Department of Biochemistry and Molecular Biology, Faculté de Médecine de Paris-Ouest, Université René Descartes, 75006 Paris, France
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ABSTRACT |
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We studied the influence of nitric oxide (NO) endogenously produced by adipocytes in lipolysis regulation. Diphenyliodonium (DPI), a nitric oxide synthase (NOS) inhibitor, was found to completely suppress NO synthesis in intact adipocytes and was thus used in lipolysis experiments. DPI was found to decrease both basal and dibutyryl cAMP (DBcAMP)-stimulated lipolysis. Inhibition of DBcAMP-stimulated lipolysis by DPI was prevented by S-nitroso-N-acetyl-penicillamine (SNAP), a NO donor. This antilipolytic effect of DPI was also prevented by two antioxidants, ascorbate or diethyldithiocarbamic acid (DDC). Preincubation of isolated adipocytes with DPI (30 min) before exposure to DBcAMP almost completely abolished the stimulated lipolysis. Addition of SNAP or antioxidant during DPI preincubation restored the lipolytic response to DBcAMP, whereas no preventive effects were observed when these compounds were added simultaneously to DBcAMP. Exposure of isolated adipocytes to an extracellular generating system of oxygen species (xanthine/xanthine oxidase) or to H2O2 also resulted in an inhibition of the lipolytic response to DBcAMP. H2O2 or DPI decreased cAMP-dependent protein kinase (PKA) activation. The DPI effect on PKA activity was prevented by SNAP, ascorbate, or DDC. These results provide clear evidence that 1) the DPI antilipolytic effect is related to adipocyte NOS inhibition leading to PKA alterations, and 2) endogenous NO is required for the cAMP lipolytic process through antioxidant-related effect.
antioxidant; adipocyte
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INTRODUCTION |
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NITRIC OXIDE (NO) is an endogenously produced free radical that controls several biological systems. NO is capable of interacting with many cellular targets, including heme and nonheme iron, thiols, oxygen, and superoxide anions (3). Reaction with these targets can result in physiological effects such as the activation of guanylate cyclase or the S-nitrosylation of proteins leading to signaling functions (21, 38, 39). Peroxynitrite is formed by the nearly diffusion-limited reaction of NO with superoxide anions (20) and thus this reaction has an important physiological role in modulating the bioavailability of both NO and superoxide anions. Although NO has an established role as cytotoxic effector molecule and mediator of tissue injury, recent studies demonstrate that NO is able to exert antioxidant and cytoprotective functions (5, 16, 23, 43-45).
NO is synthesized via L-arginine oxidation by a family of nitric oxide synthase (NOS) isoforms (12). We have previously shown that white adipose tissue expresses the NOS-II and NOS-III isoforms (36). A role for NO as a putative regulatory signal controlling lipolysis in rat fat cells was investigated using NO itself and different NO donors of various reactive nitrogen intermediates (13). These experiments provided clear evidence that exogenous NO either activates basal lipolysis or inhibits stimulated lipolysis through cGMP-independent mechanisms that are tightly linked to the redox state of NO (13).
The aim of the present study was to investigate the influence of the NO endogenously produced by adipocytes on lipolysis. For this purpose, we tested the effect of NOS inhibitors on lipolysis. The last rate-limiting step of the adipocyte lipolysis cascade is the hydrolysis of triacylglycerol by hormone-sensitive lipase (HSL), of which its activation requires HSL phosphorylation by the cAMP-dependent protein kinase (PKA). Therefore, NOS inhibitors were tested on basal and also on cAMP-stimulated lipolysis using the nonhydrolyzable cAMP analog, dibutyryl cAMP (DBcAMP).
Here we provide clear evidence that the adipocyte endogenous production of NO is required for the lipolytic activity of this cell and that prolonged NOS inhibition causes dramatic alterations of lipolysis. The prevention of these effects by antioxidants suggests the involvement of NO antioxidant-related properties.
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METHODS |
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Preparation of isolated adipocytes. Male Sprague-Dawley rats (240-250 g), fed ad libitum, were killed by decapitation, and epididymal fat pads were removed for adipocyte isolation. According to a modification (14) of the method of Rodbell (37), 1 g of adipose tissue was digested in a plastic vial with 8 ml of Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) that contained 3% (wt/vol) bovine serum albumin (BSA) and 2 mg collagenase/ml. After 15 min at 37°C under constant shaking, cells were dispersed and filtered through a nylon mesh and washed four times with KRB that contained 1% (wt/vol) BSA. Cell numbers were calculated according to Hirsh and Gallian (19).
Lipolysis studies. Fat cells (1-2 × 105/ml) were incubated during 1 h at 37°C in KRB that contained 5 mM glucose, 2% (wt/vol) BSA, and 50 mU/ml adenosine deaminase in the absence or presence of the effectors to be tested. Lipolysis was stopped by centrifugation (5,000 g, 15 min at 4°C), cells were aspirated, and glycerol release was determined following the enzymatic method of Wieland (42). Results are expressed as nanomoles of glycerol released per hour per 105 cells.
Xanthine oxidase activity.
Xanthine oxidase (XO) activity was measured spectrophotometrically by
the rate of acid uric formation in KRB that contained 2% BSA and 1 mM
xanthine at 305 nm ( = 1.1 × 10
4
M
1 · cm
1) at 37°C.
Measurement of NOS activity in intact adipocytes. NOS activity was measured as the conversion of L-[3H]arginine in L-[3H]citrulline as described by Ribière et al. (36). Fat cells (3-4 × 105/ml) were incubated for 30 min at 37°C in KRB that contained 5 mM glucose, 2% (wt/vol) BSA, 50 mU/ml adenosine deaminase, 50 mM valine (to inhibit arginase), and 1.5 µCi/ml L-[3H]arginine (specific activity: 68 Ci/mmol) in the presence or absence of the inhibitors to be tested. Reaction was stopped by addition of 250 ml ethylic alcohol, followed by 5 ml of 1:1 (vol/vol) H2O/Dowex 50W-X8 (Na+ form) resin, and was left to settle for 10 min. One milliliter of the supernatant was removed and added with liquid scintillation cocktail (Aquasafe 300 plus, Zinsser Analytic) for counting. Each experiment included blanks consisting of KRB only, the value of which was subtracted from all samples.
H2O2 measurement.
H2O2 concentration was determined according to
the method of Allen et al. (1) in which
H2O2 oxidizes I ions to form
I3
ions in the presence of ammonium molybdate
as a catalyst. Solution of H2O2 was prepared in
KRB buffer and then introduced into the adipocyte incubation medium
that contained 2% albumin. At this concentration, albumin was found to
reduce the H2O2 concentration from 1 mM to
0.750 mM.
Assay of PKA activity. Fat cells (500 µl of packed cells) were incubated for 1 h in 4.5 ml KRB that contained 5 mM glucose and 2% (wt/vol) BSA in the presence or absence of the effectors to be tested. The incubation medium was then removed and fat cells were disrupted in 50 mM Tris · HCl, pH 7.8, that contained 0.33 M sucrose, 1 mM MgCl2, and protease inhibitors (10 µM benzamidine and 100 µM phenylmethylsulfonyl fluoride). The homogenates were centrifuged for 5 min at 2,400 g to remove the fat cake. PKA activity was measured according to the method of Raynaud et al. (34) using kemptide, a specific substrate of PKA, as the phosphate acceptor. Twenty microliters of infranatant were used to catalyze the transfer of 32P from ATP (specific activity: 30 Ci/mmol; 0.5 × 106 counts per minute) to 4 µg of kemptide, in the presence of 50 mM MOPS, pH 7.0, 250 µg/ml BSA, 100 µM ATP, 10 mM MgCl2, and appropriate concentrations of cAMP in a total volume of 50 µl. The phosphorylation was allowed to proceed at 37°C for 10 min and was terminated by spotting 25 µl of the reaction mixture onto phosphocellulose P-81 strips (Whatman) that were immediately dropped into ice-cold 5% phosphoric acid (10 ml/paper strip). This was followed by three more 5% phosphoric acid washes with swirling of the strips at 200 rpm for 2 min for each wash. Radioactivity retained on the P-81 papers was determined by counting in scintillation solvent. Background counts determined in the absence of enzyme were subtracted from all experimental values. The level of PKA activity was calculated by subtracting the activity measured in the absence of cAMP from the activity measured in the presence of cAMP and was expressed as picomoles of 32P transferred to kemptide per minute per milligram of lipids in the homogenate.
Materials.
DBcAMP, cAMP, ATP, BSA fraction V (fatty acid free), XO from buttermilk
(grade III), xanthine sodium salt, diethyldithiocarbamic acid (DDC),
S-methylthiourea (SMT),
NG-nitro-L-arginine methyl ester
(L-NAME), catalase from bovine liver, diphenyliodonium
(DPI) chloride, kemptide, and superoxide dismutase (SOD) from bovine
erythrocytes were obtained from Sigma-Aldrich. Ascorbate
(L-ascorbic acid) was purchased from Merck. Adenosine deaminase and glycerol assay kits were purchased from Boehringer. Collagenase type I (267 U/mg) was obtained from Worthington
Biochemicals. H2O2 was obtained from Prolabo,
S-nitroso-N-acetyl-penicillamine (SNAP) from
Cayman (SPI-Bio), Dowex 50W-X8 resin from Bio-Rad, and
L-[3H]arginine and [-32P]ATP
from Amersham Pharmacia Biotech.
Statistical analysis. All results were expressed as the means ± SE of n (number of) separate experiments. Comparisons between groups were made using the paired Student's t-test. A value of P < 0.05 was considered significant.
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RESULTS |
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Effects of NOS inhibitors on basal lipolysis.
To evaluate the participation of endogenously produced NO in the
regulation of basal lipolysis, isolated adipocytes were incubated in
the presence of various NOS inhibitors acting through different mechanisms against NOS. L-NAME, an analog of
L-arginine, SMT, a small nonamino acid-based inhibitor, and
DPI, a flavoprotein inhibitor (40), were tested on basal
lipolysis. As shown in Fig. 1,
L-NAME (2 mM) and its inactive enantiomer
NG-nitro-D-arginine methyl ester
(D-NAME; 2 mM) both increased basal lipolysis, whereas SMT
(1 mM) had no significant effect, and DPI (30 µM) reduced lipolysis
by 20%. To ensure that these effects occurred in parallel with NOS
inhibition, we measured the formation of
L-[3H]citrulline from
L-[3H]arginine in intact adipocytes, a method
that provides a good estimation of NOS activity in intact cells
(17). Contrasting with DPI, which inhibited NOS activity
in a dose-dependent manner (Table 1),
SMT, L-NAME, and D-NAME failed to elicit any
inhibitory effect (results not shown). These findings are consistent
with other reports showing the lack of NO synthesis inhibition by
L-NAME in neutrophils and macrophages (29).
Possible explanations of the failure of L-NAME and SMT to
inhibit NOS activity could be a weak uptake of these NOS inhibitors by
the fat cells or a defective L-NAME bioactivation that is
required for its NOS inhibitory activity in cells (33). It
thus appears that the lipolytic effect of these arginine analogs,
observed regardless of their enantiomeric form, results from mechanisms
other than NOS inhibition that could be related to their oxygen
species' scavenging property (8, 35).
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Effects of DPI on stimulated lipolysis.
Effects of NOS inhibition were investigated on stimulated lipolysis
using 30 µM of DPI, which completely inhibited NOS activity (Table
1), and at first, DBcAMP, as lipolysis stimulator. A 45% decrease in
lipolysis was observed when adipocytes were incubated in the presence
of both DBcAMP and DPI (Fig. 2). It must
be noted that DPI (10 µM) was without significant effect (10%) on
DBcAMP-stimulated lipolysis. To investigate the role of endogenous NO
in this DPI antilipolytic effect, adipocytes were incubated in the
presence of both DPI and SNAP, a NO donor previously shown to unalter
DBcAMP-stimulated lipolysis (13). Under these
conditions, SNAP completely prevented the antilipolytic effect of DPI
(Fig. 2). This finding strongly suggests that the antilipolytic effect
of DPI is related to NOS inhibition. Moreover, the influence of
prolonged NOS inhibition was also investigated. Isolated adipocytes
were first exposed to DPI for 30 min before being stimulated by DBcAMP
for 1 h. Under these conditions, DBcAMP had a weak stimulatory
action on lipolysis (Fig. 3A).
Moreover, addition of SNAP during the incubation with DBcAMP failed to
restore the lipolytic activity of DBcAMP (Fig. 3A). However,
when the preincubation with DPI was performed in the presence of SNAP,
the lipolytic response of DBcAMP was unaltered (Fig. 3B). In
a second set of experiments, we investigated the influence of DPI on
lipolysis stimulated by the
-agonist isoproterenol. The
antilipolytic effect of DPI on DBcAMP-stimulated lipolysis was also
observed on isoproterenol-stimulated lipolysis. DPI inhibition rates
were almost the same with the two lipolytic agents [40% on
isoproterenol-stimulated lipolysis vs. 45% on DBcAMP-stimulated lipolysis when performed without DPI preincubation (Fig. 2) and 73 vs.
75% when adipocytes were preincubated with DPI before lipolysis stimulation (Fig. 3A)]. Prevention by SNAP of
isoproterenol-stimulated lipolysis inhibition by DPI could not be
studied, because we have previously shown that SNAP, per se, inhibits
-adrenergic-stimulated lipolysis (13). The similarity
between the DPI effects on DBcAMP- and isoproterenol-stimulated
lipolysis indicated that the DPI antilipolytic action resulted most
likely from lipolytic pathway alterations located downstream of cAMP
production. Accordingly, DBcAMP was used in all the following
experiments.
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Effects of different antioxidants on the DPI antilipolytic
property.
Involvement of the antioxidant properties of endogenous NO on
cAMP-dependent lipolysis modulation was investigated by testing the
effects of different well-known antioxidant agents on DPI antilipolytic
action. As shown in Fig. 4A,
exposure of isolated adipocytes to 10 mM ascorbate during DPI
incubation or preincubation (Fig. 4C) completely restored
the lipolytic response of these cells to DBcAMP. Moreover, 1 mM DDC, a
copper chelator that inactivates copper-zinc SOD (18),
added simultaneously (Fig. 4A) or during the preincubation
(Fig. 4C) with DPI, totally prevented the DPI inhibition of
DBcAMP-stimulated lipolysis. In contrast, addition of ascorbate or DDC
after the DPI preincubation failed to restore DPI inhibition of
cAMP-dependent lipolysis (Fig. 4B). It must be noted,
however, that ascorbate or DDC alone did not alter DBcAMP-stimulated lipolysis.
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Effects of reactive oxygen species on DBcAMP-stimulated lipolysis.
The experiments described in Fig. 4, A and C,
showed that the inhibitory effect of DPI on cAMP-stimulated lipolysis
can be prevented by some antioxidants. This finding led us to test the influence of reactive oxygen metabolites on DBcAMP-stimulated lipolysis. Exposure of adipocytes to the superoxide anion and H2O2 generating system, XO (0.06 U/ml) plus
xanthine (1 mM), resulted in a decrease in DBcAMP-stimulated lipolysis
(Fig. 5). This effect was prevented by
the addition of catalase (1,000 U/ml) but not of copper-zinc SOD (1,000 U/ml), suggesting that H2O2 was the reactive
oxygen species responsible for partial inhibition caused by the
xanthine/XO system on DBcAMP-stimulated lipolysis (Fig. 5). It is
important to note the observation that catalase or SOD alone failed to
affect DBcAMP-stimulated lipolysis. As shown in Fig. 5, when
H2O2 (750 µM) was added to isolated
adipocytes, the DBcAMP-stimulated lipolysis was decreased (43%),
confirming the modulatory influence of H2O2 on
stimulated lipolysis.
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PKA activity after incubation of isolated adipocytes with DPI or
H2O2.
As a next step, we explored the possible involvement of PKA in the
antilipolytic effect of DPI or H2O2. With the
use of kemptide as a synthetic substrate, we observed a decrease in the
ability of cAMP to stimulate PKA activity in homogenates of isolated
adipocytes after incubation with DPI or H2O2
during 1 h (Fig. 6A). Of
interest is the finding that the incubation of adipocytes with DPI in
the presence of SNAP, ascorbate, or DDC prevented the decrease in cAMP-dependent phosphotransferase activity (Fig. 6B). These
compounds were without significant effect on PKA activity when tested
alone.
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DISCUSSION |
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Although we have previously demonstrated that exogenous NO (NO donors or authentic NO) modulates lipolysis in white adipocytes, a role for endogenous NO production in lipolysis regulation was not investigated (13). The results from the present study strongly suggest that endogenous NO participates in the control of lipolysis. This view is supported by the following lines of evidence. First, with the use of DPI, a potent NOS inhibitor (40) efficient in intact cells (47), including adipocytes as presently shown, we observed inhibition of both basal and stimulated lipolysis either with isoproterenol or DBcAMP. The inhibitory effect on DBcAMP-stimulated lipolysis was prevented by SNAP, a NO donor. We have previously shown that SNAP increases basal lipolysis by phosphodiesterase inhibition and does not affect stimulated lipolysis by DBcAMP (13). With the use of DBcAMP, a nonhydrolyzable analog of cAMP (4), the prevention of the DPI antilipolytic effect by SNAP cannot be related to phosphodiesterase inhibition and indicates that the DPI effect was related to NOS inhibition. Previous reports have underlined the ability of NO to prevent oxidant-induced cell injury provided that cell exposure to oxidative stress and NO occurs simultaneously (7, 16). Our results regarding lipolysis are consistent with these observations. Second, the antilipolytic effect of DPI was also prevented by the antioxidants ascorbate and DDC when added simultaneously, suggesting that inhibition of endogenous NO production leads to an oxidative stress in adipocytes as it does in endothelial cells (32). In the latter cells, the intracellular oxidative stress following NOS inhibition was also prevented by NO donors or by antioxidants. In vitro, NO can act as an antioxidant to prevent prooxidative reactions linked to H2O2 (23). In vivo, a critical role has been postulated for NO in protecting mammalian cells from toxic reactive oxygen species (5, 16, 43-45). Furthermore, inhibition of NO production was reported to promote endothelial cell sensitization to H2O2-induced injury (16). Despite these various and convergent observations, the mechanism whereby NO elicits antioxidant properties remains yet to be fully defined. In consideration of the preventive effect of antioxidants against the DPI antilipolytic effect, it appears unsuitable to use NOS inhibitors that have oxygen-scavenging properties such the arginine analogs NAME and NG-monomethyl-L-arginine, regardless of their enantiomeric forms (8, 35).
As reported in this paper, the antilipolytic action of DPI was prevented by ascorbate. Such a prevention could be related to the well-known scavenging properties of ascorbate toward oxygen species like superoxide anions or hydroxyl radicals (15). Two recent studies reported that high ascorbate concentrations are able to scavenge superoxide anions during oxidant stress in aorta (10, 22) and thus restore the NO-dependent relaxation (10). DDC possesses antioxidant properties, as well, and one of its main properties is to inhibit copper-zinc SOD via copper chelation (18). The scavenging of H2O2 or superoxide anions by DDC has also been reported (28). For these reasons, prevention of the antilipolytic effect of DPI by antioxidants could be linked either indirectly to the inhibition of H2O2 formation, resulting from superoxide anions scavenging by ascorbate or SOD inhibition by DDC, or directly to H2O2 scavenging by DDC. The reaction rate between superoxide anions and NO occurs at the near diffusion-limited rate (20), and NO is the only biological molecule produced in high enough concentrations to outcompete SOD for superoxide anions (3). Therefore, the lipolysis control by endogenous NO production seems to be related to controlled H2O2 formation in the fat cell or to protection against H2O2 oxidant effects. A crucial role has been assigned to H2O2 in the fat cell physiology (25, 30, 31). Previous works demonstrated that rat and human adipocytes possess a plasma-membrane bound H2O2-generating system (NADPH oxidase) that is activated by the antilipolytic hormone insulin (24, 26, 31). It is noteworthy that stimulus-sensitive NADPH oxidase in fat cell plasma-membrane is not inhibited by iodonium compounds (e.g., DPI) (24) that are potent inhibitors of the respiratory burst oxidase in others cells (6). Therefore, NADPH oxidase remains a potential source of reactive oxygen species during NOS inhibition by DPI.
When fat cells were exposed to an extracellular generating system of reactive oxygen species or H2O2, we observed a reduction of the lipolytic response to DBcAMP. Although the H2O2 concentration employed here most likely exceeded the intracellular H2O2 physiopathological range, it was comparable to those used in other studies (24, 30). In isolated fat cells, such exogenously added H2O2 concentrations were reported to mimic insulin inhibition of hormone-stimulated lipolysis (24, 27, 30). The present data obtained with DBcAMP-stimulated lipolysis excludes the possibility that the antilipolytic effect of H2O2 results from cAMP-phosphodiesterase activation (41) because DBcAMP is resistant to hydrolytic cleavage catalyzed by cAMP-phosphodiesterase (4). On the other hand, H2O2 could alter the following processes: the PKA activation and/or the PKA-dependent HSL phosphorylation. Consistent with these hypotheses are recent observations showing that reactive oxygen species modified PKA activation (9, 34). The decrease in PKA activity observed after incubation of isolated adipocytes with H2O2 indicates that PKA could be a target implicated in the antilipolytic effect of H2O2. Our finding that the DPI antilipolytic effect is prevented by several antioxidant agents further suggests that PKA is a target for reactive oxygen species like H2O2 in the fat cells. The present finding that cAMP-dependent phosphotransferase activity and cAMP-dependent lipolysis are both decreased after NOS inhibition by DPI but restored by either SNAP, ascorbate, or DDC strengthens the hypothesis that the antilipolytic effect of DPI is related to oxidative alterations of PKA. The HSL activation by PKA is poorly understood but presumably involves both the translocation of HSL from the cytosol to the lipid droplet and conformational changes in the HSL molecule (11). Thus the regulation of HSL, the rate-limiting enzyme of lipolysis, involves several steps that are not evaluated by the in vitro assay of HSL activity. However, such alterations of PKA activity after NOS inhibition should indeed reduce HSL phosphorylation and its activation, and as the consequence, lipolysis.
In conclusion, the present study indicates that NO acts as a regulatory signal in the lipolysis control in fat cells. Our previous work (13) using NO donors in isolated rat adipocytes have shown that high concentrations of NO elicit a marked antilipolytic effect of stimulated lipolysis through distinct mechanisms that are tightly linked to the redox state of NO. Moreover, it was shown that in vivo NO is involved in the regulation of lipolysis in humans (2). Indeed, the inhibition of NO release by human subcutaneous adipose tissue resulted in an increased lipolysis (2). However, from these experiments, it is impossible to draw a conclusion on the role of the adipocyte endogenous NO production in the regulation of lipolysis since adipose tissue also contained nonadipose cells. Moreover, the presence of antioxidants in the extracellular fluid may also interfere with the antioxidant properties of NO and, therefore, with the NO regulation of lipolysis. As revealed by the present study, adipocyte endogenous NO production is required for the cAMP and isoproterenol-stimulated lipolytic responses. Evidence from several studies revealed that low concentrations of NO can act protectively against reactive oxygen species-associated injury, whereas high concentrations of NO may be toxic (46). Thus the participation of endogenous NO in the lipolytic process appears to be linked to its antioxidant-related activity, preventing PKA from damages caused by reactive oxygen species.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the University René Descartes (Paris V).
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. Ribière, Dept. of Biochemistry and Molecular Biology, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France.
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.
Received 17 April 2000; accepted in final form 22 June 2000.
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