[Show abstract][Hide abstract] ABSTRACT: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2-arachidonoylglycerol and anandamide (N-arachidonoylethanolamine), are up-regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids.
The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator-activated receptor (PPAR)-gamma activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high-performance liquid chromatography (HPLC)-atmospheric pressure chemical ionization (APCI)-mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme-linked immunosorbent assay (ELISA), and PPAR-gamma protein expression examined by Western blotting.
We show that 2-arachidonoylglycerol, anandamide, and both anandamide analogs, N-palmitoylethanolamine and N-oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.042+/-0.004 to 0.531+/-0.048 pM/mg lipid extract. N-palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down-regulated by leptin but not affected by adiponectin and PPAR-gamma agonist ciglitazone. N-palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR-gamma protein expression in adipocytes.
This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.
[Show abstract][Hide abstract] ABSTRACT: In this study, we have investigated the frequencies of TAP1 and TAP2 alleles in a group of 226 persons, living in La Reunion Island, consisting of 70 patients with insulin-dependent diabetes mellitus (IDDM) and most of their first degree relatives (i.e., 156 parents and full sibling subjects) and previously HLA DQB1, DQA1, and DRB1 genotyped. The population of this island is constituted by a particular structure of highly crossbreeding people. Interestingly, the new TAP2*0104 allele, previously discovered by our team in Reunion Island, was found to be increased in the IDDM population and the calculated HRR was relatively high (HRR = 3.3). This result seems to be due to a positive linkage disequilibrium between TAP2*0104 allele and the highly diabetogenous DQB1* 0201-DQA1* 0501-DRB1 0301 haplotype (HRR = 9), which suggests that TAP2*0104 cannot be considered as an additional predispositional factor, but more as a genetic susceptibility marker of IDDM. In addition, we show that minor alleles (TAP2D, *0102, *0103, *0104) are associated with a restricted number of HLA DQ-DR haplotypes and each of them exhibits a preferential linkage with one particular haplotype. In contrast with other alleles, and despite a HRR value close to 1, we show that TAP2*0102 allele contributes significantly to a drastic reduction of the diabetogenic effect of DQB1*0201-DQA1*0301.1-DRB*0701 haplotype. Indeed, this haplotype, which is usually preferentially transmitted to affected children, is dominantly transmitted to healthy children when it is associated with TAP2*0102. Therefore, this allele seems to contribute to genetic protection to IDDM.
[Show abstract][Hide abstract] ABSTRACT: The optimal temperature of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is 30 degrees C. The Arrhenius activation energy was calculated to be 5.41 kJ mol(-1), Delta H degrees of the reaction is -60.99 kJ mol(-1). At 25 degrees C, Delta G degrees and Delta S degrees were, respectively, 16.75 kJ mol(-1) and -260.87 J mol(-1) K-1. The enzyme heated at temperatures above 30 degrees C loses its activity. Fifty percent inhibition is reached in 18 min at 70 degrees C, in 8 min at 75 degrees C, and in 2.5 min at 80 degrees C. The kinetics of the thermal irreversible denaturation of this enzyme is characterized by two steps: N --> X(T-d) --> D, where N represents the native form, X represents an intermediate form, the structure of which depends on the deactivation temperature T-d, and D is the completely denatured form of the enzyme. Our experimental results rule out a two-isoenzyme (with varying heat sensitivity) model. The thermodynamic parameters of the irreversible denaturation of the intermediate form were 102.70 and 97.10 kJ mol(-1) for Delta H and Delta G, respectively, and 16.85 J mol(-1) K-1 for Delta S at 60 degrees C. Furthermore, this paper describes the effect of pH on the activity of the PPO. Studies were carried out with. 4-methylcatechol and pyrogallol as substrates. The pH profile was not a function of the nature of the substrate assayed. The pH optimum was 5.2. The plot of logV(max app) vs pH indicates that the oxidation of the substrates depended of the ionization of two groups in the enzyme-substrate complex with apparent pK values of 3.06 and 7.29 and 3.44 and 7.12, respectively, for 4-methylcatechol and pyrogallol. The very slight differences between the values suggest the existence of only one site on the molecule for both substrates.
No preview · Article · Apr 2002 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halide salts have the same inhibitory effect on the two forms of palmito PPO separated by hydrophobic chromatography. Fluoride and chloride ions showed a non-competitive, mixed type inhibition while bromide and iodide ions were found to be non-competitive inhibitors. A study of the Ki for the different halide salts showed that the smaller F- ion is a stronger inhibitor than I- and Br- and that Cl- has the highest Ki value. This suggests that the active site of the palmito PPO is not easily accessible. The inhibition by chloride and fluoride ion was found to be pH-dependent. The inhibitory effects of these ions increased with a decrease in pH. It is suggested that halide ions (X) could bind to either the protonated enzyme (EH) or the protonated substrate-enzyme complex (EHS) to yield inactive forms EHX and EHSX, respectively.
No preview · Article · Aug 1998 · Journal of enzyme inhibition
[Show abstract][Hide abstract] ABSTRACT: Several methods for protein determination have been described. In almost all cases, a detection reagent is involved (Coomassie Blue reagent, bicinchoninic acid, Folin reagent). Proteins quantification determination by measurements in the U.V. region have been abandonned because of the weak sensitivity of the apparatus (concentrations of the order of 100 μg/ml) and because of the lack of precision at low wavelengths (far U.V.).Owing to the progress in the performance of equipments, (i) we have been able to show that the reliability of current equipments allowed measurements in the more sensitivity range of proteins (190 to 220 nm) and beyond (220 to 1100 nm) and (ii) we have hence calculated the correlation factors between absorbance values and wavelengths for 17 proteins. It was found that 190 nm and 277 nm were the best wavelengths in far U.V. region for the protein quantification (correlation factor respectively of 0.69 and 0.62).The concentrations of 16 proteins were predicted at 190 nm and 277 nm, with lactoglobulin (standard range between 0 and 35 μg/ml, and 0 to 3.5 μg/ml) and trypsinogen (standard range between 0 and 35 μg/ml) respectively as standards. The results were better (especially at 190 nm) than those achieved with the BCA method (indirect quantification of proteins with the Bicinchoninic Acid method), one of the mostly used method at the moment.The main interest of such a quantification method is in its non damaging effects on proteins. Indeed, since HPLC is the mostly used purification methods, tiny quantities of proteins are recovered; it is hence necessary to save them for physico-chemical characterisation (molecular weight, secondary and quaternary structures …) and in case of enzymes, for kinetic studies.
[Show abstract][Hide abstract] ABSTRACT: The inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is reported. Recently, two forms of palmito PPO were partially purified by hydrophobic chromatography. Inhibitory effects of various carboxylic acids on these two forms have been studied. Both forms showed identical behaviour towards the inhibitors studied. Cinnamic acid was found to have the greatest inhibitory effect (Ki = 0·06 mM). When the inhibitory effects of acids from the benzoic acid family and from the cinnamic acid family were compared, it was found that acids which possess a double bond between the benzene ring and the carboxylic function showed the highest inhibitory effect. This inhibitory effect was decreased by substitutions on the benzene ring.The influence of pH on the inhibitory effect of carboxylic acids on palmito PPO has also been investigated. Ki decreased with a decrease in pH. This effect is due to the fact that it is only the undissociated form (AH) of carboxylic acids that is responsible for inhibition of PPO. Inhibition constants (Ki for the AH form have been recalculated and were found to remain constant in the pH range studied (for benzoic acid Ki = 0·14 mM, for cinnamic acid Ki = 0·019 mM, for sorbic acid Ki = 0·15 mM).
[Show abstract][Hide abstract] ABSTRACT: Polyphenol oxidases (PPO) are responsible for the oxidation of phenols into quinones that give brown or black pigments. The aim of the work presented in this paper was to study the mechanism of inhibition of palmito (Acanthophoenix rubra) PPO by metabisulfite. When monitored spectrophotometrically and with an oxymeter, a decrease in enzymatic activity was observed. When measured with the oxymeter, there was a decrease in oxygen comsumption, indicating enzyme inhibition. When monitored with the spectrophotometer, the existence of a lag phase was noted, indicating that the quinones formed either are reduced into phenols or react with metabisulfite to give a colorless complex. The oxidation of phenol took place during the lag phase, and when metabisulfite was depleted, the amount of oxygen was not enough to allow the oxidation of the substrate in the optimal conditions. The plot of inhibition versus metabisulfite showed an allosteric behavior of PPO with positive cooperativity. The incubation of PPO with metabisulfite resulted in inhibition of enzymatic activity. This inhibition was as strong as the concentration of metabisulfite was high. The plot of log(residual activity) versus incubation time showed a diphasic behavior. All of these results indicate that PPO inhibition by metabisulfite does not obey a first-order reaction and that the action of metabisulfite is accompanied by a conformational change of the enzyme. Accordingly, bisulfite would react with the enzyme and form an intermediate complex that is more stable than the native form. The decomposition of this complex would give an inactive enzymatic form (Ei) through a slower second step. Fitting experimental values to the equation describing a two-step denaturation model confirmed the structural changes observed by electrophoresis by other authors.
No preview · Article · Nov 1996 · Journal of Agricultural and Food Chemistry
[Show abstract][Hide abstract] ABSTRACT: Polyphenol oxidase (PPO) is responsible for browning reactions in fruits and vegetables. In order to inhibit these reactions during processing, the use of chemical inhibitors including thiols such as L-cysteine has been proposed. The effect of this thiol on PPO activity was studied in order to establish if it reacts with quinone and/or directly inhibits the enzyme. The inhibition of palmito PPO (catecholase activity, with 4-methylcatechol as substrate) by L-cysteine was characterized spectrophotometrically. The effect of increasing cysteine concentration with respect to incubation time was measured by polarography and the kinetic parameters calculated. Kinetic analysis using spectrophotometric assays showed a lag period suggesting that there is no quinone accumulation in the reaction mixture. The increase of absorbance at 300 nm observed with u.v.—vis spectra indicated that a colourless compound (thiol—quinone complex) was produced. The plots of log remaining enzyme activity vs incubation time were characterized by two straight lines suggesting two possible inactivation models: (i) the deactivation of one enzyme that proceeds in two steps or (ii) the existence of two isoenzymes that behave differently. We have shown that the inactivation process of PPO by cysteine followed this model: N → X[C] → I where N represents one native form, X represents an intermediate form, the structure of which depends on the cysteine concentration [C], and where I is the completely inactive form of the enzyme. These results showed that there is a direct irreversible inhibition of PPO by cysteine according to a two-step model. The analytical approach illustrated by the present study of palmito PPO inhibition by L-cysteine may be applied to other investigations of mechanisms of enzyme inhibition.
No preview · Article · Apr 1996 · The international journal of biochemistry & cell biology
[Show abstract][Hide abstract] ABSTRACT: An alternate method for enzyme study is proposed. Multidimensional statistical analysis applied on mid-infrared attenuated total reflectance spectra (Cadet et al. (1991) Appl. Spectrosc. 42, 166-172) collected during a kinetic allows a direct and fast quantification of the remaining substrate, as well as a one step enzymatic assay. Furthermore, the combination of these techniques may be used as a structural tool. The method applied to the study of beta-fructosidase is developed in this paper as an example. With appropriate calibration, the method may be extend to any enzyme.
Full-text · Article · Feb 1995 · Biochimica et Biophysica Acta
[Show abstract][Hide abstract] ABSTRACT: A fast and accurate method for determining the sucrose content of sugar cane juice has been developed. The application of principal component regression (PCR) has been proposed for the development of a prediction equation of sucrose content by mid-infrared spectroscopy. An attenuated total reflectance (ÀTR) cell is used in place of the more familiar hans-mission cell. PCR involves two steps: (1) the creation of new synthetic variables by principal component analysis (PCA) of spectral data, and (2) multiple linear regression (MLR) with these new variables. Results obtained by this procedure have been compared with those obtained by the conventional application of polarization.
Full-text · Article · Feb 1991 · Applied Spectroscopy