Exploration of the diaphorase activity of neutrophil NADPH oxidase.

In the O2- generating flavocytochrome b, the membrane-bound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPH --> FAD --> heme b -->O2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. Two series of inhibitors were used, namely the flavin analog 5-deaza FAD and the heme inhibitors bipyridyl and benzylimidazole. The following results indicate that INT reacts preferentially with the hemes rather than with the FAD redox center of flavocytochrome b and is not therefore a specific probe of the diaphorase activity of flavocytochrome b. First, in anaerobiosis, reduced heme b in activated membranes was reoxidized by INT as efficiently as by O2 even in the presence of concentrations of 5-deaza FAD which fully inhibited the NADPH oxidase activity. Second, the titration curve of dithionite-reduced heme b in neutrophil membranes obtained by oxidation with increasing amounts of INT was strictly superimposable on that of dithionite-reduced hemin. Third, INT competitively inhibited the O2 uptake by the activated NADPH oxidase in a cell-free system. Finally, the heme inhibitor bipyridyl competitively inhibited the reduction of INT in anaerobiosis, and the oxygen uptake in aerobiosis.     

Mus81-Eme1 are essential components of a Holliday junction resolvase.

Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.     

Structural analysis of hemicatenated DNA loops

Background  

We have previously isolated a stable alternative DNA structure, which was formed in vitro by reassociation of the strands of DNA fragments containing a 62 bp tract of the CA-microsatellite poly(CA)·poly(TG). In the model which was proposed for this structure the double helix is folded into a loop, the base of the loop consists of a DNA junction in which one of the strands of one duplex passes between the two strands of the other duplex, forming a DNA hemicatenane in a hemiknot structure. The hemiknot DNA structures obtained with long CA/TG inserts have been imaged by AFM allowing us to directly visualize the loops.     

Intracellular growth of Listeria monocytogenes as a prerequisite for in vivo induction of T cell-mediated immunity

The in vivo induction of T cell-mediated immunity was studied by infecting mice with two genetically closely related mutants from Listeria monocytogenes, differing only with respect to the secretion of an active SH-dependent hemolysin. It is shown that even minute doses of hemolytic bacteria capable of growing in host tissues easily induced the expression of T cell-mediated immunity, as estimated by the level of delayed sensitivity, adoptive protection and long-lasting immunological memory. On the contrary, nonhemolytic bacteria unable to multiply in host tissues totally failed to initiate the expression of T cell-mediated immunity in vivo. This failure was even observed when mice were repeatedly infected by high doses of nonhemolytic bacteria, allowing to maintain a significant amount of viable bacteria for several days in host tissues. These results mean that the presence of viable bacteria at a significant level in the host is not sufficient per se to induce detectable T cell clonal expansion in the in vivo setting, implying that the process of bacterial growth inside macrophages is required to initiate in vivo the expression of T cell-mediated immunity.     

Characterization and physico-chemical properties of human transcortin]

The molecular weight of human transcortin, calculated from the sedimentation coefficient, was found to be 49,500, thus slightly lower than previously reported values. After purification, human transcortin trended to polymerize rapidly, with participation of both non covalent bonds and one disulfide bridge per dimer. The physicochemical parameters, the amino-acid and carbohydrate composition were determined; its stability was studied under different conditions. Preliminary structural studies showed that the N-terminal sequence of the polypeptide chain was: Met-Asp-Pro-Asn-Ala-Ala-Tyr-Val and that the C-terminal amino acid was leucine.     

Organic matter mineralization in the pore water of a eutrophic lake (Aydat Lake,Puy de Dôme,France)

The chemical composition of the pore water from the sediment of a eutrophic lake is dominated by high concentrations of total dissolved CO₂ (up to 12 mM), reduced soluble iron (up to 2 mM) and dissolved silica (up to 1 mM). The pH lies within the range of 6.70 ± 0.02; this reflects that the pore water is efficiently buffered by the CO₂ acid/base system. This composition is directly related to the main diagenetic reactions which drive the organic matter mineralization i.e. methanogenesis and ferric oxides reduction. Other geochemical processes are of minor importance. A stoichiometric model based on these main reactions allow us: (i) to define a general formula for the organic matter which is close to Redfield's one for the C:N ratio, while the C:P ratio is much higher owing to a probable adsorption of phosphorus onto reactive surfaces of the solid and due to heterotrophic bacterial uptake; (ii) to calculate a global first order kinetic constant which drives the organo-polymers breakdown. Due to the strong influence on the trophic status of the lake caused by an excess of phosphate, special attention is devoted to this species. We show that the sediment-water interface is a source of dissolved phosphate when the hypolimnion is anoxic between May and November. This contribution represents about 17% of the river input and should be taken into account in any attempt toward lake restoration.     

Survival costs of reproduction vary with age in North American red squirrels

The costs of reproduction are expected to be higher under unfavourable conditions, so that breeding in years of low food supply should have important costs. In addition, the costs of reproduction may be contingent on the age of individuals, and young growing and old senescent individuals should suffer higher costs than the prime-age ones. We tested these predictions by investigating the costs of reproduction as a function of food availability and age in female North American red squirrels using the long-term data on survival and reproduction. We found that the costs of reproduction were independent of food supply, and we did not detect any trade-off between the current and future reproduction. We also did not detect any survival cost of reproduction for the prime-age females, but found evidence for survival costs in yearlings and old (6 years or above) females with successfully breeding individuals having a lower chance of survival compared with unsuccessful or non-breeding ones. These results supported our prediction that the costs of reproduction depended on the age of female red squirrels and were higher in young growing and old senescent individuals. Our study also indicated that, in contrast to large herbivores, heterogeneity in individual quality and viability selection in red squirrels do not affect the study of trade-offs and of the age variation in life-history traits.