Membrane Rigidification Research Articles

Cold sensing in grapevine-Which signals are upstream of the microtubular "thermometer".

Plants can acquire freezing tolerance in response to cold but non-freezing temperatures. To efficiently activate this cold acclimation, low temperature has to be sensed and processed swiftly, a process that is linked with a transient elimination of microtubules. Here, we address cold-induced microtubules elimination in a grapevine cell line stably expressing a green fluorescent protein fusion of Arabidopsis TuB6, which allows to follow their response in vivo and to quantify this response by quantitative image analysis. We use time-course studies with several specific pharmacological inhibitors and activators to dissect the signalling events acting upstream of microtubules elimination. We find that microtubules disappear within 30min after the onset of cold stress. We provide evidence for roles of calcium influx, membrane rigidification, and activation of NAD(P)H oxidase as factors in signal susception and amplification. We further conclude that a G-protein in concert with a phospholipase D convey the signal towards microtubules, whereas calmodulin seems to be not involved. Moreover, activation of jasmonate pathway in response to cold is required for an efficient microtubule response. We summarize our findings in a working model on a complex signalling hub at the membrane-cytoskeleton interphase that assembles the susception, perception and early transduction of cold signals.

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii.

A conserved reaction of all organisms exposed to heat stress is an increased expression of heat shock proteins (HSPs). Several studies have proposed that HSP expression in heat-stressed plant cells is triggered by an increased fluidity of the plasma membrane. Among the main lines of evidence in support of this model are as follows: (a) the degree of membrane lipid saturation was higher in cells grown at elevated temperatures and correlated with a lower amplitude of HSP expression upon a temperature upshift, (b) membrane fluidizers induce HSP expression at physiological temperatures, and (c) membrane rigidifier dimethylsulfoxide dampens heat-induced HSP expression. Here, we tested whether this holds also for Chlamydomonas reinhardtii. We show that heat-induced HSP expression in cells grown at elevated temperatures was reduced because they already contained elevated levels of cytosolic HSP70A/90A that apparently act as negative regulators of heat shock factor 1. We find that membrane rigidifier dimethylsulfoxide impaired translation under heat stress conditions and that membrane fluidizer benzyl alcohol not only induced HSP expression but also caused protein aggregation. These findings support the classical model for the cytosolic unfolded protein response, according to which HSP expression is induced by the accumulation of unfolded proteins. Hence, the membrane fluidity model should be reconsidered.

The Very Long Chain Fatty Acid (C26:25OH) Linked to the Lipid A Is Important for the Fitness of the Photosynthetic Bradyrhizobium Strain ORS278 and the Establishment of a Successful Symbiosis with Aeschynomene Legumes.

In rhizobium strains, the lipid A is modified by the addition of a very long-chain fatty acid (VLCFA) shown to play an important role in rigidification of the outer membrane, thereby facilitating their dual life cycle, outside and inside the plant. In Bradyrhizobium strains, the lipid A is more complex with the presence of at least two VLCFAs, one covalently linked to a hopanoid molecule, but the importance of these modifications is not well-understood. In this study, we identified a cluster of VLCFA genes in the photosynthetic Bradyrhizobium strain ORS278, which nodulates Aeschynomene plants in a Nod factor-independent process. We tried to mutate the different genes of the VLCFA gene cluster to prevent the synthesis of the VLCFAs, but only one mutant in the lpxXL gene encoding an acyltransferase was obtained. Structural analysis of the lipid A showed that LpxXL is involved in the transfer of the C26:25OH VLCFA to the lipid A but not in the one of the C30:29OH VLCFA which harbors the hopanoid molecule. Despite maintaining the second VLCFA, the ability of the mutant to cope with various stresses (low pH, high temperature, high osmolarity, and antimicrobial peptides) and to establish an efficient nitrogen-fixing symbiosis was drastically reduced. In parallel, we investigated whether the BRADO0045 gene, which encodes a putative acyltransferase displaying a weak identity with the apo-lipoprotein N-acyltransferase Lnt, could be involved in the transfer of the C30:29OH VLCFA to the lipid A. Although the mutant exhibited phenotypes similar to the lpxXL mutant, no difference in the lipid A structure was observed from that in the wild-type strain, indicating that this gene is not involved in the modification of lipid A. Our results advance our knowledge of the biosynthesis pathway and the role of VLCFAs-modified lipid A in free-living and symbiotic states of Bradyrhizobium strains.

The adiponectin receptor AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 prevent membrane rigidification by exogenous saturated fatty acids.

Dietary fatty acids can be incorporated directly into phospholipids. This poses a specific challenge to cellular membranes since their composition, hence properties, could greatly vary with different diets. That vast variations in diets are tolerated therefore implies the existence of regulatory mechanisms that monitor and regulate membrane compositions. Here we show that the adiponectin receptor AdipoR2, and its C. elegans homolog PAQR-2, are essential to counter the membrane rigidifying effects of exogenously provided saturated fatty acids. In particular, we use dietary supplements or mutated E. coli as food, together with direct measurements of membrane fluidity and composition, to show that diets containing a high ratio of saturated to monounsaturated fatty acids cause membrane rigidity and lethality in the paqr-2 mutant. We also show that mammalian cells in which AdipoR2 has been knocked-down by siRNA are unable to prevent the membrane-rigidifying effects of palmitic acid. We conclude that the PAQR-2 and AdipoR2 proteins share an evolutionarily conserved function that maintains membrane fluidity in the presence of exogenous saturated fatty acids.

Membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in Ganoderma lucidum.

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

The molecular mechanism by which saturated lysophosphatidylcholine attenuates the metastatic capacity of melanoma cells.

Lysophophatidylcholine (LysoPC) is an abundant constituent in human plasma. Patients with malignant cancer diseases have attenuated LysoPC plasma levels, and thus LysoPC has been examined as a metabolic biomarker for cancer prediction. Preclinical studies have shown that solid tumor cells drastically degrade LysoPCs by incorporating their free fatty acids into cell membrane phospholipids. In this way, LysoPC C18:0 reduced the metastatic spread of murine melanoma B16.F10 cells in mice. Although membrane rigidification may have a key role in the attenuation of metastasis, evidence for this has yet to be shown. Therefore, the present study aimed to determine how LysoPC reduces the metastatic capacity of B16.F10 cells. Following cellular preincubation with LysoPC C18:0 at increasing concentrations and lengths of time, cell migration was most significantly attenuated with 450 μm LysoPC C18:0 at 72 h. Biosensor measurements suggest that, despite their abundance in B16.F10 cells, LysoPC‐sensitive G protein‐coupled receptors do not appear to contribute to this effect. Instead, the attenuated migration appears to result from changes in cell membrane properties and their effect on underlying signaling pathways, most likely the formation of focal adhesion complexes. Treatment with 450 μm LysoPC C18:0 activates protein kinase C (PKC)δ to phosphorylate syndecan‐4, accompanied by deactivation of PKCα. Subsequently, focal adhesion complex formation was attenuated, as confirmed by the reduced activity of focal adhesion kinase (FAK). Interestingly, 450 μm LysoPC C18:1 did not affect FAK activity, explaining its lower propensity to affect migration and metastasis. Therefore, membrane rigidification by LysoPC C18:0 appears to prevent the formation of focal adhesion complexes, thus affecting integrin activity as a key for metastatic melanoma spread.

Investigations of the influence of liposome composition on vesicle stability and drug transfer in human plasma: a transfer study

Liposomal delivery constitutes a promising approach for i.v. administration of temoporfin (mTHPC) because lipid membranes can host these drug molecules. This study investigates the transfer and release of mTHPC to plasma proteins and stability of various liposomal formulations. To this end, we employed traces of radioactive markers and studied the effects of fatty acid chain length and the degree of saturation in the lipophilic tail, addition of cholesterol and PEGylation of the membrane surface and different drug-to-lipid ratios (DLRs). Liposomes were incubated in human plasma for various incubation times. Drawn samples were separated by asymmetrical flow field-flow fractionation (AF4). Drug was recovered in four fractions identified as albumin, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and liposomes. Our results suggest that mTHPC fits best into fluid, unmodified bilayers when the drug-to-lipid ratio is low. Membrane rigidification as well as the presence of cholesterol and PEGyated lipids reduced the ability of the membrane to accommodate the drug but simultaneously improved the vesicle stability in plasma. Both mechanisms jointly affect the total degree of mTHPC release. We analyzed our data using a kinetic model that suggests the drug to be associated with the host membrane in two distinct states of which only one interacts directly with the plasma compartment.

Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation.

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10% and 5h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

LPS-Induced Macrophage Activation and Plasma Membrane Fluidity Changes are Inhibited Under Oxidative Stress

Macrophage activation is essential for a correct and efficient response of innate immunity. During oxidative stress membrane receptors and/or membrane lipid dynamics can be altered, leading to dysfunctional cell responses. Our aim is to analyze membrane fluidity modifications and cell function under oxidative stress in LPS-activated macrophages. Membrane fluidity of individual living THP-1 macrophages was evaluated by the technique two-photon microscopy. LPS-activated macrophage function was determined by TNFα secretion. It was shown that LPS activation causes fluidification of macrophage plasma membrane and production of TNFα. However, oxidative stress induces rigidification of macrophage plasma membrane and inhibition of cell activation, which is evidenced by a decrease of TNFα secretion. Thus, under oxidative conditions macrophage proinflammatory response might develop in an inefficient manner.

Hopanoids play a role in stress tolerance and nutrient storage in the cyanobacterium Nostoc punctiforme.

Hopanes are abundant in ancient sedimentary rocks at discrete intervals in Earth history, yet interpreting their significance in the geologic record is complicated by our incomplete knowledge of what their progenitors, hopanoids, do in modern cells. To date, few studies have addressed the breadth of diversity of physiological functions of these lipids and whether those functions are conserved across the hopanoid-producing bacterial phyla. Here, we generated mutants in the filamentous cyanobacterium, Nostoc punctiforme, that are unable to make all hopanoids (shc) or 2-methylhopanoids (hpnP). While the absence of hopanoids impedes growth of vegetative cells at high temperature, the shc mutant grows faster at low temperature. This finding is consistent with hopanoids acting as membrane rigidifiers, a function shared by other hopanoid-producing phyla. Apart from impacting fitness under temperature stress, hopanoids are dispensable for vegetative cells under other stress conditions. However, hopanoids are required for stress tolerance in akinetes, a resting survival cell type. While 2-methylated hopanoids do not appear to contribute to any stress phenotype, total hopanoids and to a lesser extent 2-methylhopanoids were found to promote the formation of cyanophycin granules in akinetes. Finally, although hopanoids support symbiotic interactions between Alphaproteobacteria and plants, they do not appear to facilitate symbiosis between N.punctiforme and the hornwort Anthoceros punctatus. Collectively, these findings support interpreting hopanes as general environmental stress biomarkers. If hopanoid-mediated enhancement of nitrogen-rich storage products turns out to be a conserved phenomenon in other organisms, a better understanding of this relationship may help us parse the enrichment of 2-methylhopanes in the rock record during episodes of disrupted nutrient cycling.

Biological Activity of Japanese Quince Extract and Its Interactions with Lipids, Erythrocyte Membrane, and Human Albumin.

The aim of the study was to determine in vitro biological activity of fruit ethanol extract from Chaenomeles speciosa (Sweet) Nakai (Japanese quince, JQ) and its important constituents (−)-epicatechin (EC) and chlorogenic acid (CA). The study also investigated the structural changes in phosphatidylcholine (PC) liposomes, dipalmitoylphosphatidylcholine liposomes, and erythrocyte membranes (RBC) induced by the extract. It was found that the extract effectively inhibits oxidation of RBC, induced by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), and PC liposomes, induced by UVB radiation and AAPH. Furthermore, JQ extract to a significant degree inhibited the activity of the enzymes COX-1 and COX-2, involved in inflammatory reactions. The extract has more than 2 times greater activity in relation to COX-2 than COX-1 (selectivity ratio 0.48). JQ extract stimulated growth of the beneficial intestinal bacteria Lactobacillus casei and Lactobacillus plantarum. In the fluorimetric method by means of the probes Laurdan, DPH and TMA-DPH, and 1H-NMR, we examined the structural changes induced by JQ and its EC and CA components. The results show that JQ and its components induce a considerable increase of the packing order of the polar heads of lipids with a slight decrease in mobility of the acyl chains. Lipid membrane rigidification could hinder the diffusion of free radicals, resulting in inhibition of oxidative damage induced by physicochemical agents. JQ extract has the ability to quench the intrinsic fluorescence of human serum albumin through static quenching. This report thus could be of huge significance in the food industry, pharmacology, and clinical medicine.

Does hydrogen bonding contribute to lipoperoxidation-dependent membrane fluidity variation? An EPR-spin labeling study

This study is aimed at making clear the relationship between oxidative stress of the phospholipid bilayer and membrane fluidity. Di-(hydroperoxylinoleoyl)-phosphatidylcholine (diHpLPC) was used as a highly hydroperoxidized and unsaturated phospholipid species in order to investigate the issue. Hydrophylic Interaction Liquid Chromatography–ElectroSpray Ionization–Mass Spectrometry (HILIC-ESI-MS) and NMR spectroscopy were employed to define the structure of the peroxidized phospholipid as 1-(9-hydroperoxy-10c,12t)octadecadienoyl-2-(9t,11c-13-hydroperoxy)octadecadienoyl-sn-glycero-3-phosphorylcholine. This phospholipid's ability to form vesicular structures was confirmed by Sepharose 4B gel filtration and Dynamic Light Scattering (DLS) of its aqueous suspensions. Fatty acid misalignment and fluidity gradient were studied in the bilayer of both supported planar bilayers (SPB) and multilamellar vesicles (MLV) made of different DLPC/diHpLPC mixtures by means of spin labelling-EPR spectroscopy of either n-DSPC or 3-doxylcholestane spin labels embedded in the membranes. It was found that diHpLPC increases both fatty acid misalignment and rigidification with increasing molar ratio in spite of increasing unsaturation of the fatty acid core. Basing on our observations, the observed ability of pure diHpLPC to form rigid and disordered SPB and MLV bilayers is proposed to be dependent on the cross bridging of oxidized linoleoyl chains by mutual hydrogen bonding of hydroperoxyl groups. However, the contribution to the observed overall rigidification of the model membranes by trans double bonds in the peroxidized chains should not be neglected, as a second membrane fluidity effector also arising from lipid peroxidation.

Increased membrane surface positive charge and altered membrane fluidity leads to cationic antimicrobial peptide resistance in Enterococcus faecalis

To understand the role of cell membrane phospholipids during resistance development to cationic antimicrobial peptides (CAMPs) in Enterococcus faecalis, gradual dose-dependent single exposure pediocin-resistant (Pedr) mutants of E. faecalis (Efv2.1, Efv3.1, Efv3.2, Efv4.1, Efv4.2, Efv5.1, Efv5.2 and Efv5.3), conferring simultaneous resistance to other CAMPs, selected in previous study were characterized for cell membrane phospholipid head-groups and fatty acid composition. The involvement of phospholipids in resistance acquisition was confirmed by in vitro colorimetric assay using PDA (polydiacetylene)-biomimetic membranes. Estimation of ratio of amino-containing phospholipids to amino-lacking phospholipids suggests that phospholipids in cell membrane of Pedr mutants loose anionic character. At moderate level of resistance, the cell-membrane becomes neutralized while at further higher level of resistance, the cell-surface acquired positive charge. Increased expression of mprF gene (responsible for lysinylation of phospholipids) was also observed on acquiring resistance to pediocin in PedrE. faecalis. Decreased level of branched chain fatty acids in Pedr mutants might have contributed in enhancing rigidification of cell membrane and contributing towards resistance. The interaction of pediocin with PDA-biomimetic membranes prepared from wild-type and Pedr mutants was monitored by measuring percent colorimetric response (%CR). Increased %CR of pediocin against PDA-biomimetic membranes prepared from Pedr mutants confirmed that cell membrane phospholipids are involved in the interactions of pore formation by CAMPs. There was a direct linear relationship between percent colorimetric response and IC50 of CAMPs for wild-type and Pedr mutants. This relationship further reveals that in vitro colorimetric assay can be used effectively for quantification of resistance to CAMPs.

The influence of 1-butanol and trisodium citrate ion on morphology and chemical properties of chitosan-based microcapsules during rigidification by alkali treatment.

Linseed oil which has various biomedical applications was encapsulated by chitosan (Chi)-based microcapsules in the development of a suitable carrier. Oil droplets formed in oil-in-water emulsion using sodium dodecyl sulfate (SDS) as emulsifier was stabilized by Chi, and microcapsules with multilayers were formed by alternate additions of SDS and Chi solutions in an emulsion through electrostatic interaction. No chemical cross-linker was used in the study and the multilayer shell membrane was formed by ionic gelation using Chi and SDS. The rigidification of the shell membrane of microcapsules was achieved by alkali treatment in the presence of a small amount of 1-butanol to reduce aggregation. A trisodium citrate solution was used to stabilize the charge of microcapsules by ionic cross-linking. Effects of butanol during alkali treatment and citrate in post alkali treatment were monitored in terms of morphology and the chemical properties of microcapsules. Various characterization techniques revealed that the aggregation was decreased and surface roughness was increased with layer formation.

Cyclopropanation of unsaturated fatty acids and membrane rigidification improve the freeze-drying resistance of Lactococcus lactis subsp. lactis TOMSC161.

This work aimed at characterizing the biochemical and biophysical properties of the membrane of Lactococcus lactis TOMSC161 cells during fermentation at different temperatures, in relation to their freeze-drying and storage resistance. Cells were cultivated at two different temperatures (22 and 30°C) and were harvested at different growth phases (from the middle exponential phase to the late stationary phase). Bacterial membranes were characterized by determining the fatty acid composition, the lipid phase transition, and the membrane fluidity. Cultivability and acidification activity losses of L. lactis were quantified after freezing, drying, and 3months of storage. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to lipid composition, and it was established that the cyclopropanation of unsaturated fatty acids with concomitant membrane rigidification during growth led to an increase in the freeze-drying and storage resistance of L. lactis. As expected, cultivating cells at a lower fermentation temperature than the optimum growth temperature induced a homeoviscous adaptation that was demonstrated by a lowered lipid phase transition temperature but that was not related to any improvement in freeze-drying resistance. L. lactis TOMSC161 was therefore able to develop a combined biochemical and biophysical response at the membrane level during fermentation. The ratio of cyclic fatty acids to unsaturated fatty acids (CFA/UFA) appeared to be the most relevant parameter associated with membrane rigidification and cell resistance to freeze-drying and storage. This study increased our knowledge about the physiological mechanisms that explain the resistance of lactic acid bacteria (LAB) to freeze-drying and storage stresses and demonstrated the relevance of complementary methods of membrane characterization.

Examine the characterization of biofilm formation and inhibition by targeting SrtA mechanism in Bacillus subtilis: a combined experimental and theoretical study.

Bacillus subtilis is one of the well-known biofilm-forming organisms associated with plants, animals, and also used as a model organism for all Bacillus sp. In B. subtilis, SrtA enzyme plays the imperative roles in mechanism of signaling pathway and microbial adherence toward the host. SrtA is highly considered as a universal drug target for all Gram positive pathogens. Because of unresolved 3D structure of SrtA in Gram positive bacteria including B. subtilis, we developed a homology model protein using structural alignments of similar SrtA from B. anthracis. While the structural model of SrtA is analyzed because of its significance in biofilm formation by screening the suitable active site based compounds and analyzing the ability of bacterial biofilm inhibition. Druggability site based screening able to retrieve the active compounds against SrtA and checked the activity of the screened compounds through experimental biochemical assays and in situ microscopic analysis. Here in this study we concluded the computationally screened SrtA inhibitors showed high level of biofilm inhibition despite difficulties in bacterial membrane rigidification. Hence this study leads a way to the new compounds that may be useful to treat the bacterial infections.

Structure-dependent interactions of polyphenols with a biomimetic membrane system

Polyphenols are naturally-occurring compounds, reported to be biologically active, and through their interactions with cell membranes. Although association of the polyphenols with the bilayer has been reported, the detailed mechanism of interaction is not yet well elucidated. We report on spatio-temporal real-time membrane dynamics observed in the presence of polyphenols. Two distinct membrane dynamics, corresponding to the two classes of polyphenols used, were observed. Flavonoids (epi-gallocatechin-3-gallate, gallocatechin, theaflavin and theaflavin-3-gallate) caused lipid membrane aggregation and rigidification. As simple structural modification through opening of the aromatic C-ring into an olefin bond, present in trans-stilbenes (resveratrol and picead), completely changed the membrane properties, increasing fluidity and inducing fluctuation. There were differences in the membrane transformations within the same class of polyphenols. Structure-dependent classification of membrane dynamics may contribute to a better understanding of the physicochemical mechanism involved in the bioactivity of polyphenols. In general, an increase in the number of hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the reactivity of the polyphenols. Most notable was the difference observed through a simple addition of the gallate group. Unraveling the importance of these polyphenols, at a functional group level further opens the key to tailored design of bioactive compounds as potential drug candidates.

Membrane rigidification functions upstream of the MEKK1-MKK2-MPK4 cascade during cold acclimation in Arabidopsis thaliana.

The MEKK1-MKK2-MPK4 cascade is activated during cold acclimation. However, little is known regarding the perception of low temperature. In this study, we demonstrate that treatment of Arabidopsis with a membrane rigidifier, DMSO, caused MPK4 activation concomitantly with MEKK1 and MKK2 phosphorylation, as well as the cold-inducible gene COR15a expression. These processes are similar to the effects of cold treatment, whereas benzyl alcohol (BA), a membrane fluidizer, prevented such cold-induced events. Moreover, the DMSO-treated seedlings acquired freezing tolerance without cold acclimation. In contrast, the BA-pretreated seedlings did not show freezing tolerance. These results suggest that membrane rigidification activates this MAPK cascade and contributes to the acquisition of freezing tolerance.

The adaptation responses of bacterial cytoplasmic membrane fluidity in the presence of environmental stress factors — polychlorinated biphenyls and 3-chlorobenzoic acid

Only bacteria sufficiently resistant to the toxic compounds in their environment can be used for the efficient biodegradation process in order to eliminate a widespread contamination by polychlorinated biphenyls (PCBs). The presence of PCBs results in bacterial controlled rigidification of cytoplasmic membrane. The four bacterial isolates from long-term PCB-contaminated soil (Alcaligenes xylosoxidans, Pseudomonas stutzeri) and sediment (Ochrobactrum anthropi, Pseudomonas veronii) have been used to select the strain most adapted to the PCBs, i.e. with efficient changes in the membrane phospholipid fatty acids. PCBs and their toxic degradation products — the 3-chlorobenzoic acids (3-CBA as the most toxic one) — were added separately to the liquid medium with glucose in two experimental sets: at lag phase and in stationary phase of bacterial growth in order to evaluate the effects of chemicals to cytoplasmic membrane. The main parameter — the changes in fatty acids composition (in the total lipids and the main membrane phospholipid phosphatidyletanolamine) were studied. 3-CBA caused growth inhibition when added at lag phase. However, when added during the stationary growth, inhibition was not observed. Similarly, after addition of PCBs to the stationary growth culture, inhibition of growth was not observed with all tested strains (except for P. stutzeri). This fact indicates the importance of time contact of bacteria during growth phase with xenobiotics. O. anthropi and A. xylosoxidans appeared to be the most adapted to the presence of PCBs (with sufficient membrane adaptation), active under the adverse conditions, and able to survive in the contaminated environment.

Oxidative stress effect on progesterone-induced blocking factor (PIBF) binding to PIBF-receptor in lymphocytes

Receptor–ligand binding is an essential interaction for biological function. Oxidative stress can modify receptors and/or membrane lipid dynamics, thus altering cell physiological functions. The aim of this study is to analyze how oxidative stress may alter receptor–ligand binding and lipid domain distribution in the case of progesterone-induced blocking factor/progesterone-induced blocking factor-receptor. For membrane fluidity regionalization analysis of MEC-1 lymphocytes, two-photon microscopy was used in individual living cells. Lymphocytes were also double stained with AlexaFluor647/progesterone-induced blocking factor and Laurdan to evaluate -induced blocking factor/progesterone-induced blocking factor-receptor distribution in the different membrane domains, under oxidative stress. A new procedure has been developed which quantitatively analyzes the regionalization of a membrane receptor among the lipid domains of different fluidity in the plasma membrane. We have been able to establish a new tool which detects and evaluates lipid raft clustering from two-photon microscopy images of individual living cells. We show that binding of progesterone-induced blocking factor to progesterone-induced blocking factor-receptor causes a rigidification of plasma membrane which is related to an increase of lipid raft clustering. However, this clustering is inhibited under oxidative stress conditions. In conclusion, oxidative stress decreases membrane fluidity, impairs receptor–ligand binding and reduces lipid raft clustering.