Oxidized cholesterol bilayers. Dependence of electrical properties on degree of oxidation and aging

Black lipid membranes made from oxidized cholesterol were examined for their specific resistance, capacitance, and physical stability, as a function of cholesterol oxidation time and of age. Membranes formed from cholesterol oxidized in n-octane were not physically stable even after 7 h of oxidation unless they were aged for at least a month. Membranes formed from cholesterol oxidized in decane and tetradecane (1 : 1) were stable immediately after 2--6 h of oxidation. Oxidation times outside this range produced unstable membranes. After 1 month storage, membranes from cholesterol solutions oxidized in decane and tetradecane from 0.75--3 h were stable. After 11 months, only the 0.75 oxidation time produced stable membranes. Storage in nitrogen retarded the aging process. After initial forming of the membrane, total membrane area and capacity increased and then stabilized, although specific capacity and resistance did not change, indicating inherent stability in the bilayer's intrinsic electrical properties. Bilayers formed soon after cholesterol oxidation had membrane capacity which ranged from 0.42 to 0.55 muF/cm2. Specific membrane resistance ranged initially from 2 . 10(6) to 37 . 10(6) omega/cm2 in 0.2 M NaCl with lower resistances in the more oxidized membranes. With aging, membrane capacity decreased gradually over 11 months to values approaching 0.1 muF/cm2 indicating membrane thickening. Membrane resistance ordinarily decreases with storage time. The rate of these changes with age is dependent on the extent of initial cholesterol oxidation and subsequent oxidation, with long term stability best in the least oxidized membranes.     

Heat Injary and Changes in Electrical Resistance of Leaf Tissue

More effective means are needed for early diagnosis and quantitative evaluation of plant injury caused by various stresses. Measuring of tissue electrical resistance has been used in the research of SO2 injury recently and satisfactory results has been obtained. The present paper describes the change of tissue electrical resistance in connection with the response of plants to high temperature. Wheat and tobacco leaves were tranted with different high temperatures (30–60℃). Electrical resistance was measured during and after the treatment by means of Zhu’s newly improved method of the determination of the mid-region voltake drop on passing a stable electric current through the leaf tissue. Experimental results showed that electrical resistance changed regularly with the increase of temperature. At the range of nninjurious temperature, tissue electrical resistance kept the same level as control. Around the subinjurious temperature, electrical resistance increased remarkably and could be gradually restored after the cease of treatment. At injurious temperatures, tissue electrical resistance rised at first, and then declined. The higher the temperature, the earlier and quicker the decline, and finally it reached the lowest level showing the death of the tissue. If leaves were taken back to ordinary temperature at the rising stage or just the beginning of decrease, they could recover, in general, in their electrical resistance. They conld not recover after a substantial drop of electrical resistance, showing the oeeurence of irreversible injnry. Therefore the changes of tissue electrical resistance reflected to a certain degree the extent of heat injury. Ion leakage and ethane production were also measured parallelly. As for reflection of the extent of heat injury, changes of tissue electrical resistance is more sensitive than both ion leakage and ethane produetlon. The rise of tissue electrical resistance at thc earlier stage of heat treatment is discussed in connection with the changes of the two constituents of tissue electrical resistance-membrane resistance and apoplast resistance.     

Fluorescence polarization studies on Escherichia coli membrane stability and its relation to the resistance of the cell to freeze-thawing. I. Membrane stability in cells of differing growth phase

Physical properties of Escherichia coli membrane lipids in logarithmic- and stationary-phase cells were studied by measuring the fluorescence polarization change of cis- and trans-parinaric acid as a function of temperature. In aqueous dispersions of phospholipids extracted from cytoplasmic and outer membranes of cells of differing growth phase, a similar polarization increase was observed over the range from physiological temperature to below 0 degrees C, and nearly the same transition ratios were obtained in all samples. The cytoplasmic membrane of both of the growth-phase cells showed a higher polarization ratio above the transition temperatures, compared to that in the aqueous dispersion of phospholipids. The polarization ratios below the transition temperatures of these specimens were lower than the value obtained with the lipids, especially in the stationary-phase specimens. The outer membrane specimens showed a similar polarization change but the transition temperature ranges were considerably higher both in the logarithmic- and the stationary-phase specimens, compared to those in the cytoplasmic membrane specimens. Freeze-thawing of logarithmic-phase cells showed the emergence of activity of certain enzymes which are known to be located in the membranes. The stationary-phase cells did not suffer from any such deleterious effect and maintained a high level of cell viability in a similar treatment. These results indicate that in the stationary-phase cell membranes lipids are in a highly ordered state, and the lipid state causes a membrane stability which results in the high resistance of the cell to freeze-thawing.     

High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition.     

Temperature-dependent electrical resistivity of the hornet cuticle: Changes induced by colchicine and purine in the diet

Various cuticular areas of the Oriental hornet (Vespa orientalis) were measured for their electrical resistivity within the range of 3–40°C. It was found that: (1) the electrical resistivity is temperature dependent; (2) there are no significant differences in this respect between hornets collected in different regions; (3) there are no differences in the electrical resisitivity between the yellow and brown areas of the cuticle in control hornets; (4) there are however, significant changes in resistance between the yellow and brown strips of hornets fed on purine; (5) hornets fed on colchicine reach a minimum resistance at a higher temperature than do the control hornets; and (6) hornets fed on purine reach a minimum resistance at a lower temperature. It is suggested that the cuticular changes in resistivity at different temperatures reflect the hornets' mechanism of detecting and adjusting to changes in the ambient temperature.     

Acclimation to the growth temperature and the high-temperature effects on photosystem II and plasma membranes in a mesophilic cyanobacterium, Synechocystis sp. PCC6803.

High-temperature effects on Photosystem II and plasma membranes, temperature dependence of growth, and acclimation to the growth temperature were studied in a mesophilic cyanobacterium, Synechocystis sp. PCC6803. The following results were obtained. (1) Small but distinct temperature acclimation of the PSII reaction center activity was shown for the first time when the activity was measured at inhibitory high temperatures. However, the reaction center activity showed no apparent acclimation when it was measured at growth temperatures after heat stress. (2) Oxygen-evolving activity and the permeability of plasma membranes showed higher resistance to heat when PCC6803 cells were grown at higher temperatures. (3) Acclimation of photosynthesis to the growth temperature seemed to occur so as to maintain photosynthesis activity not at a maximum level but in a certain range at the growth temperatures. (4) Neither sensitivity to high-temperature-induced dissociation of phycobilisomes from the PSII reaction center complexes nor degradation of phycocyanin were altered by changes in the environmental temperature. (5) A close relationship between the viability of cells and the structural changes of plasma membranes (but not the inactivation of photosynthesis) was observed. The denaturation process of PSII complexes and the relationship between the temperature dependence of the growth of Synechocystis PCC6803 cells and that of the photosynthetic activity are also discussed.     

Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder

Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal Cl- concentration ([Cl]m). The effects of reduction in [Cl]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, where iodide replacements increased it. When gluconate or sulfate substitutions were brought about the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a Cl- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity.     

Electrical properties of the cellular transepithelial pathway in Necturus gallbladder. I. Circuit analysis and steady-state effects of mucosal solution ionic substitutions.

Microelectrode techniques were employed to measure the electrical resistance of the cell membranes and the shunt pathway, and the equivalent electromotive forces (EMF's) at both cell borders in Necturus gallbladder epithelium. The cell is, on the average, 57 mV negative to the mucosal solution and 59 mV negative to the serosal solution. The transepithelial potential (Vms) ranges from 0.5 to 5 mV, serosal solution positive. Assuming that the shunt EMF (Vs) is zero with standard Ringer's bathing oth sides of the tissue, both cell membrane EMF's are oriented with the negative pole toward the cell interior and are 39.9 +/- 3.6 mV (apical, Va), and 69.4 +/- 1.8 mV (basal-lateral, Vb)...     

Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 μM; (c) X-ray diffraction studies showed that PPA in the 0.1–0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.     

The effect of insulin on the permeability of phosphatidyl choline bimolecular membranes to glucose

Summary Insulin, on the outside of phosphatidyl choline bimolecular membranes, inceeased the permeability of the membranes to glucose. The magnitude of the increase in glucose permeability was dependent upon the insulin concentration and was independent of the glucose concentration over the range 2.7 to 14.4mm. The increase in the permeability of the membranes to glucose was not accompanied by a change in the direct current electrical resistance of the membranes. Ovalbumin resulted in a smaller increase in the permeability of the membranes to glucose and no change in their electrical resistance, while phloretin changed neither the permeability to glucose nor the electrical resistance. Insulin, on both sides of the bimolecular membranes, did not change the permeability to glucose from that observed when insulin was present only on the outside, nor did it change the electrical resistance.     

Lipid- and temperature-dependent structural changes in Acholeplasma laidlawii cell membranes

The lipids in cell membranes of Acholeplasma laidlawii were enriched with different fatty acids selected to produce membranes showing molecular motion discontinuities at temperatures between 10 and 35 °C. Molecular motion in these membranes was probed by ESR after labelling with 12-nitroxide stearate, and structure in these membranes was examined by electron microscopy after freeze-etching.Freeze-etching and electron microscopy showed that under certain conditions the particles in the A. laidlawii membranes aggregated, resulting in particle-rich and particle-depleted regions in the cell membrane. Depending upon the lipid content of the membrane, this aggregation could begin at temperatures well above the ESR-determined discontinuity. Aggregation increased with decreasing temperature but was completed at or near the discontinuity. However, cell membranes grown and maintained well below their ESR-determined discontinuity did not show maximum particle aggregation until after they had been exposed to temperatures at or above the discontinuity.The results show that temperatures at or near a phase transition temperature can induce aggregation of the membrane particles. This suggests that temperature-induced changes in the lipid phase of a biological membrane can induce phase separations which affect the topography of associated proteins.     

Electrical resistance changes during exposure to low temperature measure chilling and freezing tolerance in olive tree (Olea europaea L.) plants

Electrical resistance changes in different organs of four olive tree (Olea europaea L.) varieties, characterized by different tolerance to chilling and freezing, were examined, during exposure to low temperature. Apparent critical temperatures (CT) and freezing temperatures (T_fr) were identified on the basis of the electrical resistance changes. Both temperatures were lower for the more chilling‐tolerant genotypes. From the apparent critical temperatures, the absolute critical temperature (CT_abs) and the time delay of the chilling signal transduction process were calculated. In shoots, CT_abs varied from 8·8 °C for Ascolana (chilling‐tolerant variety) to 13·6 °C for Coratina (chilling‐sensitive variety). The magnitude of the transduction time was very similar (about 2 min) for the three genotypes that are more sensitive to chilling, whereas it was significantly higher (about 3 min) for the most tolerant genotype. Different freezing temperatures were observed for different organs. It would appear from this experiment that the order of sensitivity is roots > leaves > shoots > vegetative buds. Accord was found between the absolute critical temperature of electrical resistance and the critical temperature of membrane potential. The occurrence of electrical resistance changes in the tissues of the olive trees exposed to low temperature suggests the use of this experimental procedure as a quick, easy and non‐destructive tool to screen plant tissues for chilling tolerance. The strong dependence of the electrical resistance on low temperature, and the critical temperature of around 10 °C, can yield interesting information about the lowest thermal limits for the continuation of normal physiological processes and therefore about the adaptability of plants to particular environments.     

Characteristics of concanavalin A-resistant Chinese hamster ovary cells and certain revertants.

Clones of Chinese hamster ovary (CHO) cells were isolated by single-step selection for resistance to killing Concanavalin A (ConA) and certain cellular and membrane properties were examined. The ConA-resistant isolates were only about 2-fold more resistant than wild type cells to the selecting lectin, but exhibited pleiotropic temperature-sensitivity for growth, markedly altered morphology and adherence, and significant difference in susceptibility to other agents such as colchicine. Two revertants to full temperature-resistance were isolated from different ConA-resistant mutants. One revertant clone had reacquired wild type sensitivity to ConA while the other revertant remained ConA-resistant. The two series of wild typed, ConA-resistant, and temperature revertant clones were analyzed for altered mobility of cell surface glycoproteins using lactoperoxidase/125I and galactose oxidase/(3H) borohydride labelling procedures. The ConA-resistant clones showed increased mobility on polyacrylamide gels of three classes of labelled proteins, in the molecular weight ranges 225,000, 200,000, and 130,000 daltons. These changes persisted in the temperature-revertant that remained ConA-resistant, while two of the altered protein closses were restored to wild type mobility in the revertant that regained ConA-sensitivity. Cell hybridization experiments indicated that the temperature-sensitivity phenotypes of different ConA-resistant isolates are recessive and noncomplementing, implying that the same gene is affected in each case. The reversions to temperature resistance appear to be recessive suppressor mutation in different genes.     

Ripening of Nectarine Fruit (Changes in the Cell Wall,Vacuole, and Membranes Detected Using Electrical Impedance Measurements)

Electrical impedance measurements were used to characterize changes in intracellular and extracellular resistance as well as changes in the condition of membranes during ripening of nectarines (Prunus persica [L.] Batsch cv Fantasia). These measurements were related to changes in fruit texture assessed by flesh firmness and apparent juice content. An electrical model indicated that, during ripening (d 1-5) of freshly harvested fruit, the resistance of the cell wall and vacuole declined by 60 and 26%, respectively, and the capacitance of the membranes decreased by 9%. Accurate modeling of the impedance data required an additional resistance component. This resistance, which declined by 63% during ripening, was thought to be associated with either the cytoplasmic or membrane resistance. Changes in tissue resistance measured using low frequencies of alternating current were closely related to flesh firmness. After storage at 0[deg]C for 8 weeks, the nectarines developed a woolly (dry) texture during ripening at 20[deg]C. The main difference between these chilling-injured nectarines and fruit ripened immediately after harvest was the resistance of the cell wall, which was higher in woolly tissue (4435 [omega] after 5 d at 20[deg]C) than in nonwoolly tissue (2911 [omega] after 5 d at 20[deg]C). The results are discussed in relation to physiological changes that occur during the ripening and development of chilling injury in nectarine fruit.     

Hydraulic Resistance of Rough Lemon Roots

A pressure chamber technique was used to estimate hydraulic root resistance in rough lemon (Citrus jambhiri Lush.) seedlings. The effect of previous water stress on hydraulic root resistance was evaluated. A factorial 3 × 3 design with four replications was established with potted rough lemon seedlings in a growth chamber. Three water-stress treatments were applied by watering at intervals of 1, 2 and 3 days, and root resistance measurements were made after 6, 12 and 18 days of treatment. Plants that had experienced mild and severe water stress (irrigation interval of 2 and 3 days, respectively) had higher hydraulic root resistances after several drying cycles than those plants irrigated daily. Additional cycles had no significant effect. The increase in root resistance was not due to decreased root growth but apparently to changes in the permeability of the root cell membranes or to increased suberin deposition in the cell walls of the cortical cells. In a short-term experiment (1 h), temperature strongly affected water flow through rough lemon roots in the range 5 to 35°C. Temperature influenced root membrane permeability, since reduced blow could not be explained by changes in water viscosity.     

Membrane Transition Temperature Determines Cisplatin Response

Cisplatin is a classical chemotherapeutic agent used in treating several forms of cancer including head and neck. However, cells develop resistance to the drug in some patients through a range of mechanisms, some of which are poorly understood. Using isolated plasma membrane vesicles as a model system, we present evidence suggesting that cisplatin induced resistance may be due to certain changes in the bio-physical properties of plasma membranes. Giant plasma membrane vesicles (GPMVs) isolated from cortical cytoskeleton exhibit a miscibility transition between a single liquid phase at high temperature and two distinct coexisting liquid phases at low temperature. The temperature at which this transition occurs is hypothesized to reflect the magnitude of membrane heterogeneity at physiological temperature. We find that addition of cisplatin to vesicles isolated from cisplatin-sensitive cells result in a lowering of this miscibility transition temperature, whereas in cisplatin-resistant cells such treatment does not affect the transition temperature. To explore if this is a cause or consequence of cisplatin resistance, we tested if addition of cisplatin in combination with agents that modulate GPMV transition temperatures can affect cisplatin sensitivity. We found that cells become more sensitive to cisplatin when isopropanol, an agent that lowers GPMV transition temperature, was combined with cisplatin. Conversely, cells became resistant to cisplatin when added in combination with menthol that raises GPMV transition temperatures. These data suggest that changes in plasma membrane heterogeneity augments or suppresses signaling events initiated in the plasma membranes that can determine response to cisplatin. We postulate that desired perturbations of membrane heterogeneity could provide an effective therapeutic strategy to overcome cisplatin resistance for certain patients.     

Volume changes of an endothelial cell monolayer on exposure to anisotonic media

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120-630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.     

The electrical potential difference through the foot epithelium of the snail Achatina achatina, Lameere during mechanical and chemical stimulation

An important electrophysiological variable--the transepithelial potential difference reflects the electrogenic transepithelial ion currents, which are produced and modified by ion transport processes in polarized cells of epithelium. These processes result from coordinated function of transporters in apical and basolateral cell membranes and have been observed in all epithelial tissues studied so far. The experiments were performed on isolated specimens of snail foot. In the experiments, the baseline transepithelial electrical potential difference--PD, changes of transepithelial difference during mechanical stimulation--dPD and the transepithelial resistance were measured with an Ussing apparatus. A total of 60 samples of foot ventral surface of 28 snails were studied. The transepithelial electrical potential difference of isolated foot ranged from -6.0 to 10.0 mV under different experimental conditions. Mechanical stimulation of foot ventral surface caused changes of electrogenic ion transport, observed as transient hyperpolarization (electrical potential difference became more positive). When the transepithelial electrical potential difference decreased during stimulation, the reaction was described as depolarization. When amiloride and bumetanide were added to the stimulating fluid so that the sodium and chloride ion transport pathways were inhibited, prolonged depolarization occurred. Under the influence of different stimuli: mechanical (gentle rinsing), chemical (changes of ion concentrations) and pharmacological (application of ion inhibitors), transient changes of potential difference (dPD) were evoked, ranging from about -0.7 to almost 2.0 mV. Changes in transepithelial potential difference of the pedal surface of the snail's foot related to these physiological stimuli are probably involved in the locomotion of the animal and are under control of the part of the nervous system in which tachykinin related peptides (TRP) act as transmitters.     

Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion

We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of- phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell- containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time- resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.