Fluorescence depolarization studies of the phase transition in multilamellar phospholipid vesicles exposed to 1.0-GHz microwave radiation

The phase transition in multilamellar dimyristoylphosphatidylcholine (DMPC) vesicles was studied during exposure to continuous wave 1.0-GHz microwave radiation. Fluorescence depolarization measurements using a lipid-seeking molecular probe, diphenylhexatriene (DPH). were performed as a function of temperature. Semilog plots of microviscosity versus temperature illustrate the phase transition which shows a 5°C shift when the vesicles are treated with chloroform as a positive control. No shift of the phase transition was found during exposure to microwave radiation at specific absorption rates between 1 and 30 W/kg. Samples were exposed in a rectangular transmission line (TEM cell), and specific absorption rates were calculated from electrical measurements of incident, reflected, and transmitted power. Samples were exposed to increasing intensities of radiation, while the temperature was maintained at either 23.5 or 25.5 °C; these temperatures represented the two ends of the phase transition region for these vesicles. No statistically significant difference was found between exposed and control samples. These results are in contrast to those of others using laser Raman spectroscopy to measure the phase transition in similar multilamellar vesicles exposed to microwave radiation.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Involvement of Membrane Lipids in Cold Shock-induced Autolysis of Bacillus subtilis Cells

Exponentially growing Bacillus subtilis cells autolysed when exposed to cold shock treatment in minimal medium followed by incubation at 37°C. From characteristics of the lysis, it was suggested that the cold-shock-induced cell lysis resulted from the perturbation of membrane organization that is initiated by rapid changes in temperature, lipid phase transitions. For maximum lysis induction to occur, in addition to rapid cooling to 5°C or lower, retention at temperatures lower than 10°C for at least 20 min is required. The cell sensitivity to the autolysis induction by cold shock was different between cells grown at 25°C and cells grown at 37°C. Analyses of the fatty acid composition and the phase transition temperature of membrane lipids suggested that the membrane fluidity may affect the autolysis induction. Experiments to discover the effects of cerulenin treatment and lipid addition on autolysis induction and the autolysin activity level support the hypothesis that membrane lipids are involved in cold-shock-induced cell autolysis.     

Reduced lateral mobility of a fluorescent lipid probe in cholesterol-depleted erythrocyte membrane

The effect of cholesterol depletion of the human erythrocyte membrane on the lateral diffusion rate of a fluorescent lipid probe is reported. At low temperatures (?5 to 5°C), the diffusion of the probe is 50% slower in the cholesterol-depleted membrane than in non-depleted membrane. At high temperatures (30 to 40° C), probe mobility is not affected by cholesterol depletion. These results suggest that cholesterol suppresses aspects of phospholipid phase changes in animal cells in a manner consistent with its behavior in artificial bilayers and multilayers.Whole erythrocytes were depleted of 30–50% of their cholesterol by incubation with a sonicated dispersion of dipalmitoyl phosphatidylcholine. Cells were then labeled with 3,3′-dioctadecylindocarbocyanine (diI), a phospholipid-like fluorescent dye, and hemolyzed into spherical ghosts. The rate of lateral motion of diI was measured by observing the fluorescence recovery after local photobleaching with a focused laser spot.The diffusion rate of the lipid probe in both control and cholesterol-depleted erythrocyte membrane is substantially smaller than in any cell or model membrane previously measured.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

Influence of 2.45-GHz CW microwave radiation on spontaneously beating rat atria

The chronotropic and inotropic effects of 2.45-GHz continuous wave (CW) microwave radiation were investigated in the isolated spontaneously beating rat atria. Isolated atria were placed in specially designed tubes inserted into a waveguide exposure system. The atria were then irradiated for a period of 30 min, followed by a 30-min recovery period. The control atria were prepared simultaneously and sham exposed. Experiments were conducted at two temperatures, 22 and 37 °C, and two specific absorption rates, 2 mW/g and 10 mW/g. At both temperatures the rate of atrial contraction was not altered by a 30-min exposure at either 2 or 10 mW/g. The average rate (beats per min) was approximately 100 for both the control and exposed atria at 22 °C and 215 beats per min for both the control and exposed atria at 37 °C. In addition, no inotropic effects on the spontaneously beating atria were noted at any exposure level. These data suggest that 2.45-GHz CW microwave radiation at these intensities has no overt effect on these variables in isolated rat atria.     

Phase properties of binary mixtures of monogalactosyldiacylglycerols differing in hydrocarbon chain substituents dispersed in aqueous systems

Monogalactosyldiacylglycerols isolated from spinach leaves contain a high proportion of polyunsaturated fatty acyl substituents and form hexagonal-II structures when dispersed in excess water. Catalytic hydrogenation of the lipid in the presence of Adam's catalyst completely saturates the hydrocarbon chains and the lipid forms typical open sheet bilayer structures in water at 20°C. Binary mixtures of the native and hydrogenated lipid tend to phase separate at 20°C. Freeze-fracture electron microscopy reveals lamellar phase lipid indispersed with regions of hexagonal-II structure and the proportions of each reflect the composition of the mixture. X-ray diffraction in both wide- and low-angle regions show that the saturated lipid forms the typical stable gel-phase structure in mixtures that are allowed to equilibrate over three days at 20°C. The phase transition behaviour of binary mixtures of the two galactolipids was investigated by differential scanning calorimetry and fluorescence probe methods. Thermal studies indicate that the phase-separated gel structure undergoes an anomalous transition compared with the saturated pure lipid in that the transition temperature is reduced from about 57°C to 41°C and the enthalpy of the transition is also somewhat reduced. Furthermore, the transition appears to involve the conversion of the completely phase-separated system into bilayer coexisting with phases intermediate between bilayer and hexagonal-II. A homogeneous hexagonal-II phase is presumably formed at higher temperatures. The thermal and structural studies were consistent with fluorescence polarization measurements of 1,6-diphenyl-1,3,5-hexatriene interpolated into the hydrocarbon domain of the structure.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43°C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20°C, but in a liquid crystalline state when cells were grown at 37 and 43°C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Effects of X-band microwave exposure on rabbit erythrocytes

Rabbit erythrocytes were exposed in vitro to continuous wave (CW) and pulse-modulated X-band microwaves in wave guide exposure chambers. Erythrocytes were exposed as whole (hep-arinized) blood suspensions or as washed cells in 1:1 isotonic buffered K⁺-free saline suspensions. Statistically significant increases in K⁺ efflux relative to thermal controls were detected when red cells were exposed in whole blood suspensions to either CW or pulsed 8.42-GHz microwaves at SARs that resulted in equilibrium sample temperatures of approximately 24 °C. Under the same exposure conditions, no statistically significant K⁺ efflux occurred in the case of 1:1 red cell suspensions. Measured differences in sample heating rates and temperature gradients between microwave-exposed and heated control suspensions may account in part for the differential effect of microwave exposure but such effects do not appear to explain the results of this study fully.     

CHANGES IN CELLULAR PROTEINS DUE TO ENVIRONMENTAL NON-IONIZING RADIATION. I. HEAT-SHOCK PROTEINS

This paper describes the effect of weak microwave fields on the amounts of heat-shock proteins in cell cultures at various temperatures.

The field was generated by signal simulation of the Global System for Mobile communications (GSM) of 960 Mhz, used in portable phones. Transformed human epithelial amnion (AMA) cells, growing on glass coverslips, were exposed in a transverse electromagnetic (TEM) cell to a microwave field, generating a specific absorption rate (SAR) of 2.1 mW.kg^?1 in the cells. Exposure temperatures were 35, 37, and 40 ± 0.1°C, respectively, and the exposure time was 20 min.

The heat-shock proteins Hsp-70 and Hsp-27 were detected by immuno-fluorescence. Higher amounts of Hsp-70 were present in the cells exposed at 35 and 37°C than in the sham-exposed cells.

These effects can be considered to be athermal, since the field strength was much lower than the safety standard for absence of heat generation by microwave fields.

There was no significant response in the case of Hsp-27.     

The Influence of Temperature on Floral Initiation in the Olive

Floral initiation is completely inhibited when the olive is grown in a glasshouse at a minimum temperature of 16°C and a maximum temperature of 27°–30°C but occurs when it is grown at natural winter conditions in California. This study was undertaken to determine more specifically the temperature requirements for flowering and to show the relation of temperature treatment to hud development and floral initiation. Results of experiments performed with trees in containers grown at constant temperatures in controlled environment growth rooms show that the optimum temperature for flowering is 10°–13°C. Either higher (18°C) or lower (4°C) temperatures inhibit flowering completely. Morphological studies show that axis elongation and floral initiation occur in buds during temperature treatment at 10°C and 13°C but fail to occur during 4°C or 18°C treatments, or following these treatments when the temperature is raised to 21°C. When plants were exposed to 13°C for varying durations, it was found that no inflorescences formed after a 7.5 week exposure but that many formed after an 11-week exposure. A subsequent experiment showed that many more inflorescences formed after a 10-week exposure at 13°C than after 9 weeks exposure. Morphological changes in the bud seem to be associated with this increase in flowering affected by duration of treatment.     

In vitro cytogenetic effects of 2450 MHz waves on human peripheral blood lymphocytes

Cytogenetic analyses were performed on human peripheral blood lymphocytes exposed to 2450 MHz microwaves during 30 and 120 min at a constant temperature of 36.1°C (body temperature). The temperature was kept constant by means of a temperature probe put in the blood sample which gives feedback to a microcomputer that controls the microwave supply. We found a marked increase in the frequency of chromosome aberrations (including dicentric chromosomes and acentric fragments) and micronuclei. On the other hand the microwave exposure did not influence the cell kinetics nor the sister chromatid exchange (SCE) frequency. ©1993 Wiley-Liss, Inc.     

Sphingomyelin phase transition in the sheep erythrocyte membrane

The dependence of membrane dynamics on the mole ratio of lecithin to sphingomyelin (L/S) was examined by the fluorescence depolarization of the fluidity probe DPH in membranes isolated from sheep and human erythrocytes. In these membranes L/S is the main variable of lipid composition (0.02 and 1.7, respectively). The sheep erythrocyte membrane, which is rich in sphingomyelin, displays a higher lipid microviscosity than the human erythrocyte membrane in addition to a broad gel/liquid-crystal phase transition in the range of 26–35°C. Single-walled lipid vesicles of high sphingomyelin content, when studied by the same technique, exhibited dynamic characteristics similar to those found in the sheep erythrocyte membrane. Both the apparent microviscosity and the transition temperature decreased with increasing the L/S. Membrane proteins of human and sheep erythrocytes were fluorescently labeled with the sulfhydryl reagent N-dansylaziridine and the emission spectrum was recorded as a function of temperature. In the human erythrocyte membranes a gradual increase in the ratio of emission maxima at 520 and 490 nm was observed between 6 and 40°C. At this temperature range the ratio of the above emission maxima in sheep erythrocyte membranes displayed a break between 20 and 28°C, which partially overlapped the phase transition observed for the lipid core. The effect of the lipid phase transition on membrane proteins for the lipid core. The effect of the lipid phase transition on membrane proteins was further assessed by comparing the activity of the membrane bound phospholipase A2 in the intact and detergent-solubilized sheep erythrocyte membranes. Below 31°C the lipids suppress the enzyme activity by about 90%, whereas above this temperature this suppression is progressively abolished.     

Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 °C, but not at 34 °C. Its host, Pseudomonas BAL-31, grows at 34 °C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 °C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place.Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates made in its presence contain no PM2-like particles. These experiments, carried out at 25 °C, indicate that adamantanone prevents the assembly of stable PM2 virus.Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an “ordered” state at temperatures below about 33 °C and undergo a transition to a “disordered” state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 °C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at ~14°C, whereas, freeze-dried platelets exhibited a main phase transition ~12°C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42°C, whereas proteins in the rehydrated platelets denatured at 48°C.     

The effect of the lipid bilayer state on fluorescence intensity of fluorescein-PE in a saturated lipid bilayer

Fluorescein-PE is a fluorescence probe that is used as a membrane label or a sensor of surface associated processes. Fluorescein-PE fluorescence intensity depends not only on bulk pH, but also on the local electrostatic potential, which affects the local membrane interface proton concentration. The pH sensitivity and hydrophilic character of the fluorescein moiety was used to detect conformational changes at the lipid bilayer surface. When located in the dipalmitoylphosphatidylcholine (DPPC) bilayer, probe fluorescence depends on conformational changes that occur during phase transitions. Relative fluorescence intensity changes more at pretransition than at the main phase transition temperature, indicating that interface conformation affects the condition in the vicinity of the membrane. Local electrostatic potential depends on surface charge density, the local dielectric constant, salt concentration and water organisation. Initial increase in fluorescence intensity at temperatures preceding that of pretransition can be explained by the decreased value of the dielectric constant in the lipid polar headgroups region related in turn to decreased water organisation within the membrane interface. The abrupt decrease in fluorescence intensity at temperatures between 25 degrees C and 35 degrees C (DPPC pretransition) is likely to be caused by an increased value of the electrostatic potential, induced by an elevated value of the dielectric constant within the phosphate group region. Further increase in the fluorescence intensity at temperatures above that of the gel-liquid phase transition correlates with the calculated decreased surface electrostatic potential. Above the main phase transition temperature, fluorescence intensity increase at a salt concentration of 140 mM is larger than with 14 mM. This results from a sharp decline of the electrostatic potential induced by the phosphocholine dipole as a function of distance from the membrane surface.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.