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Eur. J. Biochem. 148,441 -445 (1985) 0 FEBS 1985 NADPH oxidation catalyzed by the peroxidase/H,O, system Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADP+ Jean Luc MICHOT, Alain VIRION, Daniele DEME, Simone De PRAILAUNE and Jacques POMMIER Unite de Recherche sur la Glande Thyroide et la Rtgulation Hormonale, Institut National de la Sante et de la Recherche Mtdicale, Bicttre (Received November 26, 1984/February 4, 1985) - EJB 84 1242 We have examined the respective roles played by guaiacol and scopoletin in NADPH oxidation catalyzed by the peroxidase/H202system. It was shown that NADPH was not oxidized by either the horseradish or lactoperoxidase/H202 systems alone; oxidation occurred immediately after the addition of guaiacol or scopoletin. In both cases, the oxidation product was enzymatically active NADP' . Differences were observed in the NADPH oxidation mechanism depending on whether guaiacol or scopoletin was the mediator molecule. In guaiacol-mediated NADPH oxidation, the stoichiometry between HzOz and oxidized NADPH was about 1; superoxide dismutase did not affect the oxidation rate. In scopoletin-mediated oxidation, the stoichiometry was much higher (1 : 14 in the present experiments); superoxide dismutase considerably increased the oxidation rate. It is concluded that catalysis of NADPH oxidation by the horse radish peroxidase/H202 system requires the presence of a mediator molecule. The NADPH oxidation mechanism depends on the intermediary oxidation state of this molecule. The pyridine nucleotides NADH and NADPH are known to be involved in a large number of oxidation-reduction reac- tions as well as in many biological H202-generating systems (for review, see [l]). In leukocytes [2, 31 and thyroid [4- 61, the NADPH-de- pendent H202-generatingsystem is in some way associated with a peroxidase. Although the NADPH oxidase and the peroxidase are probably compartmentalized in vivo, a linkage could exist between NADPH oxidation and the peroxidase/ H202 system. The different processes of NADPH and NADH peroxidase oxidation are of considerable biochemical interest [7]. The initial findings for peroxidase demonstrated that it can catalyze the aerobic oxidation of the two reduced pyridine nucleotides [8 - 111 and that this oxidation is stimulated by phenolic compounds [12, 131. Nucleotide oxidation has also been studied in the peroxidase/H202 system, especially with horseradish peroxidase (HRP). Whereas oxidation of NADH or NADPH in the peroxidase/H202 system was very low, it was greatly enhanced by the addition of thyroxine [9, 141 or hemato- porphyrin [15]. In the peroxidase/H202 system, nucleotide oxidation required the presence of a mediating molecule and consequently the mechanism of thyroxine-mediated NADH oxidation by HRP/H202 was studied [14]. In addition, oxida- tion of different substrates like scopoletin [16] or phenol red [17] by the peroxidase/H202system is often used to measure H202, and the presence of NADPH might interfere with such measurements [l]. Correspondence to J. Pommier, Unite de Recherche sur la Glande Thyroide et la Rtgulation Hormonale, INSERM, 78 Rue du General Leclerc, F-94270 Bicttre, France Abbreviations. HRP, horseradish peroxidase; Glc6P, glucose 6- phosphate. Enzymes. Horseradish peroxidase and lactoperoxidase (EC 1.1 1.1.7); glucose-6-phosphate dehydrogenase (EC 1 .I .I .49); glucose oxidase (EC 1.1.3.4). In the present experiments, we show that guaiacol and scopoletin are mediating molecules in NADPH oxidation catalyzed by horseradish and lactoperoxidase. Although the NADPH oxidation product is always NADP', the mech- anism of NADPH oxidation depends on the intermediary oxidation state of the mediating molecule. MATERIALS AND METHODS Products Chemicals used in the study were obtained from the following sources: NADPH, type I superoxide dismutase, lactoperoxidase (Rz = 0.706), scopoletin (7-hydroxy-6- methoxy-coumarin) and type VII ~-glucose-6-phosphate de- hydrogenase from Sigma; grade I horseradish peroxidase (HRP) and grade I glucose oxidase from Boehringer; D-glu- cose and guaiacol from Prolabo; Perhydrol (30%, H202) from Merck. Experimental procedures The assay of NADPH, guaiacol and scopoletin oxidation were carried out in a standard incubation mixture comprising 50 mM phosphate buffer, pH 7.2 at room temperature. Ex- perimental conditions are described in detail in the legends to the figures. NADPH oxidation was monitored at 340 nm, using a molar absorption coefficient of 6.2 x lo3 M-' cm-', either in a Cary model 15 or Zeiss PMQ2 spectrophotometer. Guaiacol oxidation and the resulting tetraguaiacol were assayed by the absorption of the latter at 470 nm in a Zeiss PMQ2 spectrophotometer. Oxidation of scopoletin was followed fluorometrically in a Perkin Elmer MPF 43A spectrofluorometer by measuring the decrease in scopoletin fluorescence in the presence of horseradish peroxidase [6, 161. The excitation wavelength was 360 nm and the emission wavelength 460 nm.

NADPH oxidation catalyzed by the peroxidase/H2O2 system : Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADP+

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Page 1: NADPH oxidation catalyzed by the peroxidase/H2O2 system : Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADP+

Eur. J. Biochem. 148,441 -445 (1985) 0 FEBS 1985

NADPH oxidation catalyzed by the peroxidase/H,O, system Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADP+

Jean Luc MICHOT, Alain VIRION, Daniele DEME, Simone De PRAILAUNE and Jacques POMMIER Unite de Recherche sur la Glande Thyroide et la Rtgulation Hormonale, Institut National de la Sante et de la Recherche Mtdicale, Bicttre

(Received November 26, 1984/February 4, 1985) - EJB 84 1242

We have examined the respective roles played by guaiacol and scopoletin in NADPH oxidation catalyzed by the peroxidase/H202 system.

It was shown that NADPH was not oxidized by either the horseradish or lactoperoxidase/H202 systems alone; oxidation occurred immediately after the addition of guaiacol or scopoletin. In both cases, the oxidation product was enzymatically active NADP' . Differences were observed in the NADPH oxidation mechanism depending on whether guaiacol or scopoletin was the mediator molecule. In guaiacol-mediated NADPH oxidation, the stoichiometry between HzOz and oxidized NADPH was about 1; superoxide dismutase did not affect the oxidation rate. In scopoletin-mediated oxidation, the stoichiometry was much higher (1 : 14 in the present experiments); superoxide dismutase considerably increased the oxidation rate. It is concluded that catalysis of NADPH oxidation by the horse radish peroxidase/H202 system requires the presence of a mediator molecule. The NADPH oxidation mechanism depends on the intermediary oxidation state of this molecule.

The pyridine nucleotides NADH and NADPH are known to be involved in a large number of oxidation-reduction reac- tions as well as in many biological H202-generating systems (for review, see [l]).

In leukocytes [2, 31 and thyroid [4- 61, the NADPH-de- pendent H202-generating system is in some way associated with a peroxidase. Although the NADPH oxidase and the peroxidase are probably compartmentalized in vivo, a linkage could exist between NADPH oxidation and the peroxidase/ H 2 0 2 system. The different processes of NADPH and NADH peroxidase oxidation are of considerable biochemical interest [7]. The initial findings for peroxidase demonstrated that it can catalyze the aerobic oxidation of the two reduced pyridine nucleotides [8 - 111 and that this oxidation is stimulated by phenolic compounds [12, 131.

Nucleotide oxidation has also been studied in the peroxidase/H202 system, especially with horseradish peroxidase (HRP). Whereas oxidation of NADH or NADPH in the peroxidase/H202 system was very low, it was greatly enhanced by the addition of thyroxine [9, 141 or hemato- porphyrin [15]. In the peroxidase/H202 system, nucleotide oxidation required the presence of a mediating molecule and consequently the mechanism of thyroxine-mediated NADH oxidation by HRP/H202 was studied [14]. In addition, oxida- tion of different substrates like scopoletin [16] or phenol red [17] by the peroxidase/H202 system is often used to measure H202, and the presence of NADPH might interfere with such measurements [l].

Correspondence to J. Pommier, Unite de Recherche sur la Glande Thyroide et la Rtgulation Hormonale, INSERM, 78 Rue du General Leclerc, F-94270 Bicttre, France

Abbreviations. HRP, horseradish peroxidase; Glc6P, glucose 6- phosphate.

Enzymes. Horseradish peroxidase and lactoperoxidase (EC 1.1 1.1.7); glucose-6-phosphate dehydrogenase (EC 1 .I .I .49); glucose oxidase (EC 1.1.3.4).

In the present experiments, we show that guaiacol and scopoletin are mediating molecules in NADPH oxidation catalyzed by horseradish and lactoperoxidase. Although the NADPH oxidation product is always NADP', the mech- anism of NADPH oxidation depends on the intermediary oxidation state of the mediating molecule.

MATERIALS AND METHODS

Products

Chemicals used in the study were obtained from the following sources: NADPH, type I superoxide dismutase, lactoperoxidase (Rz = 0.706), scopoletin (7-hydroxy-6- methoxy-coumarin) and type VII ~-glucose-6-phosphate de- hydrogenase from Sigma; grade I horseradish peroxidase (HRP) and grade I glucose oxidase from Boehringer; D-glu- cose and guaiacol from Prolabo; Perhydrol (30%, H202) from Merck.

Experimental procedures

The assay of NADPH, guaiacol and scopoletin oxidation were carried out in a standard incubation mixture comprising 50 mM phosphate buffer, pH 7.2 at room temperature. Ex- perimental conditions are described in detail in the legends to the figures. NADPH oxidation was monitored at 340 nm, using a molar absorption coefficient of 6.2 x lo3 M-' cm-', either in a Cary model 15 or Zeiss PMQ2 spectrophotometer.

Guaiacol oxidation and the resulting tetraguaiacol were assayed by the absorption of the latter at 470 nm in a Zeiss PMQ2 spectrophotometer. Oxidation of scopoletin was followed fluorometrically in a Perkin Elmer MPF 43A spectrofluorometer by measuring the decrease in scopoletin fluorescence in the presence of horseradish peroxidase [6, 161. The excitation wavelength was 360 nm and the emission wavelength 460 nm.

Page 2: NADPH oxidation catalyzed by the peroxidase/H2O2 system : Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADP+

442

0.60 - : 0 v m

c 0

a, U C

0 0.55 e -

m n Q

0.50 -

0 10 20 30 LO 50 60 70 lH,O,l lnmol)

B 0.10 I-

Time Imin)

Fig. 1 . Kinetic comparison of NADPH oxidation ( A ) and guaiacol oxidation ( B ) catalyzed by the H R P / H 2 0 2 system. Both assays were performed under the same experimental conditions (see Materials and Methods). Samples contained 0.1 mM NADPH, 10 pg HRP, 0.1 mM guaiacol, 6 mM glucose, 0.8 pg glucose oxidase in 1 ml of 50 mM phosphate buffer pH 7.2. The reaction was started by addition of glucose oxidase under the following different conditions: in the pres- ence of NADPH + guaiacol (0-0), in the absence of guaiacol (A-A) and in the absence of NADPH (0-0). In the inset, the H202-generating system (glucose + glucose oxidase) was replaced by H202. Different amounts of H 2 0 2 were added to the samples (1 ml) containing 0.1 mM NADPH, 10 pg HRP and 10 pM (0-0) or 0.2 mM (0-0) guaiacol. The amount of NADPH oxidized was determined by the decrease in absorbance at 340 nm

RESULTS

Guaiacol-mediated NA DPH oxidation

The data in Fig. 1 A demonstrate that NADPH oxidation can be catalyzed by horseradish peroxidase and an H202- generating system in the presence of guaiacol, a phenolic substrate for peroxidase. In the absence of guaiacol, oxidation of NADPH did not occur, suggesting that NADPH oxidation is mediated by the intermediary formation of oxidized guaiacol, as follows:

HRP + HzO2 + HRPI + HzO

HRPI + AH 4 A+ + HRP

A' + NADPH + NADP' + AH

(1)

(2) (3)

Fig. 1 B clearly shows that under the same experimental conditions, guaiacol oxidation was inhibited but not

0 . 1 1 , , , , , 0 1 2 3 L

Time Imin)

Fig. 2. Kinetics of guaiacol-mediated NADPH oxidation catalyzed by HRP in the presence (0-0) or absence (0-0) of superoxide dismutase. Samples contained 0.1 mM NADPH, 0.1 mM guaiacol, 25 l g HRP, 0.2 mM H 2 0 2 and 50 pg superoxide dismutase in 1 ml of 50 mM phosphate buffer pH 7.2. After 3 min of incubation, 5 mM glucose 6-phosphate and 5 pg glucose-6-phosphate dehydroge- nase were added to the assay (arrow). Dashed line represents the assay in the absence of H2O2

completely abolished by NADPH, suggesting competition be- tween NADPH oxidation (Eqn 3) and tetraguaiacol forma- tion.

The same conclusions can be drawn by measuring the amount of NADPH oxidized as a function of the HzOz concentration (Fig. 1 A, inset). Thus, the amount of oxidized NADPH obtained at the end of the reaction was directly proportional to the amount of HzOz added in the medium. However, the amount of oxidized NADPH was smaller than that of H 2 0 2 and depended on the guaiacol concentration, indicating that a fraction of guaiacol proportional to the H 2 0 2 concentration was also irreversibly oxidized.

In the experiment illustrated in Fig. 2, NADPH oxidation catalyzed by the HRP/HzO2 system in the presence of guaiacol was kinetically followed at 340 nm in the presence or absence of superoxide dismutase. The latter had no effect on the rate of NADPH oxidation. When most of the NADPH has been oxidized, addition of an NADPH-generating system (Glc6P + Glc6P dehydrogenase) allowed the formation of NADPH from oxidized NADPH, showing that the product of the guaiacol-mediated NADPH oxidation in the HRP/ H 2 0 2 system is NADP'. Similar results were obtained with lactoperoxidase (not shown).

Scopoletin-mediated NAD PH oxidation

As shown fdr guaiacol, the oxidation of NADPH in the HRP/HZOz system can be mediated by scopoletin. Fig. 3 shows that NADPH oxidation was obtained in a system containing HRP, an H202-generating system and scopoletin, whereas no oxidation occurred in the absence of scopoletin. 1 pM scopoletin was sufficient to produce rapid oxidation of NADPH, and when the scopoletin concentration was raised the rate of oxidation increased.

Measurement of the total amount of NADPH oxidized as a function of the H 2 0 2 concentration showed that, in contrast to the guaiacol-mediated oxidation of NADPH, much more than one mole NADPH was oxidized per mole of H 2 0 2 (Fig. 3, inset). In the present experiment the stoichiometry between oxidized NADPH and HzOz was 14:l . Similar re- sults were obtained with lactoperoxidase (not shown) except

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443

0.L -

0 1 2 3 1 5 6 7 8 IH@,l lnrnol) I I

0 0.5 1.0 1.5 Time lrnin)

Fig. 3. Kinetics of scopoletin-mediated NADPH oxidation catalyzed by the HRP/H2O2 system. Samples contained 0.1 mM NADPH, 10 pg HRP, 0.6 mM glucose, 0.4 pg glucose oxidase in 1 ml of 50 mM phosphate buffer pH 7.2. The reaction was started by addition of glucose oxidase under the following different conditions: in the absence of scopoletin (A-A) and in the presence of scopoletin 1 pM (0-O), 2 pM (0-0) or 4 pM (A-A). In the inset, the H202-generating system (glucose + glucose oxidase) was replaced by H202. Different amounts of H 2 0 2 were added to the sample (1 ml) containing 0.1 mM NADPH, 10 pg HRP and 1 pM (0-0) or 4 pM (0-0) scopoletin

that the affinity of lactoperoxidase for scopoletin was much lower.

Using a fluorescence technique, the oxidation of scopoletin was also measured as a function of the HzO2 concentration with various amounts of NADPH (Fig. 4). It appears that in the HRP/H202 system scopoletin oxidation was inhibited by NADPH. The inhibition depended on the NADPH concentration and was obtained even with an NADPH concentration as low as 0.15 pM (inset to Fig. 4). When hydrochloric acid or a-oxoglutamate + glutamate de- hydrogenase was added before H202 to the assay containing HRP, NADPH and scopoletin, NADPH was destroyed and the amount of scopoletin oxidized returned to the control value.

On the basis of these results, it is clear that, as found with guaiacol, scopoletin acts as a mediator in NADPH oxidation catalyzed by the HRP/H202 system. NADPH oxidation might take place by the reactions (2) and (3) with, in addition, irreversible oxidation of a scopoletin fraction. However, in that case one mole of H 2 0 2 would be necessary to oxidize one mole of NADPH. A much higher proportion of NADPH was oxidized per mole of H 2 0 2 in the presence of scopoletin than of guaiacol. This finding is not compatible with Eqns ( 2 ) and (3). Similar observations were made by Takayama and Nakano [14], who showed that in the HRP/H202 system, the initial step in thyroxine-mediated NADH oxidation was the oxidation of thyroxine as a phenoxy radical, which in turn oxidized NADH to NAD", which then rapidly reduced O2 to O;, thus:

A" + NADH + AH + NAD"

NAD" + O 2 + NAD+ + 0, (4)

(5)

0; +O; + 2 H f - + 0 2 + H 2 0 2 . (6)

0; undergoes spontaneous dismutation producing H 2 0 2 :

H 2 0 2 so produced permits oxidation of a second mole of NADH which in turn produces another mole of HzOZ.

If these results are applied to the present experiments, the oxidation product of NADPH would be NADP' and

- - 0 [NADPHI IpMI - E u 0

U X 0

C

aJ 0 a 0

.N 0.2 - ._

._ ..- -

0 0.1 0.2 0.3 0.L lH,O,I Inrnoll

Fig. 4. Relationship between scopoletin oxidation and the H 2 0 2 concentration. Different amounts of H z 0 2 were added to the samples containing 20 pg HRP, 0.53 pM scopoletin and various NADPH concentrations in 1 ml 50 mM phosphate buffer pH 7.2. Scopoletin oxidation was measured as the percentage of the fluorescence decrease in the absence (0-0) or presence of 1.5 pM (0-0) or 15 pM (0-0) NADPH. Inset shows the inhibition of scopoletin oxidation as a function of the NADPH concentration. The assay contained 2.6 nmol H z 0 2 and various concentration of NADPH

intermediary 0; formation would occur. To test this hypo- thesis, the kinetics of NADPH oxidation mediated by scopoletin in the HRP/H202 system were followed (Fig. 5). When most of the NADPH had been oxidized, addition of Glc6P and Glc6P dehydrogenase induced regeneration of NADPH, showing that the oxidized NADPH species is NADP'. Fur- ther, in contrast to the guaiacol-mediated oxidation of NADPH, addition of superoxide dismutase efficiently in- creased the rate of NADPH oxidation, suggesting in- termediary formation of 0;.

Page 4: NADPH oxidation catalyzed by the peroxidase/H2O2 system : Guaiacol-mediated and scopoletin-mediated oxidation of NADPH to NADP+

444

0 1 2 3 L Time (m id

Fig. 5. Kinetics of scopoletin-mediated NADPH oxidation catalyzed by HRP in the presence (0-0) or absence (.-a) of superoxide dismutase. Samples contained 0.1 mM NADPH, 25 pg HRP, 0.53 pM scopoletin, 20 pM HzOz and 50 pg superoxide dis- mutase in 1 ml of 50 mM phosphate buffer pH 7.2. After 3 min of incubation, 5 mM glucose 6-phosphate and 5 pg glucose-6-phosphate dehydrogenase were added to the assay (arrow)

Thus, the mechanism of scopoletin-mediated NADPH oxidation in the HRP/H202 system closely resembles that of thyroxine-mediated NADH oxidation.

These results imply the following sequence of events : monovalent oxidation of scopoletin by the HRP/H202 system (Eqns 7 and S), attack of NADPH by monooxidized scopoletin leading to NADP" formation (Eqn 9), and produc- tion of 0; from NADP" and O 2 (Eqn lo), the final product being H202 (Eqn 6):

HRPI + AH + A" + HRPII

HRPII + AH -+ A" + HRP

A" + NADPH + NADP" + AH

(7) (8)

(9)

(10) NADP" + O2 + NADP' + 0;

Theoretically, in this scheme, one mole of H 2 0 2 would be sufficient to oxidize NADPH completely, provided that O2 is present. However, because H 2 0 2 was used to form irreversibly oxidized scopoletin and concomitantly 0 t was trapped by free HRP, the reaction stopped before complete oxidation of NADPH. Addition of superoxide dismutase increased the rate of NADPH oxidation by increasing the rate of 0; dismuta- tion, thus preventing both the trapping of 0; by free HRP and the inhibition of HRP by 0; .

DISCUSSION The experiments described here clearly demonstrate that

the well known peroxidase substrates guaiacol and scopoletin act as catalysts for NADPH oxidation in the peroxidase/ H 2 0 2 system. Under our enzymatic conditions, NADPH was completely stable in the presence of the peroxidase/H202 system and in the absence of guaiacol or scopoletin.

NADP+ formation from NADPH requires two equiva- lents of oxidation. The two electrons can be removed simulta- neously (Eqn 11) or stepwise (Eqn 12):

- 2 e -

NADPH--NADP+ (1 1) - e -

NADPH-~NADPO-NADP+ . (12)

However, under aerobic conditions, the one-electron oxidized form of NADPH (NADP") has been shown to react with O2 [ll, 181 to form a superoxide anion radical (Eqn 10).

As it has been clearly established that the initial step in NADPH oxidation is mediator oxidation by the peroxidase/ H 2 0 z system, it is conceivable that the mechanism of this oxidation depends on the intermediary oxidation state of the mediator molecule.

According to the conventional mechanism described by George and Chance [19, 201 for horseradish peroxidase, radical forms of the substrate are produced:

HRPI + AH + HRPII + A"

HRPII + AH + HRP + A" (1 3)

(14)

2A" + A2. (15) However, if the substrate is a two-electron donor, a direct

two-electron transfer takes place, as described for iodide [21], sulfite [22] and ethanol [23] and does not involve compound 11.

HRPI + AH -+ HRP + A'.

With p-cresol [24] and guaiacol [25] another but different type of exception to the conventional mechanism was demon- strated. It was proposed that two electrons are transferred from these phenols to compound I with the intermediary formation of compound 11.

In the mechanism of guaiacol oxidation by HRP, de- scribed by Santimone [25], in addition to (Eqn 13), the following reactions were proposed:

HRPI + A" + HRPII + A' (17)

HRPII + A" -+ HRP + A'. (18) It is generally accepted that oxidized radical forms are

unstable and rapidly disappear, either by dimerization or fur- ther oxidation.

Thus, when the mediator is a two-electron donor, or as for guaiacol, an intermediary dioxidized form is produced (A'), NADPH is probably oxidized by simultaneous removal of the two electrons:

(19) Under these conditions, the stoichiometric relationship of

H20z/oxidized NADPH would be 1 : 1. In contrast, if the mediator is unable to produce a dioxidized form or if the intermediary radical form has a longer life time, NADPH is no doubt oxidized into a free radical species (Eqn 9) and the reaction (Eqn 10) occurs producing 0;.

In this mechanism, H 2 0 2 is produced and theoretically, if 0; is not completely trapped by HRP, one mole of H 2 0 2 would be sufficient to completely oxidize one mole of NADPH. Thus the mechanism of the molecule-mediated NADPH oxidization is dependent on the intermediary oxida- tion state of the mediator molecule.

In addition, while the scopoletin,,/H202 ratio was near 1 in the absence of NADPH, this ratio was greatly reduced in the presence of low NADPH concentration. A similar obser- vation was previously made with NADH [26]. NADPH is involved in many biological H202-generating systems and the most common and sensitive method for H z 0 2 measurement is based on the loss of scopoletin fluorescence in the presence of horseradish peroxidase and H 2 0 2 11 51. Our experiments show that with the scopoletin method of measuring H202, NADPH must be eliminated or diminished to an insignificant concentration to avoid interference with the assay.

(16)

A' + NADPH 4 AH + NADP'.

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445

The authors wish to thank Mrs C. Sais, A. Guedec and M. M. Bahloul for the preparation of the manuscript. This work was supported by a grant from the Fondation pour la Recherche Medicale.

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