Biomedical subjects

C Miège

Publications and source records attributed to C Miège.

10 recordsLinked to original sources

PubMed2003

Optimization, validation and comparison of various extraction techniques for the trace determination of polycyclic aromatic hydrocarbons in sewage sludges by liquid chromatography coupled to diode-array and fluorescence detection.

There is a need for a better characterization of sludges from wastewater treatment plants which are destined to be spread on agricultural lands. Inorganic pollutants are regularly controlled but organic pollutants have received few attention up to now. On this paper, we have been interested on the analysis of the 16 polycyclic aromatic hydrocarbons (PAHs) listed in the US Environmental Protection Agency (US EPA) priority list and more particularly of the six PAHs listed in the European community list (fluoranthene, benzo[b and k]fluoranthene, benzo[a]pyrene, benzo[ghi]perylene, indeno[1,2,3-cd]pyrene). The analysis step consists on liquid chromatography with both fluorescence and UV detections as described in the EPA Method 8310. As for the extraction step, several techniques such as supercritical fluid extraction, pressurized liquid extraction, focused microwave extraction in open vessels, Soxhlet and ultrasonic extractions are compared after optimization of the experimental conditions (solvent nature and quantity, temperature, pressure, duration, ... ) and validation with certified sludges. When optimized, these five extraction techniques are as much efficient with similar relative standard deviation. Whatever the extraction techniques used, the whole analysis protocol permits to quantify PAHs in the range of 0.09 to 0.9 mg/kg of dried sludges.

Chromatography, High Pressure Liquid↗

PubMed2003

Survival tests with Chironomus riparius exposed to spiked sediments can profit from DEBtox model.

DEBtox model is a biologically based model used to analyse aquatic toxicity data (The analysis of aquatic toxicity data, VU University Press, Amsterdam, The Netherlands, 1996, 149pp.). To date, it has not been used to analyse sediment survival tests, mainly because this would require daily counting of survivors, which is difficult for benthic organisms. In the present study, we adapted survival toxicity tests with the midge Chironomus riparius to permit survival data analysis with DEBtox. To validate the adaptation proposed, we exposed organisms to two chemicals, copper and methiocarb. We exposed larvae of second, third and fourth instar to different concentrations of the tested compounds and two different diets to assess the influence of diet and instar on DEBtox parameter estimates. Daily counting of organisms did not affect survival and did not lead to much more effort when compared to usual tests. Moreover, the analysis profited much from use of the DEBtox model. It was possible, with only survival data to estimate and validate the kinetics of the compounds, to predict survival during the recovery of exposed organisms and to assess the influence of food availability on toxicity. Food availability did not influence methiocarb toxicity, but copper was more toxic to food-limited organisms when a threshold concentration was exceeded. Comparison of parameters estimate also helped in understanding the differences in toxicity responses between instars. For the two compounds we studied, the difference was entirely explained by differences in threshold values.

Animals↗

PubMed1999

Selective immunoclean-up followed by liquid or gas chromatography for the monitoring of polycyclic aromatic hydrocarbons in urban waste water and sewage sludges used for soil amendment.

A selective clean-up procedure using immunoaffinity solid-phase extraction was applied for the trace-level determination of polycyclic aromatic hydrocarbons (PAHs) in urban waste water and sewage sludges used for soil amendment. Anti-pyrene antibodies have been immobilized on a silica-based sorbent and the cross-reactivity of the antibodies towards structurally related compounds were allowed to extract the whole class of priority PAHs. The selectivity of the antibodies provided clean extracts from sludges and, therefore, the identification and quantification were shown to be easier using either liquid chromatography (LC) with UV diode array and fluorescence detection in series or gas chromatography-mass spectrometry (GC-MS), although some loss of up to 50% was observed for the clean-up. The identification of the PAHs by matching of UV and MS spectra was greatly improved. The procedure, including immunoclean-up and LC coupled to diode array and fluorescence detection, was validated using certified reference materials with native PAHs of concentrations in the range of 0.57-2.16 mg/kg (dry sludges).

Chromatography, Gas↗

PubMed1999

Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in escherichia coli carrying temperature-sensitive ribosome recycling factor.

We have isolated a protein, mature RRFHCP, from chloroplasts of spinach (Spinacia oleracea L.) that shows 46% sequence identity and 66% sequence homology with ribosome recycling factor (RRF) of Escherichia coli. RRF recycles ribosomes through disassembly of the posttermination complex. From the cDNA analysis and from the amino-terminal sequencing of the isolated protein, the mature RRFHCP was deduced to have a Mr of 21,838 with 193 aa. It lacks the 78-aa chloroplast targeting sequence encoded by the RRFHCP cDNA sequence. The RRFHCP synthesized in vitro was imported into isolated chloroplasts with simultaneous conversion to the mature RRFHCP. Transcription of the gene coding for RRFHCP was not dependent on light, yet it was limited mostly to photosynthetic tissues in which only one transcript size was detected. Mature RRFHCP exerted a bactericidal effect on E. coli carrying temperature-sensitive RRF at the permissive temperature whereas wild-type E. coli was not affected.

Amino Acid Sequence↗

PubMed1999

1,2-sn-Diacylglycerol in plant cells: Product, substrate and regulator.

1,2-sn-Diacylglycerol (DAG) is a family of lipidic molecular species varying in the lengths and desaturation levels of acyl groups esterified at positions sn-1 and sn-2 of the glycerol backbone. In plant cells, DAG originating from plastid and from extraplastidial membranes have distinct molecular signatures, C18/C16 and C18/C18 structures, respectively. Under normal conditions, DAG is consumed nearly as fast as it is produced and is therefore a transient compound in the cell. In plants, DAG proved to be the most basic ingredient for cell membrane biogenesis and fat storage, but we still lack formal evidence to assert that DAG is also an intracellular messenger, as demonstrated for animals. From the biochemical and molecular comparisons of the best known DAG-manipulating proteins of prokaryotic and eukaryotic cells (phosphatidate phosphatases, diacylglycerol kinases, MGDG synthase, protein kinase C, etc.) this review aims to identify general rules driving DAG metabolism, and emphasizes its unique features in plant cells. DAG metabolism is an intricate network of local productions and utilizations: many isoenzymes can catalyse similar DAG modifications in distinct cell compartments or physiological processes. The enzymatic- or binding-specificity for DAG molecular species demonstrates that discrete DAG molecular subspecies fluxes are finely controlled (particularly for C18/C16 and C18/C18 structures in plastid membrane biogenesis). Eventually, this review stresses the diversity of structures and functioning of DAG-manipulating proteins. As a consequence, because DAG metabolism in plants is unique, the deciphering of genomic information cannot rely on homology searches using known prokaryotic, animal or yeast sequences, but requires sustained efforts in biochemical and molecular characterizations of plant DAG-manipulating proteins.

Journal Article↗

PubMed1999

Biochemical and topological properties of type A MGDG synthase, a spinach chloroplast envelope enzyme catalyzing the synthesis of both prokaryotic and eukaryotic MGDG.

MGDG synthase, the enzyme that catalyzes the synthesis of the major chloroplast membrane lipid monogalactosyldiacylglycerol (MGDG), is encoded by a multigenic family. We have analyzed the biochemical properties, subcellular localization and membrane topology of a spinach chloroplast MGDG synthase, a representative member of the type A family from Spinacia oleracea (soMGD A), using a recombinant protein that was functionally overexpressed in Escherichia coli and specific polyclonal antibodies. We demonstrated that soMGD A could catalyze the synthesis of both 'prokaryotic' and 'eukaryotic' MGDG molecular species in vitro, with a selectivity for diacylglycerol similar to that of purified chloroplast envelope MGDG synthase activity. Furthermore, soMGD A was shown to be sensitive to chemical reagents (dithiothreitol, N-ethylmaleimide and o-phenanthroline) known to affect MGDG synthesis by the partially purified enzyme, as well as in isolated chloroplast envelope membranes. In spinach chloroplasts, soMGD A was localized by Western blot analysis in the inner envelope membrane. Topological studies demonstrated that soMGD A is a monotopic enzyme, embedded within one leaflet of the inner envelope membrane from spinach chloroplasts, a structure which may involve amphipathic alpha helices. We further demonstrated that in vitro, soMGD A precursor is imported and processed to its correct mature form in intact chloroplasts. These results show that soMGD A corresponds to a mature polypeptide of approximately 45 kDa. In addition, inactivation kinetics after gamma-ray irradiation strongly suggest that both native chloroplast envelope MGDG synthase and recombinant soMGD A have a functional molecular mass of 95-100 kDa, indicating that they are probably active as homodimers made of two 45-kDa subunits. This study suggests that, in spite of the growing evidence that MGDG synthesis is catalyzed by a multigenic family of enzymes, in spinach leaves both prokaryotic and eukaryotic MGDG syntheses could be attributable to a unique dimeric enzyme, provided that diacylglycerol is transported from the outer membrane to the inner membrane of the chloroplast envelope.

Amino Acid Sequence↗

PubMed1998

Optimization and validation of solvent and supercritical-fluid extractions for the trace-determination of polycyclic aromatic hydrocarbons in sewage sludges by liquid chromatography coupled to diode-array and fluorescence detection.

There is a need for a better characterization of sludges from wastewater treatment plants which are destined to be spread on agricultural land. Inorganic pollutants are regularly controlled, but organic pollutants have received little attention up to now. The main problem for trace analysis of organic pollutants comes from the complexity of the various matrices of sludges, which depends on their origins. Therefore, methods described for soils and sediments cannot be directly applied to sludges which contain high amount of lipids. This paper provides a method for trace-determination of polycyclic aromatic hydrocarbons in the range of mg/kg of dried sludge including an extraction step, and an analysis step by liquid chromatography with fluorescence detection for sensitive quantification and UV-diode-array detection for confirmation. The extraction step was optimized by a selection of the solvent used for both Soxhlet and supercritical-fluid extraction and by setting the experimental conditions (temperature, pressure, modifier, etc.) used for SFE. Extraction recoveries were found similar and quantitative for the two procedures. However supercritical-fluid extraction appears to be a good alternative to Soxhlet extraction because of the consumption of less time and solvent. Detection limits were obtained in the range of 0.1-1 mg/kg of dried sludges, with possible confirmation by UV spectra. The whole method (extraction/quantification/confirmation) was validated using sludges which have been certified by the Community Bureau of Reference from Brussels (CRM No. 088). Application to the determination of PAHs in urban sludges is presented.

Chromatography, Liquid↗

PubMed1998

Effect of the various parameters governing solid-phase microextraction for the trace-determination of pesticides in water.

The parameters governing solid-phase microextraction (SPME) are investigated, with emphasis on the determination of the partition coefficients, K, and their use to predict the selection of a fibre, depending on the respective characteristics of the fibres and the analytes. Film thickness and stability of the compounds can interfere with the determination of K values. The time profile curves were determined for twelve pesticides having a wide range of water solubilities and polarities and using four fibres (polydimethylsiloxane, polydimethylsiloxane-divinylbenzene, Carbowax-divinylbenzene and polyacrylate). Although the affinity order was different for the four coatings, no correlation was found between the respective characteristics of the fibres and solutes. The two fibres containing divinylbenzene were shown to have the highest affinities and the polydimethylsiloxane had the lowest affinity. The polyacrylate fibre which is the more polar commercial fibre did not provide the highest affinities for the more polar and water-soluble analytes. The important parameters for quantitative analysis have been evaluated. The calibration curves were similar when one analyte of interest was present on its own in a drinking water sample, or when eleven other pesticides were present at the same concentration or when much higher concentrations of other analytes were present in the sample. Linearity was obtained over a wide range of concentrations in drinking water samples. Detection limits are in agreement with European regulatory levels in drinking water for most of the analytes using solid-phase microextraction-gas chromatography-nitrogen-phosphorus detection (SPME-GC-NPD). In contaminated surface water samples, the chromatograms are relatively clean and most of the compounds can be detected at levels lower than 0.5 microgram/l.

Calibration↗

PubMed1995

The catalytic site of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes. Biochemical analysis of the structure and of the metal content.

We have analyzed the structure of the active site of monogalactosyldiacylglycerol (MGDG) synthase from spinach chloroplast envelope. Since purification of this membrane-embedded enzyme yielded such low amounts of protein that analyses of the amino acid sequence were so far impossible, we used indirect strategies. Analyses of the inhibition of MGDG synthase by UDP and of its inactivation by citraconic anhydride first indicated that the enzyme contained two functionally independent and topologically distinct binding sites for each substrate. Whereas MGDG synthase binds both the nucleotidic part of UDP-Gal and the acyl chains of 1,2-diacylglycerol, UDP is a competitive inhibitor relatively to UDP-Gal, while it does not compete with 1,2-diacylglycerol for binding on the enzyme. The UDP-Gal-binding site contains lysine residues, as demonstrated for UDP-Gal-binding sites from all galactosyltransferases studied so far. Radiolabeling of MGDG synthase by sulfur labeling reagent, a 35S-labeled lysine-blocking reagent, confirmed that MGDG synthase was a polypeptide with a low molecular mass (around 20 kDa). The 1,2-diacylglycerol-binding site contains reduced cysteines and at least one metal. The divalent cation(s) associated to apo-MGDG synthase was not unambiguously identified, but the results suggest that it could be zinc. Therefore, MGDG synthase presents some structural features in common with diacylglycerol-manipulating enzymes, such as protein kinase C and 1,2-diacylglycerol kinase, which are characterized by the presence of a ubiquitous Cys6His2 domain involved in zinc coordination in their 1,2-diacylglycerol-binding domains.

Binding Sites↗

PubMed1994

Direction of gaze and comfort: discovering the relation for the ergonomic optimization of visual tasks.

Perceived exertion caused by the extraocular muscles was assessed as a function of the direction of gaze and recorded in 114 subjects using a psychometric method. The results confirm statements in the literature that maximum comfort is achieved when gaze is aimed downwards. We found no correlations between perceived exertion and age or viewing distance. The relationship is relevant to ergonomics, for example in lens fitting and workplace layout.

Adult↗