Calcium Enhances In Vitro Free Radical-Induced Damage to Brain Synaptosomes, Mitochondria, and Cultured Spinal Cord Neurons

Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.     

Lipoxygenase-generated hydroperoxides account for the nonphysiological features of ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes of carnations

Several lines of evidence indicate that the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by microsomal membranes from carnation flowers is attributable to hydroperoxides generated by membrane-associated lipoxygenase (EC 1.13.11.12). As the flowers senesce, the capability of isolated microsomal membranes to convert ACC to ethylene changes. This pattern of change, which is distinguishable from that for senescing intact flowers, shows a close temporal correlation with levels of lipid hydroperoxides formed by lipoxygenase in the same membranes. Specific inhibitors of lipoxygenase curtail the formation of lipid hydroperoxides and the production of ethylene from ACC to much the same extent, whereas treatment of microsomes with phospholipase A₂, which generates fatty-acid substrates for lipoxygenase, enhances the production of hydroperoxides as well as the conversion of ACC to ethylene. Lipoxygenase-generated lipid hydroperoxides mediate the conversion of ACC to ethylene in a strictly chemical system and also enhance ethylene production by microsomal membranes. The data collectively indicate that the in-vitro conversion ACC to ethylene by microsomal membranes of carnation flowers is not reflective of the reaction mediated by the native in-situ ethylene-forming enzyme.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EDTA ethylenediaminetetraacetic acid     

Softening of lipid bilayers

The softening of wet lipid bilayer membranes during their gel-to-fluid first-order phase transition is studied by computer simulation of a family of two-dimensional microscopic interaction models. The models include a variable number, q, of lipid chain conformational states, where 2q10. Results are presented as functions of q and temperature for a number of bulk properties, such as internal energy, specific heat, and lateral compressibility. A quantitative account is given of the statistics of the lipid clusters which are found to form in the neighborhood of the transition. The occurrence of these clusters is related to the softening and the strong thermal density fluctuations which dominate the specific heat and the lateral compressibility for the high-q models. The cluster distributions and the fluctuations behave in a manner reminiscent of critical phenomena and percolation. The findings of long-lived metastable states and extremely slow relaxational behavior in the transition region are shown to be caused by the presence of intermediate lipid chain conformational states which kinetically stabilize the cluster distribution and the effective phase coexistence. This has as its macroscopic consequence that the first-order transition apperas as a continuous transition, as invariably observed in all experiments on uncharged lecithin bilayer membranes. The results also suggest an explanation of the non-horizontal isotherms of lipid monolayers. Possible implications of lipid bilayer softening and enhanced passive permeability for the functioning of biological membranes are discussed.Abbreviations PC phosphatidvlcholine - DMPC dimyristoyl PC - DPPC dipalmitoyl PC - ac alternating current - DSC differential scanning calorimetry - T _m lipid gel-to-fluid phase transition temperature - TEMPO 2,2,6,6-tetramethylpiperidine-N-oxyl Supported by the Danish Natural Science Research Council and A/S De Danske Spritfabrikkers JubilæumslegatSupported in part by the NSERC of Canada and Le FCAC du Quebec     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

类脂过氧化对竹红菌甲素引起膜蛋白光敏交联的影响

本文研究了丙二醛与红细胞膜温育后所形成的交联。当丙二醛浓度在5×10~-4mol/L以上时,膜蛋白发生交联,并且在460nm处有荧光特征峰出现。而甲素光敏所致膜蛋白的交联,在460nm处没有荧光峰,光敏所产生的内源丙二醛量很少,不足以引起红细胞膜反应形成交联。我们还研究了BHT和Vit E两种抗氧化剂对甲素光敏作用的影响。BHT能抑制类脂过氧化,不能抑制膜蛋白巯基的变化和膜蛋白的交联。而Vit E仅不能抑制膜蛋白的交联。以上结果均说明甲素光敏所致膜类脂过氧化是不参与膜蛋白交联的。     

Lipid vertical motion and related steric effects in bilayer membranes

We present a theoretical model of a lipid bilayer in its gel state which explicity couples the vertical displacements of the lipid chains to their conformational state. In this model the chains are free to move longitudinally under a potential due to the neighbouring chains. The potential is due to a restoring force with a force constant k and thus acts to keep them in the local plane as defined by their nearest neighbours. It is demonstrated that the force constant k is directly related to the internal bilayer pressure, , and that if a value =33 dynes/cm is assumed then k=17.3 dynes/cm. Steric effects are explicity included by allowing chains to twist into free volume created by the vertical displacement of neighbouring chains. The Hamiltonian is expressed in terms of the projection operators, P _ij , describing the displacement of chain i relative to a neighbour j, and G _ij describing the direction of a twist in chain i. The model is solved both analytically and via Monte Carlo simulations for a one-dimensional system. The possibility of phase-transitions in two-dimensions and the relevance to the bilayer pre-transition is discussed.This work was first presented in poster form at the Canadian Biochemical Society Conference held in Banff, Alberta, Canada, April 29–May 4, 1984Work supported in part by the Natural Sciences and Engineering Research Council of Canada, Le Fonds Formation des Chercheurs et Action a la Recherche du Quebec, and the Advisory Research Council of Queen's University     

Ethanol-sensitive mutants of Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants unable to grow at ethanol concentrations at which the wild type strain S288C does grow, have been isolated. Some of them show additional phenotypic alterations in colony size, temperature sensitivity and viability in ethanol, which cosegregate with the growth sensitivity in ethanol. 21 selected monogenic ethanol-sensitive mutants define 20 complementation groups, denominated ETA1 to ETA20, which indicates that there is a high number of genes involved in the ethanol tolerance/sensitivity mechanism.Out of 21 selected monogenic mutants, 20 are not altered in the glycolytic pathway since, when maintained in glucosesupplemented medium, they can produce as much ethanol as the wild type and at about the same velocity. Nor do any of the mutants seem to be altered in the lipid biosynthetic pathway since, whether grown in the absence or in the presence of ethanol, their concentration of fatty acids and ergosterol is similar to that of the wild type under the same conditions. Therefore growth sensitivity to ethanol does not seem necessarily to be related to carbohydrate or lipid metabolism.Non-common abbreviations YP yeast extract peptone medium - YPD yeast extract peptone dextrose agar or medium - YPG yeast extract peptone glycerol agar - YPDE yeast extract peptone dextrose ethanol agar or medium - SD yeast nitrogen base dextrose agar - SPO yeast extract potassium acetate glucose agar - PD parental ditype - NPD non-parental ditype - TT tetratype     

Thermodynamic analysis of nonelectrolyte sorption in plant cuticles: The effects of concentration and temperature on sorption of 4-nitrophenol

The sorption of 4-nitrophenol (4-NP) in enzymatically isolated cuticles ofLycopersicon esculentum fruits andFicus elastica leaves was studied as a function of temperature and solute concentration. Plots of the concentrations of 4-NP sorbed in the cuticle versus the equilibrium concentrations in the aqueous phase gave linear isotherms at low concentrations that tended to approach plateaus at higher sorbate concentrations ( 10 mmol·kg^-1). At low concentrations of sorbed 4-NP, cuticles have sorptive properties similar to those of organic solvents which are able to form intermolecular hydrogen bonds, while at higher concentrations their solid nature becomes apparent. During sorption of 4-NP the cutin matrix swells and new sorption sites are successively formed. The partition coefficients of 4-NP in the system cuticle/buffer are functions of temperature and concentration. At high sorbate concentrations (approx. 1 mol·kg^-1) they approach a value of 1. Different sorptive properties were observed for the cutin regions normally encrusted with soluble cuticular lipids (SCL) and those without SCL. Increasing temperature augmented the number of sorption sites in the cutin ofLycopersicon while no effect was observed withFicus. The changes of partial molar free energy (G ^o _tr), enthalpy (H ^o _tr), and entropy (S ^o _tr) for the phase transfer of 4-NP also depended on sorbate concentration: H ^o _tr and S ^o _tr were negative and steeply decreased at high sorbate concentrations. This is due to solute-solute interactions replacing solute-cutin interactions at high concentrations resulting in solid precipitates of solute within the cutin matrix. This formation of ordered solid domaines starting from a small number of nonelectrolyte molecules interacting with the cutin is proposed as a model for the intracuticular deposition of SCL.Abbreviations CM cuticular membrane - MX polymer matrix membrane - 4-NP 4-nitrophenol - SCL soluble cuticular lipids     

Studies on the lack of cooperativity in the melting of lipid bilayers

The gel-to-fluid first-order melting transition of lipid bilayers is simulated by the use of a microscopic interaction model which includes a variable number of lipid-chain conformational states. The results suggest that the experimental observation of ‘continuous melting’ in pure wet lipid bilayers, rather than being ascribed to the presence of impurities, may be explained as a result of kinetically caused metastability of intermediate lipid-chain conformations.     

Capacitance--voltage relationship in phospholipid bilayers containing gangliosides

Changes in the position of the minimum of the parabolic capacitance-voltage curve allow the measurement of the amount of ganglioside present in artificial bilayers made with phosphatidylcholine-ganglioside mixtures and asymmetrically shielded with Ca2+. The screening effect of the ionic solution must be considered. With ganglioside/phospholipid molar ratios of up to 15%, all glycolipids can be found at the membrane surfaces.