Molecular characterisation of a versatile peroxidase from a Bjerkandera strain.

The cloning and sequencing of the rbpa gene coding for a versatile peroxidase from a novel Bjerkandera strain is hereby reported. The 1777 bp isolated fragment contained a 1698 bp peroxidase-encoding gene, interrupted by 11 introns. The 367 amino acid-deduced sequence includes a 27 amino acid-signal peptide. The molecular model, built via homology modelling with crystal structures of four fungal peroxidases, highlighted the amino acid residues putatively involved in manganese binding and aromatic substrate oxidation. The potential heme pocket residues (R44, F47, H48, E79, N85, H177, F194 and D239) include both distal and proximal histidines (H48 and H177). RBP possesses potential calcium-binding residues (D49, G67, D69, S71, S178, D195, T197, I200 and D202) and eight cysteine residues (C3, C15, C16, C35, C121, C250, C286, C316). In addition, RBP includes residues involved in substrate oxidation: three acidic residues (E37, E41 and D183)--putatively involved in manganese binding and H83 and W172--potentially involved in oxidation of aromatic substrates. Characterisation of nucleotide and amino acid sequences include RBP in versatile peroxidase group sharing catalytic properties of both LiP and MnP. In addition, the RBP enzyme appears to be closely related with the ligninolytic peroxidases from the Trametes versicolor strain.

Ground and excited state dissociation dynamics of ionized 1,1-difluoroethene.

The kinetic energy release distributions (KERDs) for the fluorine atom loss from the 1,1-difluoroethene cation have been recorded with two spectrometers in two different energy ranges. A first experiment uses dissociative photoionization with the He(I) and Ne(I) resonance lines, providing the ions with a broad internal energy range, up to 7 eV above the dissociation threshold. The second experiment samples the metastable range, and the average ion internal energy is limited to about 0.2 eV above the threshold. In both energy domains, KERDs are found to be bimodal. Each component has been analyzed by the maximum entropy method. The narrow, low kinetic energy components display for both experiments the characteristics of a statistical, simple bond cleavage reaction: constraint equal to the square root of the fragment kinetic energy and ergodicity index higher than 90%. Furthermore, this component is satisfactorily accounted for in the metastable time scale by the orbiting transition state theory. Potential energy surfaces corresponding to the five lowest electronic states of the dissociating 1,1-C2H2F2+ ion have been investigated by ab initio calculations at various levels. The equilibrium geometry of these states, their dissociation energies, and their vibrational wavenumbers have been calculated, and a few conical intersections between these surfaces have been identified. It comes out that the ionic ground state X2B1 is adiabatically correlated with the lowest dissociation asymptote. Its potential energy curve increases in a monotonic way along the reaction coordinate, giving rise to the narrow KERD component. Two states embedded in the third photoelectron band (B2A1 at 15.95 eV and C2B2 at 16.17 eV) also correlate with the lowest asymptote at 14.24 eV. We suggest that their repulsive behavior along the reaction coordinate be responsible for the KERD high kinetic energy contribution.

Qualitative comparison between the quantum calculations and electrospray mass spectra of complexes of polyammonium macrotricyclic ligands with dicarboxylic acids.

The host-guest interactions play a very important role in chemical and biological processes. It is therefore important to be able to characterize these complexes. Electrospray mass spectrometry can be used to characterize the complex formation. It provides information on the mass and the charge of these ionic complexes. In this article, we show that the use of ab initio and semiempirical calculations, in addition to the results obtained by electrospray mass spectrometry, reveal to be a promising tool for the study of these noncovalent complexes. In this article, host-guest complexes formed by macropolycyclic polyammonium host molecules and dicarboxylic acids are studied.

Structure-activity relationships on adrenoceptors and imidazoline-preferring binding sites (I(1,2)-PBSs). Part 1: Weak intramolecular H-bond and conformational flexibility in a new I1-PBS-selective imidazoline analogue, trans1-(4',5'-dihydro-1'H-imidazol-2'-yl)methyl-2-hydroxyindane (PMS 952).

The highly selective I1-PBS imidazoline analogue PMS 952 has been selected to study the incidence of intramolecular hydrogen bond and molecular flexibility on its biological activity. On one hand, the weak energy difference between three calculated conformers does not support the stabilization of one conformer by an internal hydrogen bond. The 3-D electrostatic map confirms this feature and the solvent effect does not significantly modify the relative energy of these conformers. On the other hand, the conformational spaces of the neutral and ionized forms present a great number of equilibrium structures, in a short energetic range (20 Kcal). The results are representative of an exceptional conformational flexibility due to a cooperative effect between several parts of the molecule.

Continuous profiles and within-day variations of metabolites and hormones in cows fed diets varying in alimentary supplies before short-term feed deprivation.

We investigated continuous profiles and within-day variations of some metabolites and hormones in four nonpregnant, nonlactating cows fed hay-based diets in two equal meals. Diets supplied either too much or too little N (approximately 1.3 or .8 times the maintenance requirements) and NE1 (approximately 1.2 or .8 times). Continuous collection of ruminal liquor, blood, and urine samples was performed for 42 consecutive hours, the last 16 h covering a period without feed. For twice-daily feeding, nitrogenous and energetic underfeeding decreased average ruminal propionate and the insulin:growth hormone ratio. However, only the energetic underfeeding increased plasma 3-methylhistidine and urinary excretion of 3-methylhistidine, and decreased body weight and ruminal acetate, butyrate, and total VFA. Conversely, only the nitrogenous underfeeding decreased glycemia. Whatever the dietary level, the 42-h patterns of metabolites and hormones were mainly affected by the time from the last meal. An energy deficit progressively took place during feed deprivation and the nocturnal interprandial period but not during the diurnal interprandial period. During the feed deprivation and nocturnal periods, glycemia was maintained despite a shortage in ruminal propionate. We conclude that in twice-daily fed cattle 1) the dietary supply of energy is the main trigger for an energy deficit and the subsequent muscle protein mobilization; 2) the nocturnal interprandial period may be considered as short-term feed deprivation; 3) the diurnal patterns of metabolites and hormones are not affected by the nitrogenous and(or) energetic supplies of the diet.

Response of milk yield, plasma cortisol, amino acids, urea and glucose to a single low-dose administration of adrenocorticotrophic hormone in lactating cows.

An experiment was conducted to determine the effects of a single low-dose administration of adrenocorticotrophic hormone (ACTH) on milk yield, plasma cortisol, free amino acids, urea and glucose in lactating cows. The animals were treated with either 6 IU synthetic ACTH or 5 ml physiological saline (control) administered intravenously via a jugular vein catheter. Blood was withdrawn 60 and 5 min pretreatment (baseline), and 10, 20, 30, 60, 120, 180, 240, 300, 360, and 420 min post-treatment. A rapid positive response (P < 0.05) in plasma cortisol occurred within 10 min of administration of 6 IU ACTH. The maximum increase in plasma cortisol concentration occurred at 1 h post-ACTH treatment and plasma cortisol returned to baseline 4 h later. Until 7 d after ACTH administration, no effect on milk yield was recorded. In comparison with the saline-treated group, the ACTH-treated group exhibited a significant (P < 0.05) increase in the plasma concentrations of 3-methylhistidine, glycine, histidine, isoleucine, leucine, lysine, valine, and glucose. In contrast, the concentrations of alanine, aspartate, glutamate, glutamine and proline decreased significantly (P < 0.05) after ACTH treatment. Hormone administration had no effect on the plasma arginine, asparagine, methionine, phenylalanine, serine, threonine, tyrosine, and urea. These results demonstrate that the bovine species behaves like other mammalians with respect to its metabolic response to stress. Thus, during stress, ACTH increases adrenal cortical activity which, in turn, stimulates protein catabolism in muscle and gluconeogenesis from some non-essential amino acids.

[Energetic and/or nitrogen underfeeding in the dry cow. Effects on volatile fatty acids in the rumen, some plasma metabolites and hormones and urinary excretion of 3-methylhistidine]. / La sous-alimentation énergétique et/ou azotée chez la vache tarie. Effets sur les acides gras volatils du rumen, quelques métabolites et hormones du plasma et l'excrétion urinaire de la 3-méthylhistidine.

In order to study the effects of a nitrogenous and/or energetic underfeeding on some metabolites and hormones, 4 dry cows were fed in 2 equal daily meals with hay-based rations. Four rations were distributed. They supplied too much (HN) or too little (LN) digestible protein in the small intestine and too much (HE) or too little (LE) net energy, ie the diets were HN-HE, HN-LE, LN-HE, and LN-LE. Continuous collection of rumen liquor samples (volatile fatty acids or VFA determination), venous blood samples (amino acids (alanine Ala, glutamine Gln, glycine Gly and serine Ser), urea, glucose, insulin and growth hormone (GH) measurements) and urine samples (determination of the amount of 3-methylhistidine 3-MeHi excreted) was carried out for 24 consecutive hours. By comparison with the reference diet (HN-HE), underfeeding was accompanied by a reduction in ruminal VFA concentrations, an increase in those of plasma Gly and GH, and a maintenance of glycemia levels. Furthermore, nitrogenous and energetic underfeeding (LN-LE) was accompanied by a decrease in plasma Ala, Gln and insulin concentrations. Nitrogenous underfeeding (LN-HE) caused a decrease in uremia levels, an increment in those of serinemia and no variation in circulating insulin concentrations. The energetic underfeeding (HN-LE) was accompanied by a reduction in insulinemia levels and an increase in those of alaninemia and serinemia. Such changes suggest that dry cattle adapt to underfeeding by mobilizing endogenous proteins. However, only the 2 diets with reduced energetic supplies were accompanied by an increase (P < 0.05) in the amount of the urinary 3-MeHi excreted per 24 h, suggesting that the level of energy in the diet is the main trigger for protein mobilization. Finally, the study of the 24-h evolution of the parameters revealed the influence of the feeding time (morning or evening) and the level of the dietary nitrogen and energy supplied on the variation in their concentration.

Total protozoa counts and ammonia in the rumen of mature dry Friesian cows on hay-based rations.

The present work was an attempt to determine whether the variations in ruminal ammonia concentrations could be directly correlated to corresponding changes in total protozoa numbers of cattle. Four dry Friesian cows fed with hay-based rations were used through several experiments in which the twice daily feeding (6.15-15.30 h) as well as a 30 h-fasting period were studied. Simultaneously to a continuous or a regular collection of rumen liquor (for NH3), samples of rumen contents (for total protozoa numbers) were regularly withdrawn from the ventral sac. Any definite nycthemeral cycle of the protozoa numbers could not be related to feeding time. Moreover, no significant relationship could be found between the ruminal ammonia and the corresponding total protozoa numbers measured in the nocturnal interprandial period or during starvation. The data suggest that the nycthemeral ammonia profiles recorded in cattle are not directly related to protozoal activity.

Electrostatic potential maps at the quantum chemistry level of the active sites of the serine peptidases, alpha-chymotrypsin and subtilisin.

The electronic properties of the active-sites of the structurally unrelated serine peptidases, alpha-chymotrypsin and subtilisin, have been expressed in the form of three-dimensional electrostatic potential maps derived from integrals calculated at the quantum chemistry level. As a consequence of the asymmetrical distribution of the secondary structures that occur within a 7 A sphere around the serine of the catalytic triad, the active sites are highly polarized entities and exhibit large dipole moments. One part of the active sites generates a nucleophilic suction-pump. Its isocontour at -10 kcal mol-1 defines an impressive, negatively-charged volume which bears a narrow channel in the immediate vicinity of the active-site serine 195 in alpha-chymotrypsin or 221 in subtilisin. In native alpha-chymotrypsin, there is a perfect complementation between this nucleophilic suction-pump and the positively-charged electrophilic hole that is generated by the backbone NH of Ser 195 and Gly 193. In subtilisin, generation of the complementing electrophilic hole requires binding of a carbonyl donor ligand and may be achieved by rotation of the side-chain amide of Asn 155 towards the backbone NH of Ser 221. Small variations in the atomic co-ordinates of alpha-chymotrypsin used for the calculations, the presence of water molecules in its active site and the occurrence of point mutations in the amino acid sequence of subtilisin have little effects on the shape and characteristics of the electrostatic potential.

[Nyctohemeral variations in the ammonia concentration in rumen fluid, in dairy cows during the peripartum period]. / Variations nycthémérales de la concentration en ammoniac dans le liquide du rumen, chez les vaches laitières dans la période péri-partum.

Automated and continuous recording of ammonia concentration in rumen liquor (Godeau et al., 1986) was performed on four cows around their first calving. Results suggest an insufficient concentration in early lactation.