References of "Fillet, Marianne"
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See detailDe novo C16- and C24-ceramide generation contributes to spontaneous neutrophil apoptosis.
Seumois, Gregory; Fillet, Marianne ULiege; Gillet, Laurent ULiege et al

in Journal of Leukocyte Biology (2007), 81(6), 1477-1486

Neutrophils rapidly undergo spontaneous apoptosis following their release from the bone marrow. Although central to leukocyte homeostasis, the mechanisms that regulate neutrophil apoptosis remain poorly ... [more ▼]

Neutrophils rapidly undergo spontaneous apoptosis following their release from the bone marrow. Although central to leukocyte homeostasis, the mechanisms that regulate neutrophil apoptosis remain poorly understood. We show here that apoptosis of cultured neutrophils is preceded by a substantial increase in the intracellular levels of 16 and 24 carbon atom (C(16)- and C(24))-ceramides, which are lipid second messengers of apoptosis and stress signaling. Treatment of neutrophils with fumonisin B(2), a selective inhibitor of the de novo pathway of ceramide synthesis, prevented accumulation of C(16)- and C(24)-ceramides. Moreover, fumonisin B(2) significantly reduced caspase-3, -8, and -9 activation and apoptosis in these cells. Conversely, 3-O-methylsphingomyelin and fantofarone, which are specific inhibitors of neutral and acid sphingomyelinases, respectively, neither inhibited C(16)- and C(24)-ceramide production nor decreased the apoptosis rate in neutrophils, indicating that in these cells, ceramides are not generated from membrane sphingomyelin. Further experiments showed that increasing endogenous C(16)- and C(24)-ceramide levels by using DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol and (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, two inhibitors of ceramide metabolism, enhances caspase-3, -8, and -9 activity and increases neutrophil apoptosis. Similarly, apoptosis was induced rapidly when synthetic C(16)- and/or C(24)-ceramides were added to neutrophil cultures. Finally, GM-CSF, a cytokine that delays neutrophil apoptosis, abrogated C(16)- and C(24)-ceramide accumulation totally in cultured neutrophils, whereas Fas ligation accelerated apoptosis in these cells without affecting de novo ceramide production. We conclude that de novo generation of C(16)- and C(24)-ceramides contributes to spontaneous neutrophil apoptosis via caspase activation and that GM-CSF exerts its antiapoptotic effects on neutrophils, at least partly through inhibition of ceramide accumulation. [less ▲]

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See detailDevelopment of a mass spectrometry method for the determination of a melanoma biomarker, 5-S-cysteinyldopa, in human plasma using solid phase extraction for sample clean-up.
Martin, Gaëlle ULiege; Chiap, Patrice ULiege; Paquet, Philippe ULiege et al

in Journal of Chromatography. A (2007), 1156(1-2), 141-8

5-S-cysteinyldopa is a well-known pigment intermediate and analysis of its plasma concentration is interesting for the early diagnosis, as well as for evaluation of treatment and follow-up of malignant ... [more ▼]

5-S-cysteinyldopa is a well-known pigment intermediate and analysis of its plasma concentration is interesting for the early diagnosis, as well as for evaluation of treatment and follow-up of malignant melanoma. A determination method of 5-SCD in human plasma was developed using solid phase extraction (SPE) on disposable cartridges and liquid chromatography electrospray mass spectrometry (LC-ESI-MS-MS). Compound's sensitivity to light and oxidation requires the addition of anti-oxidative agents (AO), to work in acidic media at 4 degrees C and to avoid light exposure of samples since blood collection. Different solid phases involving covalent binding to phenylboronic groups or dual retention mechanisms were evaluated and extraction cartridges containing both hydrophobic and strong cation exchange functionalities were finally selected. The LC separation of 5-SCD from endogenous catecholamines was achieved by gradient elution on a C18 stationary phase. 5-SCD was detected by multiple reaction monitoring (MRM) performed on ES(+) generated ions. Finally, the method was prevalidated in the lower ng/ml range. Good results with respect to accuracy, trueness and precision were obtained. [less ▲]

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See detailRobustness testing of a chiral NACE method for R-timolol determination in S-timolol maleate and uncertainty assessment from quantitative data.
Marini Djang'Eing'A, Roland ULiege; Rozet, Eric ULiege; Vanderheyden, Yvan et al

in Journal of Pharmaceutical & Biomedical Analysis (2007), 44(3), 640-51

A robustness test of a capillary electrophoresis method for the chiral separation of timolol in nonaqueous acidified media was performed. A two-level Plackett-Burman design was applied in which one ... [more ▼]

A robustness test of a capillary electrophoresis method for the chiral separation of timolol in nonaqueous acidified media was performed. A two-level Plackett-Burman design was applied in which one qualitative and six quantitative factors were examined. Resolution, migration times and relative migration times to pyridoxine (selected as internal standard) were examined as qualitative responses to evaluate electrophoretic performance. A quantitative response, the content of R-timolol in S-timolol maleate sample, was also considered. Even though some significant factor effects were observed on the qualitative responses, it was still possible to quantify the R-timolol in the S-timolol maleate samples properly. The quantitative response was not significantly affected by the selected factors, demonstrating the robustness of the procedure. However, the use of different HDMS-beta-CD batches seemed to affect both types of responses necessitating to introduce a warning in the procedure. Since the experiments of the Plackett-Burman design can be assimilated to laboratories in an interlaboratory study, uncertainty can be evaluated using the robustness test data. The robustness test was set-up in such a way that the required variances could be estimated. [less ▲]

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See detailEnantiomeric separation of acidic compounds using single-isomer amino cyclodextrin derivatives in nonaqueous capillary electrophoresis
Fradi, Inès; Servais, Anne-Catherine ULiege; Pedrini, Matteo et al

in Electrophoresis (2006), 27(17), 3434-3442

The enantiomeric separation of a series of acidic pharmaceuticals (mostly nonsteroidal anti-inflammatory drugs) has been investigated in NACE systems using single-isomer amino beta-CD derivatives. The ... [more ▼]

The enantiomeric separation of a series of acidic pharmaceuticals (mostly nonsteroidal anti-inflammatory drugs) has been investigated in NACE systems using single-isomer amino beta-CD derivatives. The first part of this study consisted of the selection of the basic experimental conditions to separate efficiently the enantiomers of acidic drugs. Several parameters, such as the nature of the ionic BGE components, were studied and a methanolic solution of ammonium acetate containing the cationic CD was selected as BGE. A D-optimal design with 20 experimental points was then applied and the nature and concentration of the CD were found to have a significant effect on the enantiomeric resolution for all studied compounds. Resolution (R(s)) values were always higher with 6-monodeoxy-6-mono(3-hydroxy)propylamino-beta-CD (PA-beta-CD) compared to those obtained with 6-monodeoxy-6-mono(2-hydroxy)propylamino-beta-CD (IPA-beta-CD). However, the latter led to shorter migration times. Generic NACE conditions were then selected by means of the multivariate approach in order to obtain the highest R(s) values in a minimum amount of time. Finally, dependence of separation selectivity, resolution, as well as mobility difference on chiral selector concentration was discussed and binding constants with PA-beta-CD were estimated for the two enantiomers of one of the model compounds, suprofen in these NACE systems. [less ▲]

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See detailCapillary electrophoresis-mass spectrometry, an attractive tool for drug bioanalysis and biomarker discovery
Servais, Anne-Catherine ULiege; Crommen, Jacques ULiege; Fillet, Marianne ULiege

in Electrophoresis (2006), 27(13), 2616-2629

The coupling of CE with MS detection, a relatively recent hyphenated technique, has gained increasing respect in the field of bioanalytical applications over the past few years. The first part of this ... [more ▼]

The coupling of CE with MS detection, a relatively recent hyphenated technique, has gained increasing respect in the field of bioanalytical applications over the past few years. The first part of this review presents CE-MS applications dealing with drug bioanalysis, including forensic analysis and metabolism studies. Practical considerations to achieve a robust and sensitive CE-MS coupling are also presented. It is indeed essential to strictly control some critical electrospray parameters, such as the sheath liquid composition and flow rate, the nebulizing gas pressure as well as the capillary outlet position. The second part of the review critically describes the applications of CE coupled on-line to MS for the identification of biomarkers in body fluids for diagnostic purposes. Since the sample preparation procedures strongly differ according to the intended use (drug bioanalysis or biomarker discovery), they are discussed separately, taking into account the particular properties of plasma and urine matrices. [less ▲]

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See detailOn-line coupling of cyclodextrin mediated nonaqueous capillary electrophoresis to mass spectrometry for the determination of salbutamol enantiomers in urine
Servais, Anne-Catherine ULiege; Fillet, Marianne ULiege; Mol, Roelof et al

in Journal of Pharmaceutical & Biomedical Analysis (2006), 40(3), 752-757

The usefulness of the on-line coupling of nonaqueous capillary electrophoresis (NACE) with electrospray ionization (ESI) mass spectrometry (MS) using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin ... [more ▼]

The usefulness of the on-line coupling of nonaqueous capillary electrophoresis (NACE) with electrospray ionization (ESI) mass spectrometry (MS) using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD) was demonstrated for the enantioselective determination of low concentrations of salbutamol in human urine. After optimization of several parameters, such as sheath-liquid composition and flow rate, nebulizing gas pressure, CE counter-pressure and position of the CE capillary outlet, a limit of quantification of 18 and 20 ng/ml was obtained for salbutamol enantiomers. Moreover, the relative standard deviation values for repeatability at a concentration of 30 ng/ml were below 7% for both enantiomers. Typical regression lines obtained after application of a simple linear regression model revealed a pod relationship between peak area and analyte concentration (with 0.9988 and 0.9966 as coefficients of determination). This paper proposes an easy to use and sensitive NACE-MS method to determine enantiomers of a basic chiral drug in biological fluids preceded by solid-phase extraction as sample cleanup. (c) 2005 Elsevier B.V. All rights reserved. [less ▲]

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See detailNonaqueous capillary electrophoresis method for the enantiomeric purity determination of S-timolol using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin: validation using the accuracy profile strategy and estimation of uncertainty.
Marini Djang'Eing'A, Roland ULiege; Servais, Anne-Catherine ULiege; Rozet, Eric ULiege et al

in Journal of Chromatography. A (2006), 1120(1-2), 102-11

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the enantiomeric purity determination of S-timolol maleate using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin (HDMS-beta-CD ... [more ▼]

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the enantiomeric purity determination of S-timolol maleate using heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-cyclodextrin (HDMS-beta-CD) as chiral selector. With a background electrolyte made up of a methanolic solution of 0.75 M formic acid, 30 mM potassium camphorsulfonate and containing 30 mM HDMS-beta-CD, the determination of 0.1% of R-timolol in S-timolol could be performed with an enantiomeric resolution of 8.5. Pyridoxine was selected as internal standard. The NACE method was then fully validated by applying a novel strategy using accuracy profiles. It is based on beta-expectation tolerance intervals for the total measurement error which includes trueness and intermediate precision. The uncertainty of measurements derived from beta-expectation tolerance intervals was estimated at each concentration level of the validation standards. To confirm the suitability of the developed and validated method, several real samples of S-timolol maleate containing R-timolol maleate at different concentrations were analysed and the results were compared to those obtained by liquid chromatography. [less ▲]

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See detailInhibition of ceramide-redox signaling pathway blocks glomerular injury in hyperhomocysteinemic rats.
Yi, F.; Zhang, A. Y.; Li, N. et al

in Kidney International (2006), 70(1), 88-96

Ceramide-activated NAD(P)H oxidase has been reported to participate in homocysteine (Hcys)-induced abnormal metabolism of the extracellular matrix (ECM) in rat glomerular mesangial cells. However, it ... [more ▼]

Ceramide-activated NAD(P)H oxidase has been reported to participate in homocysteine (Hcys)-induced abnormal metabolism of the extracellular matrix (ECM) in rat glomerular mesangial cells. However, it remains unknown whether this ceramide-redox signaling pathway contributes to glomerular injury induced by hyperhomocysteinemia (hHcys) in vivo. The present study was designed to address this question, defining the role of ceramide and activated NAD(P)H oxidase in the development of hHcys-induced glomerular injury. Uninephrectomized Sprague-Dawley rats were fed a folate-free diet for 8 weeks to produce hHcys and the de novo ceramide synthesis inhibitor myriocin or the NAD(P)H oxidase inhibitor apocynin was administrated. Rats with folate-free diet significantly increased plasma Hcys levels, renal ceramide levels, and NAD(P)H oxidase activity accompanied by marked glomerular injury. Treatment of rats with myriocin significantly reduced ceramide levels and improved glomerular injury, as shown by decreased urinary albumin excretion and reduced glomerular damage index. ECM components changed towards to normal levels with decreased tissue inhibitor of metalloproteinase-1 and increased matrix metalloproteinase-1 activity. NAD(P)H oxidase activity and Rac GTPase activity were reduced by 69 and 66%, respectively. In rats treated with apocynin, similar beneficial effects in protecting glomeruli from hHcys-induced injury were observed. These results support the view that de novo ceramide production is involved in Hcys-induced NAD(P)H oxidase activity in the kidney of hHcys rats and indicate the important role of ceramide-mediated redox signaling in hHcys-induced glomerular injury in rats. [less ▲]

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See detailHigh-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore.
Tokarski, Caroline; Cren-Olive, Cecile; Fillet, Marianne ULiege et al

in Electrophoresis (2006), 27(7), 1407-16

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially ... [more ▼]

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple. [less ▲]

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See detailInterlaboratory study of a NACE method for the determination of R-timolol content in S-timolol maleate: assessment of uncertainty.
Marini Djang'Eing'A, Roland ULiege; Groom, Carl; Doucet, Francois R et al

in Electrophoresis (2006), 27(12), 2386-99

Analyses of statistical variance were applied to evaluate the precision and practicality of a CD-based NACE assay for R-timolol after enantiomeric separation of R- and S-timolol. Data were collected in an ... [more ▼]

Analyses of statistical variance were applied to evaluate the precision and practicality of a CD-based NACE assay for R-timolol after enantiomeric separation of R- and S-timolol. Data were collected in an interlaboratory study by 11 participating laboratories located in Europe and North America. General qualitative method performance was examined using suitability descriptors (i.e. resolution, selectivity, migration times and S/N), while precision was determined by quantification of variances in the determination of R-timolol at four different impurity levels in S-timolol maleate samples. The interlaboratory trials were designed in accordance with the ISO guideline 5725-2. This allowed estimating for each sample, the different variances, i.e. between-laboratory (s2(Laboratories)), between-day (s2(Days)) and between-replicate (s2(Replicates)). The variances of repeatability (s2r) and reproducibility (s2R) were then calculated. The estimated uncertainty, derived from the precision estimates, seems to be concentration-dependent above a given threshold. This example of R-timolol illustrates how a laboratory can evaluate uncertainty in general. [less ▲]

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