Frankia alni Carbonic Anhydrase Regulates Cytoplasmic pH of Nitrogen-Fixing Vesicles.

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles' pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.

Towards a Non-Biased Formaldehyde Quantification in Leather: New Derivatization Conditions before HPLC Analysis of 2,4-Dinitrophenylhydrazine Derivatives.

In leathers, formaldehyde is currently analyzed according to EN ISO 17226-1 standard, by reversed phase liquid chromatography after off-line precolumn derivatization with 2,4 dinitrophenylhydrazine (DNPH) in strong acidic conditions. We first demonstrate that this standard is not adapted to leather retanned with resins likely to release formaldehyde by hydrolysis. Indeed, formaldehyde content may be largely overestimated due to concomitant resin hydrolysis (in harsh acidic conditions) that releases formaldehyde during the derivatization step and during the waiting time on autosampler before analysis. Therefore, we thoroughly studied the derivatization step in order to propose new derivatization conditions. Replacing orthophosphoric acid by less acidic buffer solutions is not enough to avoid hydrolysis. A derivatization without adding acid is realized by solubilizing DNPH in acetonitrile instead of orthophosphoric acid. These conditions lead to a complete derivatization of formaldehyde in 3 h at 50 °C (in a water bath) while avoiding the hydrolysis of co-extracted dicyandiamide and melamine resins. The as-obtained leather extracts are stable over time. Formaldehyde contents found with this method agree with the formaldehyde content measured immediately at the end of derivatization reaction in standard conditions or with formaldehyde content measured by a home-designed flow injection analysis with acetylacetone online derivatization and UV detection.

[Interest of the combination of high-sensitivity troponine and copeptin in diagnosis of non ST elevation myocardial infarction]. / Intérêt de l'association de la copeptine et de la troponine ultrasensible dans le diagnostic des syndromes coronariens aigus sans sus décalage du segment ST aux urgences.

The diagnosis of non ST elevation myocardial infarction (NSTEMI) is very important for the emergency doctor. According to the literature copeptin, a marker of the endogenous stress, combined with troponin could be of interest in this diagnosis. The objective of the study was to investigate the association of high-sensitivity (HS) troponin and copeptine to eliminate the diagnosis of NSTEMI or unstable angina (UA) in patients arriving in Casualty with a thoracic pain. This prospective study included patients showing up at Casualty with a thoracic pain less than 12 hours old. Copeptin was measured by the BRAHMS method at admission and HS troponin was measured at admission, after 2 and 6 hours. The patients also had a follow-up phone call after 3 months. The study included 114 patients with an average age of 54.6 years. NSTEMI was diagnosed for 8.8% of them and UA for 6.1%. The patients presenting NSTEMI or UA had a copeptin rate at admission higher than the others (24.7 pmol/L versus 7.1; p < 0.002). The negative predictive value of the association of HS troponin and copeptin was 95% whereas the sensitivity was 76.5% and the specificity 78.4%. The ROC curve analysis of the copeptin results brought to light a positivity limit which would have been more successful at 10.3 pmol/L than at 14.0. The association of copeptin and HS troponin can be useful to exclude the diagnosis of NSTEMI and favours faster treatment in Casualty.

Enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum explored by two-dimensional analysis: identification of seven genes encoding new dockerin-containing proteins.

The enzyme diversity of the cellulolytic system produced by Clostridium cellulolyticum grown on crystalline cellulose as a sole carbon and energy source was explored by two-dimensional electrophoresis. The cellulolytic system of C. cellulolyticum is composed of at least 30 dockerin-containing proteins (designated cellulosomal proteins) and 30 noncellulosomal components. Most of the known cellulosomal proteins, including CipC, Cel48F, Cel8C, Cel9G, Cel9E, Man5K, Cel9M, and Cel5A, were identified by using two-dimensional Western blot analysis with specific antibodies, whereas Cel5N, Cel9J, and Cel44O were identified by using N-terminal sequencing. Unknown enzymes having carboxymethyl cellulase or xylanase activities were detected by zymogram analysis of two-dimensional gels. Some of these enzymes were identified by N-terminal sequencing as homologs of proteins listed in the NCBI database. Using Trap-Dock PCR and DNA walking, seven genes encoding new dockerin-containing proteins were cloned and sequenced. Some of these genes are clustered. Enzymes encoded by these genes belong to glycoside hydrolase families GH2, GH9, GH10, GH26, GH27, and GH59. Except for members of family GH9, which contains only cellulases, the new modular glycoside hydrolases discovered in this work could be involved in the degradation of different hemicellulosic substrates, such as xylan or galactomannan.

Assessment of the phototoxic hazard of some essential oils using modified 3T3 neutral red uptake assay.

When substances are developed in the aim to be a constituent of personal care products, and to be applied on the skin, it is necessary to carry out an assessment of potential phototoxic hazard. Phototoxicity is skin reaction caused by concurrent topical or systemic exposure to specific molecule and ultraviolet radiation. Most phototoxic compounds absorb energy particularly from UVA light leading to the generation of activated derivatives which can induce cellular damage. This type of adverse cutaneous response can be reproduced in vitro using different models of phototoxicity such as the validated 3T3 Neutral Red Uptake (NRU) phototoxicity assay. In the present study we utilised two different cell lines (the murine fibroblastic cell line 3T3 and the rabbit cornea derived cell line SIRC) to compare the photo-irritation potential of a strong phototoxic compound, chlorpromazine, to a weaker composite, such as 8-methoxypsoralen and Bergamot oil. After comparison of the different systems, five other essential oils were tested with both cell lines. Cellular damage was evaluated by the NRU cytotoxicity test or by MTT conversion test.