Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Effect of the Hofmeister anions upon the swelling of a self-assembled pH-responsive hydrogel

We report the effect of a range of monovalent sodium salts on the molecular equilibrium swelling of a simple synthetic microphase separated poly(methyl methacrylate)-block-poly(2-(diethylamino)ethyl methacrylate)-block-poly(methyl methacrylate) (PMMA88-b-PDEA223-b-PMMA88) pH-responsive hydrogel. Sodium acetate, sodium chloride, sodium bromide, sodium iodide, sodium nitrate and sodium thiocyanate were selected for study at controlled ionic strength and pH; all salts are taken from the Hofmeister series (HS). The influence of the anions on the expansion of the hydrogel was found to follow the reverse order of the classical HS. The expansion ratio of the gel measured in solutions containing the simple sodium halide salts (NaCl, NaBr, and NaI) was found to be strongly related to parameters which describe the interaction of the ion with water; surface charge density, viscosity coefficient, and entropy of hydration. A global study which also included nonspherical ions (NaAce, NaNO3 and NaSCN) showed the strongest correlation with the viscosity coefficient. Our results are interpreted in terms of the Collins model,(1) where larger ions have more mobile water in the first hydration cage immediately surrounding the gel, therefore making them more adhesive to the surface of the stationary phase of the gel and ultimately reducing the level of expansion.

Mitochondrial superoxide anion radicals mediate induction of apoptosis in cardiac myoblasts exposed to chronic hypoxia

Both reactive oxygen species (ROS) and ATP depletion may be significant in hypoxia-induced damage and death, either collectively or independently, with high energy requiring, metabolically active cells being the most susceptible to damage. We investigated the kinetics and effects of ROS production in cardiac myoblasts, H9C2 cells, under 2%, 10% and 21% O2 in the presence or absence of apocynin, rotenone and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. H9C2 cells showed significant loss of viability within 30 min of culture at 2% oxygen which was not due to apoptosis, but was associated with an increase in protein oxidation. However, after 4 h, apoptosis induction was observed at 2% oxygen and also to a lesser extent at 10% oxygen; this was dependent on the levels of mitochondrial superoxide anion radicals determined using dihydroethidine. Hypoxia-induced ROS production and cell death could be rescued by the mitochondrial complex I inhibitor, rotenone, despite further depletion of ATP. In conclusion, a change to superoxide anion radical steady state level was not detectable after 30 min but was evident after 4 h of mild or severe hypoxia. Superoxide anion radicals from the mitochondrion and not ATP depletion is the major cause of apoptotic cell death in cardiac myoblasts under chronic, severe hypoxia.

Injectable hydrogels with high fixed charge density and swelling pressure for nucleus pulposus repair:biomimetic glycosaminoglycan analogues

The load-bearing biomechanical role of the intervertebral disc is governed by the composition and organization of its major macromolecular components, collagen and aggrecan. The major function of aggrecan is to maintain tissue hydration, and hence disc height, under the high loads imposed by muscle activity and body weight. Key to this role is the high negative fixed charge of its glycosaminoglycan side chains, which impart a high osmotic pressure to the tissue, thus regulating and maintaining tissue hydration and hence disc height under load. In degenerate discs, aggrecan degrades and is lost from the disc, particularly centrally from the nucleus pulposus. This loss of fixed charge results in reduced hydration and loss of disc height; such changes are closely associated with low back pain. The present authors developed biomimetic glycosaminoglycan analogues based on sulphonate-containing polymers. These biomimetics are deliverable via injection into the disc where they polymerize in situ, forming a non-degradable, nuclear "implant" aimed at restoring disc height to degenerate discs, thereby relieving back pain. In vitro, these glycosaminoglycan analogues possess appropriate fixed charge density, hydration and osmotic responsiveness, thereby displaying the capacity to restore disc height and function. Preliminary biomechanical tests using a degenerate explant model showed that the implant adapts to the space into which it is injected and restores stiffness. These hydrogels mimic the role taken by glycosaminoglycans in vivo and, unlike other hydrogels, provide an intrinsic swelling pressure, which can maintain disc hydration and height under the high and variable compressive loads encountered in vivo. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The structure and dynamics of 2-dimensional fluids in swelling clays

The interlayer pores of swelling 2:1 clays provide an ideal 2-dimensional environment in which to study confined fluids. In this paper we discuss our understanding of the structure and dynamics of interlayer fluid species in expanded clays, based primarily on the outcome of recent molecular modelling and neutron scattering studies. Counterion solvation is compared with that measured in bulk solutions, and at a local level the cation-oxygen coordination is found to be remarkably similar in these two environments. However, for the monovalent ions the contribution to the first coordination shell from the clay surfaces increases with counterion radius. This gives rise to inner-sphere (surface) complexes in the case of potassium and caesium. In this context, the location of the negative clay surface charge (i.e. arising from octahedral or tetrahedral substitution) is also found to be of major importance. Divalent cations, such as calcium, eagerly solvate to form outer-sphere complexes. These complexes are able to pin adjacent clay layers together, and thereby prevent colloidal swelling. Confined water molecules form hydrogen bonds to each other and to the clays' surfaces. In this way their local environment relaxes to close to the bulk water structure within two molecular layers of the clay surface. Finally, we discuss the way in which the simple organic molecules methane, methanol and ethylene glycol behave in the interlayer region of hydrated clays. Quasi-elastic neutron scattering of isotopically labelled interlayer CH 3OD and (CH2OD)2 in deuterated clay allows us to measure the diffusion of the CH3- and CH2-groups in both clay and liquid environments. We find that in both the one-layer methanol solvates and the two-layer glycol solvates the diffusion of the most mobile organic molecules is close to that in the bulk solution.

The pH-induced swelling and collapse of a polybase brush synthesized by atom transfer radical polymerization

We have used neutron reflectometry to characterize the swelling behaviour of brushes of poly[2-(diethyl amino)ethyl methacrylate], a polybase, as a function of pH. The brushes, synthesized by the "grafting from" method of atom transfer radical polymerization, were observed to approximately double their thickness in low pH solutions, although the pK is shifted to a lower pH than in dilute solution. The composition-depth profile obtained from the reflectometry experiments for the swollen brushes reveals a region depleted in polymer between the substrate and the extended part of the brush.

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.

Grewia gum 1:some mechanical and swelling properties of compact and film

Purpose: To study the mechanical and dynamic swelling properties of grewia gum, evaluate its compression behaviour and determine the effect of drying methods on its properties. Methods: Compacts (500 mg) of both freeze-dried and air-dried grewia gum were separately prepared by compression on a potassium bromide (KBr) press at different pressures and subjected to Heckel analysis. Swelling studies were performed using 200 mg compacts of the gum (freeze-dried or air-dried) compressed on a KBr press. The mechanical properties of the films of the gum prepared by casting 1 % dispersions of the gum were evaluated using Hounsfield tensiometer. The mechanical properties of grewia gum films were compared with films of pullulan and guar gum which were similarly prepared. The effect of temperature on the water uptake of the compacts was studied and the data subjected to Schott's analysis. Results: Drying conditions had no effect on the yield pressure of the gum compacts as both air-dried and freeze-dried fractions had a yield pressure of 322.6 MPa. The plots based on Schott's equation for the grewia gum samples showed that both samples (freeze-dried and air-dried) exhibited long swelling times. Grewia gum film had a tensile strength of 19.22±3.61 MPa which was similar to that of pullulan films (p > 0.05). It had an elastic modulus of 2.13±0.12 N/mm2 which was significantly lower (p < 0.05) than those of pullulan and guar gum with elastic moduli of 3.33±0.00 and 2.86±0.00 N/mm2, respectively. Conclusion: The type of drying method used does not have any effect on the degree of plasticity of grewia gum compacts. Grewia gum obtained by either drying method exhibited extended swelling duration. Matrix tablet formulations of the gum will likely swell slowly and promote sustained release of drug. © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City.

Short-chain fatty acid acetate stimulates adipogenesis and mitochondrial biogenesis via GPR43 in brown adipocytes

Short-chain fatty acids play crucial roles in a range of physiological functions. However, the effects of short-chain fatty acids on brown adipose tissue have not been fully investigated. We examined the role of acetate, a short-chain fatty acid formed by fermentation in the gut, in the regulation of brown adipocyte metabolism. Our results show that acetate up-regulates adipocyte protein 2, peroxisomal proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1 expression and affects the morphological changes of brown adipocytes during adipogenesis. Moreover, an increase in mitochondrial biogenesis was observed after acetate treatment. Acetate also elicited the activation of ERK and cAMP response element-binding protein, and these responses were sensitive to G(i/o)-type G protein inactivator, Gβγ-subunit inhibitor, phospholipase C inhibitor, and MAPK kinase inhibitor, indicating a role for the G(i/o)βγ/phospholipase C/protein kinase C/MAPK kinase signaling pathway in these responses. These effects of acetate were mimicked by treatment with 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide, a synthetic G protein-coupled receptor 43 (GPR43) agonist and were impaired in GPR43 knockdown cells. Taken together, our results indicate that acetate may have important physiological roles in brown adipocytes through the activation of GPR43.