Reactive oxygen and nitrogen species production in cardiomyoblasts during hypoxia and reoxygenation

Hypoxia is a stress condition in which tissues are deprived of an adequate O2 supply; this may trigger cell death with pathological consequences in cardiovascular or neurodegenerative disease. Reperfusion is the restoration of an oxygenated blood supply to hypoxic tissue and can cause more cell injury. The kinetics and consequences of reactive oxygen and nitrogen species (ROS/RNS) production in cardiomyoblasts are poorly understood. The present study describes the systematic characterization of the kinetics of ROS/RNS production and their roles in cell survival and associated protection during hypoxia and hypoxia/reperfusion. H9C2 cells showed a significant loss of viability under 2% O2 for 30min hypoxia and cell death; associated with an increase in protein oxidation. After 4h, apoptosis induction under 2% O2 and 10% O2 was dependent on the production of mitochondrial superoxide (O2-•) and nitric oxide (•NO), partly from nitric oxide synthase (NOS). Both apoptotic and necrotic cell death during 2% O2 for 4h could be rescued by the mitochondrial complex I inhibitor; rotenone and NOS inhibitor; L-NAME. Both L-NAME and the NOX (NADPH oxidase) inhibitor; apocynin reduced apoptosis under 10% O2 for 4h hypoxia. The mitochondrial uncoupler; FCCP significantly reduced cell death via a O2-• dependent mechanism during 2% O2, 30min hypoxia. During hypoxia (2% O2, 4h)/ reperfusion (21% O2, 2h), metabolic activity was significantly reduced with increased production of O2-• and •NO, during hypoxia but, partially restored during reperfusion. O2-• generation during hypoxia/reperfusion was mitochondrial and NOX- dependent, whereas •NO generation depended on both NOS and non-enzymatic sources. Inhibition of NOS worsened metabolic activity during reperfusion, but did not effect this during sustained hypoxia. Nrf2 activation during 2% O2, a sustained hypoxia and reperfusion was O2-•/•NO dependent. Inhibition of NF-?B activation aggravated metabolic activity during 2% O2, 4h hypoxia. In conclusion, mitochondrial O2-•, but, not ATP depletion is the major cause of apoptotic and necrotic cell death in cardiomyoblasts under 2% O2, 4h hypoxia, whereas apoptotic cell death under 10% O2, 4h, is due to NOS-dependent •NO. The management of ROS/RNS rather than ATP is required for improved survival during hypoxia. O2-• production from mitochondria and NOS is cardiotoxic during hypoxia/reperfusion. NF-?B activation during hypoxia and NOS activation during reperfusion is cardiomyoblast protective.

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

Phenotypic effect of mutations in evolving populations of RNA molecules

Background. The secondary structure of folded RNA sequences is a good model to map phenotype onto genotype, as represented by the RNA sequence. Computational studies of the evolution of ensembles of RNA molecules towards target secondary structures yield valuable clues to the mechanisms behind adaptation of complex populations. The relationship between the space of sequences and structures, the organization of RNA ensembles at mutation-selection equilibrium, the time of adaptation as a function of the population parameters, the presence of collective effects in quasispecies, or the optimal mutation rates to promote adaptation all are issues that can be explored within this framework. Results. We investigate the effect of microscopic mutations on the phenotype of RNA molecules during their in silico evolution and adaptation. We calculate the distribution of the effects of mutations on fitness, the relative fractions of beneficial and deleterious mutations and the corresponding selection coefficients for populations evolving under different mutation rates. Three different situations are explored: the mutation-selection equilibrium (optimized population) in three different fitness landscapes, the dynamics during adaptation towards a goal structure (adapting population), and the behavior under periodic population bottlenecks (perturbed population). Conclusions. The ratio between the number of beneficial and deleterious mutations experienced by a population of RNA sequences increases with the value of the mutation rate µ at which evolution proceeds. In contrast, the selective value of mutations remains almost constant, independent of µ, indicating that adaptation occurs through an increase in the amount of beneficial mutations, with little variations in the average effect they have on fitness. Statistical analyses of the distribution of fitness effects reveal that small effects, either beneficial or deleterious, are well described by a Pareto distribution. These results are robust under changes in the fitness landscape, remarkably when, in addition to selecting a target secondary structure, specific subsequences or low-energy folds are required. A population perturbed by bottlenecks behaves similarly to an adapting population, struggling to return to the optimized state. Whether it can survive in the long run or whether it goes extinct depends critically on the length of the time interval between bottlenecks. © 2010 Stich et al; licensee BioMed Central Ltd.

The deoxidation of steel with simple and complex deoxidisers

The deoxidation of steel with complex deoxidisers was studied at 1550°C and compared with silicon, aluminium and silicon/aluminium alloys as standards. The deoxidation alloy systems, Ca/Si/Al, Mg/Si/Al and Mn/Si/Al, were chosen for the low liquidus temperatures of many of their oxide mixtures and the potential deoxidising power of their constituent elements. Product separation rates and compositional relationships following deoxidation were examined. Silicon/aluminium alloy deoxidation resulted in the product compositions and residual oxygen contents expected from equilibrium and stoichiometric considerations, but with the Ca/Si/Al and Mg/Si/Al alloys the volatility of calcium and magnesium prevented them participating in the final solute equilibrium, despite their reported solubility in liquid iron. Electron-probe microanalysis of the products showed various concentrations of lime and magnesia, possibly resulting from reaction between the metal vapours and dissolved oxygen.The consequent reduction of silica activity in the products due to the presence of CaO and hgO produced an indirect effect of calcium and magnesium on the residual oxygen content. Product separation rates, indicated by vacuum fusion analyses, were not significantly influenced by calcium and magnesium but the rapid separation of products having a high Al2O3Si02 ratio was confirmed. Manganese participated in deoxidation, when present either as an alloying element in the steel or as a deoxidation alloy constituent. The compositions of initial oxide products were related to deoxidation alloy compositions. Separated products which were not alumina saturated, dissolved crucible material to achieve saturation. The melt equilibrated with this slag and crucible by diffusion to determine the residual oxygen content. MnO and SiO2 activities were calculated, and the approximate values of MnO deduced for the compositions obtained. Separation rates were greater for products of high interfacial tension. The rates calculated from a model based on Stoke's Law, showed qualitative agreement with experimental data when corrected for coalescence effects.