Selecting agent hygromycin B alters expression of
glucose-regulated genes in transfected Caco-2 cells
Annie
Rodolosse1,
Alain
Barbat1,
Isabelle
Chantret1,
Michel
Lacasa1,2,
Edith
Brot-Laroche1,
Alain
Zweibaum1, and
Monique
Rousset1
1 Unité de Recherches sur
la Différenciation Cellulaire Intestinale, Institut National
de la Santé et de la Recherche Médicale, Unité
178, 94807 Villejuif Cedex; and
2 Université Pierre et Marie
Curie, 75251 Paris Cedex 05, France
 |
ABSTRACT |
Incorporation into plasmids of genes conferring
resistance to aminoglycoside antibiotics such as hygromycin B is
currently utilized for selection in experiments involving gene transfer in eukaryotic cells. Using a subclone of Caco-2 cells stably
transfected with an episomal plasmid containing the hygromycin
resistance gene, we observed that transformed cells subcultured in the
presence of hygromycin B exhibit, compared with the same cells
subcultured in antibiotic-free medium, a sixfold increase in the rates
of glucose consumption and lactic acid production and dramatic changes, at mRNA and protein level, of the expressions of sucrase-isomaltase and
hexose transporter GLUT-2, which are downregulated, contrasting with an
upregulation of hexose transporter GLUT-1. This occurs without
significant modifications of the differentiation status of the cells,
as demonstrated by the normal expression of villin, ZO-1, dipeptidyl
peptidase IV, or
Na+-K+-ATPase.
The plasmid copy number is, however, the same, whether or not the cells
are cultured in the presence of hygromycin B. These results draw
attention to the need to consider antibiotic-dependent alterations of
metabolism and gene expression in transfection experiments.
glucose consumption; sucrase-isomaltase; GLUT-1; GLUT-2
 |
INTRODUCTION |
AMINOGLYCOSIDE ANTIBIOTICS (10) are highly cytotoxic to
eukaryotic cells. For this reason, incorporation into plasmids of genes
that confer resistance to these antibiotics is extensively utilized for
selection in experiments involving gene transfer in eukaryotic cells.
Among these antibiotics, hygromycin B (1, 3) has been utilized for
studying the regulation of a variety of transferred genes in several
transfected cell types, including in the enterocyte-like cell line
Caco-2. This cell line has been extensively utilized in the last decade
as an in vitro model for human intestinal enterocytes since the first
demonstration of its differentiation properties (21).
The purpose of the present report is to draw attention to the fact that
the use of hygromycin B is not neutral, since, as in Caco-2 cells, it
interferes per se with glucose utilization and expression of
glucose-regulated genes.
Previous results obtained with Caco-2 cells have shown that all
conditions of increased glucose consumption, whether obtained through
pharmacological modulation as with forskolin (5, 14, 26), monensin (5),
or inducers of cytochrome P-4501A1
(4) or by comparing clonal populations of cells selected
on the basis of different rates of glucose consumption (6), result in
marked modifications of the level of expression of a number of proteins involved in the uptake, transport or metabolism of glucose.
High-glucose-consumption-associated modifications include, for example,
a decreased expression of sucrase-isomaltase (SI) (4-6, 14, 26),
hexose transporter GLUT-2 (4, 16), fructose 1,6- diphosphatase, and pyruvate kinase (24) and an increased expression of
hexose transporters GLUT-1 and GLUT-3 (4, 16) and
phosphoenolpyruvate carboxykinase (24), occurring without modifications of the morphological
differentiation of the cells or of the expression of other
differentiation-associated proteins such as villin or dipeptidyl
peptidase IV (DPP-IV).
The results reported here were obtained during experiments conducted to
define the elements of the promoter region of the SI gene through which
glucose exerts its repressive transcriptional effect (25). For such
studies, we have used clonal populations of the enterocyte-like Caco-2
cell line, which greatly differ in their glucose utilization and their
level of SI expression (6). Among them, the TC7 clone exhibits, after
confluency, low levels of glucose consumption and GLUT-1 mRNA (4, 16) and inversely expresses high levels of SI and GLUT-2 mRNA (4, 6, 16).
We report here that, after transfection of TC7 cells with a plasmid
containing the gene that confers resistance to hygromycin B and after
selection of the stably transformed TC7 cells, the selecting agent
hygromycin B induces by itself a marked increase in the rates of
glucose consumption and lactic acid production and changes in the level
of expression of SI, GLUT-1, and GLUT-2.
 |
MATERIALS AND METHODS |
Plasmid. The plasmid used in this
study (Fig. 1) has been previously
described (25). It contains the promoter-less luciferase gene inserted
in the Nar I site of a derivative of
plasmid p205-GTI (28). This plasmid contains the Epstein-Barr virus
sequences for Ori-P and EBNA-1 that allow a stable and episomal
maintenance of the vector in human cells (32) and the hygromycin
resistance gene under control of the herpes simplex virus 1 thymidine
kinase promoter.

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Fig. 1.
Structure of plasmid used for transfection experiments. EBNA-1 and
Ori-P are Epstein-Barr virus sequences that allow a stable and episomal
maintenance of vector in human cells.
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|
Cell cultures, transfections, and selection of
transformed cells. TC7 cells, a clonal population of
the Caco-2 cell line (6), were routinely cultured in Dulbecco's
modified Eagle's medium containing 25 mM glucose (Eurobio, Paris,
France), 20% heat-inactivated (56°C, 30 min) fetal bovine serum
(Boehringer, Mannheim, Germany), and 1% nonessential aminoacids
(GIBCO-BRL, Glasgow, UK). Cultures were maintained in a 10%
CO2-90% air atmosphere. For
transfection experiments, 0.3 × 106 cells were seeded in 5 ml
medium in 25-cm2 petri dishes
(Corning Glassworks, Corning, NY). The medium was changed 2 days later,
and transfections were performed 4-5 h later by using the
CaPO4 procedure (11) and 20 µg
of plasmid DNA. The medium was changed 15 h later and then daily. For
the selection of transformed cells, hygromycin B (Sigma, St. Louis, MO)
was added at confluency at a final concentration of 210 µg/ml, which was determined to be the minimal lethal concentration for the untransformed cells. Hygromycin-resistant cells were allowed to grow
until subconfluent and were then subcultured for several passages in
the presence of hygromycin. After several passages, the cells were
either maintained in the presence of the drug or reversed to drug-free
medium. When maintained in the presence of the drug, the cells were
allowed to attach to the flask for 48 h without the drug, and then
hygromycin B was added daily at a concentration increasing
progressively from 40 to 210 µg/ml, depending on cell density. In all
cases, the medium was changed 48 h after seeding and daily thereafter.
Plasmid extraction and DNA analysis of transformed
cells. Extrachromosomal and genomic DNA were purified
by a small-scale SDS lysis procedure, adapted from the method of
Birnboim and Doly (2). DNA extracts were analyzed by slot blot and
hybridization with a luciferase probe (a 759-bp EcoR
I-Kpn I fragment from p205LUC) for
quantification of the plasmid. Probe was
32P labeled, using a megaprime DNA
labeling kit (Amersham). After 18 h of hybridization in the presence of
10% dextran sulfate (Pharmacia), the blots were washed twice in
2× standard sodium citrate (SSC)/0.1% SDS at 65°C for 30 min
and then for 15 min in 0.1× SSC at 65°C.
RNA extraction and analysis. Total RNA
was extracted by the guanidium isothiocyanate method (7). Samples of
total RNA, denatured in 1 M glyoxal, were fractionated by
electrophoresis through 1% agarose gels and transfered onto Hybond N
(Amersham) (29). Prehybridization was performed at 42°C in the
presence of 50% formamide, and hybridization was performed at 42°C
in the presence of 40% formamide and 10% dextran sulfate. The blots
were washed twice in 2× SSC/0.1% SDS for 10 min at room
temperature, once in 0.1× SSC/0.1% SDS for 15 min at 50°C,
and once in 0.1× SSC/0.1% SDS for 15 min at 65°C. The probes
used were SI2 (12) for SI mRNA, DPI-101 (9) for DPP-IV mRNA, and cDNA
V19 (22), which was obtained from D. Louvard (Institut Curie, Paris,
France), for villin mRNA. The
1-Na+-K+-ATPase
mRNA was detected with a 3.6-kb cDNA, which was a generous gift from
Dr. J. B. Lingrel (University of Cincinnati, Cincinnati, OH). The cDNA
probes pGEM4Z-HepG2GT GLUT-1 (1.75-kb insert) and pBS-HTL210/hGLUT-2
(2.4-kb insert) were obtained from Dr. G. I. Bell (Howard
Hughes Medical Institute, University of Chicago, Chicago, IL).
Electron microscopy. Transmission
electron microscopy was performed on late postconfluent
(day 20) cells grown in
25-cm2 plastic flasks. Samples
embedded in Epon were reembedded to make sections perpendicular to the
bottom of the flasks.
Immunofluorescence. Indirect
immunofluorescence was performed on frozen cryostat sections of cell
layer rolls, as already reported (6), after fixation in 3.5%
paraformaldehyde in
Ca2+/Mg2+-free
PBS (15 min at room temperature). Mouse monoclonal antibodies HBB
2/614/88, 3/775/42 (15), and N1/123/33, specific for human SI, DPP-IV,
and the
1-subunit of the
Na+-K+-ATPase,
respectively, were obtained from Dr. H. P. Hauri (Biocenter of the
University of Basel, Basel, Switzerland). Rabbit polyclonal antibodies
against porcine villin (23) and the tight-junction protein ZO-1 (31)
were obtained from Dr. D. Louvard (Institut Curie) and Dr. J. M. Anderson (Yale University, New Haven, CT) respectively. Rabbit
polyclonal antibodies (L459) against SI from Caco-2 cells were produced
in our laboratory (30). Rabbit polyclonal antibodies against GLUT-1
were from East Acres Biologicals (Southbridge, MA). Double
immunofluorescence was performed, using as second antibodies anti-mouse
and anti-rabbit fluorescein-coupled sheep antiglobulins (Institut
Pasteur Productions, Marnes-la-Coquette, France) or rhodamine-coupled
sheep antiglobulins (Boehringer).
Glycogen, glucose consumption, and lactic acid
production assays. For glycogen assays, the cells were
harvested 18 h after the medium changes for extraction and measurement
with anthrone, as previously reported (6). Results are expressed as
micrograms per 106 cells. Glucose
consumption was determined every day, 16-18 h after the medium
changes, using a Beckman glucose analyzer 2, and lactic acid production
was determined, using a L-lactic
acid measurement kit (Boehringer), as previously reported (4). Results are expressed as nanomoles per hour per
106 cells.
Sucrase and DDP-IV activity assays.
Sucrase (EC 3.2.1.48) activity was measured according to Messer and
Dahlqvist (17). DPP-IV (EC 3.4.14.5) activity was measured according to
Nagatsu et al. (20), using 1.5 mM
glycyl-L-proline-4-nitroanilide
as substrate. Results are expressed as milliunits per milligram of protein. One unit is defined as the activity that hydrolyzes 1 µmol
substrate/min at 37°C. Proteins were assayed with the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL).
 |
RESULTS |
Effect of hygromycin on rate of glucose
consumption. The presence of hygromycin has no effect
on the rate of cell division and growth curves of transformed cells
(not shown), but it deeply affects the growth-related pattern of
glucose consumption. As shown in Fig. 2,
the pattern of evolution of the rate of glucose consumption from
transformed TC7 cells grown in the absence of hygromycin is
characterized, as already reported in untransformed TC7 cells (4, 6),
by a rapid decrease starting at confluence (day
5) and reaching a plateau of low value at late
confluence. By contrast, although decreasing after confluence, the rate
of glucose consumption of transformed cells cultured in the presence of
hygromycin is maintained, at late confluency, at a level that is
sixfold higher than that observed in the cells cultured without the
drug. The hygromycin-dependent elevated rate of glucose consumption is
associated with an increased rate of lactic acid production and a lower
glycogen content (see Table 1).

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Fig. 2.
Growth-related variations of glucose consumption in transformed TC7
cells cultured in the absence (first passage) ( ) or presence ( )
of hygromycin. Results are means ± SD of 3 different cultures.
Transformed cells were maintained for 3 passages in the presence of the
drug before analysis. Analysis was done with cells from the same
corresponding passages, either subcultured in the presence of the
antibiotic or reversed for each single analyzed passage in
hygromycin-free medium. Curves of glucose consumption from
untransformed TC7 cells were undistinguishable from those of
transformed cells cultured in the absence of hygromycin (not shown).
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Effect of hygromycin on cell
morphology. Morphology of late postconfluent
(day 20) transformed TC7 cells grown
with or without hygromycin is shown in Fig.
3. As shown on thin sections, the cells
form a monolayer in both culture conditions. However, the cells
cultured in the presence of hygromycin (Fig.
3b) appear enlarged, compared with
the transformed cells cultured without the drug (Fig.
3a). At the ultrastructural level,
the brush border, which is well organized in cells cultured without the
drug (Fig. 3, c and
e), appears disorganized in the
cells cultured in the presence of hygromycin (Fig. 3,
d and
f).

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Fig. 3.
Effect of hygromycin on the morphology of postconfluent transformed TC7
cells. Cells from a same passage were analyzed after 20 days of culture
in the absence (a,
c, and
e) or presence
(b,
d, and
f) of the antibiotic.
a and
b: Thin section of the cell layer
perpendicular to the bottom of the flask (bar, 17 µm);
c and
d: transmission electron microscopy of
the same cell layers (bar, 2 µm); e
and f: higher magnification of the
apical brush border (bar, 1 µm).
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Effect of hygromycin on expression of SI, GLUT-1,
GLUT-2,
1-Na+-K+-ATPase,
DPP-IV, and villin. Because SI, GLUT-1, and GLUT-2 have been reported to show the highest variations in relation to the rate of
glucose consumption (4, 6, 16), we chose to analyze the mRNA level of
these three markers in differentiated postconfluent transformed cells
(day 20). In hygromycin-treated
cells, SI and GLUT-2 mRNA levels are considerably reduced, whereas
GLUT-1 levels are considerably increased (Fig.
4). In contrast, no significant differences
were observed between untreated and treated cells with regard to the
levels of
1-Na+-K+-ATPase,
DPP-IV, and villin mRNAs (Fig. 4).

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Fig. 4.
Northern blot analysis of sucrase-isomaltase (SI), GLUT-1, GLUT-2,
1-subunit of
Na+-K+-ATPase,
dipeptidyl peptidase IV (DPP-IV), and villin in transformed TC7 cells
analyzed after 20 days of culture in the absence ( ) or presence
(+) of hygromycin. Same quantified amount of total RNA (20 µg) was
loaded in each lane. Membrane was hybridized simultaneously with probes
for SI and GLUT-1. After dehybridization, the same membrane was
sequentially hybridized with the other probes. Similar results were
obtained in cultures from 2 different passages.
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Consistent with the results of mRNA, sucrase and DPP-IV activities
measured at late postconfluence (day
20) are elevated in transformed cells cultured in the
absence of hygromycin and similar to the values observed in
untransformed cells (Fig. 5). In contrast, in transformed cells cultured in the presence of hygromycin, the activity of sucrase is decreased by ninefold, whereas DPP-IV activity is normal (Fig. 5).

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Fig. 5.
Activity of sucrase and DPP-IV in cell homogenates from postconfluent
cultures (day 20) of control (open
bars) and transformed TC7 cells cultured without (hatched bars) or
under permanent exposure to (solid bars) hygromycin B. Results are the
means of 3 different cultures.
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Also consistent with the low levels of SI mRNA and sucrase
activity in transformed cells cultured in the presence of hygromycin is
the poor expression of the protein as detected by immunofluorescence (Fig. 6). Only a few cells show a discrete
apical expression of SI (Fig. 6d),
contrasting with the strong positivity of the entire apical domain of
the cells cultured without hygromycin (Fig.
6b). Consistent with the high level
of GLUT-1 mRNA, GLUT-1 is expressed in all hygromycin-treated cells
(Fig. 6c), whereas, in transformed cells cultured in drug-free medium, only a few cells, grouped into
clusters, express GLUT-1 (Fig. 6a).
As already observed at the mRNA level, villin (Fig. 6,
e and
g), DPP-IV (Fig. 6,
f and h), and
1-Na+-K+-ATPase
(Fig. 6, j and
l) appear similarly expressed in
both conditions, with this also being true for ZO-1 (Fig. 6,
i and
k).

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Fig. 6.
Double immunofluorescence staining of cryostat sections of the cell
layers from postconfluent cultures (day
20) of transformed TC7 cells cultured without
(a,
b, e,
f, i,
and j) or under permanent exposure
to hygromycin B (c,
d, g,
h, k,
and l);
a and
c: GLUT-1;
b and
d: SI;
e and
g: villin;
f and
h: DPP-IV;
i and
k: ZO-1,
j and
l:
Na+-K+-ATPase.
Bar (l) = 40 µm.
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Effect of hygromycin on plasmid copy
number. The plasmid copy number was assessed during the
culture of transformed TC7 cells grown in the presence or absence of
hygromycin. During the course of these experiments, we found that the
presence of the antibiotic was not necessary to maintain a similar
amount of plasmid, as the same results were obtained with or without
the drug in both dividing (day 4)
and late postconfluent (day 20)
cells (Fig. 7). It must be noted that, even
in the absence of hygromycin, the amount of plasmid was found to be
higher in postconfluent than in dividing cells.

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Fig. 7.
Slot-blot analysis of extrachromosomal DNA, as measured with the
luciferase probe (see MATERIALS AND
METHODS), in exponentially growing
(day 4) and late postconfluent
(day 20) transformed TC7 cells
cultured in the absence ( ) or presence (+) of hygromycin B.
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Reversibility of effects of hygromycin
B. Hygromycin was removed after 7 days of treatment,
i.e., at confluency, and the cells were analyzed 7 days later for the
parameters of glucose utilization. As shown in Table 1, the rates of
glucose consumption and lactic acid production are lower than in cells
maintained in hygromycin, although these rates remain still higher than
in untreated cells. Also, glycogen content is increased but is still
lower than in untreated cells. This is paralleled by an increased
activity of sucrase that even after 7 days, remains, however, much
lower than in cells cultured in the absence of drug from the beginning
of the culture (Fig. 8).

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Fig. 8.
Reversibility of the effect of hygromycin B on the activity of sucrase
in transformed TC7 cells cultured from the beginning of the culture in
the absence (hatched bars) or presence of hygromycin B (solid bars) or
switched from day 7 on from hygromycin
B treatment to drug-free medium (open bars). Activity of sucrase was
measured in the cell homogenates at the indicated days. Results are the
means ± SD of 3 different cultures.
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|
 |
DISCUSSION |
The present results show that permanent exposure to hygromycin B of
stably transformed Caco-2 cells, selected for their resistance to this
antibiotic, results, at late postconfluence (i.e., when the cells are
fully differentiated), in a dramatic increase in the rate of glucose
consumption and lactic acid production, a lower glycogen content, and
marked changes, at both mRNA and protein levels, of the expression of
SI and GLUT-2, which are downregulated, contrasting with an
upregulation of GLUT-1. This occurs without apparent modifications of
the expression of other differentiation-associated gene products, such
as villin, DPP-IV, or the
1-subunit of
Na+-K+-ATPase.
Although hygromycin B induces some degree of morphological changes,
such as a partial disorganization of the brush border, it has no effect
on the polarization of the cells, as substantiated by the apical
expression of villin, DPP-IV, and the tight junction-associated protein
ZO-1 or the basolateral expression of the
Na+-K+-ATPase.
In view of our experience with Caco-2 cells, it is likely that the
changes in the expression of SI, GLUT-2, and GLUT-1 are a consequence,
at the transcriptional level, of the effect of the drug on glucose
consumption, through a mechanism that remains to be elucidated.
Hygromycin B is a member of the aminoglycosides antibiotics, which have
been extensively studied for the mechanisms leading to their
bactericidal effect (for review, see Ref. 10). These antibiotics have
been reported to induce membrane damage in bacteria, which results in a
nonspecific increased permeability to small molecules such as anions,
cations, and small uncharged molecules (10). To date, such an effect
has not been documented in resistant transfected eukaryotic cells,
selected and maintained in the presence of the selecting agent. It has
been shown, however, that in normal kidney cells, i.e., not resistant
transfected cells, the nephrotoxic effect of aminoglycoside antibiotics
is associated with an impairment of the catabolism of phospholipids and
a change in membrane permeability and membrane aggregation (18). More
recently, it has been reported that aminoglycoside antibiotics could
exert their cytotoxic effect through a persistent activation of
phospholipase C (19). Whether alterations of the same metabolic
pathways account for the changes in glucose utilization and
glucose-related modifications of gene expression observed in our study
remains to be investigated. Also questionable is whether other
metabolic pathways or the expression of genes not analyzed here are
also modified as an effect of the drug.
Whatever the answers to these questions, it is clear that the
occurrence of changes in the metabolism of the cells induced by
aminoglycoside-selecting agents during transfection
experiments must be considered. This is particularly true for Caco-2
cells. Indeed, since the first observation of the differentiation
properties of Caco-2 cells (21), a considerable number of studies have been conducted in these cells to analyze the transcriptional regulation of genes involved in the process of intestinal cell differentiation. Because the differentiation process occurs late in the culture, stably
transformed cells obtained after selection and cloning have been
utilized in a number of studies too numerous to be referenced here.
When mentioned, the selecting agent was always present in the cultures
of the stably transformed cells. In studies concerning the effect of
the absence or overexpression of proteins potentially involved in the
differentiation of these cells, SI, because it is exclusively expressed
in the brush border of the intestine (13, 27), has been regularly taken
as a control marker of the differentiation state of Caco-2 cells, as
exemplified in Ref. 8. In these studies, metabolic properties and, more
particularly, the rates of glucose consumption and lactic acid
production of the transformed cells, were never reported. In view of
the dramatic increase of glucose consumption in hygromycin-treated
cells and the demonstrated repressive effect of glucose on the
transcription of the SI gene (25), it is clear that not taking into
account these parameters may lead to false interpretations.
This, of course, does not preclude the utilization of hygromycin B as a
selection agent in transfection experiments. It requires, however,
that, when the selection is completed, cells should be subcultured in
the absence of the drug. In such drug-free conditions, the episomal
plasmid is stably maintained in the transformed cells, as initially
reported in other systems and cell types (32), and, as soon as the
first passage, the normal metabolic status of the cells is restored. In
contrast, when the drug is removed after confluence, the reversibility
of the metabolic modifications and the restoration of gene expression,
as exemplified by the increase in sucrase activity, are very slow.
Whether these differences depend on the more rapid turnover of membrane
constituents in dividing, compared with postconfluent cells, is
questionable. Therefore, in culturing selected transformed cells in the
absence of the selecting agent from the day of seeding and after, one should avoid misleading interpretation of results, such as
assigning to the transfected cDNA or promoter an effect, which, in
fact, could be a consequence of drug-induced modifications of the
metabolic status of the cells.
 |
ACKNOWLEDGEMENTS |
A. Rodolosse was supported by a fellowship from the Ministère
de l'Enseignement Supérieur et de la Recherche. This work was
supported in part by grants from the Association pour la Recherche sur
le Cancer (Grant no. 1393) and Université Paris XI.
 |
FOOTNOTES |
Address for reprint requests: M. Rousset, INSERM U 178, 16 Ave.
Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France.
Received 19 August 1997; accepted in final form 16 January 1998.
 |
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