Modesto Carballo

Oxidative Stress Triggers STAT3 Tyrosine Phosphorylation and Nuclear Translocation in Human Lymphocytes

Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ions, that ⋅OH generated from H2O2through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway.

Characterization of Calcineurin in Human Neutrophils INHIBITORY EFFECT OF HYDROGEN PEROXIDE ON ITS ENZYME ACTIVITY AND ON NF-κB DNA BINDING

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-κB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-κB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-κB inactivation in neutrophils occurs in concert with inhibition of calcineurin.

Activation of phagocytic cell NADPH oxidase by norfloxacin: a potential mechanism to explain its bactericidal action

The mechanisms underlying the bactericidal power of fluoroquinolones against intracellular parasites in host macrophages remain poorly understood. We have analyzed the effect of norfloxacin, a fluoroquinolone antibiotic, on the production of reactive oxygen intermediates (O2•− and H2O2) and NADPH oxidase activity in mouse macrophages. The generation of anion superoxide (O2•−) was found to be significantly greater in macrophages incubated with norfloxacin than in untreated controls. This enhancing effect of norfloxacin was dose‐dependent and reached maximal values within 10 min after its addition. The O2•− generated was mainly intracellular, as determined by the use of specific dyes, such as lucigenin and luminol, and able to diffuse freely through the cell membrane. Also, the production of H2O2 was increased in macrophages in response to norfloxacin. The positive effect of norfloxacin was associated to an enhanced mobilization of NADPH oxidase subunits p47phox and p67phox from the cytosol to the plasma membrane in phagocytic cells. The effect of the antibiotic persisted in vivo for several hours. These data support the notion that norfloxacin inhibits mycobacterial growth within phagocytic cells by enhancing intracellular production of O2•− and other reactive oxygen species.

Antiproliferative and immunoactivatory ability of an enzymatic extract from rice bran.

The validation of natural products as source of functional foods or nutraceuticals has become an important issue in current health research. Thus, the present work has tested on MOLT-4 cells (human T cell acute lymphoblastic leukemic) the antiproliferative effect of a water-soluble enzymatic extract from rice bran (EERB). Present work shows that EERB induces cellular death in MOLT-4 cells in a dose-dependent way (0-10mg/mL) but not in non-tumoral lymphocytes. Flow cytometric analysis of MOLT-4 cells treated with EERB showed the presence of death cells by apoptosis rather than necrosis. Additionally, EERB also exerts an immunoactivatory effect on N13 microglia cells, by inducing TNF-alpha (tumour necrosis factor-α) expression, which plays a key role in the innate immune response to infection. Accordingly, we can propose EERB as a useful natural standardized extract with antiproliferative and immunoactivatory ability that would be beneficial to apply in the functional food field.

Draft genome of the marine gammaproteobacterium halomonas titanicae

ABSTRACT Halomonas titanicae strain BH1 is a heterotrophic, aerobic marine bacterium which was isolated from rusticles of the RMS Titanic wreck. Here we report the draft genome sequence of this halophilic gammaproteobacterium.

Macrophage inducible nitric oxide synthase gene expression is blocked by a benzothiophene derivative with anti-HIV properties.

Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNF alpha-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharide-elicited production of nitrite (NO(-)(2)) by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO(-)(2) production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFN gamma. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNF alpha, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNF alpha synthesis. Additionally, PD144795 did not block NF-kappa B nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug.

Metagenome Sequencing of Prokaryotic Microbiota from Two Hypersaline Soils of the Odiel Salt Marshes in Huelva, Southwestern Spain

ABSTRACT Two 454 shotgun metagenomes were sequenced from hypersaline soil samples collected in the Odiel salt marsh area in Huelva, southwestern Spain. Analysis of contigs and 16S rRNA-related sequences showed that Halobacteria, Balneolaeota, and Bacteroidetes were the dominant groups. Rhodothermaeota and Nanohaloarchaeota were also abundant.