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1.  Increased Fluidity of Human Platelet Membranes during Complement-Mediated Immune Platelet Injury 
Journal of Clinical Investigation  1978;61(3):582-589.
Complement appears to be involved in the destruction of platelets in certain clinical disorders, such as quinidine purpura and post-transfusion purpura. In both disorders, the classical complement sequence is activated by antigen-antibody complexes. It has been suggested that the terminal components of the complement sequence insert into the hydrophobic core of cell surface membranes and that this process leads to cell lysis. Fluidity is a fundamental property of lipids within the membrane's hydrophobic core. To examine the interaction of complement with membranes, we investigated the effect of complement activation on the fluidity of human platelet membranes. Complement was fixed to platelets using a post-transfusion purpura antibody, and membrane lipid fluidity was assessed in terms of fluorescence anisotropy using two fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene and 9-(12-anthroyl) stearic acid. Microviscosity, expressed in poise, was derived from the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene.
Post-transfusion purpura antibody plus complement made platelet membranes more fluid as evidenced by a 21% decrease in anisotropy and a 35% decrease in microviscosity of platelets at 37°C, and this was associated with platelet lysis (51Cr release). Complement damage to platelets was accompanied by a 10-15% increase in ΔE, the fusion activation energy for microviscosity, indicating that complement not only decreased membrane microviscosity but also made membrane lipids less ordered. These changes were consistent and rapid, with platelet lysis and the reduction in microviscosity being half-maximal by 6 min. They were prevented by inactivation of complement with heat or with EDTA, and they were not observed when C5-deficient plasma was used as the complement source. Qualitatively similar changes in platelet membrane fluidity were observed when complement was fixed to platelets by a quinidine-dependent anti-platelet antibody rather than by post-transfusion purpura antibody. Post-transfusion purpura antibody plus complement also decreased the microviscosity of isolated platelet membranes. Moreover, the lipids extracted from platelets lysed by complement had a 22% decrease in microviscosity (P < 0.01), with no associated changes in the amount of cholesterol relative to phospholipid or in the amounts of the various phospholipids.
These studies demonstrate that lipids within the hydrophobic core of platelet membranes damaged by complement become more fluid, and this is associated with platelet lysis. These findings are consistent with the concept that the insertion of the terminal complement components into the platelet membrane bilayer perturbs lipid-lipid interactions within the membrane's hydrophobic core.
PMCID: PMC372570  PMID: 641139
2.  Membrane Damage Induced by Supercritical Carbon Dioxide in Rhodotorula mucilaginosa 
Indian Journal of Microbiology  2013;53(3):352-358.
To clarify the mechanism of microbial inactivation by supercritical carbon dioxide (SCCO2), membrane damage of Rhodotorula mucilaginosa was investigated within specific pressure (10 Mpa), temperature (37 °C), and treatment time (10–70 min) ranges, including cell morphological structure, membrane permeability and fluidity. SEM and TEM observations showed morphological changes in the cell envelope and intracellular organization after SCCO2 treatment. Increase of membrane permeability was measured as increased uptake of the trypan blue dye with microscopy, and leakage of intracellular substances such as UV-absorbing materials and ions by determining the change of protein and electrical conductivity. The SCCO2 mediated reduction in CFU ml−1 was 0.5–1 log higher at 37 °C and 10 MPa for 60 min in Rose Bengal Medium containing 4 % sodium than a similar treatment in Rose Bengal Medium. Membrane fluidity analyzed by fluorescence polarization method using 1,6-diphenyl-1,3,5-hexatriene showed that the florescence polarization and florescence anisotropy of the SCCO2-treated cells were increased slightly and gently compared with the untreated cells. The correlation between membrane damage and death of cells under SCCO2 was clear, and the membrane damage was a key factor induced the inactivation of cells.
doi:10.1007/s12088-013-0373-4
PMCID: PMC3689409  PMID: 24426136
Membrane damage; Supercritical carbon dioxide; Rhodotorula mucilaginosa; Mechanism
3.  Influence of Anaerobiosis and Low Temperature on Bacillus cereus Growth, Metabolism, and Membrane Properties 
The impact of simultaneous anaerobiosis and low temperature on growth parameters, metabolism, and membrane properties of Bacillus cereus ATCC 14579 was studied. No growth was observed under anaerobiosis at 12°C. In bioreactors, growth rates and biomass production were drastically reduced by simultaneous anaerobiosis and low temperature (15°C). The two conditions had a synergistic effect on biomass reduction. In anaerobic cultures, fermentative metabolism was modified by low temperature, with a marked reduction in ethanol production leading to a lower ability to produce NAD+. Anaerobiosis reduced unsaturated fatty acids at both low optimal temperatures. In addition, simultaneous anaerobiosis and low temperatures markedly reduced levels of branched-chain fatty acids compared to all other conditions (accounting for 33% of total fatty acids against more 71% for low-temperature aerobiosis, optimal-temperature aerobiosis, and optimal-temperature anaerobiosis). This corresponded to high-melting-temperature lipids and to low-fluidity membranes, as indicated by differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, and infrared spectroscopy. This is in contrast to requirements for cold adaptation. A link between modification in the synthesis of metabolites of fermentative metabolism and the reduction of branched-chain fatty acids at low temperature under anaerobiosis, through a modification of the oxidizing capacity, is assumed. This link may partly explain the impact of low temperature and anaerobiosis on membrane properties and growth performance.
doi:10.1128/AEM.06410-11
PMCID: PMC3298147  PMID: 22247126
4.  Premalignant alterations in the lipid composition and fluidity of colonic brush border membranes of rats administered 1,2 dimethylhydrazine. 
Journal of Clinical Investigation  1986;77(3):831-840.
Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. To determine whether alterations in the lipid composition and fluidity of rat colonic brush border membranes existed before the development of DMH-induced colon cancer, rats were injected s.c. with this agent (20 mg/kg body weight per wk) or diluent for 5, 10, and 15 wk. Animals were killed at these time periods and brush border membranes were prepared from proximal and distal colonocytes of each group. The "static" and "dynamic" components of fluidity of each membrane were then assessed, by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of the fluorophore 1,6 diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values of DL-2-(9-anthroyl) stearic acid and DL-12-(9-anthroyl) stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. Phospholipid methylation activity in these membranes was also measured using S-adenosyl-L-methionine as the methyl donor. The results of these studies demonstrate that: the lipid composition and both components of fluidity of proximal DMH-treated and control membranes and their liposomes were similar at all time periods examined; at 5, 10, and 15 wk the "dynamic component of fluidity" of distal DMH-treated membranes and their liposomes was found to be higher, similar, and lower, respectively, than their control counterparts; the "static component of fluidity" of distal DMH-treated membranes and their liposomes, however, was similar to control preparations at all three time periods; and alterations in the lipid composition and phospholipid methylation activities appeared to be responsible for these differences in the "dynamic component of fluidity" at these various time periods.
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PMCID: PMC423469  PMID: 3949981
5.  Effects of ethanol on the Escherichia coli plasma membrane. 
Journal of Bacteriology  1984;157(1):233-239.
The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.
PMCID: PMC215157  PMID: 6360997
6.  Effects of Insecticides on the Fluidity of Mitochondrial Membranes of the Diamondback Moth, Plutella xylostella, Resistant and Susceptible to Avermectin 
The effects of various insecticides on the fluidity of mitochondrial membranes and cross-resistance were investigated in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) using strains that were both resistant and susceptible to avermectin. The resistant strain of P. xylostella, AV-R, developed 1078-fold resistance to avermetins with a high level of cross-resistance to the analogs of avermectins, ivermectin and emamectin benzoate. It had more than 1000 times greater resistance when compared with the avermectin-susceptible strain, XH-S. Mitochondrial membrane fluidity was measured by detecting fluorescence polarization using DPH (1,6-Diphenyl -1,3,5-hexatriene) as the fluorescence probe. Abamectin, emamectin benzoate, ivermectin, cypermethrin and fenvalerate decreased the fluidity of mitochondrial membranes in the XH-S strain at 25°C. However, fipronil and acephate did not change the fluidity of mitochondrial membrane when the concentration of these insecticides was 1×10-4 mol/L. Membrane fluidity increased as the temperature increased. The thermotropic effect on the polarization value of DPH increased as the insecticide concentration was increased. There was a significant difference of mitochondrial membrane fluidity between both XH-S and AV-R when temperature was less than 25°C and no difference was observed when the temperature was more than 25°C. The low-dose abamectin (0.11 mg/L) in vivo treatment caused a significant change of membrane fluidity in the XH-S strain and no change in the AV-R strain. However, a high-dose abamectin (11.86 mg/L) resulted in 100% mortality of the XH-S strain. In vivo treatment may cause a significant change of membrane fluidity in the AV-R strain
doi:10.1673/031.008.0301
PMCID: PMC3061578  PMID: 20345311
insecticide resistance; fluorescence polarization; cross-resistance
7.  The Effect of Methanol on the Structural Parameters of Neuronal Membrane Lipid Bilayers 
The structures of the intact synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortexs, and the outer and the inner monolayer separately, were evaluated with 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(1-pyrenyl)propane (Py-3-Py) as fluorescent reporters and trinitrophenyl groups as quenching agents. The methanol increased bulk rotational and lateral mobilities of SPMVs lipid bilayers. The methanol increased the rotational and lateral mobilities of the outer monolayers more than of the inner monolayers. n-(9-Anthroyloxy)stearic acid (n-AS) were used to evaluate the effect of the methanol on the rotational mobility at the 16, 12, 9, 6, and 2 position of aliphatic chains present in phospholipids of the SPMVs outer monolayers. The methanol decreased the anisotropy of the 16-(9-anthroyloxy)palmitic acid (16-AP), 12-(9-anthroyloxy)stearic acid (12-AS), 9-(9-anthroyloxy)stearic acid (9-AS), and 6-(9-anthroyloxy)stearic acid (6-AS) in the SPMVs outer monolayer but it increased the anisotropy of 2-(9-anthroyloxy)stearic acid (2-AS) in the monolayers. The magnitude of the increased rotational mobility by the methanol was in the order at the position of 16, 12, 9, and 6 of aliphatic chains in phospholipids of the outer monolayers. Furthermore, the methanol increased annular lipid fluidity and also caused membrane proteins to cluster. The important finding is that was far greater increase by methanol in annular lipid fluidity than increase in lateral and rotational mobilities by the methanol. Methanol alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that methanol, in additions to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membranes lipids.
doi:10.4196/kjpp.2012.16.4.255
PMCID: PMC3419761  PMID: 22915991
Annular lipid fluidity; Membrane protein clustering; Methanol; Neuronal membranes; Transbilayer lateral and rotational mobility
8.  Increase in lipid fluidity of cellular membranes induced by adsorption of RNA and DNA virions. 
Journal of Virology  1977;22(2):353-360.
Changes in the dynamic behavior of membrane lipids of mammalian cells induced by adsorption of animal viruses were quantitatively monitored by fluorescence polarization analysis with the aid of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene embedded in the surface membrane lipid core of intact cells. Adsorption of encephalomyocarditis, West Nile, and polyoma viruses to hamster (baby hamster kidney) and mouse (3T3) cells is accompanied by a rapid and significant increase in the degree of fluidity of membrane lipids of the infected cells. These changes in membrane fluidity, which are virus dose dependent, are inhibited by low temperature and by treatment of the cells before-hand with compounds known to block viral receptors on the cell surface. It is suggested that increase in membrane lipid fluidity, induced by the adsorption of virions, is an early event in the process of cell-virus interactions.
PMCID: PMC515726  PMID: 194057
9.  Partitioning of Homologous Nicotinic Acid Ester Prodrugs (Nicotinates) into Dipalmitoylphosphatidylcholine (DPPC) Membrane Bilayers 
The partitioning behavior of a series of perhydrocarbon nicotinic acid esters (nicotinates) between aqueous solution and dipalmitoylphosphatidylcholine (DPPC) membrane bilayers is investigated as a function of increasing alkyl chain length. The hydrocarbon nicotinates represent putative prodrugs, derivatives of the polar drug nicotinic acid, whose functionalization provides the hydrophobic character necessary for pulmonary delivery in a hydrophobic, fluorocarbon solvent, such as perfluorooctyl bromide. Independent techniques of differential scanning calorimetry and 1,6-diphenyl-1,3,5 hexatriene (DPH) fluorescence anisotropy measurements are used to analyze the thermotropic phase behavior and lipid bilayer fluidity as a function of nicotinate concentration. At increasing concentrations of nicotinates over the DPPC mole fraction range examined (XDPPC = 0.6 – 1.0), all the nicotinates (ethyl (C2H5); butyl (C4H9); hexyl (C6H13); and octyl (C8H17)) partition into the lipid bilayer at sufficient levels to eliminate the pretransition, and decrease and broaden the gel to fluid phase transition temperature. The concentration at which these effects occur is chain length-dependent; the shortest chain nicotinate, C2H5, elicits the least dramatic response. Similarly, the DPH anisotropy results demonstrate an alteration of the bilayer organization in the liposomes as a consequence of the chain length-dependent partitioning of the nicotinates into DPPC bilayers. The membrane partition coefficients (logarithm values), determined from the depressed bilayer phase transition temperatures, increase from 2.18 for C2H5 to 5.25 for C8H17. The DPPC membrane/water partitioning of the perhydrocarbon nicotinate series correlates with trends in the octanol/water partitioning of these solutes, suggesting that their incorporation into the bilayer is driven by increasing hydrophobicity.
doi:10.1016/j.colsurfb.2010.02.015
PMCID: PMC2873030  PMID: 20227859
perhydrocarbon nicotinate; DPPC bilayers; prodrug; membrane partition coefficient
10.  Structural and Functional Insights into Aeropyrum pernix OppA, a Member of a Novel Archaeal OppA Subfamily▿ † 
Journal of Bacteriology  2010;193(3):620-630.
In this study we gain insight into the structural and functional characterization of the Aeropyrum pernix oligopeptide-binding protein (OppAAp) previously identified from the extracellular medium of an Aeropyrum pernix cell culture at late stationary phase. OppAAp showed an N-terminal Q32 in a pyroglutamate form and C-terminal processing at the level of a threonine-rich region probably involved in protein membrane anchoring. Moreover, the OppAAp protein released into the medium was identified as a “nicked” form composed of two tightly associated fragments detachable only under strong denaturing conditions. The cleavage site E569-G570 seems be located on an exposed surface loop that is highly conserved in several three-dimensional (3D) structures of dipeptide/oligopeptide-binding proteins from different sources. Structural and biochemical properties of the nicked protein were virtually indistinguishable from those of the intact form. Indeed, studies of the entire bacterially expressed OppAAp protein owning the same N and C termini of the nicked form supported these findings. Moreover, in the middle exponential growth phase, OppAAp was found as an intact cell membrane-associated protein. Interestingly, the native exoprotein OppAAp was copurified with a hexapeptide (EKFKIV) showing both lysines methylated and possibly originating from an A. pernix endogenous stress-induced lipoprotein. Therefore, the involvement of OppAAp in the recycling of endogenous proteins was suggested to be a potential physiological function. Finally, a new OppA from Sulfolobus solfataricus, SSO1288, was purified and preliminarily characterized, allowing the identification of a common structural/genetic organization shared by all “true” archaeal OppA proteins of the dipeptide/oligopeptide class.
doi:10.1128/JB.00899-10
PMCID: PMC3021232  PMID: 21097609
11.  Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis. 
Journal of Bacteriology  1990;172(9):5218-5224.
A long-standing question in bacterial chemotaxis is whether repellents are sensed by receptors or whether they change a general membrane property such as the membrane fluidity and this change, in turn, is sensed by the chemotaxis system. This study addressed this question. The effects of common repellents on the membrane fluidity of Escherichia coli were measured by the fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene in liposomes made of lipids extracted from the bacteria and in membrane vesicles. Glycerol, indole, and L-leucine had no significant effect on the membrane fluidity. NiSO4 decreased the membrane fluidity but only at concentrations much higher than those which elicit a repellent response in intact bacteria. This indicated that these repellents are not sensed by modulating the membrane fluidity. Aliphatic alcohols, on the other hand, fluidized the membrane, but the concentrations that elicited a repellent response were not equally effective in fluidizing the membrane. The response of intact bacteria to alcohols was monitored in various chemotaxis mutants and found to be missing in mutants lacking all the four methyl-accepting chemotaxis proteins (MCPs) or the cytoplasmic che gene products. The presence of any single MCP was sufficient for the expression of a repellent response. It is concluded (i) that the repellent response to aliphatic alcohols can be mediated by any MCP and (ii) that although an increase in membrane fluidity may take part in a repellent response, it is not the only mechanism by which aliphatic alcohols, or at least some of them, are effective as repellents. To determine whether any of the E. coli repellents are sensed by periplasmic receptors, the effects of repellents from various classes on periplasm-void cells were examined. The responses to all the repellents tested (sodium benzoate, indole, L-leucine, and NiSO4) were retained in these cells. In a control experiment, the response of the attractant maltose, whose receptor is periplasmic, was lost. This indicates that these repellents are not sensed by periplasmic receptors. In view of this finding and the involvement of the MCPs in repellent sensing, it is proposed that the MCPs themselves are low-affinity receptors for the repellents.
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PMCID: PMC213183  PMID: 2203744
12.  Interaction of vesicular stomatitis virus with lipid vesicles: depletion of cholesterol and effect on virion membrane fluidity and infectivity. 
Journal of Virology  1978;27(2):320-329.
Interaction with excess unilamellar phosphatidylcholine (PC) vesicles resulted in depletion of as much as 90% of the cholesterol from the membrane of intact vesicular stomatitis (VS) virus. The cholesterol depletion was not significantly influenced by the proteolytic removal of virion glycoprotein spikes, but it was temperature dependent. Cholesterol depletion caused substantial reduction in anisotropy of the VS virion membrane as measured by fluorescence depolarization of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene; residual adsorbed vesicles represent a significant factor in this apparent increase in virion membrane fluidity. Interaction with PC vesicles resulted in a substantial loss of VS viral infectivity as measured by plating efficiency on L-cell monolayers. Reduction in infectivity appeared to be related to temperature-dependent depletion of virion cholesterol by PC vesicles. Interaction of VS virions with cholesterol-containing PC vesicles resulted in significantly less decline in infectivity, but attempts to restore cholesterol and infectivity to depleted VS virions were unsuccessful. Depletion of virion cholesterol apparently results through collision with PC vesicles rather than movement of cholesterol monomers or micelles through the aqueous phase, because PC vesicle-virion interaction in the presence of cholesterol oxidase did not result in substantial oxidation of translocated cholesterol.
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PMCID: PMC354170  PMID: 211263
13.  Membrane fluidity profiles as deduced by saturation-recovery EPR measurements of spin-lattice relaxation times of spin labels 
There are no easily obtainable EPR spectral parameters for lipid spin labels that describe profiles of membrane fluidity. The order parameter, which is most often used as a measure of membrane fluidity, describes the amplitude of wobbling motion of alkyl chains relative to the membrane normal and does not contain explicitly time or velocity. Thus, this parameter can be considered as nondynamic. The spin-lattice relaxation rate (T−11) obtained from saturation-recovery EPR measurements of lipid spin labels in deoxygenated samples depends primarily on the rotational correlation time of the nitroxide moiety within the lipid bilayer. Thus, T−11 can be used as a convenient quantitative measure of membrane fluidity that reflects local membrane dynamics. T−11 profiles obtained for 1-palmitoyl-2-(n-doxylstearoyl)phosphatidylcholine (n-PC) spin labels in dimyristoylphosphatidylcholine (DMPC) membranes with and without 50 mol% cholesterol are presented in parallel with profiles of the rotational diffusion coefficient, R⊥, obtained from simulation of EPR spectra using Freed's model. These profiles are compared with profiles of the order parameter obtained directly from EPR spectra and with profiles of the order parameter obtained from simulation of EPR spectra. It is shown that T−11 and R⊥ profiles reveal changes in membrane fluidity that depend on the motional properties of the lipid alkyl chain. We find that cholesterol has a rigidifying effect only to the depth occupied by the rigid steroid ring structure and a fluidizing effect at deeper locations. These effects cannot be differentiated by profiles of the order parameter. All profiles in this study were obtained at X-band (9.5 GHz).
doi:10.1016/j.jmr.2011.07.022
PMCID: PMC3214655  PMID: 21868272
Membrane fluidity; Saturation-recovery; Spin-lattice relaxation time; Spin-lattice relaxation rate; Spin labels
14.  The Effect of Lidocaine · HCl on the Fluidity of Native and Model Membrane Lipid Bilayers 
The purpose of this study is to investigated the mechanism of pharmacological action of local anesthetic and provide the basic information about the development of new effective local anesthetics. Fluorescent probe techniques were used to evaluate the effect of lidocaine·HCl on the physical properties (transbilayer asymmetric lateral and rotational mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex, and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. Lidocaine·HCl increased the bulk lateral and rotational mobility of neuronal and model membrane lipid bilayes, and had a greater fluidizing effect on the inner monolayer than the outer monolayer. Lidocaine·HCl increased annular lipid fluidity in SPMV lipid bilayers. It also caused membrane proteins to cluster. The most important finding of this study is that there is far greater increase in annular lipid fluidity than that in lateral and rotational mobilities by lidocaine·HCl. Lidocaine·HCl alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that lidocaine, in addition to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membrane lipid.
doi:10.4196/kjpp.2012.16.6.413
PMCID: PMC3526746  PMID: 23269904
Annular lipid fluidity; Lidocaine·HCl; Membrane protein clustering; Neuronal and model membranes; Transbilayer lateral and rotational mobility
15.  The kinetic aspects of intracellular fluorescence labeling with TMA-DPH support the maturation model for endocytosis in L929 cells 
The Journal of Cell Biology  1994;125(4):783-794.
TMA-DPH (1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene), a hydrophobic fluorescent membrane probe, interacts with living cells by instantaneous incorporation into the plasma membrane, where it becomes fluorescent. It then follows the intracellular constitutive membrane traffic and acts as a bulk membrane marker of the endocytic pathway (Illinger, D., P. Poindron, P. Fonteneau, M. Modolell, and J. G. Kuhry. 1990. Biochim. Biophys. Acta. 1030:73-81; Illinger, D., P. Poindron, and J. G. Kuhry. 1991. Biol. Cell. 73:131-138). As such, TMA-DPH displays particular properties mainly due to partition between membranes and aqueous media. From these properties, original arguments can be inferred in favor of the maturation model for the endocytic pathway, against that of pre-existing compartments, in L929 cultured mouse fibroblasts. (a) TMA-DPH labeling is seen to progress from the cell periphery to perinuclear regions during endocytosis without any noticeable loss in fluorescence intensity; with a vesicle shuttle model this evolution would be accompanied by probe dilution with a decrease in the overall intracellular fluorescence intensity, and the labeling of the inner (late) compartments could in no way become more intense than that of the peripheral (early) ones. (b) From TMA-DPH fluorescence anisotropy assays, it is concluded that membrane fluidity is the same in the successive endocytic compartments as in the plasma membrane, which probably denotes a similar phospholipidic membrane composition, as might be expected in the maturation model. (c) TMA-DPH internalization and release kinetics are more easily described with the maturation model.
PMCID: PMC2120073  PMID: 8188746
16.  Erythrocyte membrane microviscosity and phospholipid composition in lead workers. 
The microviscosity and fluidity of erythrocyte ghost membranes from lead workers and control subjects was measured by fluorescence polarisation using the fluorophore, 1,6-diphenyl-1,3,5-hexatriene (DPH). Increased lead was associated with a significant decrease in the average microviscosity of resealed and unsealed erythrocyte membranes. Since DPH fluorescence reflects the organisation of lipids in the central core of the membrane, two aspects of phospholipid metabolism were investigated. Phospholipids were extracted from red blood cell ghost membranes and identified by high performance liquid chromatography. The ratio of phosphatidyl choline to phosphatidyl ethanolamine, an established correlate of membrane fluidity, was significantly increased in lead workers. This is attributed to the known increases in red blood cell cholesterol in lead workers and the structural incompatibility of phosphatidyl ethanolamine and cholesterol, which result in a compensatory increase of phosphatidyl choline. Erythrocyte ghost membranes from control subjects were resealed with the intermediates in phospholipid synthesis that increase with a lead inhibited decrease in red blood cell pyrimidine 5'-nucleotidase. Membrane fluidity was not modified by incubation with cytidine triphosphate, uridine triphosphate, cytidine diphosphate choline, or cytidine diphosphate ethanolamine. Alterations in the microviscosity of the lipid regions of the hydrophobic core of the erythrocyte membrane bilayer and in the phospholipid composition of the membrane may be defects which contribute to the clinical and biochemical instability of the red blood cell on exposure to lead.
PMCID: PMC1007930  PMID: 3689721
17.  Effect of potassium perfluorooctanesulfonate, perfluorooctanoate and octanesulfonate on the phase transition of dipalmitoylphosphatidylcholine (DPPC) bilayers 
Biochimica et biophysica acta  2007;1768(5):1299-1308.
Summary
Perfluorooctanesulfonic acid (PFOS) is a persistent environmental pollutant that may cause adverse effects by inhibiting pulmonary surfactant. To gain further insights in this potential mechanism of toxicity, we investigated the interaction of PFOS potassium salt with dipalmitoylphosphatidylcholine (DPPC) – the major component of pulmonary surfactant – using steady-state fluorescence anisotropy spectroscopy and DSC (differential scanning calorimetry). In addition, we investigated the interactions of two structurally related compounds, perfluorooctanoic acid (PFOA) and octanesulfonic acid (OS) potassium salt, with DPPC. In the fluorescence experiments a linear depression of the main phase transition temperature of DPPC (Tm) and an increased peak width was observed with increasing concentration of all three compounds, both using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as fluorescent probes. PFOS caused an effect on Tm and peak width at much lower concentrations because of its increased tendency to partition onto DPPC bilayers, i.e., the partition coefficients decrease in the K(PFOS) > K(PFOA) ≫ K(OS). Similar to the fluorescence anisotropy measurements, all three compounds caused a linear depression in the onset of the main phase transition temperature and a significant peak broadening in the DSC experiments, with PFOS having the most pronounced effect of the peak width. The effect of PFOS and other fluorinated surfactants on DPPC in both mono- and bilayers may be one mechanism by which these compounds causes adverse biological effects.
doi:10.1016/j.bbamem.2007.02.003
PMCID: PMC1993895  PMID: 17349969
PFOS; PFOA; DSC; DPPC; DPH; TMA-DPH
18.  Genotype-induced changes in biophysical properties of frontal cortex lipid raft from APP/PS1 transgenic mice 
Alterations in the lipid composition of lipid rafts have been demonstrated both in human brain and transgenic mouse models, and it has been postulated that aberrant lipid composition in lipid rafts is partly responsible for neuronal degeneration. In order to assess the impact of lipid changes on lipid raft functional properties, we have aimed at determining relevant physicochemical modifications in lipid rafts purified from frontal cortex of wild type (WT) and APP/PS1 double transgenic mice. By means of steady-state fluorescence anisotropy analyses using two lipid soluble fluorescent probes, TMA-DPH (1-[(4-trimethyl-amino)phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene), we demonstrate that cortical lipid rafts from WT and APP/PS1 animals exhibit different biophysical behaviors, depending on genotype but also on age. Thus, aged APP/PS1 animals exhibited slightly more liquid-ordered lipid rafts than WT counterparts. Membrane microviscosity ηapp analyses demonstrate that WT lipid rafts are more fluid than APP/PS1 animals of similar age, both at the aqueous interface and hydrophobic core of the membrane. ηapp in APP/PS1 animals was higher for DPH than for TMA-DPH under similar experimental conditions, indicating that the internal core of the membrane is more viscous than the raft membrane at the aqueous interface. The most dramatic changes in biophysical properties of lipid rafts were observed when membrane cholesterol was depleted with methyl-β-cyclodextrin. Overall, our results indicate that APP/PS1 genotype strongly affects physicochemical properties of lipid raft. Such alterations appear not to be homogeneous across the raft membrane axis, but rather are more prominent at the membrane plane. These changes correlate with aberrant proportions of sphingomyelin, cholesterol, and saturated fatty acids, as well as polyunsaturated fatty acids, measured in lipid rafts from frontal cortex in this familial model of Alzheimer's Disease.
doi:10.3389/fphys.2012.00454
PMCID: PMC3506919  PMID: 23205014
lipid rafts; membrane viscosity; membrane thermodynamics; fluorescence anisotropy; cholesterol depletion; microdomain lipid composition
19.  Decreased Fluidity of Red Cell Membrane Lipids in Abetalipoproteinemia 
Journal of Clinical Investigation  1977;60(1):115-121.
Acanthocytic red cells in patients with abetalipoproteinemia are morphologically similar to the red cells in spur cell anemia. Fluidity of membrane lipids is decreased in spur cells due to their excess cholesterol content. Acanthocyte membranes have an increased content of sphingomyelin and a decreased content of lecithin. To assess the effect of this abnormality of acanthocyte membrane lipid composition on membrane fluidity, we studied red cells from five patients with abetalipoproteinemia and four obligate heterozygote family members.
Membrane fluidity was measured in terms of microviscosity (¯η) at 37°C, assessed by means of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. It was increased from 3.2±0.1 poise in normals to 4.01-4.14 poise in acanthocytes. This was associated with an increase in the sphingomyelin/lecithin ratio from 0.84±0.08 in normals in 1.45-1.61 in acanthocytes. The ¯η of acanthocyte membranes was not influenced by the degree of vitamin E deficiency. Similar changes in ¯η were observed in liposomes prepared from red cell lipids. Heterozygotes had normal sphingomyelin/lecithin ratios and normal values for ¯η. The flow activation energy for viscosity, a measure of the degree of order in the hydrophobic portion of the membrane, was decreased from 8.3 kcal/mole in normal red cells to 7.2 kcal/mole in acanthocytes, indicating that acanthocyte membrane lipids are more ordered. Variations in the sphingomyelin/lecithin mole ratio of liposomes prepared from brain sphingomyelin and egg lecithin with equimolar cholesterol caused similar changes in both ¯η and activation energy. The deformability of acanthocytes, assessed by means of filtration through 3-μm filters, was decreased.
These studies indicate that the increased sphingomyelin/lecithin ratio of acanthocytes is responsible for their decreased membrane fluidity. As in spur cells and in red cells enriched with cholesterol in vitro, this decrease in membrane fluidity occurs coincidentally with an abnormality in cell contour and an impairment in cell deformability.
PMCID: PMC372349  PMID: 874076
20.  Differences in Membrane Fluidity and Fatty Acid Composition between Phenotypic Variants of Streptococcus pneumoniae 
Journal of Bacteriology  2004;186(14):4638-4644.
Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P ≤ 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P ≤ 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P ≤ 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.
doi:10.1128/JB.186.14.4638-4644.2004
PMCID: PMC438610  PMID: 15231797
21.  Effect of Nedocromil Sodium on Polymorphonuclear Leukocyte Plasma Membrane 
Mediators of Inflammation  1994;3(7):S21-S24.
The effect of nedocromil sodium on the plasma membrane fluidity of polymorphonuclear leukocytes (PMNs) was investigated by measuring steady-state fluorescence anisotropy of 1-[4-trimethylammonium-phenyl]-6-phenyl- 1,3,5-hexatriene (TMA-DPH) incorporated in the membrane. Our results show that nedocromil sodium 300 μM significantly decreased membrane fluidity of PMNs. The decrease in membrane fluidity of PMNs induced by fMLP was abolished in the presence of nedocromil sodium. These data suggest that nedocromil sodium interferes with the plasma membranes of PMNs and modulates their activities.
doi:10.1155/S0962935194000700
PMCID: PMC2365593  PMID: 18475598
22.  In Vitro Low-Level Resistance to Azoles in Candida albicans Is Associated with Changes in Membrane Lipid Fluidity and Asymmetry 
The present study tracks the development of low-level azole resistance in in vitro fluconazole-adapted strains of Candida albicans, which were obtained by serially passaging a fluconazole-susceptible dose-dependent strain, YO1-16 (fluconazole MIC, 16 μg ml−1) in increasing concentrations of fluconazole, resulting in strains YO1-32 (fluconazole MIC, 32 μg ml−1) and YO1-64 (MIC, 64 μg ml−1). We show that acquired resistance to fluconazole in this series of isolates is not a random process but is a gradually evolved complex phenomenon that involves multiple changes, which included the overexpression of ABC transporter genes, e.g., CDR1 and CDR2, and the azole target enzyme, ERG11. The sequential rise in fluconazole MICs in these isolates was also accompanied by cross-resistance to other azoles and terbinafine. Interestingly, fluorescent polarization measurements performed by using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed that there was a gradual increase in membrane fluidity of adapted strains. The increase in fluidity was reflected by observed change in membrane order, which was considerably decreased (decrease in fluorescence polarization values, P value) in the adapted strain (P value of 0.1 in YO1-64, compared to 0.19 in the YO1-16 strain). The phospholipid composition of the adapted strain was not significantly altered; however, ergosterol content was reduced in YO1-64 from that in the YO1-16 strain. The asymmetrical distribution of phosphatidylethanolamine (PE) between two monolayers of plasma membrane was also changed, with PE becoming more exposed to the outer monolayer in the YO1-64 strain. The results of the present study suggest for the first time that changes in the status of membrane lipid phase and asymmetry could contribute to azole resistance in C. albicans.
doi:10.1128/AAC.46.4.1046-1052.2002
PMCID: PMC127087  PMID: 11897588
23.  Studies of Relationships among Bile Flow, Liver Plasma Membrane NaK-ATPase, and Membrane Microviscosity in the Rat 
Journal of Clinical Investigation  1979;64(6):1590-1598.
Liver plasma membrane (LPM) NaK-ATPase activity, LPM fluidity, and bile acid-independent flow (BAIF) were studied in rats pretreated with one of five experimental agents. Compared with controls, BAIF was increased 24.6% by thyroid hormone and 34.4% by phenobarbital, decreased by ethinyl estradiol, but unchanged by propylene glycol and cortisone acetate. Parallel to the observed changes in BAIF, NaK-ATPase activity also was increased by thyroid hormone (40.8%) and decreased by ethinyl estradiol (26.2%). In contrast, NaK-ATPase activity failed to increase after phenobarbital but did increase 36% after propylene glycol and 34.8% after cortisone acetate. Thus BAIF and NaK-ATPase activity did not always change in parallel. The NaK-ATPase Km for ATP was not affected by any of these agents.
LPM fluidity, measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene, was found to be increased by propylene glycol, thyroid hormone, and cortisone acetate, decreased by ethinyl estradiol, and unaffected by phenobarbital. Thus in these cases, induced changes in LPM fluidity paralleled those in NaK-ATPase activity. In no case did Mg-ATPase or 5′-nucleotidase activities change in the same direction as NaK-ATPase, and the activity of neither of these enzymes correlated with LPM fluidity, thus indicating the selective nature of the changes in LPM enzyme activity caused by the agents.
These findings indicate that LPM fluidity correlates with NaK-ATPase activity and may influence the activity of this enzyme. However, the nature of the role of LPM NaK-ATPase in bile secretion is uncertain and needs further study.
Images
PMCID: PMC371312  PMID: 227937
24.  Structural Basis of Lipid-Driven Conformational Transitions in the KvAP Voltage Sensing Domain 
Voltage-gated ion channels respond to transmembrane electric fields through reorientations of the positively charged S4 helix within the voltage-sensing domain (VSD). Despite a wealth of structural and functional data, the details of this conformational change remain controversial. Recent electrophysiological evidence showed that equilibrium between the resting (Down) and activated (Up) conformations of KvAP-VSD from Aeropyrum pernix can be biased through reconstitution in lipids with or without phosphate groups. We investigated the structural transition between these functional states using site-directed spin labeling and EPR spectroscopic methods. Solvent accessibility and inter-helical distance determinations suggest that KvAP gates through S4 movements involving a ~3 Å upward tilt and simultaneous ~2 Å axial shift. This motion leads to large accessibly changes in the intracellular water-filled crevice and supports a novel model of gating that combines structural rearrangements and electric field remodeling.
doi:10.1038/nsmb.2747
PMCID: PMC3946318  PMID: 24413055
25.  The Potent In Vitro Skin Permeation of Archaeosome Made from Lipids Extracted of Sulfolobus acidocaldarius 
Archaea  2013;2013:782012.
Archaeosomes are a new generation of liposomes that exhibit higher stabilities under different conditions, such as high temperatures, alkaline or acidic pH, and presence of bile salts in comparison with liposomes, and can be used in biotechnology including drug, gene, and vaccine delivery. The objective of this study was to prepare archaeosomes using lipid extracted from Sulfolobus acidocaldarius and evaluate their physicochemical properties. The lipids were extracted from S. acidocaldarius and assayed by High Performance Thin-Layer Chromatography (HPTLC). Archaeosomes were prepared using film method and methylene blue was used as drug model. They were characterized for their vesicle size and Differential Scanning Calorimetry (DSC) was used to investigate changes in their thermal behavior. The released amount of methylene blue was determined using a dialysis membrane and rat skin. HPTLC analysis of the extracted lipids showed that glycerol ether may be the major lipid with more than 78 percent probability. Results of particle size determination showed a mean size of 158.33 nm and the results of DSC indicated the possible interaction of methylene blue with lipids during the preparation of archaeosome. The addition of cholesterol significantly improved the encapsulation of methylene blue in the archaeosome so that the encapsulation efficiency was 61.66 ± 2.88%. The result of in vitro skin permeation showed that methylene blue could pass through skin model according to Peppas model and there was about 41.66% release after 6 h, whereas no release was observed through dialysis membrane. According to the results of the study, it is concluded that archaeosome may be successfully used as drug delivery system.
doi:10.1155/2013/782012
PMCID: PMC3888715  PMID: 24453698

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