Jean Bleton

Metabolic Origin of Carbon Isotope Composition of Leaf Dark-Respired CO2 in French Bean

The carbon isotope composition (δ13C) of CO2 produced in darkness by intact French bean (Phaseolus vulgaris) leaves was investigated for different leaf temperatures and during dark periods of increasing length. The δ13C of CO2 linearly decreased when temperature increased, from −19‰ at 10°C to −24‰ at 35°C. It also progressively decreased from −21‰ to −30‰ when leaves were maintained in continuous darkness for several days. Under normal conditions (temperature not exceeding 30°C and normal dark period), the evolved CO2 was enriched in 13C compared with carbohydrates, the most 13C-enriched metabolites. However, at the end of a long dark period (carbohydrate starvation), CO2 was depleted in 13C even when compared with the composition of total organic matter. In the two types of experiment, the variations of δ13C were linearly related to those of the respiratory quotient. This strongly suggests that the variation of δ13C is the direct consequence of a substrate switch that may occur to feed respiration; carbohydrate oxidation producing 13C-enriched CO2 and β-oxidation of fatty acids producing 13C-depleted CO2 when compared with total organic matter (−27.5‰). These results are consistent with the assumption that the δ13C of dark respired CO2 is determined by the relative contributions of the two major decarboxylation processes that occur in darkness: pyruvate dehydrogenase activity and the Krebs cycle.

Characterization of neutral sugars and uronic acids after methanolysis and trimethylsilylation for recognition of plant gums

Abstract The main standard neutral sugars and uronic acids that occur as components of plant gums were methanolysed and silylated for study by gas chromatography—electron impact and chemical ionization mass spectrometry (GC—EI-MS and GC—CI-MS). The 25 TMSi methylglycosides, components of the chromatograms obtained were studied and identified by their mass spectra and/or comparison with the corresponding standards. In addition, an unusual uronic acid (4-O-methylglucuronic acid) and the lactone forms of glucuronic acid are reported. A classification of the ions in both EI-MS and CI-MS which allows differentiation between sugar classes and their tautomeric forms is given. The sample preparation method and the results of the above identification were applied to the analysis of some plant gums and a seventeenth century ink sample.

Structural analysis of commercial ceramides by gas chromatography-mass spectrometry.

A simple method using gas chromatography-mass spectrometry was applied to analyse structures of ceramides. Identification of trimethylsilylated ceramides were obtained in short analysis times (derivatization of ceramides in 30 min at room temperature and 20 min gas chromatography mass spectrometry run) even for complex mixtures. For example in ceramide Type III, 18 peaks were observed which represent 27 various structures. The coeluted compounds were ceramides containing the same functional groups and the same carbon number but with a different distribution on the two alkyl chains of the molecule. They were accurately differentiated by mass spectrometry. Therefore, 83 structures of trimethylsilylated ceramides were identified in 11 different commercial mixtures. For 52 structures of these, mass spectral data were not described in the literature, neither full mass spectra nor characteristic fragments.

Identification of phenolic acids and inositols in balms and tissues from an Egyptian mummy

A number of samples taken from an Egyptian mummy (ca. 100 B.C.) from the Guimet Museum in Lyon have been analyzed by GC-MS. Derivatives of aromatic acids (hydroxyhydrocinnamic, vanillic, protocatechuic and gallic acids) and inositols (non-methylated and mono-O-methyl) have been found among the constituents of extracts prepared by methanolysis and trimethylsilylation. From the reported electron impact mass spectra, ion sets where proposed for a sensitive and selective profiling of these selected compounds by mass fragmentometry. The source of gallic acid and inositol was found to be a vegetable tannin, an ingredient which was not previously known to be used for mummification in ancient Egypt. The nature and abundance ratios of the detected inositols also appeared to be a promising criterion to further investigate the botanical source of the tannin employed.

Kinetic 12C/13C isotope fractionation by invertase: evidence for a small in vitro isotope effect and comparison of two techniques for the isotopic analysis of carbohydrates†

The natural (13)C/(12)C isotope composition (delta(13)C) of plants and organic compounds within plant organs is a powerful tool to understand carbon allocation patterns and the regulation of photosynthetic or respiratory metabolism. However, many enzymatic fractionations are currently unknown, thus impeding our understanding of carbon trafficking pathways within plant cells. One of them is the (12)C/(13)C isotope effect associated with invertases (EC 3.2.1.26) that are cornerstone enzymes for Suc metabolism and translocation in plants. Another conundrum of isotopic plant biology is the need to measure accurately the specific delta(13)C of individual carbohydrates. Here, we examined two complementary methods for measuring the delta(13)C value of sucrose, glucose and fructose, that is, off-line high-performance liquid chromatography (HPLC) purification followed by elemental analysis and isotope ratio mass spectrometry (EA-IRMS) analysis, and gas chromatography-combustion (GC-C)-IRMS. We also used these methods to determine the in vitro (12)C/(13)C isotope effect associated with the yeast invertase. Our results show that, although providing more variable values than HPLC approximately EA-IRMS, and being sensitive to derivatization conditions, the GC-C-IRMS method gives reliable results. When applied to the invertase reaction, both methods indicate that the (12)C/(13)C isotope effect is rather small and it is not affected by the use of heavy water (D(2)O).

Quantitative Proteomics Analysis Confirmed Oxidative Metabolism Predominates in Streptomyces coelicolor versus Glycolytic Metabolism in Streptomyces lividans

Recent physiological studies indicated that S. lividans metabolism was mainly glycolytic, whereas S. coelicolor metabolism was mainly oxidative. To determine whether such metabolic characteristics were correlated with consistent proteomics features, a comparative label-free, shotgun proteomics analysis of these strains was carried out. Among 2024 proteins identified, 360 showed significant differences in abundance between the strains. This study revealed that S. coelicolor catabolized glucose less actively than S. lividans, whereas the amino acids present in the medium were catabolized less actively by S. lividans than by S. coelicolor. The abundance of glycolytic proteins in S. lividans was consistent with its high glycolytic activity, whereas the abundance of proteins involved in the catabolism of amino acids in S. coelicolor provided an explanatory basis for its predominantly oxidative metabolism. In this study, conducted under conditions of low O2 availability, proteins involved in resistance to oxidative stress and those belonging to a DosR-like dormancy regulon were abundant in S. coelicolor, whereas tellurium resistance proteins were abundant in S. lividans. This indicated that the strains reacted differently to O2 limitation. Proteins belonging to the CDA, RED, and ACT pathways, usually highly expressed in S. coelicolor, were not detected under these conditions, whereas proteins of siderophores, 5-hydroxyectoine, and terpenoid biosynthetic pathways were present.

Establishing high temperature gas chromatographic profiles of non-polar metabolites for quality assessment of African traditional herbal medicinal products.

The quality assessment of African traditional herbal medicinal products is a difficult challenge since they are complex mixtures of several herbal drug or herbal drug preparations. The plant source is also often unknown and/or highly variable. Plant metabolites chromatographic profiling is therefore an important tool for quality control of such herbal products. The objective of this work is to propose a protocol for sample preparation and gas chromatographic profiling of non-polar metabolites for quality control of African traditional herbal medicinal products. The methodology is based on the chemometric assessment of chromatographic profiles of non-polar metabolites issued from several batches of leaves of Combretum micranthum and Mitracarpus scaber by high temperature gas chromatography coupled to mass spectrometry, performed on extracts obtained in refluxed dichloromethane, after removal of chlorophyll pigments. The method using high temperature gas chromatography after dichloromethane extraction allows detection of most non-polar bioactive and non-bioactive metabolites already identified in leaves of both species. Chemometric data analysis using Principal Component Analysis and Partial Least Squares after Orthogonal Signal Correction applied to chromatographic profiles of leaves of Combretum micranthum and Mitracarpus scaber showed slight batch to batch differences, and allowed clear differentiation of the two herbal extracts.

Challenging wax-cast figurine serial production unravelled by multi-analytical techniques

Eight complementary techniques were successfully applied to study a pair of very alike eighteenth-century colored wax figurines belonging to the Museu Nacional Machado de Castro, Coimbra (Portugal): examination under visible and ultraviolet light, X-ray radiography (XRR), neutron radiography and tomography (NR and NT), energy dispersive X-ray fluorescence (EDXRF), scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS), micro-X-ray diffraction (μ-XRD), gas chromatography coupled with mass spectrometry (GC/MS) and micro-confocal Raman spectroscopy (μ-Raman). A careful examination of the two objects provided an insight into their manufacturing and revealed that they were cast from the same molds, although details differ. The main cast material employed was a mixture of beeswax, Venice turpentine, other diterpenoid resins and a very low amount of lipids. The wax used was certainly reclaimed from a metallurgic activity involving lost-wax casting. Each figurine consists of sixteen parts, most of which consist of solid wax. The presence of fillings within the body was unexpected. The elements which remained hollowed played a fundamental role at the assembling stage. A loose wooden tenon helped to keep the head in place and metal rods were used to fasten the base to the main body. Polychromy was carried out in wax, with different pigments and opacifiers. The fabrication of the colored wax from different inorganic/organic wastes is also discussed. Textures were achieved by adding materials. The results gathered offered the unique opportunity to verify aspects inherent to the production of multiple copies in wax casting.

The celebrated écorchés of honoré Fragonard, part 2: The details of the technique used by Fragonard.

It is remarkable that the famous écorchés of Honoré Fragonard have survived the centuries to reach us today. Studies carried out by several teams have established details of the technique used by Fragonard that help to explain their longevity. The injection of the vessels was achieved by means of a mixture of mutton tallow and pine resin diluted in essence of turpentine and essential oils. This gave Fragonard a very high success rate. Above all, he did not add pigments to his mixture while injecting the veins, and this facilitated the procedure. The vessels were painted after preservation to give them the vivid colors that we can still see today. Another detail that explains their exceptional conservation is that the varnish used by Fragonard was composed of Venice turpentine, made from larch resin and known to repel insects. Clin. Anat. 23:258–264, 2010.