Claude Rouch

Identification of Endocannabinoids and Related Compounds in Human Fat Cells

Objective: 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.

Quantitative Determination of Sugar Cane Sucrose by Multidimensional Statistical Analysis of their Mid-Infrared Attenuated Total Reflectance Spectra

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 (ATR) cell is used in place of the more familiar transmission 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.

Inhibition of palmito (Acanthophoenix rubra) polyphenol oxidase by carboxylic acids

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).

A kinetic study of the inhibition of palmito polyphenol oxidase by L-cysteine

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.

Enzyme kinetics by mid-infrared spectroscopy: β-fructosidase study by a one-step assay

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.

Assessment of the C4 phosphoenolpyruvate carboxylase gene diversity in grasses (Poaceae)

Abstract.C4 phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the C4 photosynthetic pathway. To analyze the diversity of the corresponding gene in grasses, we designed PCR primers to specifically amplify C4 PEPC cDNA fragments. Using RT-PCR, we generated partial PEPC cDNA sequences in several grasses displaying a C4 photosynthetic pathway. All these sequences displayed a high homology (78–99%) with known grass C4 PEPCs. PCR amplification did not occur in two grasses that display the C3 photosynthetic pathway, and therefore we assumed that all generated sequences corresponded to C4 PEPC transcripts. Based on one large cDNA segment, phylogenetic reconstruction enabled us to assess the relationships between 22 grass species belonging to the subfamilies Panicoideae, Arundinoideae and Chloridoideae. The phylogenetic relationships between species deduced from C4 PEPC sequences were similar to those deduced from other molecular data. The sequence evolution of the C4 PEPC isoform was faster than in the other PEPC isoforms. Finally, the utility of the C4 PEPC gene phylogeny to study the evolution of C4 photosynthesis in grasses is discussed.

Inhibition of Palmito Polyphenoloxidase by Halide Salts

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.

Microquantification of Proteins by Spectrophotometry. Part I: From 190 nm to 1100 nm, Selection of Wavelengths

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. region1 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...

A new human transporter associated with antigen processing alleles encodes a large C-terminal protein domain.

The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. Five nucleotide variation sites that induce a change in amino acid residues were identified in the TAP2 gene, and seven alleles derived from the combination of these dimorphic sites have been described (Bahram et al. 1991; Colona et al. 1992; Moins-Teisserenc et al. 1994; Szaffer et al. 1994; see Table 1). In the present study, TAP2 typing was performed by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) as previously described (Powis et al. 1993). Oligonucleotide primers used for this typing are shown in Table 2. A group of 78 families living on Reunion Island, consisting of 82 insulin-dependent diabetes mellitus (IDDM) patients and most of their first-degree relatives (i. e., 154 parents and full sibling subjects), were studied. Seventeen of the 236 persons analyzed (seven IDDM patients and ten parental controls) showed a new combination of nucleotides located at polymorphic sites, allowing the definition of a new allele. This new allele, TAP2 G (see Table 1), was characterized by the presence of amino acid residues Thr on position 665 and Gln on position 687. No exception to the previously detected association between 655 Ala and 687 Gln or 665 Thr and 687 Stop has been described in Caucasians (Martinez-Laso et al. 1994; Szaffer et al. 1994). This linkage disequilibrium which was considered absolute in Caucasians (IDDM patients and control) is not recovered in the Reunion Island population, which is highly crossbred (including Caucasians, Africans, Indians, and Chinese). Sequence analysis of cDNA PCR amplified fragment, coming from the homozygous immortalized B-cell line Reu-M4-1, has confirmed the existence of this allele.

Contribution to the study of the Hatch and Slack photosynthetic cycle. Part II: Kinetic properties of the PEPcase-MDH-Malic enzyme multienzymatic complex purified from a C4 plant (sugarcane)

We found out the existence in fresh young leaves of sugarcane of a PEPcase-MDH(NADP)-Malic enzyme multienzymatic complex. We have been able to purify this complex. Its functional advantage has been investigated by comparing the kinetic properties of the constitutive enzymes in their free state with those in their complexed state.