Specific binding of Ro 09-0198 (cinnamycin) to phosphatidylethanolamine: a thermodynamic analysis

Ro 09-0198 (cinnamycin) is a tetracyclic peptide antibiotic that is used to monitor the transbilayer movement of phosphatidylethanolamine (PE) in biological membranes during cell division and apoptosis. The molecule is one of the very rare examples where a small peptide binds specifically to a particular lipid. In model membranes and biological membranes containing phosphatidylethanolamine, Ro 09-0198 forms a 1:1 complex with this lipid. We have measured the thermodynamic parameters of complex formation with high sensitivity isothermal titration calorimetry and have investigated the structural consequences with deuterium and phosphorus solid-state NMR. Complex formation is characterized by a large binding constant, K0, of 10(7) to 10(8) M(-1), depending on the experimental conditions. The reaction enthalpy, DeltaHdegrees, varies between zero at 10 degrees C to strongly exothermic -10 kcal/mol at 50 degrees C. For large vesicles with a diameter of approximately 100 nm, DeltaHdegrees decreases linearly with temperature and the molar heat capacity of complex formation can be evaluated as = -245 cal/mol, indicating a hydrophobic binding mechanism. The free energy of binding is DeltaGdegrees = -10.5 kcal/mol and shows only little temperature dependence. The constancy of DeltaGdegrees together with the distinct temperature-dependence of DeltaHdegrees provide evidence for an entropy-enthalpy compensation mechanism: at 10 degrees C, complex formation is completely entropy-driven, at 50 degrees C it is enthalpy-driven. Varying the PE fatty acid chain-length between 6 and 18 carbon atoms produces similar binding constants and DeltaHdegrees values. Addition of Ro 09-0198 to PE containing bilayers eliminates the typical bilayer structure and produces 2H- and 31P-NMR spectra characteristic of slow isotropic tumbling. This reorganization of the lipid matrix is not limited to PE but also includes other lipids.     

Specific binding of cinnamycin (Ro 09-0198) to phosphatidylethanolamine. Comparison between micellar and membrane environments

Cinnamycin (Ro 09-0198) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE). Formation of a complex with phosphatidylethanolamine follows a 1:1 stoichiometry. Using high-sensitivity isothermal titration calorimetry (ITC), we have measured the thermodynamic parameters of complex formation for two different PE environments, namely, PE dissolved either in octyl glucoside (OG) micelles or in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer membrane. We have compared diacyl-PE with lyso-PE and have varied the carbon chain length from 6 to 18. Binding requires both a PE headgroup and at least one fatty acyl chain. The optimum chain length for complex formation (n) is eight. Longer chains do not enhance the binding affinity; for shorter chains, the interaction is weakened. The cinnamycin-PE complex has a binding constant K(0) of approximately 10(7)-10(8) M(-1) in the POPC membrane and only approximately 10(6) M(-1) in the octyl glucoside micelle. The difference can be attributed to the nonspecific hydrophobic interaction of cinnamycin with the lipid membrane. Complex formation is enthalpy-driven in OG micelles, whereas enthalpy and entropy make equal contributions in bilayer membranes. However, for the optimum chain length (n) of eight, the binding reaction is also completely enthalpy-driven for the bilayer membrane.     

The structure of Ro 09-0198 in different environments.

The constitution and configuration of Ro 09-0198 (cinnamycin) have been determined in DMSO. Further investigations in aqueous solution, in SDS micelles and in a lipid bilayer have been done to study the influence of different environments on the conformation of the peptide. It turned out that in spite of the polycyclic structure of the molecule, the conformation is drastically changed going from water to SDS micelles. Ro 09-0198 orients itself in lipid bilayers as expected from its amphiphilic structure. According to a nuclear Overhauser effect spectroscopy experiment under magic angle spinning (MAS) conditions, the molecule is incorporated into the membrane with its hydrophobic part inside the bilayer.     

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphosphatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.     

Prepeptide sequence of cinnamycin (Ro 09-0198): the first structural gene of a duramycin-type lantibiotic

The tetracyclic polypeptide antibiotic cinnamycin (Ro 90-0198) belongs to the duramycin-type lantibiotics and contains the unusual amino acids threo-3-methyl-lanthionine, meso-lanthionine, lysinoalanine and 3-hydroxyaspartic acid. Its structural gene, referred to as cinA, has been identified on isolated chromosomal DNA of the Ro 09-0198-producing strain Streptoverticillium griseoverticillatum via a 39-residue oligonucleotide probe derived from fragment 7-19 of the hypothetical prolantibiotic sequence CRQSCSFGPFTFVCDGNTK. This propeptide part was then found within an open reading frame of 77 amino acids. In contrast to the nisin-type prelantibiotics, this first duramycin-type prelantibiotic has an unusually long leader sequence of 58 amino acids. it also differs in the processing site and the direction of the formation of the threo-3-methyl-lanthionine bridges is from N-terminal cysteine to C-terminal dehydrated threonine residues, whereas the meso-lanthionine and lysinoalanine bridges are formed by addition reactions from C-terminal cysteine or lysine to N-terminal dehyrated serine residues.     

Hemolytic activity of a cyclic peptide Ro09-0198 isolated from Streptoverticillium

Ro09-0198, a cyclic peptide isolated from culture filtrates of Streptoverticillium griseoverticillatum, induced lysis of erythrocytes. Preincubation of the peptide with phosphatidylethanolamine reduced the hemolytic activity, whereas other phospholipids present in erythrocytes in nature had no effect. A study of the structural requirements on phosphatidylethanolamine necessary for interaction with the peptide indicates that Ro09-0198 recognizes strictly a particular chemical structure of phosphatidylethanolamine: dialkylphosphoethanolamine as well as 1-acylglycerophosphoethanolamine showed the same inhibitory effect on hemolysis induced by Ro09-0198 as diacylphospatidylethanolamine, whereas phosphoethanolamine gave no inhibitory effect. Neither phosphatidyl-N-monomethylethanolamine nor alkylphosphopropanolamine had an inhibitory effect. Consequently, the hydrophobic chain is necessary for the interaction and the phosphoethanolamine moiety is exactly recognized by the peptide. Ro-09-0198-induced hemolysis was temperature-dependent and the sensitivity of hemolysis differed greatly among animal species.     

Interaction of a cyclic peptide, Ro09-0198, with phosphatidylethanolamine in liposomal membranes

Ro09-0198 is a cyclic peptide isolated from Streptoverticillium griseoverticillatum. This peptide caused permeability increase and aggregation of liposomes containing phosphatidylethanolamine. Liposomes containing phosphatidylserine, phosphatidylinositol or cardiolipin instead of phosphatidylethanolamine were, however, not appreciably reactive with the peptide. Among the structural analogs of phosphatidylethanolamine, dialkylphosphatidylethanolamine and 1-acylglycerophosphoethanolamine incorporated into liposomes could interact with Ro09-0198 to cause a permeability increase, whereas liposomes consisting of alkylphosphoethanolamine or phosphatidyl-N-monomethylethanolamine were insensitive to the peptide. These findings indicate that a glycerol backbone and a primary amino group of phosphatidylethanolamine are necessary for interaction with Ro09-0198 to cause membrane damage. Ro09-0198 induced a selective permeability change on liposomes. Glucose and umbelliferyl phosphate were effluxed significantly, but sucrose was only slightly permeable and inulin could not be released. Consequently, the permeability increase induced by Ro09-0198 is rather specific to molecules smaller than sucrose. Line broadening of electron spin resonance signals of spin-labeled phosphatidylethanolamine was observed upon treatment of liposomes with Ro09-0198. It was suggested from these results that Ro09-0198 can alter the physical organization of phosphatidylethanolamine in membranes, thus providing a basis for changes in membrane permeability.     

Relation between lipid polymorphism and transbilayer movement of lipid in rat liver microsomes

We have studied the effects of trinitrophenylation on the transbilayer movement of phosphatidylcholine and the macroscopic lipid structure in rat liver microsomal membranes. The transbilayer movement of phosphatidylcholine was investigated using the PC-specific transfer protein. ³¹P-NMR was employed to monitor the phospholipid organization in intact microsomal vesicles. The results indicate that modification of microsomes with trinitrobenzenesulfonic acid enhances the transbilayer movement of phosphatidylcholine at 4°C. Furthermore, phosphatidylethanolamine headgroup trinitrophenylation in microsomes increases the isotropic component in the ³¹P-NMR spectra even at 4°C, possibly representing the appearance of intermediate non-bilayer lipid structures. The observed parallel between these data suggests that phosphatidylethanolamine molecules in the microsomal membrane, probably in combination with a protein component, are able to destabilize the bilayer organization, thereby facilitating the transmembrane movement of phospholipids.     

Asymmetric addition of ceramides but not dihydroceramides promotes transbilayer (flip-flop) lipid motion in membranes

Transbilayer lipid motion in membranes may be important in certain physiological events, such as ceramide signaling. In this study, the transbilayer redistribution of lipids induced either by ceramide addition or by enzymatic ceramide generation at one side of the membrane has been monitored using pyrene-labeled phospholipid analogs. When added in organic solution to preformed liposomes, egg ceramide induced transbilayer lipid motion in a dose-dependent way. Short-chain (C6 and C2) ceramides were less active than egg ceramide, whereas dihydroceramides or dioleoylglycerol were virtually inactive in promoting flip-flop. The same results (either positive or negative) were obtained when ceramides, dihydroceramides, or diacylglycerols were generated in situ through the action of a sphingomyelinase or of a phospholipase C. The phenomenon was dependent on the bilayer lipid composition, being faster in the presence of lipids that promote inverted phase formation, e.g., phosphatidylethanolamine and cholesterol; and, conversely, slower in the presence of lysophosphatidylcholine, which inhibits inverted phase formation. Transbilayer motion was almost undetectable in bilayers composed of pure phosphatidylcholine or pure sphingomyelin. The use of pyrene-phosphatidylserine allowed detection of flip-flop movement induced by egg ceramide in human red blood cell membranes at a rate comparable to that observed in model membranes. The data suggest that when one membrane leaflet becomes enriched in ceramides, they diffuse toward the other leaflet. This is counterbalanced by lipid movement in the opposite direction, so that net mass transfer between monolayers is avoided. These observations may be relevant to the physiological mechanism of transmembrane signaling via ceramides.     

Bidirectional transbilayer lipid movement in human platelets as vizualized by the fluorescent membrane probe 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene

Transbilayer movement of the fluorescent membrane probe TMA-DPH [1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene] in the plasma membrane of human platelets was investigated by measuring fluorescence intensity and fluorescence decay. Labeling of unstimulated platelets by TMA-DPH results in a rapid increase in fluorescence intensity, leveling off within 1 min. Dilution of platelets into buffer without TMA-DPH leads to an almost complete rapid efflux of TMA-DPH, indicating that TMA-DPH labels only the outer leaflet of the plasma membrane. Transbilayer movement of the fluorescent probe in unstimulated platelets could be observed upon prolonged incubation and occurs with a t1/2 of 60-90 min. Stimulation of platelets with thrombin directly after the initial rapid uptake of TMA-DPH results in a fast increase in membrane-bound TMA-DPH, fully explained by the increase in plasma membrane caused by secretion of intracellular storage organelles. No indications for increased transbilayer movement of the probe were found, since dilution of thrombin-stimulated TMA-DPH-labeled platelets into buffer without TMA-DPH indicated no uptake of TMA-DPH by intracellular membranes. In contrast to thrombin, stimulation of TMA-DPH-labeled platelets with the Ca2(+)-ionophore ionomycin results in a much larger increase in fluorescence intensity. This process is accompanied by labeling of intracellular membranes as indicated by incomplete efflux of TMA-DPH after dilution of the stimulated platelets. Thus, stimulation of platelets by ionomycin gives rise to rapid and massive inward movement of TMA-DPH (t1/2 approximately 10-12 s). Prolonged incubation of platelets in the absence of any stimulus allows labeling of the total lipid pool, including intracellular membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of cyclodextrins to monitor transbilayer movement and differential lipid affinities of cholesterol

In view of the demonstrated cholesterol-binding capabilities of certain cyclodextrins, we have examined whether these agents can also catalyze efficient transfer of cholesterol between lipid vesicles. We here demonstrate that beta- and gamma-cyclodextrins can dramatically accelerate the rate of cholesterol transfer between lipid vesicles under conditions where a negligible fraction of the sterol is bound to cyclodextrin in steady state. beta- and gamma-cyclodextrin enhance the rate of transfer of cholesterol between vesicles by a larger factor than they accelerate the transfer of phospholipid, whereas, for alpha- and methyl-beta-cyclodextrin, the opposite is true. Analysis of the kinetics of cyclodextrin-mediated cholesterol transfer between large unilamellar vesicles composed mainly of 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC) or SOPC/cholesterol indicates that transbilayer flip-flop of cholesterol is very rapid (halftime < 1-2 min at 37 degrees C). Using beta-cyclodextrin to accelerate cholesterol transfer, we have measured the relative affinities of cholesterol for a variety of different lipid species. Our results show strong variations in cholesterol affinity for phospholipids bearing different degrees of chain unsaturation and lesser, albeit significant, effects of phospholipid headgroup structure on cholesterol-binding affinity. Our findings also confirm previous suggestions that cholesterol interacts with markedly higher affinity with sphingolipids than with common membrane phospholipids.     

Sphingomyelinase activity causes transbilayer lipid translocation in model and cell membranes

Ceramide is known to induce structural rearrangements in membrane bilayers, including the formation of ceramide-rich and -poor domains and the efflux of aqueous solutes. This report describes a novel effect of ceramide, namely the induction of transbilayer lipid movements. This effect was demonstrated in both model (large unilamellar vesicles) and cell (erythrocyte ghost) membranes in which ceramide generation took place in situ through the action of an externally added sphingomyelinase. Two different novel assays were developed to detect transbilayer lipid movement. One of the assays required the preparation of vesicles containing a ganglioside only in the outer monolayer and entrapped neuraminidase. Sphingomyelinase activity induced ganglioside hydrolysis under conditions in which no neuraminidase was released from the vesicles. The second assay involved the preparation of liposomes or erythrocyte ghosts labeled with a fluorescent energy donor in their inner leaflets. Sphingomyelin hydrolysis was accompanied by fluorescence energy transfer to an impermeable acceptor in the outer aqueous medium. Ceramide-induced transbilayer lipid movement is explained in terms of another well known property of ceramide, namely the facilitation of lamellar to non-lamellar lipid-phase transitions. Thus, sphingomyelinase generates ceramide on one side of the membrane; ceramide then induces the transient formation of non-lamellar structural intermediates, which cause the loss of lipid asymmetry in the bilayer, i.e. the transbilayer movement of ceramide together with other lipids. As direct targets for ceramide tend to be intracellular, these observations may be relevant to the mechanism of transmembrane signaling by means of the sphingomyelin pathway.     

Antiobesity effects of a novel lipid synthesis inhibitor (Ro 22-0654)

The present studies were conducted to determine the antiobesity effects of a novel inhibitor of hepatic fatty acid synthesis, 4-amino-5-ethyl-3-thiophene-carboxylic acid methyl ester hydrochloride (Ro 22-0654/001). Weight gain was decreased by treatment with Ro 22-0654 in Sprague Dawley and in lean or obese Zucker rats. Food intake was only transiently suppressed during the first few days of each study. The decreased weight gain in treated rats could be accounted for almost entirely by a decrease in total body lipid levels. Hepatic fatty acid synthesis was inhibited following two months of treatment, in rats fed either a 1% or 10% corn oil diet. It is suggested that the inhibition of hepatic fatty acid synthesis may account, in part, for the reduced weight gain.     

Pigment epithelium-derived factor (PEDF) promotes tumor cell death by inducing macrophage membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Pigment epithelium-derived factor (PEDF) is an intrinsic anti-angiogenic factor and a potential anti-tumor agent. The tumoricidal mechanism of PEDF, however, has not been fully elucidated. Here we report that PEDF induces the apoptosis of TC-1 and SK-Hep-1 tumor cells when they are cocultured with bone marrow-derived macrophages (BMDMs). This macrophage-mediated tumor killing is prevented by blockage of TNF-related apoptosis-inducing ligand (TRAIL) following treatment with the soluble TRAIL receptor. PEDF also increases the amount of membrane-bound TRAIL on cultured mouse BMDMs and on macrophages surrounding subcutaneous tumors. PEDF-induced tumor killing and TRAIL induction are abrogated by peroxisome proliferator-activated receptor γ (PPARγ) antagonists or small interfering RNAs targeting PPARγ. PEDF also induces PPARγ in BMDMs. Furthermore, the activity of the TRAIL promoter in human macrophages is increased by PEDF stimulation. Chromatin immunoprecipitation and DNA pull-down assays confirmed that endogenous PPARγ binds to a functional PPAR-response element (PPRE) in the TRAIL promoter, and mutation of this PPRE abolishes the binding of the PPARγ-RXRα heterodimer. Also, PPARγ-dependent transactivation and PPARγ-RXRα binding to this PPRE are prevented by PPARγ antagonists. Our results provide a novel mechanism for the tumoricidal activity of PEDF, which involves tumor cell killing via PPARγ-mediated TRAIL induction in macrophages.     

Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1

We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1. cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.     

Mouse fat storage‐inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

Fat storage‐inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)‐localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension‐cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER‐LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER‐vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.     

Bromolasalocid (Ro 20-0006) antihypertensive ionophore

Bromolasalocid (Ro 20-0006) is a calcium ionophore with antihypertensive activity that does not belong to any known class of antihypertensive agents. Bromolasalocid produces a relatively flat systolic blood pressure dose-response effect in the spontaneously hypertensive rat. An intensive cardiovascular evaluation of bromolasalocid at the highest dose used in the dose-response study showed full hemodynamic compensation; there was a significant decrease in both mean arterial blood pressure and peripheral resistance without a significant decrease in cardiac index. The antihypertensive action of bromolasalocid lasts many days after termination of dosing. Bromolasalocid is specifically antihypertensive and does not decrease arterial blood pressure in normotensive animals or in animal models of hypertensive cardiovascular disease with normal pulse pressures. Bromolasalocid is not a vasodilator and appears to mediate its antihypertensive action by restoring compliance of the large conduit arteries. Both the derived arterial compliance index and the blood pressure-pressor response to the carotid occlusion reflex are enhanced in the dog perinephritis model of hypertensive cardiovascular disease treated with bromolasalocid. Bromolasalocid appears to reverse the damage to cardiovascular tissue caused by prolonged hypertension via an action on calcium perturbations in large artery smooth muscle cells.     

Expression of fatty acid binding proteins inhibits lipid accumulation and alters toxicity in L cell fibroblasts

High levels of saturated,branched-chain fatty acids are deleterious to cells and animals,resulting in lipid accumulation and cytotoxicity. Although fatty acidbinding proteins (FABPs) are thought to be protective, this hypothesishas not previously been examined. Phytanic acid (branched chain,16-carbon backbone) induced lipid accumulation in L cell fibroblastssimilar to that observed with palmitic acid (unbranched,C₁₆): triacylglycerol free fatty acid > cholesterol > cholesteryl ester phospholipid. Althoughexpression of sterol carrier protein (SCP)-2, SCP-x, or liver FABP(L-FABP) in transfected L cells reduced [³H]phytanic aciduptake (57-87%) and lipid accumulation (21-27%), nevertheless [³H]phytanic acid oxidation was inhibited(74-100%) and phytanic acid toxicity was enhanced in the orderL-FABP SCP-x > SCP-2. These effects differed markedly fromthose of [³H]palmitic acid, whose uptake, oxidation, andinduction of lipid accumulation were not reduced by L-FABP, SCP-2, orSCP-x expression. Furthermore, these proteins did not enhance thecytotoxicity of palmitic acid. In summary, intracellular FABPs reducelipid accumulation induced by high levels of branched-chain but notstraight-chain saturated fatty acids. These beneficial effects wereoffset by inhibition of branched-chain fatty acid oxidation thatcorrelated with the enhanced toxicity of high levels of branched-chainfatty acid.

     

In vivo binding of (3H)Ro 15-1788 in mice: comparison with the in vivo binding of (3H)flunitrazepam

Benzodiazepine binding sites have generally been labelled with benzodiazepine agonists: (3H)flunitrazepam and (3H)diazepam in vivo. We studied the in vivo binding of the antagonist (3H)Ro 15-1788 in mice and compared it to the in vivo binding of (3H)flunitrazepam. For this in vivo labelling, mice were injected with labelled and unlabelled ligands. Animals were then sacrificed and bound radioactivity was measured after homogenization of the excised brain and filtration of the homogenate. (3H)Ro 15-1788 is a better tool than (3H)flunitrazepam for in vivo labelling of benzodiazepine receptors since 1) it labels specifically the central type binding sites, 2) injection of 4 times less (3H)Ro 15-1788 (50 microCi/kg) than (3H)flunitrazepam (200 microCi/kg) produced the same amount of bound radioactivity, 3) 70-90% of the total (3H)Ro 15-1788 present in the brain is membrane bound instead of 45-55% with (3H)flunitrazepam, 4) maximal binding of (3H)Ro 15-1788 is reached within 3 min, 5) only 5% of the membrane bound (3H)Ro 15-1788 is nonspecific instead of 15% for (3H)flunitrazepam.     

Myeloperoxidase-mediated LDL oxidation and endothelial cell toxicity of oxidized LDL: attenuation by (-)-epicatechin

Recent data suggest an inverse epidemiological association between intake of flavanol-rich cocoa products and cardiac mortality. Potential beneficial effect of cocoa may be attributed to flavanol-mediated improvement of endothelial function, as well as to enhancement of bioavailability and bioactivity of nitric oxide in vivo. ( - )-Epicatechin is one bioactive flavanol found in cocoa. This review deals with protective actions of ( - )-epicatechin on two key processes in atherogenesis, oxidation of LDL and damage to endothelial cell by oxidized LDL (oxLDL), with emphasis on data from this laboratory. ( - )-Epicatechin not only abrogates or attenuates LDL oxidation but also counteracts deleterious actions of oxLDL on vascular endothelial cells. These protective actions are only partially shared by other vasoprotective agents such as vitamins C and E or aspirin. Thus, ( - )-epicatechin appears to be a pleiotropic protectant for both LDL and endothelial cells.