Fullerenol C60(OH)36 protects human erythrocyte membrane against high-energy electrons.

Fullerenols (polyhydroxylated fullerene C60) are nanomaterial with potentially broad applicability in biomedical sciences with high antioxidant ability, thus, we investigated the radioprotecting potential of fullerenol C60(OH)36 on human erythrocytes irradiated by high-energy electrons of 6 MeV. The results demonstrate that C60(OH)36 at concentration of 150 µg/mL protects the erythrocytes against the radiation-induced hemolysis (comparing to non-protected cells, we observed 30% and 39% protection for 0.65 and 1.3 kGy irradiation doses, respectively). The protecting effect was confirmed by 32% decreased release of potassium cations comparing to the cells irradiated without C60(OH)36. Measurements of the amount of lactate dehydrogenase (LDH) released from the irradiated erythrocytes showed that the size of the pores formed by irradiation was not sufficient to release LDH across the erythrocyte membranes. We also report a significant decrease of the affinity of acetylcholinesterase (AChE) for the substrate in the presence of fullerenol, indicating the relatively strong adsorption of C60(OH)36 to components of plasma membrane. Changes in membrane fluidity detected by fluorescence spectroscopy and conformational changes in membrane proteins detected by spin labeling suggest the dose-dependent formation of disulfide groups as an effect of oxidation and this process was inhibited by C60(OH)36. We suppose that scavenging the ROS as well as adsorption of fullerenol to membrane proteins and steric protection of -SH groups against oxidation are responsible for the observed effects.

Influence of static magnetic field exposure on fatty acid composition in Salmonella Hadar.

We have been interested, in this work, to investigate the effect of the exposure to static magnetic field at 200 mT (SMF) on the fatty acid (FA) composition of Salmonella enterica subsp Enterica serovar Hadar isolate 287: effects on the proportion of saturated and unsaturated fatty acids (SFAs, UFAs), cyclopropane fatty acids (CFAs) and hydroxy fatty acids after exposure to the static magnetic field at 200 mT (SMF). Analysis with Gas Chromatography-Mass Spectrometry (GC-MS) of total lipid showed that the proportion of the most fatty acids was clearly affected. The comparison of UFAs/SFAs ratio in exposed bacteria and controls showed a diminution after 3 and 6 h of exposure. This ration reached a balance after 9 h of treatment with SMF. So we can conclude that S. Hadar tries to adapt to magnetic stress by changing the proportions of SFAs and UFAs over time to maintain an equilibrium after 9 h of exposure, thus to maintain the inner membranes fluidity. Also, a decrease in the proportion of hydroxy FAs was observed after 6 h but an increase of this proportion after 9 h of exposure. Concerning CFAs, its proportion raised after 6 h of exposure to the SMF but it decreased after 9 h of exposure. These results are strongly correlated with those of cfa (cyclopropane fatty acid synthase) gene expression which showed a decrease of its expression after 9 h of exposure.

Role of cytoskeletal mechanics and cell membrane fluidity in the intracellular delivery of molecules mediated by laser-activated carbon nanoparticles.

Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-ß-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc.

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity.

Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lß phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lß phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.

Influence of MLS laser radiation on erythrocyte membrane fluidity and secondary structure of human serum albumin.

The biostimulating activity of low level laser radiation of various wavelengths and energy doses is widely documented in the literature, but the mechanisms of the intracellular reactions involved are not precisely known. The aim of this paper is to evaluate the influence of low level laser radiation from an multiwave locked system (MLS) of two wavelengths (wavelength = 808 nm in continuous emission and 905 nm in pulsed emission) on the human erythrocyte membrane and on the secondary structure of human serum albumin (HSA). Human erythrocytes membranes and HSA were irradiated with laser light of low intensity with surface energy density ranging from 0.46 to 4.9 J cm(-2) and surface energy power density 195 mW cm(-2) (1,000 Hz) and 230 mW cm(-2) (2,000 Hz). Structural and functional changes in the erythrocyte membrane were characterized by its fluidity, while changes in the protein were monitored by its secondary structure. Dose-dependent changes in erythrocyte membrane fluidity were induced by near-infrared laser radiation. Slight changes in the secondary structure of HSA were also noted. MLS laser radiation influences the structure and function of the human erythrocyte membrane resulting in a change in fluidity.

Effect of growth temperature, surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants.

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.

Thermal effects and sensitivity of biological membranes.

Temperature is one of the key parameters that controlled the origin and evolution of life on earth and it continues to be a principal regulator of the functions of organisms. Some aspects of the response of simple and complex organisms to temperature variations are encoded in the physical properties of the cell components, with the all-important plasma membrane playing a principal role. Other responses to temperature are more specific and through evolution, specialized receptors with particular temperature sensitivities have appeared to mediate this signaling. While some of these receptors are ancient and can be found in very primitive organisms, it seems that the mechanisms used by prokaryotes and eukaryotes are very different, indicating that temperature sensitivity has evolved in more than one occasion during evolution.

In situ determination of Clostridium endospore membrane fluidity during pressure-assisted thermal processing in combination with nisin or reutericyclin.

This study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat- and pressure-induced spore inactivation and membrane fluidity.

Effects of cryoprotectants on viability of Lactobacillus reuteri CICC6226.

Freeze-drying is commonly used to preserve probiotics, but it could cause cell damage and loss of viability. The cryoprotectants play an important role in the conservation of viability during freeze-drying. In this study, we investigated the survival rates of Lactobacillus reuteri CICC6226 in the presence of cryoprotectants such as sucrose, trehalose, and reconstituted skim milk (RSM). In addition, we determined the activities of hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), and ATPases immediately following the freeze-drying. The results showed that the differences in HK and PK activities with and without the cryoprotectants during freeze-drying were not significant, but cell viability and activities of LDH and ATPase were significantly different (P<0.01) prior to and after freeze-drying. Meanwhile, the results showed that the maintenance of the membrane integrity and fluidity was improved in the presence of the 10% trehalose or 10% RSM than other treatments during freeze-drying. These results have provided direct biochemical and metabolic evidence of injured cell during freeze-drying. Freeze-drying damaged membrane structure and function of cell and inactivated enzymes (LDH and ATPases). The results imply that LDH and ATPases are key markers and could be used to evaluate the effect of cryoprotectants on viability and metabolic activities of L. reuteri CICC6226 during freeze-drying.

[Fluorescence used to investigate the sensitivity of spinach chloroplast membrane to low intensity electromagnetic radiation].

A system for studying biological effect of radio frequency electromagnetic field was developed. The system can form an area where electromagnetic wave with large frequency range is well distributed. The strength of electromagnetic wave was measured easily. Electromagnetic wave in the system did not have effect on environment. The sensitivity of spinach chloroplast membrane to low intensity electromagnetic radiation of 300 MHz under power density of 5 mW x cm(-2) was studied by the spectral analysis method of fluorescence of 8-anilino-1-naphthalene-sulfonic acid (ANS) and the changes in chlorophyll a (Chla) fluorescence parameters of spinach chloroplast membrane. The result showed that the position of spectrum of ANS fluorescence of spinach chloroplast membrane did not change, but the intensity of ANS fluorescence was obviously increased under the action of electromagnetic radiation with power density of 1-5 mW x cm(-2). There was an increase in the intensity of ANS fluorescence with the increase in electromagnetic radiation. The increase of ANS fluorescence of spinach chloroplast membrane showed that low level electromagnetic field induced the decrease in fluidity of chloroplast membrane compared with control experiment. The cause of the change in the fluidity could be related to the polarization of chloroplast membrane under the electromagnetic field. The analysis of Chla fluorescence parameters of spinach chloroplast membrane indicated that low level electromagnetic field of 300 MHz made the fluorescence parameters F0 and F(VI/)F(V) decrease, and F(V)/Fo, Fv/F(m) and deltaF(V)/T increase. It was showed that low level electromagnetic field caused the change of non-active center of photosystem II of spinach chloroplast membrane to active center and the increase in potential active and photochemical efficiency of PSII, and promoted the transmit process of electron in photosynthesis of chloroplast membrane of photosynthesis cell in spinach leaf. The study confirmed that low level electromagnetic field has non--thermal effects on photosynthesis system of spinach chloroplast membrane. The cell in spinach leaf can keep the photosynthesis through the change in heterogeneity of photosystem II and adapt to the environment of electromagnetic radiation increase.

Microwaves as modulators of membrane stability parameters during hepatic cancer.

PURPOSE: The present study explored the potential of microwaves on membrane fluidity changes in diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC), in vivo. METHODS: Rats were segregated into four groups: normal control, DEN-treated, microwave-treated, DEN+microwave-treated. Brush border membranes (BBM) were isolated from the rats and, using the membrane extrinsic fluorophore pyrene, we assessed the viscosities as well as fluidity parameters. RESULTS: DEN treatment resulted in a significant rise in lipid peroxidation (LPO). Reduced glutathione levels (GSH) and the activities of glutathione reductase (GR), glutathione transferase (GST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were found to be significantly decreased following DEN treatment. On the other hand, microwave treatment in DEN-treated rats resulted in a significant decrease in the levels of lipid peroxidation but caused a significant rise in the levels of GSH as well in the activities of GR, GST, SOD, CAT and GPx. The results further demonstrated a marked decrease in membrane microviscosity following DEN treatment. On the other hand, a significant increase was observed in the excimer/monomer ratio and fluidity parameter of DEN-treated rats when compared to normal control rats. However, the alterations in membrane microviscosity and the fluidity parameters were significantly restored after microwave treatment. CONCLUSION: The study, therefore, concludes that microwave proved quite useful in the modulation of membrane stability parameters following DEN-induced hepatic cancer.

AFM study on the electric-field effects on supported bilayer lipid membranes.

Electric-field induced changes in structure and conductivity of supported bilayer lipid membranes (SLM) have been studied at submicroscopic resolution using atomic force microscopy and electrochemical impedance spectroscopy. The SLMs are formed on gold surfaces modified with mixed self-assembled monolayers of a cholesterol-tether and 6-mercaptohexanol. At applied potentials of < or =-0.25 V versus standard hydrogen electrode, the conductance of the SLM increases and membrane areas of <150 nm in size are found to elevate from the surface up to 15 nm in height. To estimate the electric field experienced by the lipid membrane, electrowetting has been used to determine the point of zero charge of a 6-mercaptohexanol-modified surface (0.19 +/- 0.13 V versus standard hydrogen electrode). The effects of electric fields on the structure and conductance of supported membranes are discussed.

Morphological transitions of vesicles induced by alternating electric fields.

When subjected to alternating electric fields in the frequency range 10(2)-10(8) Hz, giant lipid vesicles attain oblate, prolate, and spherical shapes and undergo morphological transitions between these shapes as one varies the field frequency and/or the conductivities lambda(in) and lambda(ex) of the aqueous solution inside and outside the vesicles. Four different transitions are observed with characteristic frequencies that depend primarily on the conductivity ratio lambda(in)/lambda(ex). The theoretical models that have been described in the literature are not able to describe all of these morphological transitions.

The effect of MLS laser radiation on cell lipid membrane.

INTRODUCTION: Authors of numerous publications have proved the therapeutic effect of laser irradiation on biological material, but the mechanisms at cellular and subcellular level are not yet well understood. OBJECTIVE: The aim of this study was to assess the effect of laser radiation emitted by the MLS M1 system (Multiwave Locked System) at two wavelengths (808 nm continuous and 905 nm pulsed) on the stability and fluidity of liposomes with a lipid composition similar to that of human erythrocyte membrane or made of phosphatidylocholine. MATERIAL AND METHODS: Liposomes were exposed to low-energy laser radiation at surface densities 195 mW/cm2 (frequency 1,000 Hz) and 230 mW/cm2 (frequency 2,000 Hz). Different doses of radiation energy in the range 0-15 J were applied. The surface energy density was within the range 0.46 - 4.9 J/cm 2. RESULTS: The fluidity and stability of liposomes subjected to such irradiation changed depending on the parameters of radiation used. CONCLUSIONS: Since MLS M1 laser radiation, depending on the parameters used, affects fluidity and stability of liposomes with the lipid content similar to erythrocyte membrane, it may also cause structural and functional changes in cell membranes.

The role of cavitation in liposome formation.

Liposome size is a vital parameter of many quantitative biophysical studies. Sonication, or exposure to ultrasound, is used widely to manufacture artificial liposomes, yet little is known about the mechanism by which liposomes are affected by ultrasound. Cavitation, or the oscillation of small gas bubbles in a pressure-varying field, has been shown to be responsible for many biophysical effects of ultrasound on cells. In this study, we correlate the presence and type of cavitation with a decrease in liposome size. Aqueous lipid suspensions surrounding a hydrophone were exposed to various intensities of ultrasound and hydrostatic pressures before measuring their size distribution with dynamic light scattering. As expected, increasing ultrasound intensity at atmospheric pressure decreased the average liposome diameter. The presence of collapse cavitation was manifested in the acoustic spectrum at high ultrasonic intensities. Increasing hydrostatic pressure was shown to inhibit the presence of collapse cavitation. Collapse cavitation, however, did not correlate with decreases in liposome size, as changes in size still occurred when collapse cavitation was inhibited either by lowering ultrasound intensity or by increasing static pressure. We propose a mechanism whereby stable cavitation, another type of cavitation present in sound fields, causes fluid shearing of liposomes and reduction of liposome size. A mathematical model was developed based on the Rayleigh-Plesset equation of bubble dynamics and principles of acoustic microstreaming to estimate the shear field magnitude around an oscillating bubble. This model predicts the ultrasound intensities and pressures needed to create shear fields sufficient to cause liposome size change, and correlates well with our experimental data.

Liposomes form nanotubules and long range networks in the presence of electric field.

Liposomes are lipid bilayer-bound micron scale structures critical to therapeutic treatments, biophysical studies, cosmetics, food, constrained volume experiments, and gene transfer. Applying an electric field to separate mixtures of liposomes played a role in their discovery and is still presently used for a variety of processes. Our group has found agreement between models of electric field-induced transport and capillary electrophoresis measurements where the liposomes are described as slightly elongated with the charged lipids migrating to form a local dipole. Here we show much more diverse structures that cannot be accounted for in these models. A variety of morphologies emerge, from individual liposomes being stretched into nanotubules several microns in length to long-range organized assemblies of liposomes over tens of microns. Based in this result, existing theories for electromigration of soft particles need to be re-addressed. Also, the formation of nanoscale lipid tubules suggests that unique structures for bionanoengineering can be fabricated. Much higher intrinsic fields than those applied here are observed in biology that suggests mechanical electrostatic interaction may play role in shape and function of individual biological membranes and networks of membrane-bound structures.

Applying a potential across a biomembrane: electrostatic contribution to the bending rigidity and membrane instability.

We investigate the effect on biomembrane mechanical properties due to the presence an external potential for a nonconductive incompressible membrane surrounded by different electrolytes. By solving the Debye-Hückel and Laplace equations for the electrostatic potential and using the relevant stress-tensor we find (1) in the small screening length limit, where the Debye screening length is smaller than the distance between the electrodes, the screening certifies that all electrostatic interactions are short range and the major effect of the applied potential is to decrease the membrane tension and increase the bending rigidity; explicit expressions for electrostatic contribution to the tension and bending rigidity are derived as a function of the applied potential, the Debye screening lengths, and the dielectric constants of the membrane and the solvents. For sufficiently large voltages the negative contribution to the tension is expected to cause a membrane stretching instability. (2) For the dielectric limit, i.e., no salt (and small wave vectors compared to the distance between the electrodes), when the dielectric constant on the two sides are different the applied potential induces an effective (unscreened) membrane charge density, whose long-range interaction is expected to lead to a membrane undulation instability.

Membrane electroporation: The absolute rate equation and nanosecond time scale pore creation.

The recent applications of nanosecond, megavolt-per-meter electric field pulses to biological systems show striking cellular and subcellular electric field induced effects and revive the interest in the biophysical mechanism of electroporation. We first show that the absolute rate theory, with experimentally based parameter input, is consistent with membrane pore creation on a nanosecond time scale. Secondly we use a Smoluchowski equation-based model to formulate a self-consistent theoretical approach. The analysis is carried out for a planar cell membrane patch exposed to a 10 ns trapezoidal pulse with 1.5 ns rise and fall times. Results demonstrate reversible supraelectroporation behavior in terms of transmembrane voltage, pore density, membrane conductance, fractional aqueous area, pore distribution, and average pore radius. We further motivate and justify the use of Krassowska's asymptotic electroporation model for analyzing nanosecond pulses, showing that pore creation dominates the electrical response and that pore expansion is a negligible effect on this time scale.

Effect of extremely low frequency electromagnetic fields on bacterial membrane.

PURPOSE: The effect of extremely low frequency electromagnetic fields (ELF-EMF) on bacteria has attracted attention due to its potential for beneficial uses. This research aimed to determine the effect of ELF-EMF on bacterial membrane namely the membrane potential, surface potential, hydrophobicity, respiratory activity and growth. MATERIALS AND METHODS: Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were subjected to ELF-EMF, 50 Hz, 1 mT for 2 h. Membrane potential was determined by fluorescence spectroscopy with or without EDTA (Ethylenediaminetetraacetic acid) with DisC3(5) (3,3-dipropylthiacarbocyanine iodide), zeta potential measurements were performed by electrophoretic mobility, hydrophobicity of the membrane was measured with MATH (Microbial Adhesion to Hydrocarbons) test, respiratory activity was determined with CTC (5-Cyano-2,3-ditolyl tetrazolium chloride), colony forming unit (CFU) and DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) was used for growth determinations. RESULTS: ELF-EMF caused changes in physicochemical properties of both Gram-positive and Gram-negative bacteria. Hyperpolarization was seen in S. aureus and EDTA-treated E. coli. Surface potential showed a positive shift in S. aureus contrariwise to the negative shift seen in EDTA-untreated E. coli. Respiratory activity increased in both bacteria. A slight decrease in growth was observed. CONCLUSION: These results show that ELF-EMF affects the crucial physicochemical processes in both Gram-positive and Gram-negative bacteria which need further research.

Structural and functional damages of whole body ionizing radiation on rat brain homogenate membranes and protective effect of amifostine.

PURPOSE: To investigate the effects of whole body ionizing radiation at a sublethal dose on rat brain homogenate membranes and the protective effects of amifostine on these systems at molecular level. MATERIALS AND METHODS: Sprague-Dawley rats, in the absence and presence of amifostine, were whole-body irradiated at a single dose of 8 Gy and decapitated after 24 h. The brain homogenate membranes of these rats were analyzed using Fourier Transform Infrared (FTIR) spectroscopy. RESULTS: Ionizing radiation caused a significant increase in the lipid to protein ratio and significant decreases in the ratios of olefinic = CH/lipid, CH2/lipid, carbonyl ester/lipid and CH3/lipid suggesting, respectively, a more excessive decrease in the protein content and the degradation of lipids as a result of lipid peroxidation. In addition, radiation changed the secondary structure of proteins and the status of packing of membrane lipid head groups. Furthermore, it caused a decrease in lipid order and an increase in membrane fluidity. The administration of amifostine before ionizing radiation inhibited all the radiation-induced alterations in brain homogenate membranes. CONCLUSIONS: The results revealed that whole body ionizing radiation at a sublethal dose causes significant alterations in the structure, composition and dynamics of brain homogenate membranes and amifostine has a protective effect on these membranes.