Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells and pericytes

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P <0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal <glycated <or = minimally oxidized <glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.

Glycation and oxidation:a role in the pathogenesis of atherosclerosis

Reactions involving glycation and oxidation of proteins and lipids are believed to contribute to atherogenesis. Glycation, the nonenzymatic binding of glucose to protein molecules, can increase the atherogenic potential of certain plasma constituents, including low-density lipoprotein (LDL). Glycation of LDL is significantly increased in diabetic patients compared with normal subjects, even in the presence of good glycemic control. Metabolic abnormalities associated with glycation of LDL include diminished recognition of LDL by the classic LDL receptor; increased covalent binding of LDL in vessel walls; enhanced uptake of LDL by macrophages, thus stimulating foam cell formation; increased platelet aggregation; formation of LDL-immune complexes; and generation of oxygen free radicals, resulting in oxidative damage to both the lipid and protein components of LDL and to any nearby macromolecules. Oxidized lipoproteins are characterized by cytotoxicity, potent stimulation of foam cell formation by macrophages, and procoagulant effects. Combined glycation and oxidation, "glycoxidation," occurs when oxidative reactions affect the initial products of glycation, and results in irreversible structural alterations of proteins. Glycoxidation is of greatest significance in long-lived proteins such as collagen. In these proteins, glycoxidation products, believed to be atherogenic, accumulate with advancing age: in diabetes, their rate of accumulation is accelerated. Inhibition of glycation, oxidation, and glycoxidation may form the basis of future antiatherogenic strategies in both diabetic and nondiabetic individuals.

Identification of CO adsorbed at Ru and Pt sites on a polycrystalline Pt/Ru electrode and the observation of their oxidation and free interchange under open circuit conditions

The spontaneous oxidation of CO adsorbates on a Pt electrode modified by Ru under open circuit (OC) conditions in perchloric acid solution has been followed, for the first time, using in situ FTIR spectroscopy, and the dynamics of the surface processes taking place have been elucidated. The IR data show that adsorbed CO present on both the Ru and Pt domains and can be oxidized by the oxygen-containing adlayer on the Ru in a chemical process to produce CO under OC conditions. There is a free exchange of CO is between the Ru and Pt sites. Oxidation of CO may take place at the edges of the Ru islands, but CO is transfer, at least on the time scale of these experiments, allows the two different populations to maintain equilibrium. Oxidation is limited in this region by the rate of supply of oxygen to die surface of the catalyst. A mechanism is postulated to explain the observed behavior.

Electrochemical versus gas-phase oxidation of ru single-crystal surfaces

The electrochemical uptake of oxygen on a Ru(0001) electrode was investigated by electron diffraction, Auger spectroscopy, and cyclic voltammetry. An ordered (2 × 2)-O overlayer forms at a potential close to the hydrogen region. At +0.42 and +1.12 V vs Ag/AgCl, a (3 × 1) phase and a (1 × 1)-O phase, respectively, emerge. When the Ru electrode potential is maintained at +1.12 V for 2 min, RuO2 grows epitaxially with its (100) plane parallel to the Ru(0001) surface. In contrast to the RuO domains, the non-oxidized regions of the Ru electrode surface are flat. If, however, the electrode potential is increased to +1.98 V for 2 min, the remaining non-oxidized Ru area also becomes rough. These findings are compared with O overlayers and oxides on the Ru(0001) and Ru(101¯1) surfaces created by exposure to gaseous O under UHV conditions. On the other hand, gas-phase oxidation of the Ru(101¯0) surface leads to the formation of RuO with a (100) orientation. It is concluded that the difference in surface energy between RuO(110) and RuO(100) is quite small. RuO again grows epitaxially on Ru(0001), but with the (110) face oriented parallel to the Ru(0001) surface. The electrochemical oxidation of the Ru(0001) electrode surface proceeds via a 3-dimensional growth mechanism with a mean cluster size of 1.6 nm, whereas under UHV conditions, a 2-dimensional oxide film (1-2 nm thick) is epitaxially formed with an average domain size of 20 µm. © 2000 American Chemical Society.

In situ FTIR studies of the effect of temperature on the adsorption and electrooxidation of CO at the Ru(0001) electrode surface

The adsorption and electrooxidation of CO at a Ru(0001) electrode in perchloric acid solution have been investigated as a function of temperature, potential and time using in situ FTIR spectroscopy. This builds upon and extends previous work on the same system carried out at room temperature. As was observed at room temperature, both linear (CO) and 3-fold-hollow (CO) binding CO adsorbates (bands at 2000-2045 cm and 1768-1805 cm, respectively) were detected on the Ru(0001) electrode at 10°C and 50°C. However, the temperature of the Ru(0001) electrode had a significant effect upon the structure and behavior of the CO adlayer. At 10°C, the in-situ FTIR data showed that the adsorbed CO species still remain in rather compact islands up to ca. 1100 mV vs Ag/AgCl as the CO oxidation reaction proceeds, with oxidation occurring only at the boundaries between the CO and active surface oxide/hydroxide domains. However, the IR data collected at 50°C strongly suggest that the adsorbed CO species are present as relatively looser and weaker structures, which are more easily electro-oxidized. The temperature-, potential-, and coverage-dependent relaxation and compression of the CO adlayer at low coverages are also discussed.

Isolation of promoter for cytosolic phospholipase A2 (cPLA2)

Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid from membrane phospholipids and is believed to be the rate-limiting enzyme in the arachidonic acid pathway. We report herein the isolation of a 3 kb fragment of rodent genomic DNA containing part of the first intron, the first exon and 5'-flanking sequence. The start site of transcription was mapped by 5'-rapid amplification of cDNA ends and corroborated by ribonuclease protection assay. The gene has a TATAless promoter with no classical Sp1 binding sites or initiator element. A microsatellite series of CA repeats was noted in the 5'-flanking region of both the rodent and human promoters. Deletion constructs have been analysed for luciferase activity and confirmed promoter activity.

Identification of serum stress biomarkers in pigs housed at different stocking densities

Eight Duroc × (Landrace × Large White) male pigs housed at a stocking rate of 0.50 m2/pig were subjected to a higher stocking rate of 0.25 m2/pig (higher density, HD) for two 4-day periods over 26 days. Using biochemical and proteomic techniques serum and plasma samples were examined to identify potential biomarkers for monitoring stress due to HD housing. HD housed pigs showed significant differences (P < 0.001) in total cholesterol and low density lipoprotein-associated cholesterol, as well as in concentrations of the pig-major acute phase protein (Pig-MAP) (P = 0.002). No differences were observed in serum cortisol or other acute phase proteins such as haptoglobin, C-reactive protein or apolipoprotein A–I. HD-individuals also showed an imbalance in redox homeostasis, detected as an increase in the level of oxidized proteins measured as the total plasma carbonyl protein content (P < 0.001) with a compensatory increase in the activity of the antioxidant enzyme glutathione peroxidase (P = 0.012). Comparison of the serum proteome yielded a new potential stress biomarker, identified as actin by mass spectrometry. Cluster analysis of the results indicated that individuals segregated into two groups, with different response patterns, suggesting that the stress response depended on individual susceptibility.

Localized Flux Maxima of Arsenic, Lead, and Iron around Root Apices in Flooded Lowland Rice

In wetland-adapted plants, such as rice, it is typically root apexes, sites of rapid entry for water/nutrients, where radial oxygen losses (ROLs) are highest. Nutrient/toxic metal uptake therefore largely occurs through oxidized zones and pH microgradients. However, the processes controlling the acquisition of trace elements in rice have been difficult to explore experimentally because of a lack of techniques for simultaneously measuring labile trace elements and O2/pH. Here, we use new diffusive gradients in thin films (DGT)/planar optode sandwich sensors deployed in situ on rice roots to demonstrate a new geochemical niche of greatly enhanced As, Pb, and Fe(II) mobilization into solution immediately adjacent to the root tips characterized by O2 enrichment and low pH. Fe(II) mobilization was congruent to that of the peripheral edge of the aerobic root zone, demonstrating that the Fe(II) mobilization maximum only developed in a narrow O2 range as the oxidation front penetrates the reducing soil. The Fe flux to the DGT resin at the root apexes was 3-fold higher than the anaerobic bulk soil and 27 times greater than the aerobic rooting zone. These results provide new evidence for the importance of coupled diffusion and oxidation of Fe in modulating trace metal solubilization, dispersion, and plant uptake.

Graphene Nanoribbons as a Drug Delivery Agent for Lucanthone Mediated Therapy of Glioblastoma Multiforme

We report use of PEG-DSPE coated oxidized graphene nanoribbons (O-GNR-PEG-DSPE) as agent for delivery of anti-tumor drug Lucanthone (Luc) into Glioblastoma Multiformae (GBM) cells targeting base excision repair enzyme APE-1 (Apurinic endonuclease-1). Lucanthone, an endonuclease inhibitor of APE-1, was loaded onto O-GNR-PEG-DSPEs using a simple non-covalent method. We found its uptake by GBM cell line U251 exceeding 67% and 60% in APE-1-overexpressing U251, post 24 h. However, their uptake was ~ 38% and 29% by MCF-7 and rat glial progenitor cells (CG-4), respectively. TEM analysis of U251 showed large aggregates of O-GNR-PEG-DSPE in vesicles. Luc-O-GNR-PEG-DSPE was significantly toxic to U251 but showed little/no toxicity when exposed to MCF-7/CG-4 cells. This differential uptake effect can be exploited to use O-GNR-PEG-DSPEs as a vehicle for Luc delivery to GBM, while reducing nonspecific cytotoxicity to the surrounding healthy tissue. Cell death in U251 was necrotic, probably due to oxidative degradation of APE-1.

Graphical abstract

We used O-GNR-PEG-DSPE as a reliable, non-toxic vehicle for delivery of APE-1 inhibiting Lucanthone into GBM tumor cell lines. LUC-O-GNR-PEG-DSPE particles showed 60% or more uptake by CMV/U251 and A1-5/CMV/U251 where as the uptake by MCF7 and normal CG4 glial cells was much lower (38% and 29% respectively). Different concentrations of Luc (5–80 μM) loaded onto O-GNR-PEG-DSPE showed lower toxicity in the exposed cells compared to the free drug, due to possible slow release of the drug from this particle, which ensures minimum non-specific release of the drug from the particle once it is injected in vivo.
http://ars.els-cdn.com/content/image/1-s2.0-S1549963414004249-fx1.jpg

Mineralogical Characteristics and Transformations during Long-Term Operation of a Zerovalent Iron Reactive Barrier

Design and operation of Fe⁰ permeable reactive barriers (PRBs) can be improved by understanding the long-term mineralogical transformations that occur within PRBs. Changes in mineral precipitates, cementation, and corrosion of Fe⁰ filings within an in situ pilot-scale PRB were examined after the first 30 months of operation and compared with results of a previous study of the PRB conducted 15 months earlier using X-ray diffraction and scanning electron microscopy employing energy dispersive X-ray and backscatter electron analyses. Iron (oxy)hydroxides, aragonite, and maghemite and/or magnetite occurred throughout the cores collected 30 mo after installation. Goethite, lepidocrocite, mackinawite, aragonite, calcite, and siderite were associated with oxidized and cemented areas, while green rusts were detected in more reduced zones. Basic differences from our last detailed investigation include (i) mackinawite crystallized from amorphous FeS, (ii) aragonite transformed into calcite, (iii) akaganeite transformed to goethite and lepidocrocite, (iv) iron (oxy)hydroxides and calcium and iron carbonate minerals increased, (v) cementation was greater in the more recent study, and (vi) oxidation, corrosion, and disintegration of Fe⁰ filings were greater, especially in cemented areas, in the more recent study. If the degree of corrosion and cementation that was observed from 15 to 30 mo after installation continues, certain portions of the PRB (i.e., up-gradient entrance of the ground water to the Fe⁰ section of the PRB) may last less than five more years, thus reducing the effectiveness of the PRB to mitigate contaminants.

A Microscopic Study of Iron and Manganese Oxide Distribution in Soils from East Tennessee (U.S.A.)

Soils and saprolites developed from interbedded shales and limestones of the Conasauga Group are widespread in the Valley and Ridge Province of East Tennessee. Thin sections from four soil profiles were examined by petrographic and scanning electron microscopy including backscatter electron and energy-dispersive X-ray analyses. Iron and manganese released by weathering had migrated differentially downward and precipitated as crystalline and noncrystalline oxides. Oxides were observed as nodules, granular particulates, pore fillings, and coatings on other minerals, packing voids, vesicles, channels, and chambers. Iron oxides formed predominantly as coatings on packing-void walls and on laminated clays in vesicles and channels. Manganese oxides occurred as an early replacement phase of packing voids and of fracture-filling carbonate minerals. Iron oxides were dominant in moderately well-drained and oxidized horizons of the soil solum, whereas manganese oxides were abundant in the oxidized and moderately leached saprolite zone where the water table fluctuates seasonally. Therefore, a manganese enrichment zone, on a bulk soil basis, occurred generally below the iron oxide zone in the soil profile. Such differential migration and accumulation of iron and manganese have been controlled by localized soil microenvironments. Micromorphologic features observed in this study are important in land-use evaluation for hazardous waste disposal. © 1990.

The reaction path of CO and Fe2O3 in a chemical-looping combustion system

The reaction mechanism of CO and Fe₂O₃ in a chemical-looping combustion (CLC) was studied based on density functional theory (DFT) at B3LYP level in this paper. The structures of all reactants, intermediate, transition structures and products of this reaction had been optimized and characterized. The reaction path was validated by means of the intrinsic reaction coordinate (IRC) approach. The result showed that the reaction was divided into two steps, the adsorbed CO molecule on Fe ₂O₃ surface formed a medium state with one broken Fe-O bond in step1, and in step2, O atom broken here oxidized a subsequent CO molecule in the fuel reactor. Thus, Fe₂O₃ molecule transport O from air to oxide CO continually in the CLC process. The activation energy and rate coefficients of the two steps were also obtained.

Mechanistic studies of the photocatalytic oxidation of microcystin-LR: An investigation of byproducts of the decomposition process

Microcystins (cyclic heptapeptides) are produced by a number of freshwater cyanobacteria and cause concern in potable water supplies due to their acute and chronic toxicity. The present study reports the structural characterization of the degradation products of the photocatalytic oxidation of microcystin-LR, so aiding the mechanistic understanding of this process. TiO₂ photocatalysis is a promising technology for removal of these toxins from drinking water. However, before it can be adopted in any practical application it is necessary to have a sufficient knowledge of degradation byproducts and their potential toxicity. Liquid chromatography-mass spectrometry analysis demonstrated that the major destruction pathway of microcystin appears to be initiated via three mechanisms: UV irradiation, hydroxyl radical attack, and oxidation. UV irradiation caused geometrical isomerization of microcystin converting the (4E), (6E) of the Adda configuration to (4E), 6(Z) or 4(Z), 6(E). Hydroxyl radical attack on the conjugated diene structure of Adda moiety produced dihyroxylated products. Further oxidation cleaved the hydroxylated 4-5 and/or 6-7 bond of Adda to form aldehyde or ketone peptide residues, which then were oxidized into the corresponding carboxylic acids. Photocatalysis also hydrolyzed the peptide bond on the ring structure of microcystin to form linear structures although this appeared to be a minor pathway.

Regulating macrophage phenotype and function by retinal pigment epithelial cells

Purpose: We have shown previously that macrophages/microglia accumulate in the subretinal space and express CD68 and Arginase-1 in the aging eye. Subretinal macrophages are in close contact with retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may play an important role in regulating macrophage/microglial phenotype and function. The aim of this study was to investigate the effect of RPE cells on the phenotype and function of bone marrow–derived macrophages (BM-DMs).
Methods: BM-DM from C57BL/6J mice were cultured in DMEM supplemented with 20%L929 cell supernatant for 5 days. The phenotype of BM-DMs was confirmed by flow cytometry as CD11b+F4/80+. Primary RPE cells were cultured from C57BL/6J mice and confirmed by RPE65 and cytokeratin staining. BMDMs were co-cultured with different types of RPE cells (healthy, oxidized, and apoptotic RPE) and then isolated from the co-culture system for phenotypic and functional assays.
Results: Co-culture of BM-DMs with RPE cells results in a time-dependent down-regulation of MHC-II expression and the generation of CD11b+F4/80+Ly6G+ myeloid-derived suppressor cells (MDSC). qRT-PCR analysis showed that RPE-induced MDSCs expressed high levels of IL-6, IL-1β, and Arginase-1, but lower levels of IL-12p40 and TNF-a compared to naïve BM-DMs. The expression levels of iNOS, TGF-β and Ym1 did not differ 207 between naive BMDMs and RPE-induced MDSCs. Furthermore, functional studies showed that these cells had reduced phagocytic activity and lower ability to stimulate T cell activation and proliferation. When RPE cells were pre-treated with oxidized photoreceptor outer segments before co-culturing with BMDMs, the expression of IL-1β and IL-6 in BMDMs was increased whereas the expression of Arginase-1 was decreased. 
Conclusion: Our results suggest that healthy RPE cells can convert BMDMs into myeloid-derived suppressor cells under in vitro culture conditions, RPE-induced myeloid-derived suppressor cells are CD11b+F4/80+Ly6G+MHCIIlowIL6+IL1b+Arg-1+. The ability of RPE cells is reduced when suffering from oxidative insults.

Application of Asymmetric Marcus-Hush Theory to Voltammetry in Room-Temperature Ionic Liquids

Asymmetric MarcusHush (AMH) theory is applied for the first time in ionic solvents to model the voltammetric reduction of oxygen in 1-butyl-1-methylpyrrolidinium bis-(trifluoromethylsulfonyl)-imide and of 2-nitrotoluene (2-NT), nitrocyclopentane (NCP), and 1-nitro-butane (BuN) in trihexyltetradecylphosphonium tris(pentafluoroethyl)trifluorophosphate on a gold microdisc electrode. An asymmetry parameter, gamma, was estimated for all systems as -0.4 for the reduction of oxygen and -0.05, 0.25, and 0 +/- 0.05 for the reductions of 2-NT, NCP, and BuN, respectively, which suggests equal force constants of reactants and products in the case of 2-NT and BuN and unequal force constants for oxygen and NCP where the force constants of the oxidized species are greater than the reduced species in the case of oxygen and less than the reduced species in the case of NCP. Previously measured values for a, the Butler-Volmer transfer coefficient, reflect this in each case. Where appreciable asymmetry occurs, AMH theory was seen to parametrize the experimental data better than either Butler-Volmer or symmetric Marcus-Hush theory, allowing additionally the extraction of reorganization energy. This is the first study to provide key physical insights into electrochemical systems in room-temperature ionic liquids using AMH theory, allowing elucidation of the reorganization energies and the relative force constants of the reactants and products in each reaction.