DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway

The DNA damage response (DDR) cascade and ROS (reactive oxygen species) signaling are both involved in the induction of cell death after DNA damage, but a mechanistic link between these two pathways has not been clearly elucidated. This study demonstrates that ROS induction after treatment of cells with neocarzinostatin (NCS), an ionizing radiation mimetic, is at least partly mediated by increasing histone H2AX. Increased levels of ROS and cell death induced by H2AX overexpression alone or DNA damage leading to H2AX accumulation are reduced by treating cells with the antioxidant N-Acetyl-L-Cysteine (NAC), the NADP(H) oxidase (Nox) inhibitor DPI, expression of Rac1N17, and knockdown of Nox1, but not Nox4, indicating that induction of ROS by H2AX is mediated through Nox1 and Rac1 GTPase. H2AX increases Nox1 activity partly by reducing the interaction between a Nox1 activator NOXA1 and its inhibitor 14-3-3zeta. These results point to a novel role of histone H2AX that regulates Nox1-mediated ROS generation after DNA damage

Exclusive neuronal expression of SUCLA2 in the human brain

SUCLA2 encodes the ATP-forming subunit (A-SUCL-) of succinyl-CoA ligase, an enzyme of the citric acid cycle. Mutations in SUCLA2 lead to a mitochondrial disorder manifesting as encephalomyopathy with dystonia, deafness and lesions in the basal ganglia. Despite the distinct brain pathology associated with SUCLA2 mutations, the precise localization of SUCLA2 protein has never been investigated. Here we show that immunoreactivity of A-SUCL- in surgical human cortical tissue samples was present exclusively in neurons, identified by their morphology and visualized by double labeling with a fluorescent Nissl dye. A-SUCL- immunoreactivity co-localized >99% with that of the d subunit of the mitochondrial F0-F1 ATP synthase. Specificity of the anti-A-SUCL- antiserum was verified by the absence of labeling in fibroblasts from a patient with a complete deletion of SUCLA2. A-SUCL- immunoreactivity was absent in glial cells, identified by antibodies directed against the glial markers GFAP and S100. Furthermore, in situ hybridization histochemistry demonstrated that SUCLA2 mRNA was present in Nissl-labeled neurons but not glial cells labeled with S100. Immunoreactivity of the GTP-forming subunit (G-SUCL-) encoded by SUCLG2, or in situ hybridization histochemistry for SUCLG2 mRNA could not be demonstrated in either neurons or astrocytes. Western blotting of post mortem brain samples revealed minor G-SUCL- immunoreactivity that was however, not upregulated in samples obtained from diabetic versus non-diabetic patients, as has been described for murine brain. Our work establishes that SUCLA2 is expressed exclusively in neurons in the human cerebral cortex

Phagocytosis of Streptococcus pyogenes by all-trans retinoic acid-differentiated HL-60 cells: roles of azurophilic granules and NADPH oxidase.

BACKGROUND: New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules. CONCLUSIONS/SIGNIFICANCE: The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion

Translating the oxidative stress hypothesis into the clinic: NOX versus NOS

Cardiovascular diseases remain the leading cause of death in industrialised nations. Since the pathomechanisms of most cardiovascular diseases are not understood, the majority of therapeutic approaches are symptom-orientated. Knowing the molecular mechanism of disease would enable more targeted therapies. One postulated underlying mechanism of cardiovascular diseases is oxidative stress, i.e. the increased occurrence of reactive oxygen species such as superoxide. Oxidative stress leads to a dysfunction of vascular endothelium-dependent protective mechanisms. There is growing evidence that this scenario also involves impaired nitric oxide (NO)-cyclic GMP signalling. Out of a number of enzyme families that can produce reactive oxygen species, NADPH oxidases stand out, as they are the only enzymes whose sole purpose is to produce reactive oxygen species. This review focuses on the clinically validated targets of oxidative stress, NO synthase (NOS) and the NO receptor, soluble guanylate cyclase as well as the source of ROS, e.g. NADPH oxidases. We place recent knowledge in the function and regulation of these enzyme families into clinical perspective. For a comprehensive overview of the biology and pharmacology of oxidative stress and possible other sources and targets, we refer to other literature overviews

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

<p>Abstract</p> <p>Background</p> <p>Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^stand 2^ndEF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.</p> <p>Results</p> <p>The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵and 5.0 × 10²M^-1with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^stnon-canonical EF-hand is largely enhanced by the binding of Ca²⁺to the 2^ndcanonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.</p> <p>Conclusions</p> <p>We demonstrated that the 1^stnon-canonical EF-hand of NOX5 has very weak Ca²⁺binding affinity compared with the 2^ndcanonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺specific.</p> <p>Graphical abstract</p> <p><display-formula><graphic file="1752-153X-6-29-i1.gif"/></display-formula></p

Role of cellular senescence and NOX4-mediated oxidative stress in systemic sclerosis pathogenesis.

Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by progressive fibrosis of skin and numerous internal organs and a severe fibroproliferative vasculopathy resulting frequently in severe disability and high mortality. Although the etiology of SSc is unknown and the detailed mechanisms responsible for the fibrotic process have not been fully elucidated, one important observation from a large US population study was the demonstration of a late onset of SSc with a peak incidence between 45 and 54 years of age in African-American females and between 65 and 74 years of age in white females. Although it is not appropriate to consider SSc as a disease of aging, the possibility that senescence changes in the cellular elements involved in its pathogenesis may play a role has not been thoroughly examined. The process of cellular senescence is extremely complex, and the mechanisms, molecular events, and signaling pathways involved have not been fully elucidated; however, there is strong evidence to support the concept that oxidative stress caused by the excessive generation of reactive oxygen species may be one important mechanism involved. On the other hand, numerous studies have implicated oxidative stress in SSc pathogenesis, thus, suggesting a plausible mechanism in which excessive oxidative stress induces cellular senescence and that the molecular events associated with this complex process play an important role in the fibrotic and fibroproliferative vasculopathy characteristic of SSc. Here, recent studies examining the role of cellular senescence and of oxidative stress in SSc pathogenesis will be reviewed

Is music enriching for group-housed captive chimpanzees (Pan troglodytes)?

Many facilities that house captive primates play music for animal enrichment or for caregiver enjoyment. However, the impact on primates is unknown as previous studies have been inconclusive. We conducted three studies with zoo-housed chimpanzees (Pan troglodytes) and one with group-housed chimpanzees at the National Centre for Chimpanzee Care to investigate the effects of classical and pop/rock music on various variables that may be indicative of increased welfare. Study one compared the behaviour and use of space of 18 animals when silence, classical or pop/rock music was played into one of several indoor areas. Overall, chimpanzees did not actively avoid the area when music was playing but were more likely to exit the area when songs with higher beats per minute were broadcast. Chimpanzees showed significantly fewer active social behaviours when music, rather than silence, was playing. They also tended to be more active and engage in less abnormal behaviour during the music but there was no change to either self-grooming or aggression between music and silent conditions. The genre of music had no differential effects on the chimpanzees’ use of space and behaviour. In the second study, continuous focal observations were carried out on three individuals with relatively high levels of abnormal behaviour. No differences in behaviour between music and silence periods were found in any of the individuals. The final two studies used devices that allowed chimpanzees to choose if they wanted to listen to music of various types or silence. Both studies showed that there were no persistent preferences for any type of music or silence. When taken together, our results do not suggest music is enriching for group-housed captive chimpanzees, but they also do not suggest that music has a negative effect on welfare

A Model for Damage Load and Its Implications for the Evolution of Bacterial Aging

Deleterious mutations appearing in a population increase in frequency until stopped by natural selection. The ensuing equilibrium creates a stable frequency of deleterious mutations or the mutational load. Here I develop the comparable concept of a damage load, which is caused by harmful non-heritable changes to the phenotype. A damage load also ensues when the increase of damage is opposed by selection. The presence of a damage load favors the evolution of asymmetrical transmission of damage by a mother to her daughters. The asymmetry is beneficial because it increases fitness variance, but it also leads to aging or senescence. A mathematical model based on microbes reveals that a cell lineage dividing symmetrically is immortal if lifetime damage rates do not exceed a threshold. The evolution of asymmetry allows the lineage to persist above the threshold, but the lineage becomes mortal. In microbes with low genomic mutation rates, it is likely that the damage load is much greater than the mutational load. In metazoans with higher genomic mutation rates, the damage and the mutational load could be of the same magnitude. A fit of the model to experimental data shows that Escherichia coli cells experience a damage rate that is below the threshold and are immortal under the conditions examined. The model estimates the asymmetry level of E. coli to be low but sufficient for persisting at higher damage rates. The model also predicts that increasing asymmetry results in diminishing fitness returns, which may explain why the bacterium has not evolved higher asymmetry

Role of endothelial Nox2 NADPH oxidase in angiotensin II-induced hypertension and vasomotor dysfunction

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are known to be involved in angiotensin II-induced hypertension and endothelial dysfunction. Several Nox isoforms are expressed in the vessel wall, among which Nox2 is especially abundant in the endothelium. Endothelial Nox2 levels rise during hypertension but little is known about the cell-specific role of endothelial Nox2 in vivo. To address this question, we generated transgenic mice with endothelial-specific overexpression of Nox2 (Tg) and studied the effects on endothelial function and blood pressure. Tg had an about twofold increase in endothelial Nox2 levels which was accompanied by an increase in p22phox levels but no change in levels of other Nox isoforms or endothelial nitric oxide synthase (eNOS). Basal NADPH oxidase activity, endothelial function and blood pressure were unaltered in Tg compared to wild-type littermates. Angiotensin II caused a greater increase in ROS production in Tg compared to wild-type aorta and attenuated acetylcholine-induced vasorelaxation. Both low and high dose chronic angiotensin II infusion increased telemetric ambulatory blood pressure more in Tg compared to wild-type, but with different patterns of BP change and aortic remodeling depending upon the dose of angiotensin II dose. These results indicate that an increase in endothelial Nox2 levels contributes to angiotensin II-induced endothelial dysfunction, vascular remodeling and hypertension