Polyunsaturated Membrane Research Articles

Structural characteristics of thylakoid membranes of Arabidopsis mutants deficient in lipid fatty acid desaturation

The ultrastructure of thylakoid membranes from Arabidopsis thaliana wild-type, JB67 and LK3 fatty acid desaturation deficient mutants was studied by thin-section and freeze-fracture electron microscopy. There was a decrease in the amount of the appressed and non-appressed membranes in JB67 and LK3 Arabidopsis mutants when compared to the wild type, resulting in a reduction in the length of photosynthetic membrane per plastid. The results from freeze-fracture showed a decrease in size and a marked increase in packing density of membrane-associated particles on the exo- and endoplasmic fracture faces of the mutants. In addition, areas of the appressed membranes of the mutants contained particles in regular arrays under conditions where no such arrays were observed in wild-type thylakoid membranes. These observations suggest, that the decreased level of lipid fatty acid unsaturation affects the ability of the lipid matrix to mediate the assembly of chloroplast membrane components. The role of polyunsaturated membrane lipids is considered in terms of their ability to promote functional oligomeric assemblies of components of the photosynthetic apparatus.

Arabidopsis requires polyunsaturated lipids for low-temperature survival.

Mutants of Arabidopsis that contain reduced levels of polyunsaturated fatty acids showed growth characteristics at 22 degrees C that were very similar to wild type. By contrast, at 12 degrees C, the mutants failed to undergo stem elongation during reproductive growth although they produced normal flowers and fertile seeds. After transfer to 6 degrees C, rosette leaves of the mutants gradually died, and the plants were inviable. These different responses of the mutant plants at 12 degrees C and 6 degrees C suggest that distinct functions may be affected at these two temperatures. The gradual development of symptoms at 6 degrees C and other lines of evidence argue against a general collapse of membrane integrity as the cause of the lethal phenotype. Rather, they indicate that the decrease in polyunsaturated membrane lipids may initially have relatively limited effects in disrupting cellular function.

Lipid peroxidation during myocardial ischaemia induced by pacing.

Oxygen derived free radical generation can be shown in experimental models of myocardial ischaemia and reperfusion and may cause cellular damage by peroxidizing polyunsaturated membrane phospholipids. An attempt was made to quantify human intracardiac lipid peroxidation during transient myocardial ischaemia by measuring the aortic and coronary sinus concentrations of malondialdehyde (a marker of lipid peroxidation) before, during, and after incremental pacing. Twenty six patients were paced until they had severe chest pain or 2 mm ST segment depression or they reached a paced rate of 180 beats/min. They were divided into two groups according to whether or not lactate was produced during pacing. Twelve patients (group 1), all with coronary artery disease, produced myocardial lactate during pacing. None of the other 14 patients (group 2), half of whom had coronary disease, produced lactate during pacing. Concentrations of malondialdehyde in the aorta and coronary sinus were significantly higher in group 1 than in group 2. Five minutes after the end of pacing coronary sinus malondialdehyde concentrations in group 1 had increased significantly from baseline values. There were no changes with time in the coronary sinus concentration of malondialdehyde in group 2 or in the aorta in either group. The negative malondialdehyde extraction ratio in group 1 suggests that intracardiac lipid peroxidation occurs during transient human myocardial ischaemia.

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65 35 ( v v ) or acetonitrile/water 40 60 ( v v ) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.

Membrane radiosensitivity of fatty acid supplemented fibroblasts as assayed by the loss of intracellular potassium.

Leakage of potassium from mouse fibroblast LM cells, X-irradiated at 0 degrees C with doses up to 400 Gy is shown to be related to plasma membrane lipid composition. Fatty acid supplemented cells, containing about 40 per cent polyunsaturated fatty acids (PUFA) in their membranes were much more sensitive to radiation, as measured by increased permeability, than normal cells, which contained 7 per cent PUFA. The damage observed after irradiation at 0 degrees C was partially repaired during a post-irradiation incubation at 22 degrees C. The o.e.r. for potassium leakage was about 4 for normal fibroblasts and 8 for the PUFA-supplemented cells. No oxygen-dependent radiation damage could be observed in cells treated with high amounts of vitamin E. Depletion of glutathione in PUFA cells sensitized oxic cells to radiation damage, resulting in an increase of the o.e.r. from 8 to 17. No lipid peroxidation (malondialdehyde production and disappearance of fatty acyl chains) could be demonstrated. While PUFA, normal and vitamin E grown cells showed a differential sensitivity in radiation-induced potassium leakage and trypan blue uptake (high doses, interphase death), no difference in radiation-induced clonogenic ability (reproductive death) could be observed after the different cell treatments. The experiments reported are supportive of a role of membranes in the mechanism of radiation-induced interphase death and show that increased damage may be expected when high amounts of polyunsaturated membrane lipids are present under conditions of low amounts of appropriate antioxidants.

Production of free radicals and lipid peroxides in early experimental myocardial ischemia

Free radicals and lipid peroxides have recently been identified by us [ 1, 2, 3] as metabolic intermediates during acute myocardial ischemia. The mechanisms by which evolving myocardial ischemia initiates free radical production are not clear. Based on studies in vitro, it is feasible to consider the following possibilities: (a) dissociation of intramitochondrial electron support system and altered phospholipid integrity with inactivation of cytochrome oxidase, which results in release of ubisemiquinone, flavoprotein and superoxide radicals; (b) accumulation and increased release of intra/extracellular metabolites like NADH, lactate flavoproteins and catecholamines which react either with themselves or with O 2 and ascorbic acid; (c) interaction of the metabolic product hypoxanthine with O 2 in the presence of xanthine oxidase and (d) activation of phospholipase by calcium influx with enhanced arachidonic acid metabolism and superoxide radical production. Detailed in vitro radiobiological studies [ 4] have demonstrated that free radical reactions occur even at very low O 2 tensions (83% of maximum rate of P o 2∼6 mmHg and 50% at P o 2∼1 mmHg), and Smith [ 5] has demonstrated that free radical peroxidation takes place quite rapidly in rat brain homogenates incubated in gas mixtures containing only 5% O 2. Thus, the low oxygen tensions in ischemic tissue are adequate to support free radical reactions. The free radicals thus produced may initiate and enhance lipid peroxidation by attacking polyunsaturated membrane lipids.

The mechanism of linuron phytotoxicity in maize

Summary:The mechanism whereby Iinuron changes the functional and structural characteristics of chloroplast membranes was studied. The primary effect of the herbicide, the inhibition of phoiosynihetic electron transport results in an immuediatecessa‐tion of carbon dioxide fixation and alterations in the membrane energization. These are accompanied by secondary processes contributing to phytotoxicity. The fundamental action in its phytotoxicity, as shown in a model system of isolated chloro‐plasts, is the peroxidaiive breakdown of the polyunsaturated membrane lipids. This leads to destruction of the pigments, changes in the energy transfer, disorganization of the chloro‐plast membranes, and accumulation of toxic organic peroxides.

Effect of glutathione peroxidase activity on lipid peroxidation in biological membranes

Results are presented indicating that, although glutathione peroxidase activity inhibits lipid peroxidation in membranes, it does not appear to do so by reducing membrane lipid peroxides to lipid alcohols, as has been shown by others to be the case for free fatty acid peroxides in solution. Lipid peroxidation was studied in an enzymic system (microsomal NADPH oxidase) and in a non-enzymic system (mitochondria plus ascorbate). A study of the fatty acids in the phospholipids of microsomes and mitochondria demonstrated that detectable amounts of hydroxy fatty acids were not formed in the membranes when the latter were incubated in the presence of the glutathione peroxidase system even under conditions known to have generated significant levels of lipid peroxides in the membrane. Fatty acid analyses of the microsomal and mitochondrial particles indicated that glutathione peroxidase activity inhibited loss of polyunsaturated fatty acids when these organelles were exposed to peroxidizing conditions. If glutathione peroxidase activity were inhibiting the formation of malondialdehyde (a product of lipid peroxidation) by converting peroxide groups to alcohols, the loss of the constitutive polyunsaturated fatty acids in the membrane should not have been appreciably affected by addition of the peroxidase system. The protective effect cannot be due to quenching of an autocatalytic type of lipid peroxidation (at least in the microsomal system) since it has been established that the microsomal enzyme system (NADPH oxidase) catalyzes a continuous attack on microsomal polyunsaturated fatty acyl groups during the reaction and that the peroxidative process is not autocatalytic in nature. It appears, therefore, that glutathione peroxidase activity must exert its effect on this system by preventing free radical attack on the polyunsaturated membrane lipids in the first place. A possible mechanism for the interruption of a free radical attack on the lipids is proposed.