Role Of Redox Chemistry Research Articles

Abstract 488: Glutathione-CaSR Interaction Contributes To Glutathione Regulation Of Angiogenesis

Introduction: In addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium-sensing receptor (CaSR). CaSR has been found in a variety of cell types including endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH-CaSR interaction and regulation of angiogenesis. We hypothesize a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of physiological and pathological angiogenesis. Methods: Matrigel tube formation and wound healing assays were used to measure EC activity. RGECO1.2 was used to measure changes in intracellular calcium. Cell surface expression assay was used to measure surface CaSR expression. Cell counts and flow cytometry were used to monitor cell proliferation and cell cycle progression. Endothelial and mesenchymal markers were measured using real-time PCR. Results: Extracellular GSH increased endothelial tube length and branching in tube formation assay, while a selective CaSR inhibitor abrogated these effects. GSH also influenced EC migration as measured by wound healing assay. GSH or a CaSR agonist (R-568) reduced surface CaSR expression, with no changes in intracellular calcium. Extracellular GSH alters EC proliferation and cell cycle progression, coinciding with alterations in endothelial phenotype along the endothelial-mesenchymal spectrum. Conclusion: In addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by tube formation assay. The use of a selective CaSR inhibitor confirms the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in wound healing assays. These results could be explained by GSH-CaSR-mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G-protein coupled receptor, representing a novel mechanism of action for physiological and pathological blood vessel development.

Investigating a Ligand‐receptor Role for Glutathione in Angiogenesis

IntroductionIn addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium‐sensing receptor (CaSR). CaSR expression has been found in a variety of cell types including vascular smooth muscle and endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH‐CaSR interaction and regulation of blood vessel development. We hypothesize that a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of normal physiology, with potential extensions to pathological angiogenesis.MethodsMatrigel tube formation and under agarose migration assays were used to measure EC activity in primary human aortic endothelial cells (HAECs). Fura‐2 AM was used with cells from an hTERT immortalized aortic endothelial cell line (tHAECs) to measure changes in intracellular calcium. Cell surface expression assay (CelLISA) and flow cytometry were used to measure surface CaSR expression. Endothelial and endothelial‐mesenchymal transition (EndoMT) markers were measured using real‐time PCR.ResultsExtracellular application of GSH increased endothelial tube length and branching in tube formation assay, while treating cells with selective CaSR inhibitor (NPS‐2143) abrogated these effects, indicating a role for CaSR in vascular modeling. Extracellular GSH influenced EC migration as measured by under agarose migration assay. GSH or a CaSR agonist (R‐568) reduced surface CaSR expression, with little/no change in intracellular calcium. RT‐PCR results suggest that extracellular GSH alters endothelial phenotype along the endothelial‐mesenchymal spectrum, promoting endothelial differentiation.ConclusionIn addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by the tube formation assay. With the use of a selective CaSR inhibitor, we were able to confirm the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in the under agarose assay. These results could be explained by GSH‐CaSR‐mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G‐protein coupled receptor, which represents a novel mechanism of action for physiological and pathological blood vessel development.

The sulfur formation system mediating extracellular cysteine-cystine recycling in Fervidobacterium islandicum AW-1 is associated with keratin degradation.

Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe-S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe-S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW-1. Comparative genomics of the order Thermotogales revealed that the feather-degrading F. islandicum AW-1 has a complete Suf-like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S0 ). On the other hand, F. islandicum AW-1 exhibited a significant retardation in the Suf system-mediated keratin degradation in the presence of S0 . Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS-SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf-like machinery including the SufS-SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.

The mineralogy of suspended matter, fresh and Cenozoic sediments in the fluvio-deltaic Rhine–Meuse–Scheldt–Ems area, the Netherlands: An overview and review

AbstractMinerals are the building blocks of clastic sediments and play an important role with respect to the physico-chemical properties of the sediment and the lithostratigraphy of sediments. This paper aims to provide an overview of the mineralogy (including solid organic matter) of sediments as well as suspended matter as found in the Netherlands (and some parts of Belgium). The work is based on a review of the scientific literature published over more than 100 years. Cenozoic sediments are addressed together with suspended matter and recent sediments of the surface water systems because they form a geoscientific continuum from material subject to transport via recently settled to aged material. Most attention is paid to heavy minerals, clay minerals, feldspars, Ca carbonates, reactive Fe minerals (oxides, siderite, sulphides, glauconite) and solid organic matter because they represent the dominant minerals and their properties form a main issue in subsurface and water management. When possible and relevant, the amounts, provenance, relationship with grain size distribution, early diagenesis and palaeohydrological evolution are described. Tables with statistical data about the mineral contents and isotopic composition of carbonates and organic matter are presented as overviews. The review on the mineralogy of Dutch fluvial and marine environments is more extensive than that for the other sedimentary environments because the first two have been studied much more intensively than the others and they also form the larger part of the Dutch deposits. The focus is on the natural background mineralogy of Dutch sediments, but this is hard for recent sediments, largely because the massive hydraulic infrastructure present in the Netherlands has probably also affected the mineralogy and geochemistry of sediments deposited in recent centuries. Many findings are summarised, several of which lead to more general insights for the Dutch situation. Ca carbonates in sediments often have several provenances and thus must be considered as mixtures. Dolomite is commonly present in addition to calcite. The importance of biotite as weatherable mica is unclear. Weathering of heavy minerals plays some role but it is unclear in which way it affects the heavy mineral associations. Clays are usually dominated by illite, smectite and their interstratified variant, while kaolinite is usually below 20% and chlorite below 5%. Vermiculite is a minor constituent in fluvial clays and its illitisation presumably happens during early diagenesis in the marine environment. Opaque Fe hydroxides can be present in addition to Fe oxyhydroxide coatings and both will play a role in redox chemistry as reactive Fe minerals. Feldspars in marine sediments must be present but they have not been properly studied. The genesis of rattle stones and carbonate concretions has not been completely elucidated. The fraction of terrigeneous organic matter in estuarine and coastal marine sediments is substantial. The available data and information are spread irregularly over the country and the reviewed information discussed in this paper is derived from relatively small-scale studies dealing with a limited amount of analysed samples. Much information is available from the Scheldt estuaries in the southwestern part of the Netherlands partly due to the severe contamination of the Western Scheldt in recent decades.

Arsenic(III) and iron(II) co-oxidation by oxygen and hydrogen peroxide: Divergent reactions in the presence of organic ligands

Iron-catalyzed oxidation of As(III) to As(V) can be highly effective for toxic arsenic removal via Fenton reaction and Fe(II) oxygenation. However, the contribution of ubiquitous organic ligands is poorly understood, despite its significant role in redox chemistry of arsenic in natural and engineered systems. In this work, selected naturally occurring organic ligands and synthetic ligands in co-oxidation of Fe(II) and As(III) were examined as a function of pH, Fe(II), H2O2, and radical scavengers (methanol and 2-propanol) concentration. As(III) was not measurably oxidised in the presence of excess ethylenediaminetetraacetic acid (EDTA) (i.e. Fe(II):EDTA<1:1), contrasting with the rapid oxidation of Fe(II) by O2 and H2O2 at neutral pH under the same conditions. However, partial oxidation of As(III) was observed at a 2:1 ratio of Fe(II):EDTA. Rapid Fe(II) oxidation in the presence of organic ligands did not necessarily result in the coupled As(III) oxidation. Organic ligands act as both iron speciation regulators and radicals scavengers. Further quenching experiments suggested both hydroxyl radicals and high-valent Fe species contributed to As(III) oxidation. The present findings are significant for the better understanding of aquatic redox chemistry of iron and arsenic in the environment and for optimization of iron-catalyzed arsenic remediation technology.

Substrate profiling and aldehyde dismutase activity of the Kvβ2 subunit of the mammalian Kv1 potassium channel

Voltage-dependent potassium channels (Kv) are involved in various cellular signalling processes by governing the membrane potential of excitable cells. The cytosolic face of these α subunit-containing channels is associated with β subunits that can modulate channel responses. Surprisingly, the β subunit of the mammalian Kv1 channels, Kvβ2, has a high level of sequence homology with the aldo-keto reductase (AKR) superfamily of proteins. Recent studies have shown that Kvβ2 can catalyze the reduction of aldehydes and, most significantly, that channel function is modulated when Kvβ2-bound NADPH is concomitantly oxidized. As a result, the redox chemistry of this subunit is crucial to understanding its role in K(+) channel modulation. The present study has extended knowledge of the substrate profile of this subunit using a single turnover fluorimetric assay. Kvβ2 was found to catalyse the reduction of aromatic aldehyde substrates such as 2, 3 and 4-nitrobenzaldehydes, 4-hydroxybenzaldehyde, pyridine 2-aldehyde and benzaldehyde. The presence of an electron withdrawing group at the position para to the aldehyde in aromatic compounds facilitated reduction. Aliphatic aldehydes proved to be poor substrates. We devised a simple HPLC-based assay to identify Kvβ2 reaction products. Using this assay we showed, for the first time, that Kvβ2 can catalyze a slow aldehyde dismutation reaction using 4-nitrobenzaldehyde as substrate and have identified the products of this reaction. The ability of Kvβ2 to carry out both an aldehyde reduction and a dismutation reaction is discussed in the light of current thinking on the role of redox chemistry in channel modulation.

The influence of aggregation on the redox chemistry of humic substances

Environmental context. The ability of humic substances (decaying plant and animal matter) to partake in redox reactions in the environment depends on the extent to which the various humic polymers aggregate in solution to form larger particles. This aggregation, in turn, is predicated on the solution conditions, especially ionic strength, the pH, and the types of cations present. Abstract. Aggregation and conformation play an important role in the aqueous redox chemistry of humic substances (HS). The reduction potentials of dissolved humic and fulvic acids vary with pH, ionic strength, and type of humate used, and depending on the solution conditions, they can abiotically reduce various species. Changes in HS reduction potential ranged from 60 to 140 mV on addition of divalent cations, whereas no significant changes were observed with equivalent additions of monovalent cations. Dynamic light scattering measurements showed that this behaviour paralleled the size changes obtained with humic aggregates under the same conditions. The effect was more pronounced at higher pH, where divalent cations caused a significant decrease in the average hydrodynamic radius, whereas monovalent cations did not. At pH 4, neither mono- nor divalent cations substantially affected aggregate sizes. Quinoid moieties, which are known to play an important role in the redox chemistry of HS, displayed fluorescence excitation–emission matrices with features related to changes in the reduction potential of HS. An increase in the reduction potential (Eh) induced by the addition of Ca2+, for instance, caused a red shift in the excitation–emission matrix maximum.

Biochemical entities involved in reactive oxygen species generation by human spermatozoa.

Spermatozoa were the first cell type suggested to generate reactive oxygen species. However, a lack of standardization in sperm preparation techniques and the obfuscating impact of contaminating cell types in human ejaculates have made it difficult to confirm that mammalian germ cells do, in fact, make such reactive metabolites. By identifying, on a molecular level, those entities involved in reactive oxygen species generation and demonstrating their presence in spermatozoa, the role of redox chemistry in the control of sperm function can be elucidated. Two major proteins have apparently been identified in this context, namely, NOX5, a calcium-activated NADPH oxidase, and nitric oxide synthase. Understanding the involvement of these enzymes in sperm physiology is essential if we are to understand the causes of oxidative stress in the male germ line.

Iron-sulfur cluster biosynthesis. Kinetic analysis of [2Fe-2S] cluster transfer from holo ISU to apo Fd: role of redox chemistry and a conserved aspartate.

ISU-type proteins mediate cluster transfer to apo protein targets. Rate constants have been determined for cluster transfer from ISU to apo Fd for both Homo sapiens and Schizosaccharomyces pombe proteins, and cross reactions have also been examined. Substitution of a key aspartate residue of ISU is found to decrease the rate of cluster transfer by at least an order of magnitude (for wild-type Hs ISU cluster transfer to Hs apo Fd, k(2) approximately 540 M(-1) min(-1), relative 56 M(-1) min(-1) for D37A ISU). This change in rate constant does not reflect any change in binding affinity of the ISU and Fd proteins. The pH dependencies of cluster transfer rates are similar for WT and D37A ISU, arguing against a role for Asp37 as a catalytic base, although evidence for general base catalysis mediating deprotonation of Cys from the apo target is supported by an observed pK(a) of 6.9 determined from the pH profiles for both WT and D37A ISU. Such a pK(a) value is at the lower limit for Cys and is common for solvent-accessible Cys thiols. The temperature dependence of the rate constant defining the cluster transfer reaction for wild type versus the aspartate derivative is distinct. Thermal activation parameters (DeltaH and DeltaS) are consistent with a solvent-accessible ISU-bound cluster, with desolvation as a principle barrier to cluster transfer. Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer to be rate-limiting. Fully oxidized cluster appears to be the natural state for transfer to target proteins. Reduced Fd does not readily reduce ISU-bound [2Fe-2S](2+) and does not promote cluster transfer to an apo Fd target.

The role of redox chemistry in scanning tunneling microscopy imaging of electroactive films

The dependence of tunneling current I on bias voltage Vb in scanning tunneling microscopy (STM) measurements of multilayer electroactive films is interpreted in terms of a redox mechanism involving electron transfer reactions at the STM tip|film and electrode|film interfaces. Analysis suggests that a symmetrical I-Vb response is anticipated when the molecular film is sufficiently thick (ca. 2–3 monolayers) that the electron-transfer steps at the STM tip and electrode can be considered as discrete steps in the current-carrying processes. A macroscopic two-electrode thin-layer electrochemical cell is used to mimic the response of an electrode|redox film|STM tip junction. Steady-state symmetrical I-V responses are obtained for the thin-layer cell, even when the solution initially contains only one half of an electroactive redox couple. Tunneling spectroscopic measurements, using a STM, of dry films of protoporphyrin(IX)Fe(III)Cl deposited on highly oriented pyrolytic graphite electrodes are interpreted in terms of the proposed redox mechanism.