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Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and 'moonlighting' activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H 2 O 2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H 2 O 2 - and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation. [Display omitted] • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has an oxidant-sensitive cysteine (Cys) in its active site. • Effects of macromolecular crowding on GAPDH oxidation by H 2 O 2 and SIN-1 was investigated. • Dextran differentially affects H 2 O 2 - and SIN-1-mediated GAPDH cross-linking. • Higher yields of active site Cys-derived sulfonic acids were detected with crowding. [ABSTRACT FROM AUTHOR]

Biological milieus are highly crowded and heterogeneous systems where organization of macromolecules within nanodomains (e.g. membraneless compartments) is vital to the regulation of metabolic processes. There is an increasing interest in understanding the effects that such packed environments have on different biochemical and biological processes. In this context, the redox biochemistry and redox signaling fields are moving towards investigating oxidative processes under conditions that exhibit these key features of biological systems in order to solve existing paradigms including those related to the generation and transmission of specific redox signals within and between cells in both normal physiology and under conditions of oxidative stress. This review outlines the effects that crowding, nanodomain formation and altered local viscosities can have on biochemical processes involving proteins, and then discusses some of the reactions and pathways involving proteins and oxidants that may, or are known to, be modulated by these factors. We postulate that knowledge of protein modification processes (e.g. kinetics, pathways and product formation) under conditions that mimic biological milieus, will provide a better understanding of the response of cells to endogenous and exogenous stressors, and their role in ageing, signaling, health and disease. [Display omitted] • Biological systems are heavily crowded and heterogeneous environments. • These factors may influence protein oxidation, glycation and redox signaling. • Crowding modulates reaction kinetics, and the extent and sites of modification. • Crowding enhances chain reactions, and alters product yields and cross-linking. • Nanodomains and membraneless organelles can facilitate redox signaling. [ABSTRACT FROM AUTHOR]

Recent clinical trials in hypertrophic cardiomyopathy (HCM) have focused on whether treatments can improve symptom burden and quality of life. Sodium glucose cotransporter (SGLT) inhibitors, including sotagliflozin, improve symptoms in patients with heart failure (including HFpEF), and may provide similar benefits in HCM. The SONATA-HCM is a Phase 3, randomized, double-blind, placebo-controlled multicenter trial that will evaluate efficacy of sotagliflozin, a dual SGLT1 and SGLT2 inhibitor, on symptoms, function, and other patient-reported outcomes (PROs), as well as safety in patients with symptomatic HCM. The study will be conducted in 130 sites in 20 countries and will include approximately 500 adults aged 18 years or older diagnosed with symptomatic HCM, either obstructive (oHCM) or non-obstructive (nHCM). Key inclusion criteria include Kansas City Cardiomyopathy Questionnaire (KCCQ-23) Clinical Summary Scale (CSS) score of less than 85 and a New York Heart Association (NYHA) functional class II or III. Participants must be on stable background therapy for HCM. Eligible participants will be randomly assigned in a 1:1 ratio to receive either sotagliflozin 400 mg once daily or a matching placebo for 26 weeks (Figure). Stratification factors are HCM subtype and NYHA class. Randomization will be conducted using a centralized web-based system to ensure allocation concealment. Both participants and investigators will be blinded to the treatment assignments. Adherence to medication will be monitored through pill counts and patient diaries. The primary endpoint is the change from baseline to week 26 in the KCCQ CSS score. Secondary endpoints include the proportion of participants achieving at least a one-class improvement in NYHA functional class at week 26 and the change from baseline in the KCCQ Total Symptom Score. Exploratory endpoints include change from baseline in echocardiographic parameters, biomarkers including N-terminal pro-B-type natriuretic peptide and high-sensitivity cardiac troponin I, and additional PROs (eg, Patient Global Impression of Severity, Patient Global Impression of Change, Work Productivity and Impairment Questionnaire, and EuroQol 5-Dimension, 5-Level Questionnaire). Treatment-emergent adverse events (TEAEs), serious AEs, clinical laboratory, vital signs, and echocardiographic assessments (eg, LV outflow tract gradient) will be monitored. Data will be reviewed by an independent Data Safety Monitoring Board to ensure participant safety throughout the study. The findings could introduce dual SGLT1 and SGLT2 inhibition as a therapeutic option to improve disease-specific symptoms and physical limitations in patients with HCM. [ABSTRACT FROM AUTHOR]

The heme peroxidase family generates a battery of oxidants both for synthetic purposes, and in the innate immune defence against pathogens. Myeloperoxidase (MPO) is the most promiscuous family member, generating powerful oxidizing species including hypochlorous acid (HOCl). Whilst HOCl formation is important in pathogen removal, this species is also implicated in host tissue damage and multiple inflammatory diseases. Significant oxidant formation and damage occurs extracellularly as a result of MPO release via phagolysosomal leakage, cell lysis, extracellular trap formation, and inappropriate trafficking. MPO binds strongly to extracellular biomolecules including polyanionic glycosaminoglycans, proteoglycans, proteins, and DNA. This localizes MPO and subsequent damage, at least partly, to specific sites and species, including extracellular matrix (ECM) components and plasma proteins/lipoproteins. Biopolymer-bound MPO retains, or has enhanced, catalytic activity, though evidence is also available for non-catalytic effects. These interactions, particularly at cell surfaces and with the ECM/glycocalyx induce cellular dysfunction and altered gene expression. MPO binds with higher affinity to some damaged ECM components, rationalizing its accumulation at sites of inflammation. MPO-damaged biomolecules and fragments act as chemo-attractants and cell activators, and can modulate gene and protein expression in naïve cells, consistent with an increasing cycle of MPO adhesion, activity, damage, and altered cell function at sites of leukocyte infiltration and activation, with subsequent tissue damage and dysfunction. MPO levels are used clinically both diagnostically and prognostically, and there is increasing interest in strategies to prevent MPO-mediated damage; therapeutic aspects are not discussed as these have been reviewed elsewhere. [Display omitted] • Myeloperoxidase (MPO) is released extracellularly from leukocytes at sites of inflammation. • MPO generates multiple oxidants to kill pathogens, but also induces host tissue damage. • MPO binds to extracellular matrix proteins, glycosaminoglycans and plasma (lipo)proteins. • Binding via ionic interactions results in site-specific effects and localized modifications. • Biological effects are induced by both oxidant formation and ionic interactions. [ABSTRACT FROM AUTHOR]

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Exposure of biological molecules to oxidants is inevitable and therefore commonplace. Oxidative stress in cells arises from both external agents and endogenous processes that generate reactive species, either purposely (e.g. during pathogen killing or enzymatic reactions) or accidentally (e.g. exposure to radiation, pollutants, drugs, or chemicals). As proteins are highly abundant and react rapidly with many oxidants, they are highly susceptible to, and major targets of, oxidative damage. This can result in changes to protein structure, function, and turnover and to loss or (occasional) gain of activity. Accumulation of oxidatively- modified proteins, due to either increased generation or decreased removal, has been associated with both aging and multiple diseases. Different oxidants generate a broad, and sometimes characteristic, spectrum of post-translational modifications. The kinetics (rates) of damage formation also vary dramatically. There is a pressing need for reliable and robust methods that can detect, identify, and quantify the products formed on amino acids, peptides, and proteins, especially in complex systems. This review summarizes several advances in our understanding of this complex chemistry and highlights methods that are available to detect oxidative modifications--at the amino acid, peptide, or protein level--and their nature, quantity, and position within a peptide sequence. Although considerable progress has been made in the development and application of new techniques, it is clear that further development is required to fully assess the relative importance of protein oxidation and to determine whether an oxidation is a cause, or merely a consequence, of injurious processes. [ABSTRACT FROM AUTHOR]

Left ventricular hypertrophy (LVH) is associated with negative cardiovascular (CV) outcomes. LVH without typical etiology such as hypertension or aortic stenosis is common in patients with hypertrophic cardiomyopathy (HCM). The clinical burdens of HCM include heart failure (HF), atrial fibrillation, and stroke, which have been shown to be reduced with SGLT inhibitors. However, no clinical study to date has evaluated the effects of SGLT inhibitors on CV outcomes in patients with HCM. The objective of this post hoc analysis from SCORED was to evaluate the effects of sotagliflozin, a dual SGLT1 and SGLT2 inhibitor, on CV outcomes in patients with LVH and no hypertension. In SCORED, patients with type 2 diabetes, chronic kidney disease, and additional CV risk factors were randomized to sotagliflozin or placebo and followed for a median of 15.9 months. The primary endpoint was a composite of the number of CV deaths and hospitalizations (HHF)/urgent visits for heart failure (UVHF). The present post hoc analysis identified patients who had reported LVH and no hypertension. Diagnosis of LVH was among the CV risk factors collected during screening. Hypertension was reported as part of medical history. Uncontrolled BP was determined as a CV risk factor in patients by assessing SBP >140 and DBP >90 mmHg despite antihypertensive treatment. Event rates were calculated for the primary endpoint, 3-point MACE (CV death, myocardial infarction, and stroke), and MACE plus HHF and compared between sotagliflozin and placebo. Of the 10,584 randomized patients, 418 (3.9%) reported LVH and no hypertension. At baseline, these patients had a mean age of 68 years, BMI of 31.8 kg/m2, eGFR of 46 mL/min/1.73 m2, LVEF of 55%, SBP of 134 mmHg, and DBP of 77 mmHg and 44% had a history of CV disease and 10% were hospitalized for HF within the 2 years prior to screening. In this cohort, treatment with sotagliflozin was associated with a significantly reduced risk of CV death and HF-related events and the combined composite of MACE plus HHF (p<0.05; Table). Similar results were found in a sensitivity analysis removing the 56 patients with uncontrolled BP per CV risk factor assessment or aortic stenosis and, also, in the cohort of patients with LVH and hypertension. Sotagliflozin significantly reduced the risk of CV events in a cohort of high-risk patients with LVH without hypertension. Although this cohort may share phenotypic and physiological traits of patients with HCM, clinical studies are needed to determine the efficacy and safety of sotagliflozin in patients with HCM. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) affects around 10% of women of reproductive age and is most common in developed countries. The aetiology of PCOS is not completely understood. Current evidence suggests that the syndrome results from a genetic predisposition interacting with developmental events during fetal or perinatal life that together increase susceptibility in some individuals. This implies that environmental factors influence the initiation of PCOS in the fetus or infant, either directly or via the mother. PCOS is often considered to be an ancient disorder but there is no direct proof of this in the medical or historic record. One of the cardinal features, polycystic ovaries, was first described only in the early 1900s, despite reports of many thousands of autopsies recorded earlier. This conundrum could be explained by postulating that polycystic ovaries were rare before the 1900s and have become more common over the last 100 years. The hypothesis that PCOS is a syndrome of the 20th Century would eliminate the need to explain the paradox of why there exists a genetic predisposition to subfertility syndrome. [ABSTRACT FROM AUTHOR]

SGLT inhibitors, including sotagliflozin, have been shown to consistently reduce the risk of heart failure (HF) and renal-related events. Their effects on major adverse cardiovascular events (MACE: myocardial infarction [MI] and stroke) have been variable. Sotagliflozin was the first SGLT inhibitor to demonstrate a significant reduction in both MI and stroke in adults with type 2 diabetes (T2D) and chronic kidney disease. The objective of this analysis was to evaluate the effect of sotagliflozin on MACE across sotagliflozin clinical program, which included Phase 2 and Phase 3 clinical trials with 20,292 patients with T2D or type 1 diabetes (T1D). SCORED and SOLOIST were Phase 3, randomized, double-blind, studies evaluating sotagliflozin and placebo in 10,584 patients with T2D, chronic kidney disease, and other CV risk factors and 1,222 patients with T2D admitted for a worsening heart failure event, respectively. In addition, three Phase 3 placebo-controlled trials were conducted in 3,000 patients with T1D. Nine Phase 3 placebo- and active controlled studies evaluated sotagliflozin in 5,100 patients with T2D. The present analysis focuses on 3-point MACE (CV death, non-fatal MI, and non-fatal stroke). Meta-analysis was performed using a fixed effects model. SCORED contributed the most events to the analysis. MACE results for each dataset significantly or numerically favored sotagliflozin. Overall, the composite of 3-point MACE was lower with sotagliflozin compared with comparator (HR [95% CI] = 0.79 [0.68, 0.90]) (Table). In summary, treatment with sotagliflozin was associated with a significant reduction in MACE in patients with T1D and T2D. [ABSTRACT FROM AUTHOR]

Objective Type 2 diabetes and obesity are pro-inflammatory states associated with increased risk of cardiovascular disease. Canagliflozin, an SGLT2 inhibitor, demonstrated superiority in lowering HbA1c versus glimepiride with less hypoglycemia and greater weight reduction via loss of fat mass in a 52-week trial of type 2 diabetes patients. This post hoc, exploratory analysis assessed the effects of canagliflozin versus glimepiride on select adipokines, inflammatory biomarkers, and chemokines. Methods Changes from baseline to Week 52 in serum leptin, adiponectin, IL-6, TNFα, CRP, PAI-1, VCAM-1, and MCP-1 were measured in a randomly selected subset of type 2 diabetes patients on metformin receiving canagliflozin 300 mg ( n  = 100) or glimepiride ( n  = 100) in the overall study. Correlations between change in biomarkers and change in select metabolic and anthropometric variables were assessed. Results At Week 52, canagliflozin decreased median serum leptin by 25% (95% CI: −34%, −15%) and increased median serum adiponectin by 17% (95% CI: 11%, 23%) compared with glimepiride. There was a 22% reduction in median serum IL-6 (95% CI: −34%, −10%) and a 7% increase in median serum TNFα (95% CI: 1%, 12%) with canagliflozin versus glimepiride. No between-group differences were observed with the other biomarkers. The decrease in serum leptin with canagliflozin was correlated with change in weight ( r  ≥ 0.3) only; the increase in adiponectin and decrease in IL-6 with canagliflozin occurred independently of changes in HbA1c, weight, or lipids. Conclusions These results indicate that canagliflozin may improve adipose tissue function and induce changes in serum leptin, adiponectin, and IL-6 that favorably impact insulin sensitivity and cardiovascular disease risk. [ABSTRACT FROM AUTHOR]

Growth control of container-grown hardy nursery stock generally requires substantial labour investment. Therefore the possibility of alternative growth control using deficit irrigation is appealing. Increasing water costs and limited availability of abstraction licences have added further incentives for nursery stock producers to use deficit irrigation. There are still, however, concerns that inherent non-uniformity of water uptake under commonly used overhead irrigation, and differing irrigation requirements of diverse crops and substrates, may limit the commercial relevance of a protocol developed for single crops growing in 100% peat and irrigated with a high precision drip system. The aim of this research was to determine whether growth control of hardy nursery stock is possible using deficit irrigation applied with conventional overhead irrigation. Over two years, crop growth under an overhead irrigation system was compared under full irrigation and two severities of deficit irrigation. Initially, two crops of contrasting canopy structure i.e. Cornus alba and Lonicera periclymenum were grown. In a subsequent experiment one crop ( Forsythia × intermedia ) was grown in two substrates with contrasting quantities of peat (60 and 100%). Deficit irrigation was found to be highly effective in controlling vegetative growth when applied using overhead irrigation—with similar results as when drip irrigation was used. This comparable response suggests that deficit irrigation can be applied without precision drip irrigation. Scheduling two very different crops with respect to their water use and uptake potential, however, highlighted challenges with respect to application of appropriate deficits for very different crops under one system; responses to deficit irrigation will be more consistent where nursery management allows for scheduling of crops with very different architecture and water use under different regimes. The effectiveness of deficit irrigation in controlling the growth of Forsythia was similar when a reduced peat based substrate was compared with pure peat; additionally, flowering was enhanced. [ABSTRACT FROM AUTHOR]

Activated white cells use oxidants generated by the heme enzyme myeloperoxidase to kill invading pathogens. This enzyme utilizes H2O2 and Cl−, Br−, or SCN− to generate the oxidants HOCl, HOBr, and HOSCN, respectively. Whereas controlled production of these species is vital in maintaining good health, their uncontrolled or inappropriate formation (as occurs at sites of inflammation) can cause host tissue damage that has been associated with multiple inflammatory pathologies including cardiovascular diseases and cancer. Previous studies have reported that sulfur-containing species are major targets for HOCl but as the reactions are fast the only physiologically relevant kinetic data available have been extrapolated from data measured at high pH (>10). In this study these values have been determined at pH 7.4 using a newly developed competition kinetic approach that employs a fluorescently tagged methionine derivative as the competitive substrate (k(HOCl + Fmoc-Met), 1.5×108M−1s−1). This assay was validated using the known k(HOCl + NADH) value and has allowed revised k values for the reactions of HOCl with Cys, N-acetylcysteine, and glutathione to be determined as 3.6×108, 2.9×107, and 1.24×108M−1s−1, respectively. Similar experiments with methionine derivatives yielded k values of 3.4×107M−1s−1 for Met and 1.7×108M−1s−1 for N-acetylmethionine. The k values determined here for the reaction of HOCl with thiols are up to 10-fold higher than those previously determined and further emphasize the critical importance of reactions of HOCl with thiol targets in biological systems. [ABSTRACT FROM AUTHOR]

Abstract: Background: Electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, spectroscopy) is widely considered to be the “gold standard” for the detection and characterisation of radicals in biological systems. Scope of review: The article reviews the major positive and negative aspects of EPR spectroscopy and discusses how this technique and associated methodologies can be used to maximise useful information, and minimise artefacts, when used in biological studies. Consideration is given to the direct detection of radicals (at both ambient and low temperature), the use of spin trapping and spin scavenging (e.g. reaction with hydroxylamines), the detection of nitric oxide and the detection and quantification of some transition metal ions (particularly iron and copper) and their environment. Major conclusions: When used with care this technique can provide a wealth of valuable information on the presence of radicals and some transition metal ions in biological systems. It can provide definitive information on the identity of the species present and also information on their concentration, structure, mobility and interactions. It is however a technique that has major limitations and the user needs to understand the various pitfalls and shortcoming of the method to avoid making errors. General significance: EPR remains the most definitive method of identifying radicals in complex systems and is also a valuable method of examining radical kinetics, concentrations and structure. This article is part of a Special Issue entitled Current methods to study reactive oxygen species — pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. [Copyright &y& Elsevier]

Abstract: Purpose: This study aims to crystallise salbutamol sulphate beneath simulated pulmonary surfactant monolayers. Such ensembles serve as heterogeneous nucleating sites to direct crystallisation. This contribution builds upon previous work to confirm the suitability of Langmuir monolayers in supporting the rational generation of respirable therapeutic material. Methods: Langmuir monolayers (i.e. DPPC or a ‘mixed’ system) were supported on a subphase containing the extremely water soluble model drug (2.5g/ml) and compressed to 5mNm−1 or 35mNm−1 whilst experiencing a temperature reduction and positioned within a humid environment. Control samples were produced via batch crystallisation. Analysis involved scanning electron microscopy (SEM), atomic force microscopy (AFM), powder X-ray diffraction (PXRD) and inverse gas chromatography (IGC). Results: Expanded Langmuir isotherms confirmed drug–surfactant interaction; crystal growth was inhibited at high surface pressure. Resultant crystals exhibited a range of morphologies, dependent upon the crystallisation route. AFM analysis highlighted nanoscale surface undulations. IGC data confirmed sample surface energy profiles were variable and influenced by crystallisation route. Conclusions: Principal modes of drug–surfactant interaction are proposed as hydrogen bond and ion–dipole associations. A range of pharmaceutical approaches have been applied to understand drug–surfactant complementarity. The results strengthen the argument for the use of Langmuir monolayers in drug particle engineering. [Copyright &y& Elsevier]

Abstract: Cultivation of strawberry (Fragaria × ananassa) requires irrigation. Improving crop water use efficiency (WUE) is important for future production. Fragaria chiloensis, a progenitor of cultivated strawberry, grows in sandy soils, and may prove useful in breeding for improved WUE. Little, however, is known about variation in drought tolerance within this species. This research explores drought tolerance in a range of F. chiloensis and F. × ananassa genotypes. Four cultivars of F. × ananassa and four accessions of F. chiloensis were compared when well watered, and when subjected to a water deficit (65% of evapotranspiration). New leaf production, stomatal conductance, and photosynthetic rate were significantly reduced under water deficit, and also significantly differed between genotypes. A significant interaction of genotype and irrigation was found for transpiration rate, leaf area and dry mass, production of runners, predawn water potential, a measure of transpiration efficiency (shoot biomass produced per litre water transpired), and carbon isotope composition, indicating that some genotypes were more severely affected by water deficit than others. The South American F. chiloensis accession ‘Manzanar Alto’ had a similar rate of transpiration to the commercial cultivars, but the remaining (North American) F. chiloensis accessions used far less water than the F. × ananassa. Well-watered F. chiloensis plants used less water than water-limited plants of the F. × ananassa cultivar ‘Florence’. Transpiration efficiency of the F. chiloensis accession ‘BSP14’ was improved by water deficit: this was the only genotype not to show a reduction in leaf area and dry mass under water deficit. Greater drought resistance in three F. chiloensis accessions compared to F. × ananassa results from a conservative vegetative growth strategy, reducing loss of water. [Copyright &y& Elsevier]

Abstract: Cultivars with low stomatal conductance (g s) may show high water use efficiency (WUE) under drought conditions, but under optimal conditions low g s may result in low vigour. A combination of thermal imaging and carbon isotope composition (δ13C) analysis offers potential for screening simultaneously for both high g s and high WUE. Ten cultivars of strawberry (Fragaria × ananassa Duch.) were grown in well watered or water limited conditions. Thermal images were taken of the plants, with various approaches to determine the optimal protocol for detecting variation in g s, including use of reference leaves, grids to maintain leaves horizontal, and collection of meteorological data in synchrony with thermal images. δ13C of leaves, fruit, and crowns was determined. An index of g s derived from the temperature of horizontal leaves and the temperature of wet and dry references showed significant differences between cultivars and between well watered and water limited plants, as did g s estimated from leaf temperature, the temperature of a dry reference, and humidity. Thermal imaging indicated low g s in ‘Elsanta’ and ‘Totem’ and relatively high g s in well watered ‘Elvira’, ‘Florence’ and ‘Cambridge Favourite’. δ13C of all plant material was higher in water limited than well watered plants and showed significant differences between cultivars. In one experiment leaf δ13C indicated lowest WUE in ‘Elvira’ and highest WUE in ‘Totem’. δ13C was inversely correlated with an index of g s derived from thermal imaging. Although the results indicate substantial variation in g s and WUE between cultivars, generally all cultivars responded to water deficit by lowering g s and hence increasing WUE. [Copyright &y& Elsevier]

Abstract: Selenium is a critical trace element, with deficiency associated with numerous diseases including cardiovascular disease, diabetes, and cancer. Selenomethionine (SeMet; a selenium analogue of the amino acid methionine, Met) is a major form of organic selenium and an important dietary source of selenium for selenoprotein synthesis in vivo. As selenium compounds can be readily oxidized and reduced, and selenocysteine residues play a critical role in the catalytic activity of the key protective enzymes glutathione peroxidase and thioredoxin reductase, we investigated the ability of SeMet (and its sulfur analogue, Met) to scavenge hydroperoxides present on amino acids, peptides, and proteins, which are key intermediates in protein oxidation. We show that SeMet, but not Met, can remove these species both stoichiometrically and catalytically in the presence of glutathione (GSH) or a thioredoxin reductase (TrxR)/thioredoxin (Trx)/NADPH system. Reaction of the hydroperoxide with SeMet results in selenoxide formation as detected by HPLC. Recycling of the selenoxide back to SeMet occurs rapidly with GSH, TrxR/NADPH, or a complete TrxR/Trx/NADPH reducing system, with this resulting in an enhanced rate of peroxide removal. In the complete TrxR/Trx/NADPH system loss of peroxide is essentially stoichiometric with NADPH consumption, indicative of a highly efficient system. Similar reactions do not occur with Met under these conditions. Studies using murine macrophage-like J774A.1 cells demonstrate a greater peroxide-removing capacity in cells supplemented with SeMet, compared to nonsupplemented controls. Overall, these findings demonstrate that SeMet may play an important role in the catalytic removal of damaging peptide and protein oxidation products. [Copyright &y& Elsevier]

Abstract: Purpose: To generate theophylline monohydrate crystals underneath Langmuir monolayers composed of material expressed at the alveolar air–liquid interface. Such monolayers can act as nucleation sites to direct crystallisation. The approach offers a novel route to rationally engineer therapeutic crystals and thereby optimise inhaled drug delivery. Methods: Langmuir monolayers consisting of either dipalmitoylphosphatidylcholine (DPPC) or a surfactant mix reflecting pulmonary surfactant were supported on an aqueous theophylline (5.7mg/ml) subphase. The monolayers were compressed to surface pressures reflecting inhalation and exhalation (i.e. 5mNm−1 or 55mNm−1) with a period of 16h to allow crystallisation. Analysis involved scanning electron microscopy (SEM), atomic force microscopy (AFM) and powder X-ray diffraction (PXRD). Results: Condensed isotherms were acquired, which signified surfactant–theophylline interaction. Theophylline monohydrate crystals were obtained and exhibited needle-like morphology. SEM and AFM data highlighted regions of roughened growth along with smooth, stepwise growth on the same crystal face. The surfactant monolayers appeared to influence crystal morphology over time. Conclusions: The data indicate a favourable interaction between each species. The principal mechanism of interaction is thought to be an ion–dipole association. This approach may be applied to generate material with improved complementarity with pulmonary surfactant thus enhancing the interaction between inhaled drug particles and internal lung surfaces. [Copyright &y& Elsevier]

Abstract: Glycosaminoglycans (long-chain polysaccharides) are major components of the extracellular matrix, glycocalyx, and synovial fluid. These materials provide strength and elasticity to tissues and play a key role in regulating cell behavior. Modifications to these materials have been linked to multiple human pathologies. Although modification may occur via both enzymatic and nonenzymatic mechanisms, there is considerable evidence for oxidant-mediated matrix damage. Peroxynitrite (ONOO−/ONOOH) is a potential mediator of such damage, as elevated levels of this oxidant are likely to be present at sites of inflammation. In this study we demonstrate that hyaluronan and chondroitin sulfate are extensively depolymerized by HO⋅ and CO3 ⋅−, but not NO2 ⋅, which may be formed from peroxynitrite. Polymer fragmentation is shown to be dependent on the radical flux, to be O2-independent, and to occur in a site-selective manner as indicated by the detection of disaccharide fragments. EPR spin trapping experiments with polymers, oligomers, and component monosaccharides, including 13C-labeled materials, have provided evidence for the formation of specific carbon-centered sugar-derived radicals. The time course of formation of these radicals is consistent with these species being involved in polymer fragmentation. [Copyright &y& Elsevier]

Abstract: The extracellular matrix (ECM) provides strength and elasticity to tissues and plays a key role in regulating cell behavior; damage to this material is believed to be a major factor in many inflammatory diseases. Peroxynitrite/peroxynitrous acid, which is generated at elevated levels at sites of inflammation, is believed to play a role in ECM damage; however, the mechanisms involved are poorly understood. Here we examined the reactions of bolus peroxynitrite, and that generated in a time-dependent manner by SIN-1 decomposition, with ECM isolated from a vascular smooth muscle cell line and porcine thoracic aorta. Bolus peroxynitrite caused the release of ECM glycosaminoglycans and proteins, the formation of 3-nitroTyr, and the detection of ECM-derived radicals (by immuno-spin trapping) in a concentration-dependent manner. Release and nitration of ECM components were modulated by the local pH and bicarbonate. SIN-1 caused the release of glycosaminoglycan, but not protein, from vascular smooth muscle cell-derived ECM in a concentration-, time-, and pH-dependent manner. The data presented here suggest that peroxynitrite-mediated damage to ECM occurs via a radical-mediated pathway. These reactions may contribute to ECM damage at sites of inflammation and play a role in disease progression, including rupture of atherosclerotic lesions. [Copyright &y& Elsevier]

Abstract: Coadministration of fenofibrate and ezetimibe (FENO + EZE) produced complementary and favorable effects on the major lipids and lipoproteins, low-density lipoprotein cholesterol (LDL-C), triglycerides, high-density lipoprotein cholesterol (HDL-C), and non–HDL-C levels, and was well tolerated in patients with mixed hyperlipidemia. The current analysis evaluates the effects of FENO and EZE, as monotherapies and in coadministration, on lipoprotein subfractions and LDL particle size distributions in these patients. In a 12-week, multicenter, randomized, double-blind, placebo-controlled, parallel-group study, patients with mixed hyperlipidemia were randomized in a 1:3:3:3 ratio to one of 4 treatment groups: placebo, FENO 160 mg/day, EZE 10 mg/day, or FENO 160 mg/day + EZE 10 mg/day. At baseline and study end point, the Vertical Auto Profile II method was used to measure the cholesterol associated with 2 very low-density lipoprotein (VLDL) subfractions (VLDL-C1 + 2 and VLDL-C3), intermediate-density lipoproteins (IDL-C), and 4 LDL subfractions (LDL-C1 through LDL-C4, from most buoyant to most dense), lipoprotein (Lp) (a), and 2 HDL-C subfractions (HDL-C2 and HDL-C3). The LDL particle size was determined using segmented gradient gel electrophoresis. Fenofibrate reduced cholesterol mass within VLDL, IDL, and dense LDL (primarily LDL-C4) subfractions, and increased cholesterol mass within the more buoyant LDL-C2 subfraction, consistent with a shift to a more buoyant LDL peak particle size. Ezetimibe reduced cholesterol mass within all of the apolipoprotein B–containing particles (eg, VLDL-C, IDL-C, and LDL-C) but did not lead to a shift in the LDL particle size distribution profile. Coadministration of FENO and EZE promoted more pronounced reductions in VLDL-C, IDL-C, and LDL-C, and a preferential decrease in dense LDL subfractions. Fenofibrate and FENO + EZE promoted similar increases in HDL-C2 and HDL-C3. Coadministration of FENO + EZE produced complementary and favorable changes in lipoprotein fractions and subfractions, as assessed by the Vertical Auto Profile II method, in patients with mixed hyperlipidemia. These changes reflected the combined effects of FENO in reducing triglycerides-rich lipoproteins and promoting a shift in the LDL particle distribution profile toward larger, more buoyant particles and of EZE in promoting reductions in cholesterol mass across the apolipoprotein B particle spectrum. [Copyright &y& Elsevier]

Abstract: Reaction of radicals in the presence of O2, or singlet oxygen, with some amino acids, peptides, and proteins yields hydroperoxides. These species are key intermediates in chain reactions and protein damage. They can be detected in cells and are poorly removed by enzymatic defenses. Previously we have shown that peptide and protein hydroperoxides react rapidly with thiols, with this resulting in inactivation of some thiol-dependent enzymes. In light of these data, we hypothesized that inactivation of protein tyrosine phosphatases (PTPs), by hydroperoxides present on oxidized proteins, may contribute to cellular and tissue dysfunction by modulation of phosphorylation-dependent cell signaling. We show here that PTPs in cell lysates, and purified PTP-1B, are inactivated by amino acid, peptide, and protein hydroperoxides in a concentration- and structure-dependent manner. Protein hydroperoxides are particularly effective, with inhibition occurring with greater efficacy than with H2O2. Inactivation involves reaction of the hydroperoxide with the conserved active-site Cys residue of the PTPs, as evidenced by hydroperoxide consumption measurements and a diminution of this effect on blocking the Cys residue. This inhibition of PTPs, by oxidized proteins containing hydroperoxide groups, may contribute to cellular dysfunction and altered redox signaling in systems subject to oxidative stress. [Copyright &y& Elsevier]