Paul Dumas

Brachypodium distachyon grain: characterization of endosperm cell walls

The wild grass Brachypodium distachyon has been proposed as an alternative model species for temperate cereals. The present paper reports on the characterization of B. distachyon grain, placing emphasis on endosperm cell walls. Brachypodium distachyon is notable for its high cell wall polysaccharide content that accounts for ∼52% (w/w) of the endosperm in comparison with 2-7% (w/w) in other cereals. Starch, the typical storage polysaccharide, is low [<10% (w/w)] in the endosperm where the main polysaccharide is (1-3) (1-4)-β-glucan [40% (w/w) of the endosperm], which in all likelihood plays a role as a storage compound. In addition to (1-3) (1-4)-β-glucan, endosperm cells contain cellulose and xylan in significant amounts. Interestingly, the ratio of ferulic acid to arabinoxylan is higher in B. distachyon grain than in other investigated cereals. Feruloylated arabinoxylan is mainly found in the middle lamella and cell junction zones of the storage endosperm, suggesting a potential role in cell-cell adhesion. The present results indicate that B. distachyon grains contain all the cell wall polysaccharides encountered in other cereal grains. Thus, due to its fully sequenced genome, its short life cycle, and the genetic tools available for mutagenesis/transformation, B. distachyon is a good model to investigate cell wall polysaccharide synthesis and function in cereal grains.

Multimodal Spectroscopy Combining Time-of-Flight-Secondary Ion Mass Spectrometry, Synchrotron-FT-IR, and Synchrotron-UV Microspectroscopies on the Same Tissue Section

Mass spectrometry and spectroscopy-based approaches can provide an overview of the chemical composition of a tissue sample. This opens up the possibility to investigate in depth the subtle biochemical changes associated with pathological tissues. In this study, time-of-flight secondary ion mass spectrometry (TOF-SIMS) and synchrotron-FT-IR and -UV imaging were applied to the same tissue section by using the same sample holder. The tested sample involved liver cirrhosis, which is characterized by regeneration nodules surrounded by annular fibrosis. A tissue section from a cirrhotic liver was deposited on a gold coated glass slide and was initially analyzed by FT-IR microspectroscopy in order to image the distribution of lipids, proteins, sugars, and nucleic acids. This technique has identified collagen enrichment in fibrosis whereas esters were mostly distributed into the cirrhotic nodules. The exact same section was investigated using TOF-SIMS demonstrating that some molecular lipid species were differentially distributed into the fibrosis areas or cirrhotic nodules. Spectra of UV microspectroscopy obtained from the same section allowed visualizing high autofluorescence from fibrous septa confirming the presence of collagen. Altogether, these results demonstrated that TOF-SIMS and FT-IR/UV microspectroscopy analyses can be successfully performed on the same tissue section.

Aleurone Cell Walls of Wheat Grain: High Spatial Resolution Investigation Using Synchrotron Infrared Microspectroscopy

Infrared microspectroscopy and immunolabeling techniques were employed in order to obtain deeper insight into the biochemical nature of aleurone cell walls of wheat grain. The use of a synchrotron source, thanks to its intrinsic brightness, has provided unprecedented information at the level of a few micrometers and has allowed the discrimination of various polysaccharides in cell walls. The high spectral quality obtained in the small analyzed domain has been beneficial in estimating the relative proportions of β-glucan and arabinoxylan, through the use of principal component analysis (PCA). The highest amount of β-glucan is found in periclinal cell walls close to the starchy endosperm. The junction regions between aleurone cells are enriched in arabinoxylan. At the early stage of wheat grain development (271°D), the chemical composition along the cell walls is more heterogeneous than at the mature stage. Both synchrotron infrared microspectroscopy and immunolabeling experiments made it possible to reveal the spatial heterogeneity of the various chemical compositions of aleurone cell walls.

Photosensitizer effects on cancerous cells: A combined study using synchrotron infrared and fluorescence microscopies

Hypocrellin A (HA), a lipid-soluble peryloquinone derivative, isolated from natural fungus sacs of Hypocrella bambusae, has been reported to be a highly potential photosensitizer in photodynamic therapy (PDT). It has been studied increasingly because of its anticancer activities when irradiated with light. We have studied the interaction mechanisms of HA with HeLa cells as a function of incubation time. Fluorescence microscopy confirmed that HA localisation is limited in the cytoplasm before eventually concentrating in clusters around the nucleus. The IR spectra of HA-treated, PDT-treated and control HeLa cells were recorded at the ESRF Infrared beamline (ID21). Principal component analysis has been used to assess the IR spectral changes between the various HeLa cells spectral data sets (The Unscrambler software, CAMO). PCA revealed that there is a frequency shift of protein amide I and amide II vibrational bands, indicating changes in the protein secondary structures of the HA-treated and PDT-treated cancer cells compared to the control cells. In addition, the relative DNA intensity in HA-treated cells decreases gradually along the incubation time. The use of synchrotron infrared microscopy is shown to be of paramount importance for targeting specifically the biochemical modification induced in the cell nucleus.

Profiling pluripotent stem cells and organelles using synchrotron radiation infrared microspectroscopy

FTIR micro-spectroscopy is a sensitive, non-destructive and label-free method offering diffraction-limited resolution with high signal-to-noise ratios when combined with a synchrotron radiation source. The vibrational signature of individual cells was used to validate an alternative strategy for reprogramming induced pluripotent stem cells generated from amniocytes. The iPSC lines PB09 and PB10, were reprogrammed from the same amniocyte cell line using respectively the Oct54, Sox2, Lin28, and Nanog and the Oct4 and Sox2 transcription factor cocktail. We show that cells reprogrammed by the two different sets of transfection factors have similar spectral signatures after reprogramming, except for a small subpopulation of cells in one of the cell lines. Mapping HeLa cells at subcellular resolution, we show that the Golgi apparatus, the cytoplasm and the nucleus have a specific spectral signature. The CH(3):CH(2) ratio is the highest in the nucleus and the lowest in the Golgi apparatus/endoplasmic reticulum, in agreement with the membrane composition of these organelles. This is confirmed by specific staining of the organelles with fluorescent dyes. Subcellular differentiation of cell compartments is also demonstrated in living cells.

In Situ Chemical Composition Analysis of Cirrhosis by Combining Synchrotron Fourier Transform Infrared and Synchrotron X-ray Fluorescence Microspectroscopies on the Same Tissue Section

Liver is subject to various chronic pathologies, progressively leading to cirrhosis, which is associated with an increased risk of hepatocellular carcinoma. There is an urgent need for diagnostic and prognostic markers of chronic liver diseases and liver cancer. Spectroscopy-based approaches can provide an overview of the chemical composition of a tissue sample offering the possibility of investigating in depth the subtle chemical changes associated with pathological states. In this study, we have addressed the composition of cirrhotic liver tissue by combining synchrotron Fourier transform infrared (FTIR) microspectroscopy and synchrotron micro-X-ray fluorescence (XRF) on the same tissue section using a single sample holder in copper. This allowed investigation of the in situ biochemical as well as elemental composition of cells and tissues at high spatial resolution. Cirrhosis is characterized by regeneration nodules surrounded by annular fibrosis. Hepatocytes within cirrhotic nodules were characterized by high content in esters and sugars as well as in phosphorus and iron compared with fibrotic septa. A high heterogeneity was observed between cirrhotic nodules in their content in sugars and iron. On fibrosis, synchrotron XRF revealed enrichment in calcium compared to cirrhotic hepatocytes. Careful scrutiny of tissue sections led to detection of the presence of microcrystals that were demonstrated as precipitates of calcite using synchrotron FTIR. These results demonstrated that synchrotron FTIR and synchrotron XRF microspectroscopies provide complementary information on the chemical composition of cirrhotic hepatocytes and fibrotic septa in cirrhosis.

Action du manganese methylcyclopentadienyle tricarbonyle (MMT) sur les mitochondries I. Effects du MMT, in vitro, sur la phosphorylation oxydative des mitochondries hepatiques de rats

Abstract Effects of methylcyclopentadienyl manganese tricarbonyl (MMT) of rat liver mitochondria. I. Effects, in vitro, on the oxidative phosphorylation . Methylcyclopentadienyl manganese tricarbonyl (MMT) is an effective inhibitor of mitochondrial respiration associated with NAD + -linked substrates. At concentrations which inhibit glutamate-malate oxidation (over 80% inhibition) succinate and tetramethylphenylenediamine (TMPD)-ascorbate are inhibited less than 25%. MMT inhibits both electron and energy transfer in mitochondria as revealed by the partial release of MMT inhibition by 2,4-dinitrophenol (DNP). The data indicated that the effects of MMT are supported by energy conservation at site I of the respiratory chain.

Specificite de L'Iodotyrosine desiodase des microsomes thyroïdiens et hepatiques

Abstract The deiodinating activity of thyroid and liver microsomes was studied with 19 compounds including 3-iodo- l -tyrosine and 3:5-diiodo- l -tyrosine and their analogs. While l -iodotyrosines were almost completely dehalogenated, d -iodotyrosines, α-methyl- dl -iodotyrosines and 3:5-diiodo-4-hydroxyphenyl- dl -lactic acid were poor substrates for the deiodinase. All the other compounds tested remained unchanged including 3:5-diiodo-4-hydroxyphenyl-α guanidyl propionic acid, 3:5-diiodo-4-hydroxyphenyl propionic acid and 3:5-diiodotyramine. The phenol group played a major role since 3-iodo-5-nitro- l -tyrosine and 3-iodo- l -phenylalanine were not decomposed. In conclusion, thyroid and liver deiodinase are highly specific enzymes. It appears probable that the enzymatic site requires the carboxyl, amino and phenol groups of the substrate to exert its activity.Abstract The deiodinating activity of thyroid and liver microsomes was studied with 19 compounds including 3-iodo- l -tyrosine and 3:5-diiodo- l -tyrosine and their analogs. While l -iodotyrosines were almost completely dehalogenated, d -iodotyrosines, α-methyl- dl -iodotyrosines and 3:5-diiodo-4-hydroxyphenyl- dl -lactic acid were poor substrates for the deiodinase. All the other compounds tested remained unchanged including 3:5-diiodo-4-hydroxyphenyl-α guanidyl propionic acid, 3:5-diiodo-4-hydroxyphenyl propionic acid and 3:5-diiodotyramine. The phenol group played a major role since 3-iodo-5-nitro- l -tyrosine and 3-iodo- l -phenylalanine were not decomposed. In conclusion, thyroid and liver deiodinase are highly specific enzymes. It appears probable that the enzymatic site requires the carboxyl, amino and phenol groups of the substrate to exert its activity.

In situ thermal denaturation of myofibre sub-type proteins studied by immunohistofluorescence and synchrotron radiation FT-IR microspectroscopy.

The thermal denaturation of proteins in skeletal muscle was studied and characterised for the first time taking into account the in situ metabolic and contractile fibre types. From serial histological sections, collagen, elastin, various type I, IIa and IIx fibres and type I-IIa and IIa-IIx hybrids were identified by immunohistofluorescence. Histological sections were incubated in buffer solutions at increasing temperatures (40, 50, 60, 70 and 80°C). Protein secondary structure was investigated by synchrotron radiation FT-IR microspectroscopy on connective tissue and in muscle fibres rigorously identified for sub-type. Whatever the target protein components, increasing temperature resulted in a decrease in α-helix secondary structure and an increase in β-sheet structure. This phenomenon was more pronounced for intracellular proteins than for connective tissue. Although hybrid fibres were generally somewhat less sensitive to unfolding than the pure types, the amplitude of the thermal denaturation of intracellular proteins was practically independent of fibre type.

Deshalogenation de divers derives iodes phenoliques chez le rat normal et thyroidectomise

Abstract Deiodination of eleven compounds including, 3-iodo- l -tyrosine, 3,5-diiodo- l -tyrosine and their analogs were studied in rats in vivo . While l -iodotyrosines were almost completely dehalogenated in normal rats, d -iodotyrosines, 3,5-diiodo-4-hydroxyphenyl- dl -lactic acid, 3,5-diiodo-4-hydroxyphenyl acetic acid and 3,5-diiodotyramine were poorly deiodinated. All the other products remained unchanged. l - and d -3-iodotyrosines were more rapidly deiodinated than l - and D-3,5-diiodotyrosines. Similar results were found with thyroldectomized rats, except that the d -iodotyrosines were deiodinated to a lesser extent than in normal rats. Metabolism in vivo of iodophenolic compounds is in accord with specificity of the thyroidal and hepatic microsomal iodotyrosine-deiodinase.