[Show abstract][Hide abstract] ABSTRACT: A recurring question in membrane biological chemistry is whether bioactive signaling lipids act only as second messenger ligands or also through an effect on bilayer physical properties. Sphingosine (Sph) and sphingosine-1-phosphate (S1P) are single-chained charged sphingolipids that have antagonistic functions in the "sphingolipid rheostat" which determines cell fate. Sph and S1P respectively promote apoptosis and cell growth. In the present study, potential effects of these bioactive lipids on physicochemical properties of the lipid bilayer of cell membranes were evaluated. We have investigated the effect of both sphingolipids, incorporated separately or, for the first time, together, in large or giant phosphadidylcholine (PC) unilamellar vesicles. Three bilayer properties were examined: membrane surface charge, lipid packing, and formation of membrane microdomains. Sph and S1P appear to have distinct, when not inverse, effects on all three properties. Besides, when both sphingolipids are mixed together, their effects on lipid packing are synergistic, whereas their effects on microdomain formation and zeta-potential are mostly antagonistic. These results are interpreted as arising from different electrostatic interactions between lipid headgroups. In particular, Sph and S1P may interact together electrostatically and form a complex. These mostly inverse and opposing effects of both single-chain phospholipids on membrane physical properties might be involved in their antagonistic role in regulating cell fate. Particularly, the mutual interaction between Sph and S1P as a complex might be able to sequester both molecules in a biologically inactive form and therefore to promote a mutual regulation of their biological activities, depending on their ratio, consistent with the sphingolipid rheostat.
[Show abstract][Hide abstract] ABSTRACT: In a previous work, we have shown that a spatially localized transmembrane pH gradient, produced by acid micro-injection near the external side of cardiolipin-containing giant unilamellar vesicles, leads to the formation of tubules that retract after the dissipation of this gradient. These tubules have morphologies similar to mitochondrial cristae. The tubulation effect is attributable to direct phospholipid packing modification in the outer leaflet, that is promoted by protonation of cardiolipin headgroups. In this study, we compare the case of cardiolipin-containing giant unilamellar vesicles with that of giant unilamellar vesicles that contain phosphatidylglycerol (PG). Local acidification also promotes formation of tubules in the latter. However, compared with cardiolipin-containing giant unilamellar vesicles the tubules are longer, exhibit a visible pearling, and have a much longer lifetime after acid micro-injection is stopped. We attribute these differences to an additional mechanism that increases monolayer surface imbalance, namely inward PG flip-flop promoted by the local transmembrane pH gradient. Simulations using a fully nonlinear membrane model as well as geometrical calculations are in agreement with this hypothesis. Interestingly, among yeast mutants deficient in cardiolipin biosynthesis, only the crd1-null mutant, which accumulates phosphatidylglycerol, displays significant mitochondrial activity. Our work provides a possible explanation of such a property and further emphasizes the salient role of specific lipids in mitochondrial function.
[Show abstract][Hide abstract] ABSTRACT: Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting "raft-like" Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including of C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A "morphological" phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and to a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, "arresting" domain growth and thereby stabilizing Lo nanodomains.
[Show abstract][Hide abstract] ABSTRACT: Microbiological investigations of cultural heritage may be helpful in the choice of possible intervention for the prevention of biological degradation of works of art. Fungi presented in stone, fretwork and fresco samples from different objects in Rila monastery were isolated and characterized. Pure cultures of 9 strains were obtained and tested for their ability to grow (mycelia mat radial extension) on four agar media at temperature 30°C. The best growth occurred on Sabouraud agar, beer agar and potato glucose agar.
[Show abstract][Hide abstract] ABSTRACT: Antioxidant enzymes are essential for living cells, producing protection from reactive oxygen species such as superoxide, which cause oxidative damage to cell structures. Superoxide dismutase (SOD) and catalase (CAT) activities were determined for three Vibrio strains (Vibrio cholerae non O1/29, V. cholerae non O1/29-T and V. cholerae non O1/26) aerobically grown at 30°C (optimal level) and 10 °C (cold stress). All strains tested expressed both antioxidant enzymes under normophysiological and stress conditions, but the cell response is more strain-dependent than dependent on temperature. Levels of SOD in cultures of V. cholerae non O1/26 grown at 30°C were about 2.5 to 9.5-times higher than those in the cultures grown at 10°C. In contrast, SOD activity in V. cholerae non O1/29 increased by 7.5-fold under stress conditions in comparison to that at optimal temperature. The strain V. cholerae non O1/29-T did not show any significant difference in the cell response depending on the growth temperature. CAT activity in cells of V. cholerae non O1/26 and V. cholerae non O1/29-T exhibited a similar tendency suggesting that this enzyme is not included in antioxidant response against cold stress. Contrary to the above observations, V. cholerae non 01/29 demonstrated higher CAT activity in response to temperature downshift. Only one SOD isoenzyme was detected in each of the three Vibrio strains by native PAGE analysis.
[Show abstract][Hide abstract] ABSTRACT: Light and electron microscopy of immobilized system of the Claviceps sp. strain in Ca-alginate reveal some peculiarities in the growth and morphology of the cells as compared to the free ones. The immobilized cells develop mainly in the surface and subsurface layer of the alginate beads. Cells of differing physiological state are observed. Higher is the number of the sclerotia-like cells and of the arthrosporoid-like structures.
[Show abstract][Hide abstract] ABSTRACT: Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, while Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles, indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells.
[Show abstract][Hide abstract] ABSTRACT: We study the deformation of a lipid membrane in response to a local pH
modification. Experimentally, a basic solution is microinjected close to a
giant unilamellar vesicle. A local deformation appears in the zone of the
membrane that is closest to the micropipette, and relaxes when the injection is
stopped. A theoretical description of this phenomenon is provided. It takes
fully into account the spatiotemporal evolution of the concentration of
hydroxide ions during and after the microinjection, as well as the linear
dynamics of the membrane. This description applies to a local injection of any
substance that reacts reversibly with the membrane lipids. We compare
experimental data obtained in the domain of small deformations to the results
of our linear description, and we obtain a good agreement between theory and
experiments. In addition, we present direct experimental observations of the pH
profile on the membrane during and after the microinjection, using pH-sensitive
[Show abstract][Hide abstract] ABSTRACT: To investigate the relationship among the intracellular antioxidant activity (superoxide dismutase, SOD; catalase, CAT), the apoptosis of granulose luteinized cells (GLCs) and the controlled ovarian hyperstimulation/ in-vitro- fertilization (COH/IVF) outcome in infertile women. The GLCs were obtained from follicular fluids (FFls) of 42 infertile women undergoing COH/IVF by gradient centrifugation. The activity of SOD, CAT and the level of GLCs apoptosis were measured using the Beaushap and Fridovich; Beers and Sizer and DNA-DAPI staining respectively. The statistical analysis was made with ANOVA test and Correlation matrices. The activity of the intracellular antioxidant enzymes was significantly higher in the group of infertile women with a low level of apoptosis compared to the infertile women with a high level of apoptosis (P < 0.05). A positive relationship of SOD activity with retrieved and fertilized oocytes was documented (P < 0.05). A negative correlation between the level of apoptotic GLCs and COH/IVF outcome was found (P < 0.05). The higher activity of intracellular SOD and CAT in human GLCs was related with their low level of apoptosis and with better results of COH/IVF outcome.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is a degenerative disease of the central nervous system which causes irreversible damage to neuron structure and function. The main hypothesis concerning the cause of AD is excessive accumulation of amyloid-β peptides (Aβ). There has recently been a surge in studies on neuronal morphological and functional pathologies related to Aβ-induced mitochondrial dysfunctions and morphological alternations. What is the relation between the accumulation of Aβ in mitochondria, decreased production of ATP, and the large number of mitochondria with broken or scarce cristae observed in AD patients' neurons? The problem is complex, as it is now widely recognized that mitochondria function determines mitochondrial inner membrane (IM) morphology and, conversely, that IM morphology can influence mitochondrial functions. In our previous work, we designed an artificial mitochondrial IM, a minimal model system (giant unilamellar vesicle) mimicking the IM. We showed experimentally that modulation of the local pH gradient at the membrane level of cardiolipin-containing vesicles induces dynamic membrane invaginations similar to the mitochondrial cristae. In the present work we show, using our artificial IM, that Aβ renders the membrane unable to support the formation of cristae-like structures when local pH gradient occurs, leading to the failure of this cristae-like morphology. Fluorescent probe studies suggest that the dramatic change of membrane mechanical properties is due to Aβ-induced lipid bilayer dehydration, increased ordering of lipids, loss of membrane fluidity, and possibly to Aβ-induced changes in dynamic friction between the two leaflets of the lipid membrane.
[Show abstract][Hide abstract] ABSTRACT: Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.
[Show abstract][Hide abstract] ABSTRACT: Deleterious effects of free radicals do not only result from the amount of free radicals produced but also are related to the efficiency and to the activities of enzymatic antioxidant systems. We investigated the effect of exogenous superoxide dismutase (SOD1) or Cu-chelating agent diethyldithiocarbamate (DDC) on the apoptosis (caspase-3 activity) of human granulosa luteinized cells (hGLC) in vitro.
The effects of SOD1 and DDC were studied using in vitro culture system, caspase-3 and the total SOD activity in hGLCs were measured using AcDEVD-PNA substrate and Beaushap and Fridovich methods, respectively, after 48 h of the culture period.
The activity of SOD1 was the lowest in GLCs treated with 100 µM DDC as compared to control cells and to the cells supplemented with Cu, Zn-SOD or DDC (10 µM). The effect of DDC was associated with elevated caspase-3 activity as compared to control cells.
It was demonstrated for the first time that the supplementation of cultured hGLCs with Cu2+, Zn2+-SOD (200 U/ml) maintains the viability of hGLCs via caspase-3 suppression.
[Show abstract][Hide abstract] ABSTRACT: Cardiolipin is a four-tailed acidic lipid found predominantly within the inner membrane of mitochondria, and is thought to be a key component in determining inner membrane properties and potential. Thus, cardiolipin may be involved in the dynamics of the inner membrane characteristic invaginations (named cristae) that protrude into the matrix space. In previous studies, we showed the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid model membrane mimicking the inner mitochondrial membrane. In addition, we reported a theoretical model describing the dynamics of a chemically driven membrane shape instability caused by a modification of the plane-shape equilibrium density of the lipids in the membrane. In the present work, we focus on the lipid-packing modifications observed in a model cardiolipin-containing lipid membrane submitted to pH decrease because this is the driving force of the instability. Laurdan fluorescence and ζ-potential measurements show that under pH decrease, membrane surface charge decreases, but that significant modification of the lipid packing is observed only for CL-containing membranes. Our giant unilamellar vesicle experiments also indicate that cristae-like morphologies are only observed for CL-containing lipid membranes. Taken together, these results highlight the fact that only a strong modulation of the lipid packing of the exposed monolayer leads to membrane shape instability and suggest that mitochondrial lipids, in particular the cardiolipin, play a specific role under pH modulation in inner mitochondrial membrane morphology and dynamics.
[Show abstract][Hide abstract] ABSTRACT: We study a dynamical curvature instability caused by a local chemical modification of a phospholipid membrane. In our experiments, a basic solution is microinjected close to a giant unilamellar vesicle, which induces a local chemical modification of some lipids in the external monolayer of the membrane. This modification causes a local deformation of the vesicle, which then relaxes. We present a theoretical description of this instability, taking into account both the change of the equilibrium lipid density and the change of the spontaneous membrane curvature induced by the chemical modification. We show that these two types of changes of the membrane properties yield different dynamics. In contrast, it is impossible to distinguish them when studying the equilibrium shape of a vesicle subjected to a global modification. In our model, the longest relaxation timescale is related to the intermonolayer friction, which plays an important part when there is a change in the equilibrium density in one monolayer. We compare our experimental results to the predictions of our model by fitting the measured time evolution of the deformation height to the solution of our dynamical equations. We obtain good agreement between theory and experiments. Our fits enable us to estimate the intermonolayer friction coefficient, yielding values that are consistent with previous measurements.
[Show abstract][Hide abstract] ABSTRACT: The members of Verbascum L. (Scrophulariaceae) are known to be rich in phenylethnoid glycosides,
and among them Verbascum xanthophoeniceum is an endemic plant species for the Balkan region,
Northwestern, and Southern Turkey. A scheme was developed for the isolation of the main active
constituents that accumulate in plant aerial parts. The antioxidant activities of total methanol
extracts, collected phenylethanoid glycosides fractions and specific active constituents (forsythoside B,
verbascoside and leucosceptoside B) were then evaluated in 2,20-diphenyl-1-picrylhydrazyl (DPPH�),
oxygen radical absorbance capacity (ORACFL), hydroxyl radical averting capacity (HORACFL), ferric-reducing
antioxidant power (FRAP), and superoxide anion ðO�� 2 Þ radical scavenging assays. In vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BChe) inhibitory activities of abovementioned extracts, fractions and isolated pure compounds were also examined. Depending on the method used, forsythoside B, verbascoside and leucosceptoside B proved to be effective radical scavengers and cholinesterases inhibitors. On the basis of these findings it can be proposed that in addition to providing a potent source of antimicrobial and anti-inflammatory compounds, Verbascum plants could serve as attractive mines of powerful antioxidants for various purposes.