Membrane properties of structurally modified ceramides : effects on lipid lateral distribution and sphingomyelin-interactions in artificial bilayer membranes

Ceramides comprise a class of sphingolipids that exist only in small amounts in cellular membranes, but which have been associated with important roles in cellular signaling processes. The influences that ceramides have on the physical properties of bilayer membranes reach from altered thermodynamical behavior to significant impacts on the molecular order and lateral distribution of membrane lipids. Along with the idea that the membrane physical state could influence the physiological state of a cell, the membrane properties of ceramides have gained increasing interest. Therefore, membrane phenomena related to ceramides have become a subject of intense study both in cellular as well as in artificial membranes. Artificial bilayers, the so called model membranes, are substantially simpler in terms of contents and spatio-temporal variation than actual cellular membranes, and can be used to give detailed information about the properties of individual lipid species in different environments. This thesis focuses on investigating how the different parts of the ceramide molecule, i.e., the N-linked acyl chain, the long-chain sphingoid base and the membrane-water interface region, govern the interactions and lateral distribution of these lipids in bilayer membranes. With the emphasis on ceramide/sphingomyelin(SM)-interactions, the relevance of the size of the SMhead group for the interaction was also studied. Ceramides with methylbranched N-linked acyl chains, varying length sphingoid bases, or methylated 2N (amide-nitrogen) and 3O (C3-hydroxyl) at the interface region, as well as SMs with decreased head group size, were synthesized and their bilayer properties studied by calorimetric and fluorescence spectroscopic techniques. In brief, the results showed that the packing of the ceramide acyl chains was more sensitive to methyl-branching in the mid part than in the distal end of the N-linked chain, and that disrupting the interfacial structure at the amide-nitrogen, as opposed to the C3-hydroxyl, had greater effect on the interlipid interactions of ceramides. Interestingly, it appeared that the bilayer properties of ceramides could be more sensitive to small alterations in the length of the long-chain base than what was previously reported for the N-linked acyl chain. Furthermore, the data indicated that the SM-head group does not strongly influence the interactions between SMs and ceramides. The results in this thesis illustrate the pivotal role of some essential parts of the ceramide molecules in determining their bilayer properties. The thesis provides increased understanding of the molecular aspects of ceramides that possibly affect their functions in biological membranes, and could relate to distinct effects on cell physiology.

Use of blends of bioabsorbable poly(L-lactic acid)/poly(hydroxybutyrate- co-hydroxyvalerate) as surfaces for Vero cell culture

Vero cells, a cell line established from the kidney of the African green monkey (Cercopithecus aethiops), were cultured in F-10 Ham medium supplemented with 10% fetal calf serum at 37°C on membranes of poly(L-lactic acid) (PLLA), poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and their blends in different proportions (100/0, 60/40, 50/50, 40/60, and 0/100). The present study evaluated morphology of cells grown on different polymeric substrates after 24 h of culture by scanning electron microscopy. Cell adhesion was also analyzed after 2 h of inoculation. For cell growth evaluation, the cells were maintained in culture for 48, 120, 240, and 360 h. For cytochemical study, the cells were cultured for 120 or 240 h, fixed, processed for histological analysis, and stained with Toluidine blue, pH 4.0, and Xylidine ponceau, pH 2.5. Our results showed that cell adhesion was better when 60/40 and 50/50 blends were used although cells were able to grow and proliferate on all blends tested. When using PLLA/PHBV (50/50) slightly flattened cells were observed on porous and smooth areas. PLLA/PHBV (40/60) blends presented flattened cells on smooth areas. PLLA/PHBV (0/100), which presented no pores, also supported spreading cells interconnected by thin filaments. Histological sections showed that cells grew as a confluent monolayer on different substrates. Cytochemical analysis showed basophilic cells, indicating a large amount of RNA and proteins. Hence, we detected changes in cell morphology induced by alterations in blend proportions. This suggests that the cells changed their differentiation pattern when on various PLLA/PHBV blend surfaces.

Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellataroot extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

Caractérisation du co-transporteur Na+/myo-inositol SMIT2 dans les membranes en bordure en brosse de rein de lapin et d’intestin de rat

Le myo-inositol (MI) est un soluté organique impliqué dans diverses fonctions physiologiques de la cellule dont la signalisation cellulaire. Il est également un osmolyte compatible reconnu. Trois co-transporteurs de type actif secondaire responsables de son absorption ont été identifiés. Deux d’entre eux sont couplés au transport du sodium (SMIT1 et SMIT2) et le troisième est couplé au transport de protons (HMIT). L’objectif de cette étude a été la caractérisation du transport du MI par SMIT2 dans des membranes en bordure en brosse (BBMv) issues du rein de lapin et de l’intestin de rat ainsi qu’après expression dans les ovocytes de Xenopus laevis. La quantification de l’ARNm de SMIT1 et de SMIT2 dans le rein nous a appris que SMIT1 est majoritairement présent dans la médullaire alors que SMIT2 est principalement localisé dans le cortex. Ces résultats ont été confirmés par immunobuvardage en utilisant un anticorps dirigé contre SMIT2. Grâce à l’inhibition sélective de SMIT1 par le L-Fucose et de SMIT2 par le D-chiro-inositol (DCI), nous avons démontré que SMIT2 semble le seul responsable du transport luminal de MI dans le tubule contourné proximal avec un Km de 57 ± 14 µM. Pour ce qui est de l’intestin, des études de transport de MI radioactif ont démontré une absence de transport de MI chez le lapin alors que l’intestin de rat présente un transport de MI très actif. Une quantification par qRT-PCR nous a permis de constater que l’intestin de lapin ne semble pas posséder les transporteurs de MI nécessaires. Comme pour le rein, SMIT2 semble le seul transporteur de MI présent au niveau du pôle apical des entérocytes intestinaux chez le rat. Il est chargé du prélèvement du MI de l'alimentation avec un Km de 150 ± 40 µM. Les analyses fonctionnelles exécutées sur SMIT2 de rat en électrophysiologie après expression dans les ovocytes de Xenopus laevis donnent sensiblement les mêmes résultats que pour les BBMv de rein de lapin et d’intestin de rat. Dans les ovocytes, SMIT2 présente une grande affinité pour le MI (270 ± 19 µM) et le DCI (310 ± 60 µM) et aucune affinité pour le L-fucose. Il est ii également très sensible à la phlorizine (16 ± 7 µM). Une seule exception persiste : la constante d’affinité pour le glucose dans les BBMv d’intestin de rat est 40 fois plus petite que celle observée sur les ovocytes de Xenopus laevis. Nous avons également testé la capacité de certains transporteurs de sucre présents à la surface des membranes apicales des entérocytes à prélever le MI. Vu que l'inhibition de ces transporteurs (SGLT1 et GLUT5) ne changeait rien au taux de MI radioactif transporté, nous en avons conclu qu'ils ne sont pas impliqués dans son transport. Finalement, l’efflux de MI à partir du pôle basolatéral des entérocytes n’est pas effectué par GLUT2 puisque ce dernier lorsqu'il est exprimé dans des ovocytes, est incapable de transporter le MI.

Biochemical Investigations on the Stability of Biological Membranes

In this project, an attempt has been made to study the stability of erythrocyte and lysosomal membranes biochemically. Erythrocytes were chosen for the study because of their ready availability and relative simplicity. Biological membranes forming closed boundaries between compartments of varying composition consist mainly of proteins and lipids. They are asymmetric, fluid structures that are thermodynamically stable and metabolically active. Normal cellular function begins with normal membrane structure and any variation in it may upset the normal functions. The degree of fluidity of a membrane depends on the chain length of its lipids and degree of unsaturation of constituent fatty acids. In response to environmental changes, many cells can regulate composition of their membranes to maintain the overall semi fluid environment necessary for many membrane associated functions. The assembly and Maintenance of membrane structures in cells is a dynamic process. The components are not only synthesized and inserted into a growing membrane but are also continuously degraded at a slower rate. This turnover process varies with each individual molecule.Lysosomes are important in the catabolic processes occurring in the cell. Lysosomes contain hydrolytic enzymes and are stable under normal conditions. In certain pathological conditions, the lysosomal membrane may rupture, releasing the hydrolytic enzymes into the cell and digestion of cell takes place as a whole. This is very dangerous. In normal life processes of multi cellular organisms, lysosomes rupture following the death of a cell and it may have some value as a built in mechanism for selfremoval of dead cells.An attempt has also been made in this project towards developing lysosome membrane stability as an index of fish spoilage during storage. Different membranes within the cell and between cells have different compositions as reflected in the ratio of protein to lipid. The difference is not surprising given the very different functions of membranes

Glucosinolate-accumulating S-cells in Arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation

Plant secondary metabolites glucosinolates (GSL) have important functions in plant resistance to herbivores and pathogens. We identified all major GSL that are accumulated in S-cells in Arabidopsis by MALDI-TOF MS, and estimated by LC-MS that the total GSL concentration in these cells is above 130 mM. The precise locations of the S-cells outside phloem bundles in rosette and cauline leaves and in flower stalks were visualised using sulphur mapping by cryo-SEM/EDX. S-cells contain up to 40% of total sulphur in flower stalk tissues. S-cells in emerging flower stalks and developing leaf tissues show typical signs of Programmed Cell Death (PCD) or apoptosis, such as chromatin condensation in the nucleus and blebbing of the membranes. TUNEL staining for DNA double strand breaks confirmed PCD in S-cells in postmeristematic tissues in the flower stalk as well as in the leaf. Our results show that S-cells in postmeristematic tissues proceed to an extreme degree of metabolic specialisation besides PCD. Accumulation and maintenance of a high concentration of GSL in these cells are accompanied by degradation of a number of cell organelles. The substantial changes in the cell composition during S-cell differentiation indicate the importance of this particular GSL-based phloem defence system. The specific anatomy of the S-cells and ability to accumulate specialised secondary metabolites is similar to that of the non-articulated laticifer cells in latex plants and thus indicates a common evolutionary origin.

Microblock ionomers: a new concept in high temperature, swelling-resistant membranes for PEM fuel cells

A novel series of polyaromatic ionomers with similar equivalent weights but very different sulphonic acid distributions along the ionomer backbone has been designed and prepared. By synthetically organising the sequence-distribution so that it consists of fully defined ionic segments (containing singlets, doublets or quadruplets of sulphonic acid groups) alternating strictly with equally well-defined nonionic spacer segments, a new class of polymers which may be described as microblock ionomers has been developed. These materials exhibit very different properties and morphologies from analogous randomly substituted systems. Progressively extending the nonionic spacer length in the repeat unit (maintaining a constant equivalent weight by increasing the degree of sulphonation. of the ionic segment) leads to an increasing degree of nanophase separation between hydrophilic and hydrophobic domains in these materials. Membranes cast from ionomers with the more highly phase-separated morphologies show significantly higher onset temperatures for uncontrolled swelling in water. This new type of ionomer design has enabled the fabrication of swelling-resistant hydrocarbon membranes, suitable for fuel cell operation, with very much higher ion exchange capacities (>2 meq g(-1)) than those previously reported in the literature. When tested in a fuel cell at high temperature (120 degrees C) and low relative humidity (35% RH), the best microblock membrane matched the performance of Nafion 112. Moreover, comparative low load cycle testing of membrane -electrode assemblies suggests that the durability of the new membranes under conditions of high temperature and low relative humidity is superior to that of conventional perfluorinated materials.

Ion-conducting polymers and membranes comprising them

An ion-conducting polymer wherein at least 80% of the repeat units comprise an ion-conducting region and a spacer region is disclosed. The ion-conducting region has an aromatic backbone of one or more aromatic groups, wherein at least one ion-conducting functional group is attached to each aromatic group. The spacer region has an aromatic backbone of at least four aromatic groups, wherein no ion-conducting functional groups are attached to the aromatic backbone. The polymer is suitable for use as a fuel cell membrane, and can be incorporated into membrane electrode assemblies.

Characterisation of cell death in bagged baby salad leaves

Baby leaf salads are gaining in popularity over traditional whole head lettuce salads in response to consumer demand for greater variety and convenience in their diet. Baby lettuce leaves are mixed, washed and packaged as whole leaves, with a shelf-life of approximately 10 days post-processing. End of shelf-life, as determined by the consumer, is typified by bruising, water-logging and blackening of the leaves, but the biological events causing this phenotype have not been studied to date. We investigated the physiological and ultrastructural characteristics during postharvest shelf-life of two lettuce varieties with very different leaf morphologies. Membrane disruption was an important determinant of cell death in both varieties. although the timing and characteristics of breakdown was different in each with Lollo rossa showing signs of aging such as thylakoid disruption and plastoglobuli accumulation earlier than Cos. Membranes in Lollo rossa showed a later, but more distinct increase in permeability than in Cos. as indicated by electrolyte leakage and the presence of cytoplasmic fragments in the vacuole, but Cos membranes show distinct fractures towards the end of shelf-life. The tissue lost less than 25% fresh weight during shelf-life and there was little protein loss compared to developmentally aging leaves in an ambient environment. Biophysical measurements showed that breakstrength was significantly reduced in Lollo rossa, whereas irreversible leaf plasticity was significantly reduced in Cos leaves. The reversible elastic properties of both varieties changed throughout shelf-life. We compared the characteristics of shelf-life in both varieties of bagged lettuce leaves with other leafy salad crops and discuss the potential targets for future work to improve postharvest quality of baby leaf lettuce. (C) 2007 Elsevier B.V. All rights reserved.

Apolipoprotein E genotype and alpha-tocopherol modulate amyloid precursor protein metabolism and cell cycle regulation

Apolipoprotein E4 (apoE4) genotype is associated with an increased risk for Alzheimer's disease (AD). This is thought to be in part attributable to an impact of apoE genotype on the processing of the transmembrane amyloid precursor protein (APP) thereby contributing to amyloid beta peptide formation in apoE4 carriers, which is a primary patho-physiological feature of AD. As apoE and alphato-copherol (alpha-toc) have been shown to modulate membrane bilayer properties and hippocampal gene expression, we studied the effect of apoE genotype on APP metabolism and cell cycle regulation in response to dietary a-toc. ApoE3 and apoE4 transgenic mice were fed a diet low (VE) or high (+VE) in vitamin E (3 and 235 mg alpha-toe/kg diet, respectively) for 12 weeks. Cholesterol levels and membrane fluidity were not different in synaptosomal plasma membranes isolated from brains of apoE3 and apoE4 mice (-VE and +VE). Non-amyloidogenic alpha-secretase mRNA concentration and activity were significantly higher in brains of apoE3 relative to apoE4 mice irrespective of the dietary a-toe supply, while amyloidogenic beta-secretase and gamma-secretase remained unchanged. Relative mRNA concentration of cell cycle related proteins were modulated differentially by dietary a-toc supplementation in apoE3 and apoE4 mice, suggesting genotype-dependent signalling effects on cell cycle regulation.

Role of rpoS in the Development of Cell Envelope Resilience and Pressure Resistance in Stationary-Phase Escherichia coli

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure-sensitive and took up more PI than the parent strains with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure-sensitive, was unaffected in membrane resealing implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.

The Bone Formation Capabilities of the Anorganic Bone Matrix-Synthetic Cell-Binding Peptide 15 Grafts in an Animal Periodontal Model: A Histologic and Histomorphometric Study in Dogs

Background: The aim of this study is to verify the regenerative potential of particulate anorganic bone matrix synthetic peptide-15 (ABM-P-15) in class III furcation defects associated or not with expanded polytetrafluoroethylene membranes. Methods: Class III furcation defects were produced in the mandibular premolars (P2, P3, and P4) of six dogs and filled with impression material. The membranes and the bone grafts were inserted into P3 and P4, which were randomized to form the test and control groups, respectively; P2 was the negative control group. The animals were sacrificed 3 months post-treatment. Results: Histologically, the complete closure of class III furcation defects was not observed in any of the groups. Partial periodontal regeneration with similar morphologic characteristics among the groups was observed, however, through the formation of new cementum, periodontal ligament, and bone above the notch. Histologic analysis showed granules from the bone graft surrounded by immature bone matrix and encircled by newly formed tissue in the test group. The new bone formation area found in the negative control group was 2.28 +/- 2.49 mm(2) and in the test group it was 6.52 +/- 5.69 mm(2), which showed statistically significant differences for these groups considering this parameter (Friedman test P <0.05). There was no statistically significant difference among the negative control, control, and test groups for the other parameters. Conclusions: The regenerative potential of ABM-P-15 was demonstrated through new bone formation circumscribing and above the graft particles. The new bone also was accompanied by the formation of new cementum and periodontal ligament fibers. J Periodontol 2010;81:594-603.

Interaction of oligonucleotide-based amphiphilic block copolymers with cell membrane models

Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides. (C) 2010 Elsevier Inc. All rights reserved.

Comparative study of liponucleosides in Langmuir monolayers as cell membrane models

Liponucleosides may assist the anchoring of nucleic acid nitrogen bases into biological membranes for tailored nanobiotechnological applications. To this end precise knowledge about the biophysical and chemical details at the membrane surface is required. In this paper, we used Langmuir monolayers as simplified cell membrane models and studied the insertion of five lipidated nucleosides. These molecules varied in the type of the covalently attached lipid group, the nucleobase, and the number of hydrophobic moieties attached to the nucleoside. All five lipidated nucleosides were found to be surface-active and capable of forming stable monolayers. They could also be incorporated into dipalmitoylphosphatidylcholine (DPPC) monolayers, four of which induced expansion in the surface pressure isotherm and a decrease in the surface compression modulus of DPPC. In contrast, one nucleoside possessing three alkyl chain modifications formed very condensed monolayers and induced film condensation and an increase in the compression modulus for the DPPC monolayer, thus reflecting the importance of the ability of the nucleoside molecules to be arranged in a closely packed manner. The implications of these results lie on the possibility of tuning nucleic acid pairing by modifying structural characteristics of the liponucleosides. (C) 2010 Elsevier B.V. All rights reserved.

pH-Sensitive Binding of Cytochrome c to the Inner Mitochondrial Membrane. Implications for the Participation of the Protein in Cell Respiration and Apoptosis

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pK(a) values and site L containing ionizable groups with pK(aobs),values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, We demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pi-I-independent binding (microscopic dissociation constant K(sapp2), similar to 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pK(a) of similar to 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on K(sapp1), was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed K(sapp1) values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site Lionization influences the participation of cytochrome c in the respiratory chain and apoptosis.