Bromopyruvate, a potential affinity label for octopine dehydrogenase

Bromopyruvate, an analogue of pyruvate, one of the substrates of octopine dehydrogenase, was tested as an inhibitor of the enzyme. Provided both the coenzyme and the second substrate, arginine, were present, bromopyruvate rapidly inactivated the enzyme. This inactivation was irreversible, obeyed pseudo-first order kinetics and exhibited a rate saturation effect. Pyruvate protected the enzyme against inactivation by bromopyruvate and these compounds competed for the same site. Bromopyruvate also behaved as a true substrate for the enzyme. This reagent thus exhibits the kinetic characteristics of a good affinity label for octopine dehydrogenase.     

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.     

Implication of glutamate, isocitrate and malate deshydrogenases in nitrogen assimilation in the cadmium-stressed tomato

Tomato seedlings grown on nitric medium and treated with various cadmium concentrations (0 to 50 microM) were used. Results obtained show that cadmium remains predominantly located in the roots, which then seem to play the role of trap-organs. Increasing cadmium concentration in the medium leads particularly to a decrease in NO3- accumulation, together with a decrease in the activity of glutamine synthetase and in the quantity of plastidic isoform ARNm (GS2), and, on the contrary, to an increase of the cytosolic isoform ARNm (GS1). On the other hand, stimulations were observed for NADH-dependent glutamate synthase, NADH-dependent glutamate dehydrogenase, ARNm quantity of this enzyme, ammonium accumulation, and protease activity. In parallel, stimulations were observed for NAD+ and NADP+-dependent malate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. These results were discussed in relation to the hypothesis attributing to the dehydrogenase enzymes (GDH, MDH, ICDH) an important role in the plant defence processes against cadmium-induced stresses.     

Generation of hydrogen peroxide during the oxidation of L-phenylalanine by Proteus mirabilis isolated membranes

In the process of L-phenylalanine oxidation by Proteus mirabilis cytoplasmic membrane, hydrogen peroxide was produced at a rate corresponding to 1-3 per cent of the total electron flow (30-110 nmoles min-1mg-1). Peroxide was estimated using a fluorimetric assay with horseradish peroxidase, or by anodic oxidation on a platinum electrode. When using the former method, superoxide dismutase decreased the apparent yield of peroxide, a fact suggesting that H2O2 was in part the dismutation product of superoxide radicals. However the superoxide dismutase effect could be an artefact due to the generation of some superoxide during the peroxidatic reaction in the assay. Adrenaline was the reagent used for the detection of superoxide. There was no significant emergence of superoxide as the result of phenylalanine oxidation by the membrane (specific activity lower than 1-2 nmoles min-1mg-1). Thus it seemed that superoxide was not an intermediate for the bulk of H2O2 formed in this system. According to the results, peroxide was probably formed at a stage of electron transport earlier than the cytochrome level. The membrane phenylalanine dehydrogenase could be a site where peroxide was evolved in these experiments.     

Premiers cas de brachyptérisme dans le genre Sisyphus Latreille (Coleoptera,Scarabaeidae, Sisyphini)

Les espèces du genre Sisyphus Latreille, 1807, présentant des touffes de soies sur les élytres sont étudiées. Sept nouvelles espèces appartenant à ce groupe sont décrites, illustrées et comparées aux espèces proches: deux espèces aux ailes normalement développées, S. manni n. sp. et S. perissinottoi n. sp., et cinq espèces brachyptères, S. jossoi n. sp., S. janssensi n. sp., S. bouyeri n. sp., S. felschei n. sp. et S. usambaricus n. sp. Une autre espèce appartenant au groupe étudié, S. alveatus Boucomont, 1935, se révèle également être brachyptère. Elles constituent les premiers exemples de brachyptérisme au sein de ce genre. Une clé d’identification des espèces à touffes de soies sur les élytres est donnée.     

Indépendance des formes spectrales de chlorophylle a et des holochromes chlorophylliens

Independence of special forms of chlorophyll a and chlorophyll holochromesZea mays L. seedlings were cultivated for 10 days with submission to 4 s illumination periods interspersed with dark periods varying in length from 30 min to 6 h depending on the lot analyzed. The results show that, for the case in which the dark periods were shorter than 1 h, the relative proportions of different spectroscopic chlorophyll forms (maxima at 662, 670, 677.5, and 684 nm) were constant. For longer durations of darkness between illuminations, the relative proportion of the form Ca₆₇₀ increases, while that of Ca₆₈₄ diminishes with the length of darkness; to a lesser extent, the relative proportion of Ca₆₆₂ increases and a form Ca₆₉₂ disappears. A scheme is proposed to explain the evolution of the relative proportions of the different spectral forms.The different chlorophyll holochromes present in the chloroplasts were also analysed. If the dark period was longer than 1 h, chlorophyll was associated with peptide chains of molecular weights 21 000 and 29 000. If the dark period was shorter than 1 h chlorophyll was associated with four peptide chains of molecular weights 21 000, 25 000, 29 000 and 70 000.The results taken together demonstrate that a given spectral chlorophyll a form cannot be associated with a definite chlorophyll holochrome.     

Ammonium ions enhance the flow through the pyruvate dehydrogenase in Ehrlich ascites tumor cells

The flow through pyruvate dehydrogenase was assayed in glycolysing cells by the evolution of 14CO2 from [1-14C] pyruvate. Parallel incubations were carried out in high bicarbonate buffer (25 mM) and in bicarbonate-free buffer. The activation of the complex by NH+4 was only observed in high bicarbonate buffer, because the dilution of labelled CO2 in the presence of an excess of bicarbonate enables the quantitative determination of labelled CO2 evolved from pyruvate in the decarboxylase step. In the bicarbonate-free buffer the activation of the complex was not observed, because the 14CO2 evolved from pyruvate was consumed by biosynthetic processes inside the cell. On the contrary in isolated hepatocytes the NH+4 activation of the pyruvate dehydrogenase was observed in both buffers. In Ehrlich ascites cells, in common with other mammalian tissues, pyruvate dehydrogenase activity was found to be inversely correlated to the intramitochondrial ATP/ADP ratio.     

Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase

Present knowledge of the different enzymatic steps of the denitrification chains in various bacteria, particularly Paracoccus denitrificans and Pseudomonas aeruginosa has been briefly reviewed. The question whether nitric oxide (NO), nitrous oxide (N2O) and other nitrogen derivatives are obligatory intermediates has been discussed. The second part is an extensive review of the structure and the function of a key enzyme in denitrification, cytochrome c551-nitrite-oxidoreductase from P. aeruginosa. Recent results on the stoichiometry of nitrite reduction have been discussed.     

The interconversion between monomeric and dimeric bovine heart cytochrome c oxidase

Monomers and dimers of bovine heart cytochrome c oxidase (EC 1.9.3.1.) were separated by gel filtration chromatography on Ultrogel AcA 34 or by sucrose gradient centrifugation. Factors influencing the interconversion of the two aggregation states of this enzyme were analyzed. At very low ionic strength, in the presence of dodecyl maltoside, monomers were the main species. Salts appeared to stabilize the dimeric form, divalent cations being more efficient than monovalent. High enzyme concentrations favoured the formation of dimers, also at low ionic strength. The type of detergent had a strong influence on the monomer-dimer interconversion; in Triton X-100 and dodecyl maltoside (at high ionic strength) cytochrome c oxidase was homogenously dispersed in its dimeric form, while in Tween-80 gel filtration showed only large particles eluting in the void volume. In cholate monomers and aggregates were observed but no dimers. The aggregation state had an influence on the steady state kinetics of the ferrocytochrome c oxidase activity. Monomers showed linear Eadie-Hofstee plots, whilst the dimeric and aggregated enzyme gave nonlinear Eadie-Hofstee plots. Ionic strength, enzyme concentration and type of detergent were affecting the enzyme's kinetics in a way consistent with the molecular form obtained by the gel filtration or sedimentation analysis. The data support a negative cooperative mechanism for the interaction of cytochrome c with the dimeric enzyme, as proposed earlier (K.A. Na?ecz et al., (1983) Biochem. Biophys. Res. Commun., 114, 822-828).     

Experimental study on the sensory capabilities of coral-reef fish larvae for the recognition of their settlement location

Some experiments in aquarium and in situ have been carried out to investigate the sensory abilities of coral reef fish larvae in the recognition of their settlement location. Ten out 12 species studied detected their settlement location due to the presence of conspecifics and not by habitat characteristics. Larvae use three senses in this detection: sight, smell and vibratory sense.     

Purification and properties of aromatic L-amino acid decarboxylase (4.1.1.28) of rat brain]

L-aromatic aminoacid decarboxylase has been purified more than thousand times from homogenates of rat brain, in several steps : centrifugation, DEAE-cellulose, CM cellulose, hydroxylapatite, DEAE sephadex. Its properties have been studied, most of them on an intermediate fraction of the purification, because of the instability of the purified enzyme in spite of the addition of different stabilizing agents : the enzyme decarboxylates 5-hydroxytryptophan (5 HTP) and DOPA in a ratio constant throughout the purification but does not decarboxylate tryptophan, tyrosine, histidine at a measurable rate. Optimum pH, Km, Vm, have been measured with 5 HTP and DOPA as substrates. The enzyme has a molecular weight of 115.000, an apparent isoelectric point of 6,4-6,5. It is inhibited by serotonin, dopamine, some cations : Cu++, Fe++, Ni++ by N-ethylmaleimide, sodium dodecylsulfate. Some pyridoxal-5 phosphate (PLP) remains strongly bound to the enzyme. For relatively weak concentrations of substrate, the enzyme is inhibited by an excess of PLP ; for weak concentrations of PLP, the enzyme in inhibited by an excess of substrate, particularly of DOPA. We also observe a spontaneous decarboxylation of the substrates that reaches a plateau and is enhanced by high concentrations of PLP, by serotonin, dopamine, Cu++ and reduced by mercaptoethanol and the presence of crude or boiled homogenates. Several possible explanations of the spontaneous decarboxylation and of the enzymic inhibitions by an excess of PLP and by the substrates are given.     

Effect of organic and mineral fertilizers on N-use by wheat under different irrigation frequencies

A field trial was established in Errachidia, southern Morocco, to investigate the interaction between wheat residue management and mineral 15N-labelled ammonium sulphate, under different irrigation treatments, applied to wheat (Triticum durum var. Karim). In treatments I1, I2, I3 and I4, plots were irrigated every 10, 15, 21 and 30 days. Each plot contained three sub-plots that received three fertilization treatments: T1 received 42 kg N ha-1 of ammonium sulphate before seedling, 42 kg N ha-1 of ammonium sulphate labelled with 9.764 at % 15N excess at tillering and 84 N kg ha-1 of ammonium sulphate at flowering; T2 received 42 kg N ha-1 of ammonium sulphate labelled with 9.764 at % 15N excess at seedling, 42 kg N ha-1 at tillering and 42 kg N ha-1 at flowering; T3 received 4800 kg ha-1 of wheat residue labelled with 1.504 at % 15N excess and 42 kg N ha-1 of ammonium sulphate before seedling and 42 kg N ha-1 of ammonium sulphate at flowering. Nitrogen fertilization with 168 kg N ha-1 did no significantly increase grain and straw yields in comparison to the 126 kg N ha-1 application. The combination of the organic input and supplementary application of mineral fertilizer N has been found as a more attractive management option. For all irrigation treatments, the % recovery of N in the whole plant was higher in plants that received 15N at tillering (63%, 49% respectively for irrigation intervals between 10 and 30 d) than in plants that received 15N just after seeding (28% for irrigation each 10- and 30-d intervals). For the irrigation treatment each 10 and 15 days, the 15N was mainly recovered by the grain for all fertilization treatments, whereas for irrigation treatment each 30 days, the grain and straw recovered nearly equal amounts of fertilizer. For grain and straw of wheat, nitrogen in the plant derived from the fertilizer was low, while most of the N was derived from the soil for all irrigation and fertilization treatments. The % nitrogen in the plant derived from the fertilizer values showed no significant difference between the different plant parts. The results suggested a dominant influence of moisture availability on the fertilizer N uptake by wheat. Under dry conditions the losses of N can be allotted to denitrification and volatilisation.     

Non-linear elastic deformable models for real-time surgery simulation

In this article, we describe the latest developments of the minimally invasive hepatic surgery simulator prototype developed at INRIA. A key problem with such a simulator is the physical modelling of soft tissues. We propose a new deformable model based on non-linear elasticity and the finite element method. This model is valid for large displacements, which means in particular that it is invariant with respect to rotations. This property improves the realism of the deformations and solves the problems related to the shortcomings of linear elasticity, which is only valid for small displacements. We also address the problem of volume variations by adding to our model incompressibility constraints. Finally, we demonstrate the relevance of this approach for the real-time simulation of laparoscopic surgical gestures on the liver.     

Zein of maize grain: II — the charge heterogeneity of free subunits

The subunits present as monomers in unreduced zein and isolated as fraction M by gel filtration, were chromatographed on sulfoethyl-cellulose. Three major subfractions were detected and characterized. Each of them, submitted to electrophoresis at pH 3.5, migrated as a single band corresponding to each of the three major electrophoretic forms seen in fraction M at the same pH. The presence of lysine in some polypeptides, suggested by amino acid composition data, was confirmed by electrophoretic analysis of carbamylated subfractions at pH 4.5. At pH 8.9 each subfractions was further resolved into three cationic bands in starch gel and three (or more) anionic bands in polyacrylamide gel. The same fractionation was also obtained by submitting the major electroforms of fraction M, as isolated at pH 3.5, to isoelectric focusing. Based on these observations, the most probable distributions of basic amino acids in subunits detected by electrophoresis at pH 8.9 were specified and compared to those recently published for several zein clones. The presence per polypeptide chain of three carboxyl groups and occasionally of one lysine would be a feature of zein originating from maize hybrid Inra 260.     

Defensins: antimicrobial peptides of vertebrates

This review, based on my presentation at the French Academy of Sciences on May 19, 2003, describes recent progress in the study of antimicrobial peptides, mediators of innate immunity in plants and animals. The main focus is on vertebrate defensins, a family of cysteine-rich antimicrobial peptides abundantly represented in human cells and tissues.