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Objectives Osteoarthritis (OA) is a common degenerative joint disease with the pathological features of the reduced cartilage cellularity. Celastrol, a compound from Tripterygium wilfordii, exerted therapeutic effects on arthritis, but the potential mechanism remains unclear. Methods Tunicamycin was used to establish a model of OA in vitro, and ACLT surgery model in rats was applied to verify the mechanism. Chondrocytes were isolated from the knee articular cartilage of rabbit. MTT and flow cytometry assay were used to detect cell viability and apoptosis rate. Haematoxylin–eosin staining was used to assess for the histopathological changes. The activity and expression of apoptosis-related factors and ERs (endoplasmic reticulum stress)-related factors were detected by ELISA, WB, PCR and IHC, respectively. Key findings Celastrol exhibited significant enhancement on cell viability and reduced the rate of apoptosis in Tm-exposed chondrocytes. Celastrol reduced enzyme activity and protein expression of caspase-3, caspase-6 and caspase-9, decreased Bip, Atf6, Chop and Xbp-1 expression both at protein and mRNA levels. Celastrol showed a more significant effect on cell apoptosis rate and mRNA expression in the combination with 4-PBA. Conclusions This study reveals that celastrol may prevent OA by inhibiting the ERs-mediated apoptosis. All these might supply beneficial hints for celastrol on OA treatment.

The nuclear magnetic resonance study of site-specific natural isotope fractionation (SNIF-NMR) produced in the glycolytic conversion of glucose into ethanol and glycerol provides isotopic transfer coefficients, a ij , which relate sites in the products to sites j in the reactants. The isotopic connection between the carbon-bound hydrogens of glycerol and those of glucose and water in fermentation reactions carried out with Saccharomyces cerevisiae has been investigated. The a ij coefficients provide mechanistic information on the genealogy of the glycerol hydrogens, on the relative rates of triose phosphate isomerization and reduction of dihydroxyacetone phosphate into glycerol 3-phosphate, on the stereospecificity of the reduction, on the percentages of intra- and intermolecular hydrogen transfer occurring in the course of the reaction, and on the order of magnitude of the active overall kinetic and thermodynamic isotope effects. Thus a close connection is determined between sites 3 pro-R of glycerol (stereospecific numbering) and H 6 pro-R and to a lesser extent H 2 of glucose and between site 3 pro-S of glycerol and H 6 pro-S and H 1 of glucose. Moreover site 2 of glycerol, which exhibits a strong correlation with water is also partly connected with glucose. Possible changes in the values of the isotopic transfer coefficients, as a function of the composition of the medium or of the environmental conditions, enable mechanistic perturbations such as variations in the percentage of intermolecular transfers to be detected. Analyzed in terms of stereospecificity of the reduction step the isotopic results provide a direct chemical shift assignment of the enantiomeric pairs (1 S , 3 R ) and (1 R , 3 S ) of glycerol triacetate. The influence of added bisulfite, which strongly increases the yield in glycerol is estimated. The isotopic characterization of the bioconversion producing both ethanol and glycerol has been extended to the determination of the carbon isotopic parameters by isotope ratio mass spectrometry. Although it usually occurs as a by-product of the fermentation, glycerol can be considered as a useful complementary probe for characterizing the glycolytic pathway and for inferring various properties of the carbohydrate precursors.

The isotopic parameters of fermentation products provide reliable criteria for characterizing their carbohydrate precursors on the condition that the isotopic coefficients which relate individual sites in the products and in the reactants have strictly reproducible or predictible values. Since fermentation may be performed either in natural media (such as fruit juices) which exhibit variable compositions or in “synthetic” media prepared from appropriate nutrients, it is necessary to estimate the influence of the medium on the carbon and hydrogen isotope ratios. By combining isotope ratio mass spectrometry and nuclear magnetic resonance investigations it has been shown that variations in the reaction rates induced by the medium are not connected with isotopic fractionation effects. At natural concentrations the nutrients present in juices, for instance, ensure a significant acceleration of the bioconversion with respect to pure water without introducing undesirable isotopic effects. The role of different nutrients added in relatively large amounts to synthetic media has been analyzed. It has been shown in particular that a significant decrease in the isotope ratio of the methylenic site of ethanol is due to the combined influence of both phosphates and assimilable nitrogen which are also the key components governing the growth and activity of yeasts. For a given nutrient composition the effect of the medium on the isotopic coefficients which connect the individual sites of ethanol to the starting water can be estimated. The observed behavior can be explained by modifications in the percentages of intra- and intermolecular hydrogen transfers (methyl site) and by variations in the kinetic isotope effects and exchange rates involving the hydrogens originating from water. Carbohydrates from different origins (beet and cane) have been fermented in different “natural” media prepared by eliminating sugars from orange, apple, and grape juices. In spite of very different fermentation rates the isotopic ratios of the products are then nearly independent of the composition of the medium. These results support the use of the ethanol probe for characterizing endogenous or exogenous sugars contained in different fruit juices.

For a given biochemical transformation, such as the fermentation reaction, the redistribution coefficients, which relate the natural site-specific isotope contents in end products to those of their precursors, are a source of mechanistic information. These coefficients characterize the traceability of specific hydrogens in the products (ethanol and water) to their parent hydrogens in the starting materials (glucose and water). In conditions of complete transformation, they also enable intermolecular exchanges with the water medium to be estimated. Thus it is directly confirmed that hydrogens 1, 2, 6, and 6′ of glucose are strongly connected to the methyl site I of ethanol obtained by fermentation by Saccharomyces cerevisiae. However, whereas hydrogens 6 and 6′ are transferred to a great extent, transfer is only partial for hydrogen 2, and it is even less for hydrogen 1. Because the two moieties of glucose corresponding to carbons 1-2-3 and 4-5-6 are scrambled by the aldolase and triose-phosphate isomerase reactions, additional exchange of hydrogens at positions 1 and 2 must have occurred before these steps. The value of the coefficient that relates site 2 of glucose to site I of ethanol in particular can be used to quantify the contribution of intermolecular exchange occurring in the course of the transfer from site 2 of glucose 6-phosphate to site 1 of fructose 6-phosphate mediated by phosphoglucoisomerase. The average hydrogen isotope effects associated with the transfer of hydrogen from the water pool to the methyl or methylene site of ethanol are estimated. In contrast to conventional experiments carried out in strongly deuterium-enriched media where metabolic switching may occur, the NMR investigation of site-specific natural isotope fractionation, which operates at tracer isotopic abundance, faithfully describes the unperturbed metabolic pathways.

Several series of wines were prepared in standard conditions from well defined varieties (Cabernet-Sauvignon, Carignan, Chasselas, Chardonay, Grenache Noir, Riesling, Sauvignon, Syrah et Ugni Blanc) harvested in 1991 in three different regions of production in France (Alsace, Bordeaux and Languedoc). In order to study the influence of the year of production as well, the same varieties grown in Anjou were considered for three different periods of time : 1983, 1984 and 1988. The stable isotope composition of these wines was determined by 2H-NMR spectroscopy ((D/H)I and (D/H)II isotope ratios of ethanol) and by Mass Spectrometry for water (2H and 18O) and ethanol (13C). The variations observed for the wines of the different varieties are explained in terms of the climatic conditions (temperature, precipitation and insolation) which governed the regions of production during the vine vegetation cycles considered. It is shown that similar behaviour is observed for the 2H and 18O contents of the water of musts and wines but they differ from the Craig relationship existing in meteorological waters. The early vine varieties cultivated in the different regions considered give wines with a higher concentration in the heavy isotopes than the later varieties as far as the ethanol (D/H)I parameter is concerned.

Cyanide is one of the common poisons in murders. When cyanide has been used, to identify the origin of cyanide may be necessary in the forensic investigation. We have examined the possibility of distinguishing different commercial cyanide samples through the δ 13 C and δ 15 N values and developed a protocol for the isotope analysis of cyanide extracted from several matrices as food and medicine. Several cyanide precipitates were tested for the isotope analysis. The results show that cupric ferrocyanide Cu 2 [Fe(CN) 6 ] is the most appropriate precipitate for the analysis. Thirteen batches of KCN and nine batches of NaCN chemicals were randomly chosen from different suppliers. The cyanides were converted to cupric ferrocyanide and then analysed by isotope ratio mass spectrometry coupled to elemental analysis (EA-IRMS). The isotopic signature of the commercial samples varied from −51.96 to −25.77‰ for δ 13 C and from −4.51 to +3.81‰ for δ 15 N, highlighting the potential of applying EA-IRMS technique to identify cyanide from different batches and sources. The influence of the cyanide extraction and isolation from spiked matrix on the isotopic analysis was also studied. Three matrices: orange juice, yogurt drink and a medicine were tested. In many cases, the isotopic analysis results obtained from the original cyanides precipitates and those isolated from the matrices showed a good accordance, especially for δ 15 N. In some matrices, the 13 C analysis was interfered by co-precipitates. With carefully elaborated working protocol, determining the isotope ratio of N and C in cyanide by EA-IRMS is a promising method for forensic investigations.

International audience; The anaerobic fermentation of glucose by Leuconostoc mesenteroides via the reductive pentose phosphate pathway leads to the accumulation of lactic acid and ethanol. The isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom in ethanol in relation to the non-exchangeable hydrogen atoms in glucose and the medium water have been determined using quantitative H-2 NMR. First, it is confirmed that the hydrogens of the methylene group are related only to the 1 and 3 positions of glucose via the NAD(P)H pool and not to the 4 position, in contrast to ethanol produced by Saccharomyces cerevisiae. Second, it is found that the conversion factors (C-f) for the transfer of hydrogen to the pro-S and pro-R positions of the methylene group are not equivalent: the C-f- 1-R: C-f-1-S ratio is 2.1, whereas the Cf-3-R: Cf-3-S ratio is 0.8. It is shown that this non-equivalence is not determined by the stereochemistry of the terminal NADH- and NADPH-dependent alcohol dehydrogenases, but is dependent on the cofactor selectivities of the reductive and oxidative steps of the reduced nucleotide cycle.

The hydrogen transfer mechanism of cofactor reduction and recycling processes in the yeast reduction of alpha,beta-unsaturated ketone was studied by using quantitative isotope tracing close to natural abundance measured by (2)H NMR. In the reaction, the active cofactor is NADPH. The cofactor-transferred hydride attacks the beta sp(2) carbon of the enone carbonyl while water hydrogen is transferred to the alpha position. The reductant involved in the reaction depends on the quantity of yeast. When the amount of yeast is very large, the enzymes use preferentially certain unidentified substance stored in the yeast cells rather than the added glucose as electron donor. In this case, the hydrogen transferred by the cofactor is mainly of water origin. When the yeast amount is low, the added glucose is more efficiently used by the enzymes as electron donor and its hydrogen atoms bound to C-1 and C-3 are delivered to the substrate.

For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined. By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid. Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used. Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.

Pure d (−) and l (+) enantiomers of lactic acid were prepared by fermentation reactions with specific bacteria. In addition, naturally deuterated ethanol was prepared and converted into diastereoisomers using mandelic acid. Various sugars and nutrients were fermented into lactic acid in water having different deuterium contents and ethanol samples were obtained from yeast fermentation of sugars from different botanical origins. The methine and methylene groups in lactic acid and ethanol respectively show similar deuterium contents which are related to that found in the fermentation water. However, the methyl groups of both molecules are significantly different whatever the botanical origin of the carbon source in the fermentation medium.

Le role nucleophile du solvant dans l'etape d'ionisation de bromation via des intermediaires bromonium n'est pas negligeable. Ce resultat est deduit de la comparaison des effets polaires et steriques de groupes alkyles dans le methanol et l'acide acetique

Naturel abundance deuterium NMR has been applied to the quantitative determination of natural methylene chirality in amino acids of different configurations and in ethanols of different origins. An enantiomeric enrichment is detected for aspartic acid of the L series obtained from fermentation and the technique therefore provides an absolute method for identifying the natural origin of a given substance. In ethanols of different origins the pro-R and pro-S positions appear as naturally equally populated at the present level of precision of the experiments. These results provide a new source of information about reaction mechanisms in the natural conditions.