Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.     

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins

A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.     

Proline betaine accumulation and metabolism in alfalfa plants under sodium chloride stress. Exploring its compartmentalization in nodules

The osmoprotectant Pro betaine is the main betaine identified in alfalfa (Medicago sativa). We have investigated the long-term responses of nodulated alfalfa plants to salt stress, with a particular interest for Pro betaine accumulation, compartmentalization, and metabolism. Exposure of 3-week-old nodulated alfalfa plants to 0.2 m NaCl for 4 weeks was followed by a 10-, 4-, and 8-fold increase in Pro betaine in shoots, roots, and nodules, respectively. Isotope-labeling studies in alfalfa shoots indicate that [14C]Pro betaine was synthesized from l-[14C]Pro. [14C]Pro betaine was efficiently catabolized through sequential demethylations via N-methylPro and Pro. Salt stress had a minor effect on Pro betaine biosynthesis, whereas it strongly reduced Pro betaine turnover. Analysis of Pro betaine and Pro compartmentalization within nodules revealed that 4 weeks of salinization of the host plants induced a strong increase in cytosol and bacteroids. The estimated Pro betaine and Pro concentrations in salt-stressed bacteroids reached 7.4 and 11.8 mm, respectively, compared to only 0.8 mm in control bacteroids. Na+ content in nodule compartments was also enhanced under salinization, leading to a concentration of 14.7 mm in bacteroids. [14C]Pro betaine and [14C]Pro were taken up by purified symbiosomes and free bacteroids. There was no indication of saturable carrier(s), and the rate of uptake was moderately enhanced by salinization. Ultrastructural analysis showed a large peribacteroid space in salt-stressed nodules, suggesting an increased turgor pressure inside the symbiosomes, which might partially be due to an elevated concentration in Pro, Pro betaine, and Na+ in this compartment.     

Effect of high-fat diet on the expression of proteins in muscle, adipose tissues, and liver of C57BL/6 mice

In the present study, the effect of a high fat diet on the expression of proteins in insulin target tissues was analyzed using a proteomic approach. Gastrocnemius muscle, white and brown adipose tissue, and liver were taken from C57BL/6 mice either fed on a high-fat or a chow diet. Expression levels of approximately 10 000 polypeptides for all the four tissues were assessed by two-dimensional gel electrophoresis (2-DE). Computer-assisted image analysis allowed the detection of 50 significantly (p < 0.05) differentially expressed proteins between obese and lean mice. Interestingly, more than half of these proteins were detected in the brown adipose tissue. The differentially expressed proteins were identified by tandem mass spectrometry. Several stress and redox proteins were modulated in response to the high-fat diet. A key glycolytic enzyme was found to be downregulated in adipose tissues and muscle, suggesting that at elevated plasma fatty acid concentrations, fatty acids compete with glucose as an oxidative fuel source. Furthermore, in brown adipose tissue there were significant changes in mitochondrial enzymes involved in the Krebs tricarboxylic acid (TCA) cycle and in the respiratory chain in response to the high-fat diet. The brown adipose tissue is an energy-dissipating tissue. Our data suggest that the high-fat diet treated mice were increasing energy expenditure to defend against weight gain.     

Structural variations of 1-(4-(phenoxymethyl)benzyl)piperidines as nonimidazole histamine H3 receptor antagonists

Recent bioisoteric replacements in histamine H3 receptor ligands with an exchange of the imidazole moiety by a piperidino group as well as of the trimethylene chain in 4-((3-phenoxy)propyl)-lH-imidazole derivatives (proxifan class) by an alpha,alpha'-xylendiyl linker represents the starting point in the development of 1-(4-(phenoxymethyl)benzyl)piperidines as a new class of nonimidazole histamine H3 receptor antagonists. According to different strategies in optimization of imidazole-containing antagonists the central benzyl phenyl ether moiety was replaced by numerous other polar functionalities. Additionally, the ortho- and meta-analogues of the lead were synthesized to determine the influence of the position of the piperidinomethyl substituent. The new compounds were tested in an in vitro binding assay for their affinities for cloned human H3 receptors stably expressed in CHO-K1 cells and for their oral in vivo potencies brain in a functional screening assay in the brain of mice. Additionally, activities of selected compounds were determined in the guinea-pig ileum functional test model. In contrast to the analogues ortho-substituted compounds all other compounds maintained respectable affinities for the human H3 receptor (-log Ki values 6.3-7.5). Despite the results from other classes of compounds the 4-methyl substituted derivatives generally displayed higher affinities than the corresponding 4-chloro substituted compounds. In vivo only the inverse phenyl benzyl ether (3) showed worthwhile antagonist potencies.     

RNA interference inhibition of Mus81 reduces mitotic recombination in human cells

Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex. In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function. Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo. We show that recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro. By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1. Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo. Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells. The recombination defect is rescued by expression of a bacterial Holliday junction resolvase. These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo.     

A revised annotation and comparative analysis of Helicobacter pylori genomes

Huge amounts of genomic information are currently being generated. Therefore, biologists require structured, exhaustive and comparative databases. The PyloriGene database (http://genolist.pasteur.fr/PyloriGene) was developed to respond to these needs, by integrating and connecting the information generated during the sequencing of two distinct strains of Helicobacter pylori. This led to the need for a general annotation consensus, as the physical and functional annotations of the two strains differed significantly in some cases. A revised functional classification system was created to accommodate the existing data and to make it possible to classify coding sequences (CDS) into several functional categories to harmonize CDS classification. The annotation of the two complete genomes was revised in the light of new data, allowing us to reduce the percentage of hypothetical proteins from approximately 40 to 33%. This resulted in the reassignment of functions for 108 CDS (approximately 7% of all CDS). Interestingly, the functions of only approximately 13% of CDS (222 out of 1658 CDS) were annotated as a result of work done directly on H.pylori genes. Finally, comparison of the two published genomes revealed a significant amount of size variation between corresponding (orthologous) CDS. Most of these size variations were due to natural polymorphisms, although other sources of variation were identified, such as pseudogenes, new genes potentially regulated by slipped-strand mispairing mechanism, or frame-shifts. 113 of these differences were due to different start codon assignments, a common problem when constructing physical annotations.     

A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells

Homologous gene targeting is the ultimate tool for reverse genetics, but its use is often limited by low efficiency. In a number of recent studies, site- specific DNA double-strand breaks (DSBs) have been used to induce efficient gene targeting. Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. In this study, we present two novel, chimeric meganucleases, derived from homing endonucleases. The first one is able to induce recombination in yeast and mammalian cells, whereas the second cleaves a novel (chosen) DNA target site. These results are a first step toward the generation of custom endonucleases for the purpose of targeted genome engineering.     

Influence of oxygen partial pressures on protein synthesis in feeding crabs

Many water-breathing animals have a strategy that consists of maintaining low blood PO2 values in a large range of water oxygenation level (4-40 kPa). This study examines the postprandial changes in O2 consumption, arterial blood PO2, and tissue protein synthesis in the shore crab Carcinus maenas in normoxic, O2-depleted, and O2-enriched waters to study the effects of this strategy on the O2 consumption and peptide bond formation after feeding. In normoxic water (21 kPa), the arterial PO2 was 1.1 kPa before feeding and 1.2 kPa 24 h later. In water with a PO2 of 3 kPa (arterial PO2 0.6 kPa), postprandial stimulation of protein synthesis and O2 consumption were blocked. The blockade was partial at a water PO2 of 4 kPa (arterial PO2 0.8 kPa). An increase in environmental PO2 (60 kPa, arterial PO2 10 kPa) resulted in an increase in protein synthesis compared with normoxic rates. It is concluded that the arterial PO2 spontaneously set in normoxic Carcinus limits the rates of protein synthesis. The rationale for such a strategy is discussed.