Oxidation-Induced Increase In Photoreactivity of Bovine Retinal Lipid Extract

Open access original paper Source : 10th EPR Workshop on Applications of EPR in Biology and Medicine, 2016 in Krakow, POLAND.International audienceThe mammalian retina contains a high level of polyunsaturated fatty acids, including docosahexaenoic acid (22:6) (DHA), which are highly susceptible to oxidation. It has been shown that one of the products of DHA oxidation-carboxyethylpyrrole (CEP), generated in situ, causes modifications of retinal proteins and induces inflammation response in the outer retina. These contributing factors may play a role in the development of age-related macular degeneration (AMD). It is also possible that some of the lipid oxidation products are photoreactive, and upon irradiation with blue light may generate reactive oxygen species. Therefore, in this work we analysed oxidation-induced changes in photoreactivity of lipids extracted from bovine neural retinas. Lipid composition of bovine neural retinas closely resembles that of human retinas making the bovine tissue a convenient model for studying the photoreactivity and potential phototoxicity of oxidized human retinal lipids. Lipid composition of bovine neural retinas Folch' extracts (BRex) was determined by gas chromatography (GC) and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS) analysis. Liposomes prepared from BRex, equilibrated with air, were oxidized in the dark at 37 A degrees C for up to 400 h. The photoreactivity of BRex at different stages of oxidation was studied by EPR-oximetry and EPR-spin trapping. Photogeneration of singlet oxygen (O-1(2), (1)Delta(g)) by BRex was measured using time-resolved detection of the characteristic phosphorescence at 1270 nm. To establish contribution of lipid components to the analysed photoreactivity of Folch' extract of bovine retinas, a mixture of selected synthetic lipids in percent by weight (w/w %) ratio resembling that of the BRex has been also studied. Folch's extraction of bovine neural retinas was very susceptible to oxidation despite the presence of powerful endogenous antioxidants such as alpha-tocopherol and zeaxanthin. Non-oxidized and oxidized BRex photogenerated singlet oxygen with moderate quantum yield. Blue-light induced generation of superoxide anion by Folch' extract of bovine neural retinas strongly depended on the oxidation time. The observed photoreactivity of the studied extract gradually increased during its in vitro oxidation

Synthetic connectivity, emergence, and self-regeneration in the network of prebiotic chemistry

The challenge of prebiotic chemistry is to trace the syntheses of life's key building blocks from a handful of primordial substrates. Here we report a forward-synthesis algorithm that generates a full network of prebiotic chemical reactions accessible from these substrates under generally accepted conditions. This network contains both reported and previously unidentified routes to biotic targets, as well as plausible syntheses of abiotic molecules. It also exhibits three forms of nontrivial chemical emergence, as the molecules within the network can act as catalysts of downstream reaction types; form functional chemical systems, including self-regenerating cycles; and produce surfactants relevant to primitive forms of biological compartmentalization. To support these claims, computer-predicted, prebiotic syntheses of several biotic molecules as well as a multistep, self-regenerative cycle of iminodiacetic acid were validated by experiment

The role of plasmalogens in the photoreactivity of human retinal lipid extracts of different age groups

Purpose: Plasmalogens (PLs) constitute a specific subclass of phospholipids characterized by a vinyl-ether bond in SN-1 position of the glycerol backbone and account for 10% of total phospholipids in the retina, where are considered as sacrificial antioxidants. PLs above 50% of other phospholipids present in the retina contain polyunsaturated fatty acid (PUFA) chain in SN-2 position. In this work we analyzed the influence of PLs on of phospholipids naturally occurring in the human retina (SRL) in a model system and in retinal pigment epithelium cells in vitro. Methods: Lipid composition of human retinal extracts (RLE) has been determined by GC and LC/MS analysis. RLE and SRL with and without PLS were oxidized in the dark in liposomes equilibrated with air at 37°C. Blue-light induced photoreactivity of the oxidized lipid samples was measured in model systems and in ARPE-19 cells in vitro using time-resolved singlet oxygen phosphorescence at 1270nm, electron paramagnetic resonance (EPR) -oximetry, EPR-spin trapping and electrochemical detection of cholesterol hydroperoxides.Results: Human RLE are very sensitive to oxidation despite the presence of endogenous hydrophobic antioxidants. Both oxidised RLE and SRL generate singlet oxygen and superoxide anion upon irradiation with blue light. The observed photoreactivity of lipids extracted from human retinas increases with age of the donor. It also becomes more pronounced with autooxidation in case of both RLE and SRL.Conclusions: Our results indicate that plasmalogens mediate photoreactivity of retinal lipids in a complex way. It is detected by an increase in the photouptake of oxygen and decreased yields of singlet oxygen photogeneration by oxidized phospholipids

High-Throughput Screening Assays for Lipolytic Enzymes

Screening is defined as the identification of hits within a large library of variants of an enzyme or protein with a predefined property. In theory, each variant present in the respective library needs to be assayed; however, to save time and consumables, many screening regimes involve a primary round to identify clones producing active enzymes. Such primary or prescreenings for lipolytic enzyme activity are often carried out on agar plates containing pH indicators or substrates as triolein or tributyrin. Subsequently, high-throughput screening assays are usually performed in microtiter plate (MTP) format using chromogenic or fluorogenic substrates and, if available, automated liquid handling robotics. Here, we describe different assay systems to determine the activity and enantioselectivity of lipases and esterases as well as the synthesis of several substrates. We also report on the construction of a complete site saturation library derived from lipase A of Bacillus subtilis and its testing for detergent tolerance. This approach allows for the identification of amino acids affecting sensitivity or resistance against different detergents