Outer Monolayer Research Articles

Effects of Thimerosal on Lipid Bilayers and Human Erythrocytes: An In Vitro Study.

Thimerosal (THI, ethyl-mercury thiosalicylate) is added to vaccines as a preservative; as a consequence, infants may have been exposed to bolus doses of Hg that collectively added up to nominally 200µgHg during the first 6months of life. While several studies report an association between THI-containing vaccines and neurological disorders, other studies do not support the causal relation between THI and autism. With the purpose to understand the molecular mechanisms of the toxic effect of THI it was assayed on human red cells and in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids found in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of THI to interact with DMPC and DMPE was determined by X-ray diffraction and differential scanning calorimetry, whereas intact human erythrocytes were observed by optical, defocusing and scanning electron microscopy. The experimental findings of this study demonstrated that THI interacted in a concentration-dependent manner with DMPC and DMPE bilayers, and in vitro interacted with erythrocytes inducing morphological changes. However, concentrations were considerable higher than those present in vaccines.

Block copolymer morphologies confined by square-shaped particle: Hard and soft confinement**Project supported by the National Natural Science Foundation of China (Grant No. 20804060) and the Research Foundation of Chongqing University of Science and Technology, China (Grant No. CK2013B16).

The self-assembly of diblock copolymers confined around one square-shaped particle is studied systematically within two-dimensional self-consistent field theory (SCFT). In this model, we assume that the thin block copolymer film is confined in the vicinity of a square-shaped particle by a homopolymer melt, which is equivalent to the poor solvents. Multiple sequences of square-shaped particle-induced copolymer aggregates with different shapes and self-assembled internal morphologies are predicted as functions of the particle size, the structural portion of the copolymer, and the volume fraction of the copolymer. A rich variety of aggregates are found with complex internal self-assembled morphologies including complex structures of the vesicle, with one or several inverted micelle surrounded by the outer monolayer with the particle confined in the core. These results demonstrate that the assemblies of diblock copolymers formed around the square-shaped particle in poor solvents are of immediate interest to the assembly of copolymer and the morphology of biomembrane in the confined environment, as well as to the transitions of vesicles to micelles.

Potential of Salvinia auriculata biomass as biosorbent of the Cr(III): directed chemical treatment, modeling and sorption mechanism study

ABSTRACTIn this work, the mechanism of the Cr(III) sorption by Salvinia auriculata biosorbent was studied in two stages. To understand the influence of the sorption parameters on the Cr(III) uptake, preliminary tests were performed. First, S. auriculata biomass was separately treated with base and acid solutions. Second, acid and base treatment of samples was performed based on the knowledge data base of our group. It was achieved a higher Cr(III) sorption capacity above 15 mg g-1 as associated to an increase of the micro-pores specific area and biosorbent volume. The obtained kinetic data of raw and treated biosorbents were well described by the intra-particle diffusion model. In this model, Cr(III) adsorption onto treated biomass is progressively improved with appearing of different mass transfer zones from out layer up to micro-porous layers. The equilibrium data of raw biomass were best described by the Langmuir isotherm, whereas the equilibrium data of the treated biomass were best fit by a combination of both Langmuir and Dubinin–Radushkevich isotherms. At low concentrations the adsorption most likely occurred on the outer monolayer, as proposed by the Langmuir model, followed by the adsorption on the micro-porous layers, as validated by the Dubinin–Radushkevich isotherm.

Antioxidant Capacity of Gallic Acid in vitro Assayed on Human Erythrocytes.

Gallic acid (GA) is a polyphenol present in many plants. This study was aimed to investigate the molecular interaction of GA with the human erythrocyte membrane and to determine its antioxidant capacity. The molecular interaction with the membrane of human red cells and the antioxidant property was assayed on both human red cells and molecular models of its membrane. Observations by optical, scanning electron, and defocusing microscopy demonstrated that GA is capable to convert red cells from their normal biconcave shape to crenated echinocytes. This result indicates that GA molecules are positioned in the outer monolayer of the red cell membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were selected as classes of phospholipids found in the outer and inner monolayers of the red cell membrane, respectively. X-ray diffraction and differential scanning calorimetry showed that GA was preferentially bound to DMPC bilayers. Experiments related to the antioxidant capacity of GA indicated that this compound offsets HClO oxidative capacity on DMPE bilayers. In addition, optical, scanning, defocusing microscopy, and hemolysis assays confirmed the protective capacity of GA against HClO deleterious effects on human red cells. As a conclusion, GA would be capable to block the access of oxidants into the lipid bilayer, and thus avoid their access into red cells.

Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles.

The cell-penetrating peptide R9, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R9 (CF-R9) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R9-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R9 with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R9 (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R9 entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R9 at 6 min in the absence of pore formation, Pentry (6 min), increased with an increase in CF-R9 concentration, but the CF-R9 concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of Pentry (6 min) of CF-R9 for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R9 concentrations. In contrast, a high concentration of CF-R9 induced pores in DLPG/DTPC (4/6) GUVs through which CF-R9 entered the GUV lumen, so the CF-R9 concentration in the lumen was higher. However, CF-R9 could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R9 was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R9 enters GUVs of various lipid compositions.

GsMTx4 Mutagenesis Reveals Dynamic Membrane Binding Properties that Confer Inhibition of Piezo1

GsMTx4 is a spider venom derived peptide selective for cationic mechanosensitive channels (MSCs). It has six lysine residues that have been proposed to affect membrane binding. We created six lysine to glutamate mutants and tested them against Piezo1 channels in outside-out patches and measured lipid binding using a variety of physicochemical assays. Most mutant peptides had lower efficacy (0-40%) compared to wild type (WT). Increased association rates for some of the mutants did not correlate with increased inhibition. And tryptophan fluorescence quenching and isothermal titration calorimetry showed that the free energy of binding to lipid vesicles was largely unaffected by the mutations, suggesting channel inhibition was not a simple function of peptide-lipid association strength. Fluorescence quenching assays showed that WT and mutant peptides bound near the lipid-water interface, but the mutants penetrated deeper than WT. Peptide association as a function of lipid tension was investigated in Langmuir monolayers. The peptides occupied a large fraction of the expanded monolayer area, but that fraction was reduced by peptide expulsion as the pressure approached the monolayer-bilayer equivalence pressure (indicated by a shallow slope of the pressure-area isotherms). Mutants with compromised efficacy had steeper slopes in this region suggesting tighter association. These data place GsMTx4 at the membrane surface where it is stabilized by the lysines, and occupying a small fraction of membrane area at normal tension. As surface pressure decreases and free volume increases during membrane stretching, available peptides penetrate deeper acting as outer monolayer “area reservoirs”, transferring tension to the inner monolayer.

Morphological Effects and Antioxidant Capacity of Solanum crispum (Natre) In Vitro Assayed on Human Erythrocytes.

In order to gain insight into the molecular mechanism of the antioxidant properties of Solanum crispum, aqueous extracts of its leaves were assayed on human erythrocytes and molecular models of its membrane. Phenolics and alkaloids were detected by HPLC-MS. Scanning electron and defocusing microscopy showed that S. crispum changed erythrocytes from the normal shape to echinocytes. These results imply that molecules present in the aqueous extracts were located in the outer monolayer of the erythrocyte membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction showed that S. crispum preferentially interacted with DMPC bilayers. Experiments regarding its antioxidant properties showed that S. crispum neutralized the oxidative capacity of HClO on DMPE bilayers; defocusing microscopy and hemolysis assays demonstrated the protective effect of S. crispum against the oxidant effects of HClO on human erythrocytes.

Spheroid-Based In Vitro Angiogenesis Model.

In vitro models mimicking capillary sprouting are important tools to investigate the tumor angiogenesis, developmental blood vessel formation, and pathophysiological remodeling processes of the capillary system in the adult. With this focus, in 1998 Korff et al. introduced endothelial cell (EC) spheroids as a three-dimensional in vitro model resembling angiogenic responses and sprouting behavior [1]. As such, EC spheroids are capable of giving rise to capillary-like sprouts which are relatively close to the physiologically and genetically programmed arrangement of endothelial cells in vessels. Co-culture spheroids consisting of endothelial cells and smooth muscle cells form a spheroidal core composed of smooth muscle cells and an outer monolayer of endothelial cells, similar to the physiological architecture of larger blood vessels. In practise, a defined number of endothelial cells are cultured in a round-bottom well plate or in "hanging drops" to allow the formation and arrangement of the spheroidal three-dimensional structure. Subsequently, they are harvested and embedded in a collagen gel to allow outgrowth of endothelial cell sprouts originating from each spheroid. To evaluate the pro- or antiangiogenic impact of a cytokine or compound, the number and length of sprouts is determined.

Structural and functional effects of benzimidazole/thioether–copper complexes with antitumor activity on cell membranes and molecular models

Two cytotoxic copper(II) complexes with N–H and N-methylated benzimidazole-derived ligands (Cu-L2 and Cu-L2Me) were synthesized and made to interact with human erythrocytes and molecular models of their plasmatic membranes. The latter consisted in lipid bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), lipids of the types present in the outer and inner monolayers of the human erythrocyte membrane, respectively. Initial assessment of the interaction of the complexes with DMPC and DMPE consisted of X-ray diffraction studies, which showed preferential interactions with the former. Scanning electron microscopy (SEM) of erythrocytes incubated with solutions of the Cu(II) complexes evidenced deformation of the cells to stomatocytes and knizocytes by Cu-L2 and Cu-L2Me due to interactions with the inner and outer leaflets of the cell membranes, respectively. This was further confirmed by real-time observation of the dose-dependent effects of the complexes on live erythrocytes by defocusing microscopy (DM). The combined observations, including the increased antiproliferative activity of the N-methylated complex Cu-L2Me over that of Cu-L2 is rationalized based on the higher lipophilicity of the former. This property would facilitate passive diffusion of Cu-L2Me through the cell membrane, particularly in the initial stages when the DMPC-rich outer leaflet is involved. In contrast, the benzimidazole N–H groups of Cu-L2 may participate in hydrogen bonding with DMPE polar groups; this result is consistent with the formation of stomatocyte induced by the latter complex.

STUDY OF CRYOINJURIES OF FREE AND IMMOBILIZED IN SODIUM ALGINATE GEL SACCHAROMYCES CEREVISIAE YEAST CELLS

The aim of this study was the revealing of the peculiarities of cryoinjuries of free and immobilized in sodium alginate gel Saccharomyces cerevisiae cells after cryopreservation. Methods. The research object was the Saccharomyces cerevisiae yeast cells. By means of fluorescent dyes Annexin V and 7AAD with flow cytometry the injuries of frozen-thawed Saccharomyces cerevisiae cells were assessed. Results. The characteristic features of negative effect of low temperatures were impairments in the integrity and lipid asymmetry of cytoplasm membranes (CPM), DNA fragmentation. The performed analysis of asymmetric distribution of phospholipids in membrane of Saccharomyces cerevisiae yeast cells has shown that the rearrangements in lipid membrane structure during cryopreservation may occur not only under the effect of physical and chemical factors but also due to cryoprotectants. The yield of phosphotidylserine in an outer monolayer layer of CPM was observed in 2.5% of immobilized cells (group 2). The number of Annexin V – labeled cells in group 4 was twice higher relative to the amount of intact cells after freezing. A similar tendency was observed with regard to this kind of damage in the samples of free cells. However, it should be noted that the number of AnnexinV – labeled cells in group 1 was significantly lower and it is comparable with the controls. We believe that the reduction in the number of cells with disordered asymmetric distribution of phospholipids in group 1 was due to increase in the number of completely destroyed (not subjected to analysis) cells in these samples. It has been found that all the tested samples, including controls, there were the cells being at different stages of apoptosis/necrosis. The difference in the samples consists in the value of the percentage of cells eliminated from the analysis of flow cytometry. The number of completely destroyed cells in immobilized samples was significantly lower than in the group of free cells in a suspension. It has been established that there are the cells being at different stages of apoptosis, in all the studied samples including the control ones. The difference in the samples consists in the value of the percentage of the cells eliminated from the process of flow cytometry analysis. The number of completely destroyed cells in immobilized samples was statistically and significantly lower than in the groups of free in suspension cells. Conclusions. It has been established that under the influence of cryopreservation damaging factors in a part of free and immobilized in alginate gel S. cerevisiae cells there were developed the disorders in an integrity and lipid asymmetry of the cytoplasmic membranes, DNA fragmentation. The presence of DMSO as a cryoprotectant in preserving medium has been shown to lead to an increase in the number of cryopreserved cells with an asymmetric distribution of phospholipids in cell membranes. The number of cells in early apoptosis after freezing with DMSO as a cryoprotectant in 2–2.5 times exceeded the number of similar cells frozen without cryoprotectant. Cryopreservation of yeasts in polysaccharide gel of sodium alginate may be an alternative way to preservation of biological objects under the protection with traditional cryoprotective agents, allowing a high degree of viability and preservation of cells with no cryoprotectants.

Antimicrobial Peptide Lactoferricin B-Induced Rapid Leakage of Internal Contents from Single Giant Unilamellar Vesicles.

Enzymatic digestion of bovine lactoferrin generates lactoferricin B (Lfcin B), a 25-mer peptide with strong antimicrobial activity of unknown mechanism. To elucidate the mechanistic basis of Lfcin B bactericidal activity, we investigated the interaction of Lfcin B with Escherichia coli and liposomes of lipid membranes. Lfcin B induced the influx of a membrane-impermeant fluorescent probe, SYTOX green, from the outside of E. coli into its cytoplasm. Lfcin B induced gradual leakage of calcein from large unilamellar vesicles (LUVs) of dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) membranes. To clarify the cause of Lfcin B-induced leakage of calcein from the LUVs, we used the single giant unilamellar vesicle (GUV) method to investigate the interaction of Lfcin B with calcein-containing DOPG/DOPC-GUVs. We observed that a rapid leakage of calcein from a GUV started stochastically; statistical analysis provided a rate constant for Lfcin B-induced pore formation, kp. On the other hand, phase-contrast microscopic images revealed that Lfcin B induced a rapid leakage of sucrose from the single GUVs with concomitant appearance of a spherical GUV of smaller diameter. Because of the very fast leakage, and at the present time resolution of the experiments (33 ms), we could not follow the evolution of pore nor the process of the structural changes of the GUV. Here we used the term "local rupture" to express the rapid leakage of sucrose and determined the rate constant of local rupture, kL. On the basis of the comparison between kp and kL, we concluded that the leakage of calcein from single GUVs occurred as a result of a local rupture in the GUVs and that smaller pores inducing leakage of calcein were not formed before the local rupture. The results of the effect of the surface charge density of lipid membranes and that of salt concentration in buffer on kp clearly show that kp increases with an increase in the extent of electrostatic interactions due to the surface charges. Analysis of Lfcin B-induced shape changes indicated that the binding of Lfcin B increased the area of the outer monolayer of GUVs. These results indicate that Lfcin B-induced damage of the plasma membrane of E. coli with its concomitant rapid leakage of internal contents is a key factor for the bactericidal activity of LfcinB.

An in vitro study on the antioxidant capacity of usnic acid on human erythrocytes and molecular models of its membrane

Usnic acid (UA) has been associated with chronic diseases through its antioxidant action. Its main target is the cell membrane; however, its effect on that of human erythrocytes has been scarcely investigated. To gain insight into the molecular mechanisms of the interaction between UA and cell membranes human erythrocytes and molecular models of its membrane have been utilized. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. Results by X-ray diffraction showed that UA produced structural perturbations on DMPC and DMPE bilayers. DSC studies have indicated that thermotropic behavior of DMPE was most strongly distorted by UA than DMPC, whereas the latter is mainly affected on the pretransition. Scanning electron (SEM) and defocusing microscopy (DM) showed that UA induced alterations to erythrocytes from the normal discoid shape to echinocytes. These results imply that UA molecules were located in the outer monolayer of the erythrocyte membrane. Results of its antioxidant properties showed that UA neutralized the oxidative capacity of HClO on DMPC and DMPE bilayers; SEM, DM and hemolysis assays demonstrated the protective effect of UA against the deleterious oxidant effects of HClO upon human erythrocytes.

Mixed SAMs of backbone-functionalized tribenzotriquinacenes and alkanethiols: Synthesis, preparation and STM-investigations

We present the synthesis of a backbone-functionalized tribenzotriquinacene (TBTQ) 3, serving as a prototype for TBTQ-based stators comprising thioether anchor groups for self-assembled monolayers on Au-(111) surfaces. Self-assembled monolayers (SAM) consisting of isolated molecules of the minor component 3 embedded in a close-packed matrix of decanethiol (C10-SH) were prepared, leading to stable monolayers which were characterized by scanning tunneling microscopy (STM) under ambient conditions. The structural features of the highly ordered mixed monolayers point to the fact that isolated TBTQ-headgroups are exposed to the outer monolayer surface, making them available for the interaction with shape-complementary guest molecules.

Features of inhibition of glycerol-3-phosphate oxidase activity of liver mitochondria by palmitic acid in the presence of ATP and tert-butylhydroperoxide

The effect of palmitic acid on glycerol-3-phosphate oxidation, i.e., on the glycerol-3-phosphate oxidase (GP oxidase) activity of liver mitochondria was investigated in the presence and absence of ATP, as well as under conditions of tert-butylhydroperoxide (TBH)-induced oxidative stress. It was found that palmitic and lauric acids inhibit GP oxidase activity of deenergized liver mitochondria formally in the competitive manner. In this case lauric acid, which is less hydrophobic than palmitic acid, inhibits GP oxidase activity of mitochondria significantly weaker. It is noted that the competitive inhibition of mitochondrial GP oxidase activity by these saturated fatty acids may be related to their ability, as amphiphilic compounds, to increase the negative charge density on the outer surface of the inner mitochondrial membrane. It is shown that ATP at a concentration of 2 mM eliminates the ability of palmitic acid to inhibit GP oxidase activity of mitochondria. This effect of ATP is not observed in the presence of F O F 1-ATP synthase inhibitor oligomycin. Apparently, in the case of vector transport of H+ from the matrix to the intermembrane space of mitochondria induced by ATP hydrolysis there is occurs protonation of palmitic acid anions on the outer surface of the inner membrane and subsequent movement of its neutral molecules to the inner monolayer of the inner membrane. It was found that TBH at a concentration of 300 µM in the presence of ATP and palmitic acid inhibits their GP oxidase activity formally in the competitive manner without significant effect on ATPase activity of liver mitochondria. Thiourea antioxidant eliminates this effect of TBH. It is assumed that TBH-induced oxidative stress in the case of ATP-energized mitochondria results in an increase in transport rate of palmitic acid anions from the inner monolayer of the inner membrane to its outer monolayer. This, in turn, is accompanied by an increase in the density of negative charges on the outer surface of the inner mitochondrial membrane and inhibition of GP oxidase activity of liver mitochondrial formally in the competitive manner.

Surface Density-Induced Pleating of a Lipid Monolayer Drives Nascent High-Density Lipoprotein Assembly

Biogenesis of high-density lipoproteins (HDL) is coupled to the transmembrane protein, ATP-binding cassette transporter A1 (ABCA1), which transports phospholipid (PL) from the inner to the outer membrane monolayer. Using a combination of computational and experimental approaches, we show that increased outer lipid monolayer surface density, driven by excess PL or membrane insertion of amphipathic helices, results in pleating of the outer monolayer to form membrane-attached discoidal bilayers. Apolipoprotein (apo)A-I accelerates and stabilizes the pleats. In the absence of apoA-I, pleats collapse to form vesicles. These results mimic cells overexpressing ABCA1 that, in the absence of apoA-I, form and release vesicles. We conclude that the basic driving force for nascent discoidal HDL assembly is a PL pump-induced surface density increase that produces lipid monolayer pleating. We then argue that ABCA1 forms an extracellular reservoir containing an isolated pressurized lipid monolayer decoupled from the transbilayer density buffering of cholesterol.

PROTECTIVE EFFECT OF PHILESIA MAGELLANICA (COICOPIHUE) FROM CHILEAN PATAGONIA AGAINST OXIDATIVE DAMAGE

Philesia magellanica (P. magellanica) is a plant collected in the Chilean Patagonia. Its antioxidant properties were assessed in human erythrocytes exposed in vitro to oxidative stress induced by HClO. Scanning electron microscopy (SEM) observations showed that HClO induced a morphological alteration in the red blood cells from a normal discoid to a spherocytic form, and cells of unequal size. However, a concentration as low as 1 mM gallic acid equivalents (GAE) of P. magellanica aqueous extract neutralized the change effects of 50 µM HClO. On the other hand, 20 µM (GAE) of the extract considerably reduced the deleterious capacity of 0.25 mM HClO to induce hemolysis in red blood cells. In addition, X-ray diffraction experiments were performed in molecular models of the human erythrocyte membrane. These consisted in multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of lipids preferentially located in the outer and inner monolayers, respectively of the human erythrocyte membrane. It was observed that P. magellanica only interacted with DMPC affecting its multilayer structure. It was also observed that 0.1 µM (GAE) of P. magellanica neutralized the structural perturbation induced by 0.05 and 0.5 mM HClO. These experiments confirmed the antioxidant properties of P. magellanica aqueous extracts.

APPLICATION OF ALKYL SULFATES AND HEAT TREATED ERYTHROCYTES IN HYPERTONIC CRYOHEMOLYSIS

The research aim was to study the application efficiency of alkyl sulfates and heat treatment of erythrocytes of mammals (human, horse, bull and rabbit) in order to increase their resistance to hypertonic cryohemolysis. The hemolysis rate for erythrocytes was recorded by spectrophotometry; the efficiency of alkyl sulfates was assessed on the values of maximum antihemolytic activity and the ones of effective concentrations; mammalian erythrocytes were morphologically analyzed by light microscopy. It has been found that anionic amphiphiles (sodium decyl and dodecyl sulfates) exhibit an antihemolytic activity in hypertonic cryohemolysis of erythrocytes. There was shown the transformation of erythrocytes on the “discocyte-echinocyte” type in the presence of alkyl sulfates, indicating to the distribution of amphiphilic molecules in an outer monolayer of erythrocyte membrane bilayer. It has been revealed that pre-incubation of the cells at 49 C increases the resistance to hypertonic cryohemolysis effect for human and equine erythrocytes and as well as it reduces the one for bovine and rabbit cells. Sodium decyl and sodium dodecyl sulfates exhibit an antihemolytic activity under hypertonic cryohemolysis of heat treated mammalian erythrocytes, but it is lower if compared with the control cells. The findings about distribution of alkyl sulfates in membranes and their antihemolytic activity under conditions of hypertonic cryohemolysis of heat treated erythrocytes testify to the perspective of using these substances as the tool for assessing the state of erythrocyte membranes when changing the temperatureosmotic environment.

Structures of Bacteroides fragilis uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (BfLpxA).

Uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes a reversible reaction for adding an O-acyl group to the GlcNAc in UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A constitutes a major component of lipopolysaccharides, also referred to as endotoxins, which form the outer monolayer of the outer membrane of Gram-negative bacteria. Ligand-free and UDP-GlcNAc-bound crystal structures of LpxA from Bacteroides fragilis NCTC 9343, the most common pathogenic bacteria found in abdominal abscesses, have been determined and are presented here. The enzyme crystallizes in a cubic space group, with the crystallographic threefold axis generating the biological functional homotrimer and with each monomer forming a nine-rung left-handed β-helical (LβH) fold in the N-terminus followed by an α-helical motif in the C-terminus. The structure is highly similar to LpxA from other organisms. Yet, despite sharing a similar LβH structure with LpxAs from Escherichia coli and others, previously unseen calcium ions are observed on the threefold axis in B. fragilis LpxA to help stabilize the trimeric assembly.

Interactions of the antiviral and antiparkinson agent amantadine with lipid membranes and human erythrocytes

Aimed to better understand the molecular mechanisms of its interactions with cell membranes, human erythrocyte and molecular models of the red cell membrane were utilized. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of amantadine to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, fluorescence spectroscopy and differential scanning calorimetry (DSC). In an attempt to further elucidate its effects on cell membranes, the present work also examined amantadine influence on the morphology of intact human erythrocytes by means of scanning electron microscopy (SEM). Results indicated that amantadine induced morphological changes to human erythrocytes and interacted in a concentration-dependent manner with DMPC bilayers in contrast to DMPE that was hardly affected by the presence of the drug.

Morphological Effects Induced In Vitro by Propranolol on Human Erythrocytes.

Despite the extended use and well-documented information, there are insufficient reports concerning the effects of propranolol on the structure and functions of cell membranes, particularly those of human erythrocytes. Aimed to better understand the molecular mechanisms of its interactions with cell membranes, human erythrocyte and molecular models of the red cell membrane were utilized. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of propranolol to perturb the multibilayer structures of DMPC and DMPE was evaluated by X-ray diffraction. Moreover, we took advantage of the capability of differential scanning calorimetry to detect the changes in the thermotropic phase behavior of lipid bilayers resulting from propranolol interaction with DMPC and DMPE multilamellar vesicles. In an attempt to further elucidate their effects on cell membranes, the present work also examined their influence on the morphology of intact human erythrocytes by means of defocusing and scanning electron microscopy. Results indicated that propranolol induced morphological changes to human erythrocytes and interacted in a concentration-dependent manner with phospholipid bilayer.