
1) with high capacity
(n = 114 ± 13 fmol/mg protein)
were found. Similarly, a single population of low-affinity binding
sites (Ka = 0.3 ± 0.1 × 109
M
1) was found in
leiomyoma (Fig. 3). Their concentration was two- to threefold greater
(n = 273 ± 38 fmol/mg protein)
than the concentration in the nonpregnant
(P < 0.001) or pregnant
(P < 0.003) myometria.
|
|
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DISCUSSION |
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This study describes the specific phenotypes of human myometria in
pregnancy and leiomyoma, in terms of ER subtypes. The distributions of
ER and ER
mRNAs and the binding properties of the resulting ER in
late-pregnancy myometria and in leiomyoma reveal certain similarities
and differences between the normal and pathological growth of the human myometrium.
We first analyzed the ER status according to the physiological
development of the human myometrium in pregnancy. mRNAs for both the
classical ER and the new ER
were detected in the myometria of
pregnant and nonpregnant women, but their patterns differed with the
tissue studied. The RT-PCR analysis clearly indicated that the
myometria of pregnant women contains little mRNA encoding ER
,
whereas the amounts of ER
mRNA in pregnant and nonpregnant tissues
appear to be similar. The binding characteristics of the ER emphasize
the changes occurring in the myometrium from pregnant women. Thus the
nonpregnant myometria contains two specific 17
-estradiol-binding sites, one with a high affinity and low capacity and a second with a
lower affinity and greater capacity. In contrast, the myometria of
pregnant women appear to contain no high-affinity binding sites and
only the lower-affinity sites at the same concentration as in
nonpregnant myometria. These data are compatible with previous results
obtained using sucrose density gradient centrifugation and titration
analyses (18, 29, 31).
Our second interest is in a physiopathological development of the human
myometrium, the leiomyoma, which is the most common uterine tumor and a
major public health problem since it is often an indication for
hysterectomy. The increases in ER mRNA and ER protein in leiomyoma
are controversial (4, 24). In agreement with previous studies (4) and
although there are wide variations in ER mRNA levels in myometrial
samples from different patients, our results indicate that the amounts
of ER
mRNA may well be enhanced in leiomyoma. There also appears to
be more ER
mRNA in leiomyoma. These observations might be explained
by the fact that the region 14q-22
24 of chromosome 14, which includes
the ER
gene, is involved in rearrangements in uterine leiomyoma
(13). The ER-binding studies revealed only low-affinity
estrogen-binding sites in leiomyoma. The number of these low-affinity
binding sites is two- to threefold greater in leiomyoma than in normal
nonpregnant myometria (P < 0.001). Previous reports also indicate an increase in estrogen-binding
sites in leiomyoma (4). The increase in the number of ER in leiomyoma
may account for their enhanced sensitivity to estrogen (26).
Recent studies indicate that the ER subtype differs from the ER
subtype in the COOH-terminal ligand binding (58% amino acid sequence
homology) and the NH2-terminal
transactivation domains, although the two ER subtypes have nearly
identical DNA binding domains (96% homology; see Refs. 13, 19, 22, 23,
27). However, the ER
and ER
subtypes can give opposing regulatory signals to 17
-estradiol from the same DNA response element at activator protein 1 sites. Thus 17
-estradiol bound to ER
activates transcription, whereas this natural hormone bound to ER
inhibits transcription (30). There are also differences in the binding affinities of the ER subtypes for estrogenic substances. ER
receptors have a lower affinity for 17
-estradiol than do ER
receptors (22). Our findings show not only parallels between the little mRNA encoding ER
and the lack of high-affinity binding sites in the
myometria of pregnant women but also between the presence of ER
mRNA
and the persistence of low-affinity estrogen-binding sites in this
tissue. It is thus possible that the ER
corresponds to the classical
17
-estradiol high-affinity binding sites (15) and that ER
is
equivalent to the low-affinity binding sites. This notion is also
consistent with the apparent elevated ER
mRNA and the increased
number of low-affinity estradiol-binding sites in leiomyoma compared
with nonpregnant myometrium. Nevertheless, the discrepancy between the
presence of substantial ER
mRNA in leiomyoma and the apparent lack
of high-affinity estradiol-binding sites in this tumor must be
clarified. This might result from inappropriate relative proportions of
the two ER subtypes in leiomyoma. The range of ligand concentrations
used in this study was wide and normally sufficient to distinguish
between a mixed population of receptor subtypes with sufficiently
different affinities (10- to 100-fold), but it is possible that the
overabundance of low-affinity sites in leiomyoma may mask the presence
of high-affinity binding sites. In addition, there could be ER
variants lacking part of the hormone-binding domain in leiomyoma, as
shown recently in breast cancer (28). Last, posttranscriptional
modification or faulty translation of the ER
mRNA into functional
protein is always possible.
Hence, what is the functional significance of the presence of ER
mRNA and the low-affinity ER subtype in pregnant myometria and
leiomyoma? The role of the ER
may well differ depending on the
presence or absence of the classical ER
. Recent studies indicate that ER
/ER
heterodimers are apparently formed in the preference to homodimers (11). This opens the possibility that ER
and ER
act
synergistically or as antagonists via (homo or hetero) dimerization and
activation of a common responsive element. The relative importance of
the ER
and ER
subtypes in uterine development and function is
shown by studies using ER
knockout (ERKO; see Refs. 11 and 21) mice.
The ERKO mice, in which the amount of ER
is not significantly
altered, survive but are infertile. ERKO mice also have abnormally high
baseline plasma levels of gonadal hormones (10-fold higher
17
-estradiol than in wild mice; see Ref. 11). The weak ER
mRNA
signal and the presence of ER
mRNA, plus the high circulating
estrogen concentration in women in term pregnancy, appear to be
remarkably similar to the endocrine situation in ERKO mice.
Another question is how does estrogen still exert its physiological
action, despite the apparent lack of high-affinity binding sites? There
is still no clear consensus as to how steroid receptors act or the
precise function of the low-affinity binding sites. These latter sites
have been found in normal and malignant mammalian tissues (36) and have
been proposed to be involved in estrogen-induced cell growth (25). It
has also been suggested that they bind not only estrogen but other
ligands and inhibitors of cell proliferation as well (25). Recent
reports indicate that ER preferentially binds environmental
phytoestrogens like genestein (22), and several studies indicate that
genestein may regulate cell growth (1, 3, 35) and mainly binds to these
sites in uterine and mammary tumor cells (3). The effect of such
environmental estrogens on the reproductive ability of mammalians is
also debated, and there has been speculation that some of their claimed
effects on fertility are mediated via ER
(13). It is, perhaps, not so surprising that low-affinity binding sites and ER
mRNA are substantially found during pregnancy and in leiomyoma, since both situations lead to a relative infertility and involve myometrial hypertrophy and/or hyperplasia. The growth of the myometrium during periods of increased estrogen production, such as pregnancy, is thought
to be primarily due to cell hypertrophy, resulting in an increase in
cell volume (6, 8). Thus the hypertrophy of the smooth muscle cells
that occurs mainly during pregnancy is accompanied by an increase in
contractile proteins and reorganization of intracellular organelles
(8). On the other hand, the increased myometrial growth in response to
estrogen in leiomyoma is mainly due to proliferation of smooth muscle
cells and could reflect altered estrogen responsiveness (6).
The influences of ER and ER
and their relative proportions in
sustaining myometrial hypertrophy and proliferation, together with the
actions of other effectors, such as progesterone and growth factors,
require further clarification. However, the changes in the ER
and
ER
balance during pregnancy and in leiomyoma described in this study
shed some light on the long-standing puzzle of the uterotropic response
to steroid hormones mediated via specific steroid-regulated genes.
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ACKNOWLEDGEMENTS |
---|
We thank G. Delrue [SC6 Institut National de la Santé et de la Recherche Médicale (INSERM)] for photographic work and M. Verger for skillfull secretarial assistance. We also thank Dr. O. Parkes for editorial help.
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FOOTNOTES |
---|
This work was supported by INSERM and grants from University René Descartes Paris V (UFR Cochin-Port Royal).
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. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: C. Benassayag, INSERM, Unité 361, Université René Descartes Paris V, Pavillon Baudelocque, 123 Bvd de Port Royal, 75014 Paris, France (E-mail: u361{at}cochin.inserm.fr).
Received 30 November 1998; accepted in final form 23 February 1999.
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