Changes In Membrane Stability Research Articles

Murine Norovirus Interaction with Enterobacter cloacae Leads to Changes in Membrane Stability and Packaging of Lipid and Metabolite Vesicle Content.

Outer membrane vesicles (OMVs) are a primary means of communication for Gram-negative bacteria. The specific role of vesicle components in cellular communication and how components are packaged are still under investigation, but a correlation exists between OMV biogenesis and content. The two primary mechanisms of OMV biogenesis are membrane blebbing and explosive cell lysis, and vesicle content is based on the biogenesis mechanism. Hypervesiculation, which can be induced by stress conditions, also influences OMV content. Norovirus interaction with Enterobacter cloacae induces stress responses leading to increased OMV production and changes in DNA content, protein content, and vesicle size. The presence of genomic DNA and cytoplasmic proteins in these OMVs suggests some of the vesicles are formed by explosive cell lysis, so reduction or loss of these components indicates a shift away from this mechanism of biogenesis. Based on this, further investigation into bacterial stability and OMV content was conducted. Results showed that norovirus induced a dramatic shift in OMV lipid content. Specifically, the increased accumulation of phospholipids is associated with increased blebbing, thereby supporting previous observations that noroviruses shift the mechanism of OMV biogenesis. Slight differences in OMV metabolite content were also observed. While norovirus induced changes in OMV content, it did not change the lipid content of the bacterial outer membrane or the metabolite content of the bacterial cell. Overall, these results indicate that norovirus induces significant changes to OMV lipid architecture and cargo, which may be linked to a change in the mechanism of vesicle biogenesis. IMPORTANCE Extracellular vesicles from commensal bacteria are recognized for their importance in modulating host immune responses, and vesicle content is related to their impact on the host. Therefore, understanding how vesicles are formed and how their content shifts in response to stress conditions is necessary for elucidating their downstream functions. Our recent work has demonstrated that interactions between noroviruses and Enterobacter cloacae induce bacterial stress responses leading to hypervesiculation. In this article, we characterize and compare the lipid and metabolomic cargo of E. cloacae vesicles generated in the presence and absence of norovirus and show that viral interactions induce significant changes in vesicle content. Furthermore, we probe how these changes and changes to the bacterial cell may be indicative of a shift in the mechanism of vesicle biogenesis. Importantly, we find that noroviruses induce significant changes in vesicle lipid architecture and cargo that may be responsible for the immunogenic activity of these vesicles.

Sodium Taurocholate Stimulates Campylobacter jejuni Outer Membrane Vesicle Production via Down-Regulation of the Maintenance of Lipid Asymmetry Pathway.

Campylobacter jejuni outer membrane vesicles (OMVs) contain numerous virulence-associated proteins including the cytolethal distending toxin and three serine proteases. As C. jejuni lacks the classical virulence-associated secretion systems of other enteric pathogens that deliver effectors directly into target cells, OMVs may have a particularly important role in virulence. C. jejuni OMV production is stimulated by the presence of physiological concentrations of the bile salt sodium taurocholate (ST) through an unknown mechanism. The maintenance of lipid asymmetry (MLA) pathway has been implicated in a novel mechanism for OMV biogenesis, open to regulation by host signals. In this study we investigated the role of the MLA pathway in C. jejuni OMV biogenesis with ST as a potential regulator. OMV production was quantified by analyzing protein and lipid concentrations of OMV preparations and OMV particle counts produced by nanoparticle tracking analysis. Mutation of mlaA which encodes the outer membrane component of the MLA pathway significantly increased OMV production compared to the wild-type strain. Detergent sensitivity and membrane permeability assays confirmed the increased OMV production was not due to changes in membrane stability. The presence of 0.2% (w/v) ST increased wild-type OMV production and reduced OMV size, but did not further stimulate mlaA mutant OMV production or significantly alter mlaA mutant OMV size. qRT-PCR analysis demonstrated that the presence of ST decreased expression of both mlaA and mlaC in C. jejuni wild-type strains 11168 and 488. Collectively the data in this study suggests C. jejuni can regulate OMV production in response to host gut signals through changes in expression of the MLA pathway. As the gut bile composition is dependent on both diet and the microbiota, this study highlights the potential importance of diet and lifestyle factors on the varying disease presentations associated with gut pathogen infection.

Ion content and its correlation with some physiological parameters in olive cultivars in response to salinity

ABSTRACT- Olive (Olea europaea L.) is one of the most valuable and widespread fruit trees in Iran. Salt stress-induced changes in membrane stability, photosynthesis and antioxidant enzyme activity were examined on four olive cultivars (Dakal, Shiraz, Zard and Amigdalifolia) by emphasizing the correlation between measured parameters and ion (K+, Na+) accumulation. Plants were subjected to four salt treatments (0, 100, 150 or 200 mM NaCl) under greenhouse conditions. The exposure of the olive plants to increased salinity resulted in a decline in relative leaf chlorophyll content (RLCC), photosynthesis rate (Pn), transpiration rate (E) and leaf and root K+ content. NaCl increased superoxide dismutase (SOD) and peroxidase (POX) activity of olive leaves. Increasing the concentrations of NaCl in soil increased the concentration of Na+ in the leaves and roots. Differences in the effectiveness of Na+ exclusion mechanism among cultivars at high salinity reflected differences in salt tolerance. ‘Zard’, the better-adapted cultivar, displayed tolerance to high internal salt concentrations without apparent cell damage. Relationships between parameters involved in salinity response are discussed in relation to ion accumulation in leaves and roots of olive cultivars.

Effects of sex and age on the osmotic stability of Sahel goat erythrocytes

Adaptation of goats to the arid and semi-arid environments may involve adjustments in erythrocyte osmoregulation. Sexual dimorphic and age-related effects on osmotic stability of the goat erythrocyte were investigated to determine physiological variations. Apparently healthy male (n = 23) and non-pregnant dry female (n = 24) Sahel goats, aged 1½–2, >2–2½, and >2½ years with mean body weights of 10.35–27.21 kg, were selected for the study from a semi-intensively managed flock. Heparinised venous blood of each goat was used to determine packed cell volume, erythrocyte count, mean corpuscular volume and osmotic fragility. Erythrocyte parameters did not vary with sex or age of the animal. Erythrocyte osmotic fragility (EOF), determined in hypotonic buffered saline, did not show sexual difference, but age elicited significant (p 1½–2 years, respectively. Body weights increased (p < 0.05; r = 0.96) with age, and 85.8 % weight gain occurred in the transition from age of <1 to 1–1½ years, whereas subsequent weight gains in other age transitions were 8.3, 18.8 and 9.9 %, respectively. Sex and age did not influence saline tonicities at the median erythrocyte fragility. The effect of age on EOF at minimal hypotonicity, occurring after a period of rapid growth, seemed to associate the change in membrane stability with actions of growth-promoting hormones on lipid metabolism which could have altered the lipid content and permeability of erythrocyte membrane.

Osmolyte Accumulation, Membrane Stability and ABA Profiles in Rice Genotypes Exposed to Heat and Drought Stress

The effect of combined heat and drought stress on proline, chlorophyll stability index, membrane thermal stability and abscisic acid (ABA) contents were investigated to screen and study the physiological basis of heat and drought tolerance in three rice genotypes (ADT 43, TKM 9 and N 22). The stresses were imposed at panicle initiation (PI) and anthesis stages of crop growth. The genotype N 22 was found to be the tolerant followed by TKM 9 while ADT 43 was observed to the susceptible genotype based on the above physiological and biochemical traits. Irrespective of genotypes, anthesis stage stress brought about accumulation of osmolyte and ABA contents with membrane stability changes compared to stress at PI stage. ADT 43 recorded low proline content of (201 μg g−1), CSI of (52.5%), MTS of (24.39%) and ABA content of (0.587 μg g−1). While, N 22 recorded higher proline content of (252.6 μg g−1), CSI of (89.8%), MTS of (33.08%) and ABA content of (0.685 μg g−1). Higher proline and ABA contents with improved membrane stability was clearly demonstrated in the tolerant genotype (N 22) compared to susceptible genotype (ADT 43). This conforms that these biochemical traits can be used to screen rice genotypes to combined stresses and understand the mechanism underlying stress tolerance.

Reduced ABCA1-Mediated Cholesterol Efflux and Accelerated Atherosclerosis in Apolipoprotein E–Deficient Mice Lacking Macrophage-Derived ACAT1

Macrophage acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) and apolipoprotein E (apoE) have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation. Deletion of either ACAT1 or apoE results in increased atherosclerosis in hyperlipidemic mice, possibly as a consequence of altered cholesterol processing. We have studied the effect of macrophage ACAT1 deletion on atherogenesis in apoE-deficient (apoE-/-) mice with or without the restoration of macrophage apoE. We used bone marrow transplantation to generate apoE-/- mice with macrophages of 4 genotypes: apoE+/+/ACAT1+/+ (wild type), apoE+/+/ACAT1-/- (ACAT-/-), apoE-/-/ACAT1+/+ (apoE-/-), and apoE-/-/ACAT1-/- (2KO). When macrophage apoE was present, plasma cholesterol levels normalized, and ACAT1 deficiency did not have significant effects on atherogenesis. However, when macrophage apoE was absent, ACAT1 deficiency increased atherosclerosis and apoptosis in the proximal aorta. Cholesterol efflux to apoA-I was significantly reduced (30% to 40%; P<0.001) in ACAT1-/- peritoneal macrophages compared with ACAT1+/+ controls regardless of apoE expression. 2KO macrophages had a 3- to 4-fold increase in ABCA1 message levels but decreased ABCA1 protein levels relative to ACAT1+/+ macrophages. Microarray analyses of ACAT1-/- macrophages showed increases in proinflammatory and procollagen genes and decreases in genes regulating membrane integrity, protein biosynthesis, and apoptosis. Deficiency of macrophage ACAT1 accelerates atherosclerosis in hypercholesterolemic apoE-/- mice but has no effect when the hypercholesterolemia is corrected by macrophage apoE expression. However, ACAT1 deletion impairs ABCA1-mediated cholesterol efflux in macrophages regardless of apoE expression. Changes in membrane stability, susceptibility to apoptosis, and inflammatory response may also be important in this process.

Water immersion wrinkling--physiology and use as an indicator of sympathetic function.

Water immersion skin wrinkling is an indicator of limb sympathetic function. Routine clinical usage of this enigmatic phenomenon is hampered by poor endpoint quantification, which involves counting skin folds. The recent discovery of the importance of vasoconstriction in immersion wrinkling suggests digital blood flow or volume changes as better endpoints. Water probably initiates the wrinkling process by altering epidermal electrolyte homeostasis as it diffuses into the porous skin of the hands and soles via its many sweat ducts. Altered epidermal electrolyte homeostasis would lead to a change in membrane stability of the surrounding dense network of nerve fibers and trigger increased vasomotor firing with subsequent vasoconstriction. Vasoconstriction, through loss of volume, leads to negative digit pulp pressure resulting in a downward pull on the overlying skin, which wrinkles as it is distorted. The degree of wrinkling would directly depend on the change in digit tip volume and implies any process inducing loss of digit volume will precipitate wrinkling. This review discusses the physiology of water immersion wrinkling and explores its potential as an indicator of limb sympathetic dysfunction.

Role of GPI-anchored enzyme in liposome detergent-resistance.

In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45 degrees C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.

Novel studies on membrane extraction of bioactive components of green tea in organic solvents: part I

Membrane extraction of the bioactive components of green tea in an organic solvent has been investigated using different nanofiltration membranes. Different concentrations of ethanol (10%, 20%, 50% and 80%) were used as the organic solvent. Membrane separation performance of bioactive components of green tea showed some changes in flux and solute rejection with negligible change in membrane stability. Special attention was focused on the removal of caffeine from the solution mixture of the green tea extract while retaining the group of catechins known as polyphenols. A series of tests with various membranes using higher ethanol concentrations (e.g. 80%) gave highest rejection of catechins with lower rejection of caffeine for G-10 and G-20 membranes, respectively. The rejection using HC-50 and HR98PP membranes were fairly good but the pure ethanol fluxes under the same pressure (5.0 MPa) were not promising for this solvent.

Ectoplasmic insertion of a glycosylphosphatidylinositol-anchored protein in glycosphingolipid- and cholesterol-containing phosphatidylcholine vesicles.

Glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and glycosphingolipids (GlySphs). We have prepared biomimetic membranes in order to study the possible phenomena of surface aggregation of these membrane components. Phosphatidylcholine liposomes were treated by octylglucoside to insert a glycosyl-PtdIns-protein, alkaline phosphatase (ALP), some cholesterol, and a GlySph, the lactocerebroside. The association of these compounds was shown by centrifugation on a density gradient. The presence of ALP on the surface of the vesicles was shown by the action of a phospholipase, and the presence of the lactocerebroside was shown by the use of a galactose-specific tetravalent lectin. Our data show that total alkaline phosphatase and half to total lactocerebroside were ectoplasmically inserted in the vesicles membrane. In addition, we observed that the presence of small amounts of ALP in the liposomes led to significant changes in membrane stability with regard to detergent, as shown by the changes in the solubilization process monitored by turbidimetry. Furthermore, we have built an original method to study the cohesion of the vesicles membrane, in which some magnesium ions were trapped in the luminal space of the liposomes during several days. The ALP is magnesium-dependent for its catalytic activity and was inhibited after incubation of ALP-containing liposomes in a magnesium-free buffer. The ALP activity was restored by the addition of detergent to the liposomes, due to the release of the luminal magnesium ions. Surface aggregation phenomena will now be investigated by atomic force microscopy.

Parallel study of thermal resistance and permeability barrier stability of Enterococcus faecalis as affected by salt composition, growth temperature and pre-incubation temperature

In this study Enterococcus faecalis cells were grown to stationary phase in various conditions resulting in strong but similar variations in both cellular thermoresistance and permeability barrier stability (the temperature T m that induced rapid dissipation of the ion concentration gradient during constant heating). Cells grown at 17–22°C were heat sensitive and barrier labile whilst cells grown at 10–13°C and 42–47°C were heat resistant and barrier stable. The thermal resistance and barrier stability in heat-sensitive cells, compared to the same parameters in heat-resistant cells, remained low after an additional culture at 43–47°C, indicating a persistent effect of culture at 17–22°C. In cells grown at 10–13°C, these parameters were as low as they were in the heat-sensitive cells, provided the growth media contained an ammonium salt (1%) which thus abolished the cold acclimation. Both parameters were reduced in cells growth at increased salinity (1–3% Na and K salts) and the reduction was more pronounced during growth at 17–22°C. Moreover, cells pre-cultured at 21°C with increased salinity (3% NaCl) displayed strong phenotypic effect during subsequent culturing which reflected in a 6°C decrease in both the optimal temperature and maximal temperature of growth. Compared to other bacterial strains, only a part of the change in membrane stability could be related to the variations in fatty acid composition. The index of unsaturation changed in accordance with the barrier stability and survival of cells. These findings support the conclusion that stability of permeability barrier as affected by the growth temperature, presence of ammonium and cultural conditions of progenitor cells was involved in thermal sensitivity and temperature-acclimation of E. faecalis.

Evaluation of plasma membrane stability by detergent-induced rupture of osmotically swollen sperm

A general assay for plasma membrane stability was developed and tested. Osmotically swollen spermatozoa were ruptured with detergents and their volume distribution was monitored with resistance pulse spectroscopy. The extent of cell breakage was determined and expressed as [D]50, the concentration of detergent necessary to lyse 50% of the initially intact cells. Preliminary experiments established the degree to which spermatozoa could be swollen without lysis (no detergent) and the ability of the method to detect known mixtures of intact and membrane disrupted spermatozoa. [D]50 values were determined for caput (immature) and cauda (mature) ram epididymal spermatozoa with four detergents (cetyltrimethylammonium bromide, sodium dodecylsulfate, Zwittergent 3–14, and sodium deoxycholate). [D]50 values for caput spermatozoa were higher than those for cauda spermatozoa (P < 0.05) for all detergents but cetyltrimethylammonium bromide. Theese changes are consistent with a qualitative model of membrane structure and stability based on lipid shape and composition and with the compositional changes known to occur during epididymal maturation. Additional studies using rooster spermatozoa established that a typical cryopreservation protocol leaves the surviving spermatozoa with membranes with greater sensitivity to detergent-induced stress. Since osmotic swelling has been microscopically localized to the tail plasma membrane, the changes in membrane stability can be assigned specifically to that region.

Regulation of red cell membrane deformability and stability by skeletal protein network

The skeletal protein network of the red blood cell is thought to be important in regulating such membrane functions as deformability and stability. In the present study, we measured membrane deformability and stability of the resealed ghosts using an ektacytometer, a laser diffraction method, and identified the functional role of protein 4.1 and that of Ca2+ and calmodulin in maintaining membrane stability. To obtain direct evidence for a crucial role of protein 4.1 in maintaining membrane stability, we reconstituted protein 4.1-deficient membranes with purified protein 4.1. Although native membranes deficient in protein 4.1 had marked reduction in membrane stability, reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability, providing direct evidence that protein 4.1 is essential for normal membrane stability. To determine if Ca2+ and calmodulin could modulate membrane properties, we measured membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and physiologic concentrations of calmodulin. Our data show that Ca2+ concentrations in the range of 1 to 100 microM can markedly decrease membrane stability only in the presence of calmodulin, but not in its absence. In contrast, deformability decreased only at Ca2+ concentrations higher than 100 microM, and calmodulin had no effect. Examination of the the effects of Ca2+ and calmodulin on various membrane protein interactions has enabled us to suggest that the observed changes in membrane stability may be partly related to the effects of Ca2+ and calmodulin on spectrin-protein 4.1-actin interaction.

Modulation of erythrocyte membrane material properties by Ca2+ and calmodulin. Implications for their role in regulation of skeletal protein interactions.

Skeletal proteins of the red blood cell apparently play an important role in regulating membrane material properties of deformability and stability. However, the role of various intracellular constituents in regulating membrane properties has not been clearly defined. To determine whether Ca2+ and calmodulin might play a role in this regulation, we measured the membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and calmodulin (CaM). For membranes resealed in the presence of Ca2+ and physiologic concentrations of CaM (2-8 microM), membrane stability decreased with increasing Ca2+ concentrations (greater than 1.0 microM). Moreover, Ca2+ and CaM-induced alterations in membrane stability were completely reversible. In the absence of CaM, an equivalent decrease in membrane stability was seen only when Ca2+ concentration was two orders of magnitude higher (greater than 100 microM). Calmodulin did not alter membrane stability in the absence of Ca2+. Compared with these changes in membrane stability, membrane deformability decreased only at Ca2+ concentrations greater than 100 microM, and calmodulin had no effect on Ca2+-induced decrease in membrane deformability. Examination of the effects of Ca2+ and CaM on various membrane interactions have enabled us to suggest that spectrin-protein 4.1-actin interaction may be one of the targets for the effect of Ca2+ and CaM. These results imply that Ca2+ and calmodulin can regulate membrane stability through modulation of skeletal protein interactions, and that these protein interactions are of a dynamic nature on intact membranes.

Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations.

Skeletal proteins play an important role in determining erythrocyte membrane biophysical properties. To study whether membrane deformability and stability are regulated by the same or different skeletal protein interactions, we measured these two properties, by means of ektacytometry, in biochemically perturbed normal membranes and in membranes from individuals with known erythrocyte abnormalities. Treatment with 2,3-diphosphoglycerate resulted in membranes with decreased deformability and decreased stability, whereas treatment with diamide produced decreased deformability but increased stability. N-ethylmaleimide induced time-dependent changes in membrane stability. Over the first minute, the stability increased; but with continued incubation, the membranes became less stable than control. Meanwhile, the deformability of these membranes decreased with no time dependence. Biophysical measurements were also carried out on pathologic erythrocytes. Membranes from an individual with hereditary spherocytosis and a defined abnormality in spectrin-protein 4.1 association showed decreased stability but normal deformability. In a family with hereditary elliptocytosis and an abnormality in spectrin self-association, the membranes had decreased deformability and stability. Finally, membranes from several individuals with Malaysian ovalocytosis had decreased deformability but increased stability. Our data from both pathologic membranes and biochemically perturbed membranes show that deformability and stability change with no fixed relationship to one another. These findings imply that different skeletal protein interactions regulate membrane deformability and stability. In light of these data, we propose a model of the role of skeletal protein interactions in deformability and stability.

On membrane phospholipids and protein-lipid association

Abstract

Effect of membrane modification on cell fusion of hen erythrocytes induced by dimethyl sulfoxide

Fusion of hen erythrocytes is inhibited by millimolar concentrations of bifunctional reagents glutaraldehyde, formaldehyde and dimethyl suberimidate and by low concentrations of detergents Triton X-100 and sodium dodecylsulfate, whereas fusion is activated by organomercurials p -chloromercuribenzene sulfonate and p -chloromercuribenzoate. The effects are interpreted in terms of changes in membrane stability produced by these reagents.

The modification of water structure by divalent cations as a mechanism of membrane permeability control.

During the past two decades the emphasis of research in cryobiology has shifted away from the mechanical effects of ice crystals on cells and has focused increasingly on the dehydration and solute concentration which result when water is frozen out of solution. Our own studies have led us to two tentative conclusions regarding freezing injury in living cells (1, 2). First, the primary injury is to the cell membrane and is associated with an osmotic dehydration and volume reduction of the cell beyond a tolerable limit. Second, membrane dehydration is probably responsible for the changes in membrane stability that are seen. The mechanism by which the loss of membrane water influences membrane properties has been of particular interest to us. Our concern with the role of water and its influence on membrane structure and permeability has led us to the experiments, observations and speculations that are summarized in this paper.