Progesterone induces transient changes in plasma membrane fluidity of amphibian oocytes during the first meiotic division

Progesterone acts at the surface of the amphibian oocyte to induce resumption of the meiotic divisions. Progesterone binding leads to a transient dose-dependent decrease in the fluidity (increase in order parameter) of the Rana oocyte plasma membrane, which was detected by electron spin resonance in isolated plasma membranes using either 5- or 16-DOXYL stearic acid probes. The 5-DOXYL probe, which inserts into the membrane with the spin label nearest the surface, showed an increase in the order parameter within minutes, a maximum change by 2 h, and a return to control levels by 6 h. The order parameter for the 16-DOXYL probe, which reflects the fluidity deeper within the plasma membrane, increased slowly and remained elevated during the first meiotic division. RU 38486, a synthetic steroid that blocks progesterone receptors, prevents progesterone-induced fluidity changes. These findings indicate that the binding of progesterone to its receptor changes the oocyte plasma membrane structure resulting in a differential decrease in mobility near the membrane surface compared to that deeper in the membrane.     

Lipid fluidity at different regions in LDL and HDL of beta-thalassemia/Hb E patients

Atherosclerosis-related vascular complications in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients may result from iron induced oxidation of lipoproteins. To identify the specific site of oxidative damage, changes in lipid fluidity at different regions in LDL and HDL particle were investigated using two fluorescence probes and two ESR spin probes. The magnitude of increased lipid fluidity in thalassemic lipoproteins was dependent on the location of the probes. In hydrophobic region, the rotational correlation times for 16-doxyl stearic acid and DPH anisotropy were markedly changed in LDL and HDL of the patients. In the surface region, there was only a slight change in the order parameter (S) for 5-doxyl stearic acid and TMA-DPH anisotropy. Lipid fluidity at the core of LDL and HDL showed good correlation with oxidative stress markers, the ratio of CL/CO, and the level of alpha-tocopherol, suggesting that hydrophobic region of thalassemic lipoprotein was a target site for oxidative damage.     

Radiation-induced association of beta-glucuronidase with purified nuclei from irradiated MOLT-4 and HeLa cells

Beta-glucuronidase, a lysosomal marker enzyme, associates with purified nuclei from HeLa and MOLT-4 cell lines in a radiation dose-dependent manner, up to 300 cGy in MOLT-4 cells, and 1000 cGy in HeLa cells. In MOLT-4 cells (200-cGy exposure), there is a significant increase in beta-glucuronidase activity detected in the nuclear fraction 24 h postirradiation with a maximum association occurring at 72 h. In HeLa cells (1000-cGy exposure), a significant association is first detected 24 h postirradiation with a maximum association at 48 h. The association is not the result of nonspecific contamination occurring during nuclei purification since nuclei from irradiated cells show no greater levels of plasma membrane marker and mitochondrial marker than controls. The nature of the association remains unclear, but activity is not removed by detergents used in the nuclei isolation procedure, and incubation of the nuclei with EDTA reverses the association only modestly. Exposure of nuclei from irradiated cells to anisotonic buffers also results in only a small decrease in beta-glucuronidase activity associated with the nuclei. These observations suggest that lysosomal hydrolases become intimately associated with the nuclei of irradiated cells.     

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.     

Effects of pH on membrane fluidity of human erythrocytes

The effects of pH on the membrane fluidity of intact human erythrocytes, ghosts, and their lipid vesicles were studied by spin label techniques in the range of pH 3.0 to 9.1. Two fatty acid spin labels, 5-nitroxide stearic acid (5NS) and 12-nitroxide stearic acid (12NS), and a maleimide spin label were used for the labeling of the membrane lipids and proteins, respectively. The outer hyperfine splitting (T parallel) was measured as a parameter of membrane fluidity. In the case of 5NS, the T parallel values for intact erythrocytes and ghosts remained almost constant over the entire pH range at 22 degrees C but those for their lipid vesicles changed slightly, indicating the vertical displacement of the labels in lipid bilayers. On the other hand, the ESR spectra of 12NS incorporated into intact erythrocytes and ghosts, as compared with their lipid vesicles, showed marked pH dependence. By means of spin labeling of membrane proteins, the conformational changes of the proteins were observed in the pH range mentioned above. These results suggest a possible association between the strong pH dependence of the T parallel values and the conformation changes of membrane proteins. The pH dependence of the membrane fluidity was also investigated in cholesterol-enriched and -depleted erythrocytes. The effects of cholesterol demonstrated that the membrane fluidity was significantly mediated by cholesterol at low pH, but not at high pH.     

Radiation-induced structural alterations in fish red blood cells

The structural properties of gamma-irradiated fish red blood cells were studied using a spin labelling method. The gradient increase of lipid fluidity with the increasing gamma radiation doses was indicated by methyl 5-doxylpalmitate and methyl 12-doxylstearate spin labels spectra. In turns, the spectra of maleimide spin label (4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl) indicated a modification of the internal proteins and the increased internal viscosity of red blood cells. The results encourage the conclusion that the increase in membrane fluidity may result from theernations in lipid-protein interactions rather than lipid peroxidation.     

EPR spin labeling measurements of thylakoid membrane fluidity during barley leaf senescence

Physical properties of thylakoid membranes isolated from barley were investigated by the electron paramagnetic resonance (EPR) spin labeling technique. EPR spectra of stearic acid spin labels 5-SASL and 16-SASL were measured as a function of temperature in secondary barley leaves during natural and dark-induced senescence. Oxygen transport parameter was determined from the power saturation curves of the spin labels obtained in the presence and absence of molecular oxygen at 25 °C. Parameters of EPR spectra of both spin labels showed an increase in the thylakoid membrane fluidity during senescence, in the headgroup area of the membrane, as well as in its interior. The oxygen transport parameter also increased with age of barley, indicating easier diffusion of oxygen within the membrane and its higher fluidity. The data are consistent with age-related changes of the spin label parameters obtained directly by EPR spectroscopy. Similar outcome was also observed when senescence was induced in mature secondary barley leaves by dark incubation. Such leaves showed higher membrane fluidity in comparison with leaves of the same age, grown under light conditions. Changes in the membrane fluidity of barley secondary leaves were compared with changes in the levels of carotenoids (car) and proteins, which are known to modify membrane fluidity. Determination of total car and proteins showed linear decrease in their level with senescence. The results indicate that thylakoid membrane fluidity of barley leaves increases with senescence; the changes are accompanied with a decrease in the content of car and proteins, which could be a contributing factor.     

Heavy ion induced membrane damage: Hemolysis of erythrocytes and changes in erythrocyte membrane fluidity

Human erythrocytes were irradiated with heavy ions of energies between 4 and 18 MeV/u having linear energy transfer (LET) values between 92 and 14000 keV/µm. Hemolysis has been studied as a macroscopic parameter for membrane damage and changes of the fluidity as a more microscopic parameter. The membrane fluidity changed in a characteristic dose-dependent manner as detected by electron spin resonance employing 12-doxylstearic acid methyl ester spin label (SL 12). Lysis cross sections and RBE values were determined from dose effect curves. The results demonstrate a high hemolytic efficiency of heavy ions compared to X rays. With increasing LET values the measured relative biological efficiency (RBE) values increase continuously. In the complete LET range the cross sections formed one common curve as function of LET and no saturation effects are observed. This is in direct contrast to other biological endpoints such as cell inactivation or DNA damage.     

The effects of verbascoside on plasma lipid peroxidation level and erythrocyte membrane fluidity during immobilization in rabbits: a time course study

We studied the changes in the level of plasma lipid peroxidation indicated by malondialdehyde (MDA) level and erythrocyte membrane fluidity expressed by the value of order parameter (S) during single hindlimb immobilization of 21 days using thiobarbituric acid - reactive substances method and spin label electron spin resonance method, respectively. The impacts of verbascoside that has been proved its antioxidative activity on the measured parameters were examined. 11 New Zealand white rabbits were immobilized and divided into two groups. The rabbits in the verbascoside group were administrated with 0.8 mg/kg of verbascoside twice a day orally throughout the immobilization. The rabbits in the placebo group were treated with normal saline. In placebo group, the results showed that the level of MDA significantly increased on day 3, peaked on day 7, and was still significantly higher on day 14 of immobilization, compared with the value measured before immobilization. The value of S reached the highest on day 7 and subsequently lowered but still higher on day 14 than those measured before immobilization. Compared with placebo group, there were lower MDA level (P < 0.05, 0.001, and 0.05 for days 3, 7, and 14, respectively) and higher erythrocyte membrane fluidity (P < 0.05, 0.001, and 0.001 for days 3, 7, and 14, respectively) in verbascoside group. The data indicated that immobilization caused temporal changes of increase in plasma lipid peroxidation and decrease in erythrocyte membrane fluidity. Verbascoside might have the effects to moderate oxidative stress and erythrocyte membrane fluidity during immobilization.     

Membrane dynamics of differentiating cultured embryonic chick skeletal muscle cells by fluorescence microscopy techniques

Changes in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FDR), with the use of a lipidsoluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.     

Electron spin resonance studies of starch-water-probe interactions

Interactions between starch, water and stable nitroxide radicals were studied by electron spin resonance. The motional properties of TEMPO, 4-(2-bromoacetamido) TEMPO (BrAcTEMPO), 5-DOXYL-stearic acid and 16-DOXYL-stearic acid probes as well as a label covalently attached to amylopectin were investigated in concentrated (10–50%) starch-water systems as a function of temperature, concentration of polymer and storage period. Compared with the free probes in solution, TEMPO and BrAcTEMPO showed slower tumbling rates in starch-water dispersions or gels, suggesting a higher microviscosity in the probe's environment. The spectra, however, remained motionally narrowed. In contrast, the three line spectra of the fatty acid probes in solution became highly anisotropic in the presence of starch. The results indicated that these probes were highly immobilized at room temperature by the starch granules or by the polysaccharide gel matrix. These interactions are weakened at elevated temperatures where the spectra revealed the presence of both motionally narrowed and motionally slowed spin populations. The nitroxide label on the amylopectin exhibited a much slower mobility than the corresponding free probe as well as being found to be more motionally sensitive to temperature changes; such motional behavior was interpreted as reflecting contributions from rotation of the label around the chain backbone as well as local segmental motion of the polymer chain itself. Starch gels doped with free probes or the spin labelled amylopectin displayed no change in the motion of the nitroxide group upon storage, i.e. the tumbling rates did not follow the time-dependent conformational changes associated with the retrogradation phenomenon.     

Effect of hyperthermia and ionizing radiation on the erythrocyte membrane

Spin-label studies of the effects of hyperthermia on the erythrocyte membrane revealed a decrease in the fluidity of lipids and changes in the state of membrane proteins. The rate of haemolysis in iso-osmotic glycerol solution was increased. Changes of most of the parameters studied when plotted in Arrhenius coordinates revealed a discontinuity (critical hyperthermic transition in the membrane) between 46 and 50 degrees C. Studies of the combined action of ionizing radiation (100 Gy) and hyperthermia (43 degrees C) showed the same direction of changes for (Na-K-Mg)-ATPase activity and spectra of membrane-bound maleimide spin label for both agents, but the additivity of changes depended on the parameter studied.     

Ascorbic acid radicals induced by the action of radiation in tissues from rat organs frozen at 77 K

Subsequent annealing technique and computer assistant analysis of EPR spectra were used to isolate an asymmetric EPR signal Rs(g = 2,0051; delta H = 0.8 mT) from the EPR spectrum of rat spleen gamma-irradiated at 77 K. Radicals with the same EPR spectrum were registered in: 1) water solution of ascorbic acid (2.10(-2) M, pH 3.4) frozen and irradiated at 77 K and 2) water-glycerol solution of ascorbic acid (10(-2) M, pH 10.3) frozen rapidly at the moment of intensive autooxidation. These model experiments allow to conclude that Rs signal is caused by the radicals of semidehydroascorbic acid. Radiochemical yield of these radicals as well as of all the radicals induced by gamma radiation in the whole rat tissues were measured. The EPR signal (Rs) is equivalent to the well known "artifact" signal of lyophilized tissues. The explanation of the mechanism of the radicals formation taking place under annealing of the frozen and irradiated tissues was suggested.     

Effect of Mg2+ on membrane fluidity and K+ transport in rat liver mitochondria

1. The effects of Mg2+ on the fluidity and on the transport properties of mitochondrial inner membrane were compared in parallel experiments. The fluidity was measured by intercalated fatty acid spin probes. Valinomycin-induced K+ uptake was followed using an ion-selective electrode. 2. The rotational diffusion rate of lipids was very slightly affected by Mg2+, whereas the ordering of the probed region of the inner membrane increased considerably above 30 degrees C in the presence of Mg2+. Mg2+ strongly inhibited K+ transport, particularly above 30 degrees C. 3. In the presence of different concentration of MgCl2 (0--30 mM) the order parameter showed no significant variation, whereas the rotational correlation time had essentially biphasic character with a minimum (i.e., faster diffusion rate) at 10 mM MgCl2. 4. We conclude that Mg2+ induces structural changes in the mitochondrial inner membrane and concomitant changes in its functional properties. The term 'fluidity' is inadequate for the interpretation of the data, since changes in the order parameter and in the characteristic correlation time of the inner membrane upon addition of Mg2+ did not show parallel tendencies.     

Radiation-induced changes in permeability in unilamellar phospholipid liposomes

Gamma-radiation-induced oxidative damage in unilamellar dipalmitoylphosphatidylcholine liposomes was investigated using a fluorescence technique. Liposomal changes in permeability induced by gamma radiation were monitored by measuring the leakage of pre-encapsulated 6-carboxyfluorescein, and alterations in lipid bilayer fluidity were determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. The changes in permeability and fluidity in the bilayer were found to be dependent on the radiation dose in a biphasic fashion. The results are interpreted in terms of lipid bilayer fluidization after exposure to doses up to 1 kGy, but rigidization of the bilayer at higher doses. These results indicate a relationship between alterations in permeability and fluidity in the lipid bilayer after irradiation. The vesicles were protected significantly against radiation-induced oxidative damage in the presence of alpha-tocopherol and ascorbic acid. Radiation-induced changes in the permeability of the liposomes after exposure to gamma radiation and their modification by antioxidants indicate the involvement of a free radical mechanism in the production of damage, which may offer new insights in to the modification of cellular radiosensitivity by modulation of membrane damage.     

Non-disjunction and chromosome loss in gamma-irradiated human lymphocytes: a fluorescence in situ hybridization analysis using centromere-specific probes

Centromere-specific DNA probes for chromosomes 4, 7 and 18 were used to simultaneously analyze chromosome loss, non-disjunction, breaks within the labeled region, and nucleoplasmic bridges induced by gamma rays in binucleated human lymphocytes. The doses used were 0, 1, 2 and 4 Gy, and approximately 1000 cells were scored per dose. Micronucleus frequency increased in a linear-quadratic fashion. For chromosome loss, significant increases were observed at 2 and 4 Gy, whereas for non-disjunction significant increases were observed at 1 Gy; thus non-disjunction allowed us to detect the effects of radiation at a lower dose than chromosome loss. The use of centromere-specific probes allowed discrimination between the clastogenic and aneugenic effects of ionizing radiation. The analysis of chromosome loss, not taking fragmented signals into account, ensures the detection of an aneugenic effect, which was not possible using pancentromeric probes. The frequency of chromosome breakage within the labeled regions was higher in nuclei than in micronuclei, suggesting an increase in the engulfment of chromosomal material by nuclei as a consequence of the presence of cytochalasin B in the cultures. Chromatin filaments connecting main nuclei, the so-called nucleoplasmic bridges, were observed in irradiated samples, and are a manifestation of rearranged chromosomes producing anaphase bridges.     

Effect of lipid composition on rat liver nuclear membrane fluidity

Nuclear membrane fluidity is measured in rat liver by use of the fluorescence anisotropy of two probes: diphenylhexatriene and its cationic derivative trimethylammonium-diphenylhexatriene. It has been shown that, in 2-month-old rat liver cells, the bilayer surface is less fluid than the hydrophobic core. The fluidity was higher in 6-day-old rat liver nuclei, in which both the amount of cholesterol and the cholesterol/phospholipid ratio decreased. The influence of the single phospholipids, and in particular of phosphatidylcholine, has been studied by increasing the phosphatidylcholine with a choline base exchange reaction in isolated nuclear membranes. After this reaction, the fluorescence anisotropy of the bilayer surface increased, whereas at the hydrophobic core it decreased. Analysis of fatty acid composition shows an increase of phosphatidylcholine unsaturated fatty acids. The results show that the fluidity of nuclear membranes changes in relation to the lipid content and to the fatty acid composition. The role of nuclear membrane fluidity in cell function is discussed. © 1997 John Wiley & Sons, Ltd.     

The Negative Effect of Soy Extract on Erythrocyte Membrane Fluidity: An Electron Paramagnetic Resonance Study

A decrease of erythrocyte membrane fluidity can contribute to the pathophysiology of hypertension. Soy products, which are used as alternative therapeutics in some cardiovascular conditions, contain various isoflavones (genistein, daidzein, and their glucosides, genistin and daidzin), which can incorporate cellular membrane and change its fluidity. The aim of this study was to examine the effects of soy extract (which generally corresponds to the soy products of isoflavone composition) on erythrocyte membrane fluidity at graded depths. We used electron paramagnetic resonance spectroscopy and fatty acid spin probes (5-DS and 12-DS), the spectra of which are dependent on membrane fluidity. After being treated with soy extract, erythrocytes showed a significant (P = 0.016) decrease of membrane fluidity near the hydrophilic surface, while there were no significant changes of fluidity in deeper hydrophobic membrane regions. These results suggest that soy products containing high levels of genistein and isoflavone glucosides may not be suitable for use in hypertension because they decrease erythrocyte membrane fluidity.     

Comparison of structural changes in skin and amnion tissue grafts for transplantation induced by gamma and electron beam irradiation for sterilization

Sterilization is an important step in the preparation of biological material for transplantation. The aim of the study is to compare morphological changes in three types of biological tissues induced by different doses of gamma and electron beam radiation. Frozen biological tissues (porcine skin xenografts, human skin allografts and human amnion) were irradiated with different doses of gamma rays (12.5, 25, 35, 50 kGy) and electron beam (15, 25, 50 kGy). Not irradiated specimens served as controls. The tissue samples were then thawn and fixed in 10 % formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid Schiff reaction, phosphotungstic acid hematoxylin, Sirius red and silver impregnation. The staining with hematoxylin and eosin showed vacuolar cytoplasmic changes of epidermal cells mainly in the samples of xenografts irradiated by the lowest doses of gamma and electron beam radiation. The staining with orcein revealed damage of fine elastic fibers in the xenograft dermis at the dose of 25 kGy of both radiation types. Disintegration of epithelial basement membrane, especially in the xenografts, was induced by the dose of 15 kGy of electron beam radiation. The silver impregnation disclosed nuclear chromatin condensation mainly in human amnion at the lowest doses of both radiation types and disintegration of the fine collagen fibers in the papillary dermis induced by the lowest dose of electron beam and by the higher doses of gamma radiation. Irradiation by both, gamma rays and the electron beam, causes similar changes on cells and extracellular matrix, with significant damage of the basement membrane and of the fine and elastic and collagen fibers in the papillary dermis, the last caused already by low dose electron beam radiation.     

Neisseria gonorrhoeae membrane microenvironment studied by spin-label electron spin resonance: comparison of colony types.

Spin-label electron spin resonance was used to characterize the microenvironment around spin probes which localize (i) in membranes, (ii) at the membrane surface, or (iii) in the cytoplasm of living Neisseria gonorrhoeae. Four colony types (T1, T2, T3, and T4) of gonococci were compared on the basis of the electron spin resonance parameters 2T parallel to, S (order parameter), and tau c (microviscosity). The concentration of spin label used had little or no effect on viability. T1 and T2 gonococci were found to have a more restricted environment for molecular motion of a membrane surface spin label than did T3 and T4. The membrane fluidity, as measured by a membrane lipid spin label, of T4 (S = 0.571) was significantly greater than that of T1 or T3 (S = 0.580). This difference was detected at 37 degrees C, at 25 degrees C, in agar-grown bacteria, and in exponential-phase cells. Studies using spin labels which probe different levels of the membrane indicated the presence of a membrane flexibility gradient. Cytoplasmic spin-label studies indicated that the cytoplasm of all gonococcal colony types was three to five times more viscous than water.