Diphenylhexatriene Anisotropy Research Articles

A small HSP, Lo18, interacts with the cell membrane and modulates lipid physical state under heat shock conditions in a lactic acid bacterium

The small heat shock proteins (sHSP) are characterized by a chaperone activity to prevent irreversible protein denaturation. This study deals with the sHSP Lo18 induced by multiple stresses in Oenococcus oeni, a lactic acid bacterium. Using in situ immunocytochemistry and cellular fractionation experiments, we demonstrated the association of Lo18 with the membrane in O. oeni cells submitted to heat shock. The same result was obtained after exposure of cells to ethanol or benzyl alcohol, agents known to have an influence on membranes. For the different stresses, the protein was located on the periphery of the cell at membrane level and was also found within the cytoplasm. In order to determine if Lo18 could interact with the phospholipids, we used model membranes made of lipids extracted from O. oeni cells. Using fluorescence anisotropy of diphenylhexatriene (DPH) and generalized polarization of Laurdan, we showed that purified Lo18 interacts with these liposomes, and increases the molecular order of the lipid bilayer in these membranes when the temperature reaches 33.8 °C. All these data suggest that Lo18 could be involved in an adaptive response allowing the maintenance of membrane integrity during stress conditions in O. oeni cells.

Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus

Changes in water permeability and membrane packing were measured in cells of Lactobacillus bulgaricus and in vesicles prepared with lipids extracted from them. The osmotic response of whole cells and vesicles is compared with the one of bacteria grown in a high osmolal medium. Both bacteria and vesicles, behave as osmometers. This means that the volume decrease is promoted by the outflow of water, driven by the NaCl concentration difference, arguing that neither Na + nor Cl − permeates the cell or the lipid membrane in these conditions. Therefore, the volume changes can be correlated with the rate of water permeation across the cell or the vesicle membranes. The permeation of water was analyzed as a function of the lipid species by measuring the volume changes and the saturation ratio of the lipids. To put into relevance the membrane processes, the permeation properties of lipid vesicles prepared with lipids extracted from bacteria grown in normal and high osmolality conditions were also analyzed. The permeation response was correlated with the physical properties of the membrane of whole cells and vesicles, by means of fluorescence anisotropy of diphenyl hexatriene (DPH). The modifications in membrane properties are related with the changes in the membrane composition triggered by the growth in a high osmolal medium. The changes appear related to an increase in the sugar content of the whole pool of lipids and in the saturated fatty acid residues.

Thyroid hormones stimulate Na+?Pi transport activity in rat renal brush-border membranes: Role of membrane lipid composition and fluidity

Antecedent studies have suggested that lipid composition and fluidity of cellular membranes of various organs are altered in response to thyroid hormone status. To date, the effects of thyroid hormone status on these parameters have not been examined in rat renal apical membrane in regard to sodium-dependent phosphate transport. In the present study, we determined the potential role of alterations in cortical brush-border membrane lipid composition and fluidity in modulation of Na+-Pi transport activity in response to thyroid hormone status. Thyroid hormone status influences the fractional excretion of Pi, which is associated with alteration in renal brush-border membrane phosphate transport. The increment in Na+-Pi transport in renal BBMV isolated from Hyper-T rats is manifested as an increase in the maximal velocity (Vmax) of Na+-Pi transport. Further, the cholesterol content was significantly increased in renal BBM of Hypo-T rats and decreased in Hyper-T rats as compared to the Eu-T rats. The molar ratio of cholesterol/phospholipids was also higher in renal BBM from hypo-T rats. Subsequently, fluorescence anisotropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the renal BBM of the Hyper-T rats and increased in the Hypo-T rats as compared to Eu-T rats. The result of this study, therefore, suggest that alteration in renal BBM cholesterol, cholesterol/phospholipid molar ratio, and membrane fluidity play an important role in the modulation of renal BBM Na+-Pi transport in response to thyroid hormone status of animals.

Nicotinic Acetylcholine Receptor Induces Lateral Segregation of Phosphatidic Acid and Phosphatidylcholine in Reconstituted Membranes

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.

Dipole potential and head-group spacing are determinants for the membrane partitioning of pregnanolone.

The membrane interactions of pregnanolone, an intravenous general anesthetic steroid, were characterized using fluorescence spectroscopy and monolayer technique. di-8-ANEPPS [4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium], a membrane dipole potential (Psi)-sensitive probe, revealed pregnanolone to decrease Psi similarly as reported previously for other anesthetics. The decrement in Psi was approximately 16 and 10 mV in dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol (90:10, mol/mol) vesicles, respectively. Diphenylhexatriene anisotropy indicated pregnanolone to have a negligible effect on the acyl chain order. In contrast, substantial changes were observed for the fluorescent dye Prodan, thus suggesting pregnanolone to reside in the interfacial region of lipid bilayers. Langmuir balance studies indicated increased association of pregnanolone to DPPC monolayers containing cholesterol or 6-ketocholestanol at surface pressures pi > 20 mN/m as well as to monolayers of the unsaturated 1-palmitoyl-2-oleoylphosphatidylcholine. In the same surface pressure range, the addition of phloretin, which decreases Psi, reduced the penetration of pregnanolone into the monolayers. These results suggest that membrane partitioning of pregnanolone is influenced by the spacing of the phosphocholine head groups as well as by membrane dipole potential. The latter can be explained in terms of electrostatic dipole-dipole interactions between pregnanolone and the membrane lipids with their associated water molecules. Considering the universal nature of these interactions, they are likely to affect membrane partitioning of most, if not all, weakly amphiphilic drugs.

Structural changes in DODAC unilamellar liposomes by addition of sucrose esters monitored by using fluorescent techniques

The effect of the incorporation of the non-ionic surfactant sucrose palmitate in dioctadecyldimethylammonium chloride (DODAC) large unilamellar vesicles was studied by using several spectroscopic techniques. Significant changes in the liposome bilayer were detected prior to the formation of mixed micelles. Turbidity experiments show major growth in vesicle size. This behavior is rarely observed in large unilamellar vesicles. The vesicle size increase is accompanied by several alterations of the lipidic bilayer. Fluorescence of 6-dodecanoyl-2-(dimethylamino)-naphthalene (Laurdan) and pyrene incorporated in liposomes shows a dependence on sucrose palmitate addition, this is compatible with modifications of polarity and fluidity of its surroundings, probably due to a decrease in the bilayer water content. In contrast, diphenylhexatriene anisotropy changes indicate that deeper microenvironments are not substantially affected. In addition, changes in the quenching constant of pyrene and its derivatives by oxygen are indicative of the increase in oxygen solubility when the sucrose ester is incorporated in the liposome. These changes in the structure-dependent properties of DODAC liposome bilayer are explained by the location of the voluminous co-surfactant sucrose head groups on the liposome surface.

Variations in the lipid profile of patients with chronic renal failure, treated with folic acid.

Dyslipidemia and increases in plasma homocysteine usually occur at end-stage renal disease; both are recognized as risk factors for atherosclerosis. Folate administration reduces homocysteine concentration. In this study we determined the effect of a high dose of folic acid (40 mg intravenous injection three times a week) on plasma and red blood cell lipid profiles in twelve chronic renal failure patients on regular hemodialysis. Fasting blood samples were taken at the beginning of the study (baseline) and after 21, 42, and 64 days of treatment. Folic acid supplementation decreased plasma homocysteine. Plasma triglyceride levels decreased whereas polyunsaturated fatty acid values increased after 21 days; then they returned to baseline levels at the end of treatment. Total cholesterol and low-density lipoprotein (LDL) cholesterol were higher than those of the baseline during all the study, whereas high-density lipoprotein (HDL) cholesterol was reduced. In erythrocyte membranes, folic acid therapy enhanced cholesterol/phospholipid ratios and the fluorescence anisotropy of diphenyl-hexatriene. We conclude that large doses of folic acid produce a favorable effect, reducing plasma homocysteine levels and protecting patients from atherosclerosis. However, as this therapy induces significant alterations in both plasma and erythrocyte membrane lipid profiles, plasma lipid values should be controlled throughout the treatment of patients with renal failure.

PH, serum proteins and ionic strength influence the uptake of merocyanine 540 by WiDr cells and its interaction with membrane structures

It has been suggested that selective uptake of photosensitizers is due to significantly lower pH of the interstitial fluid in tumors compared to normal tissue. Therefore, the cellular uptake of merocyanine 540 (MC 540) was examined at two pH values: 6.8±0.1 and 7.4±0.1. There was no difference in spectral properties (absorption and fluorescence maxima positions, fluorescence intensity) of the drug in the presence of increasing amounts of either human blood plasma or FCS (0–2%) at the two pH values investigated. Nevertheless, significantly higher amounts of the drug were taken up by WiDr cells at pH 6.8±0.1, both in the presence of 10% FCS and in the absence of FCS. The absorption spectra of MC 540 in the presence of egg phosphatidylcholine (PC) liposomes turned out to be NaCl concentration-dependent (0.00–0.30 mol l −1). Membrane fluidity, as measured by fluorescence anisotropy of diphenylhexatriene (DPH), was unchanged within the experimental error in the NaCl concentration range 0.01–0.30 mol l −1. The spectral changes indicated an enhancement of the incorporation of MC 540 into lipid membranes with increasing ionic strength. Such a salt concentration dependence suggests a possible involvement of the surface potential in the interaction of MC 540 with lipid membranes. The results might provide an explanation of the pH dependency of the cellular uptake of MC 540 observed in this study.

Interaction of the antimalarial agents halofantrine and lumefantrine with lipid bilayers.

The effects of antimalarial drugs halofantrine and lumefantrine on the fluoresence anisotropy of diphenylhexatriene (DPH)-containing phospholipid vesicles have been examined. Lumefantrine increases DPH anisotropy, indicating a condensing effect on bilayers of dipalmitoylphosphatidyl choline (DPPC), dioleoylphosphatidylcholine (DOPC), egg lecithin and mouse erythrocyte membranes (including membranes isolated from plasmodial-infected mice). Its condensing effect is more pronounced in bilayers of lower microviscosity. In contrast, increases or decreases in DPH anisotropy are observed with halofantrine, depending on the nature of the lipid. Decreases in anisotropy, which reflect a perturbing effect, are observed in bilayers of high microviscosity (for example, gel state of DPPC bilayers). Increases in anisotropy are observed in bilayers of low microviscosity (such as DOPC and egg lecithin bilayers). The perturbing effect of halofantrine is further confirmed by the increases in permeability of calcein-containing DPPC vesicles in the presence of the drug. However the perturbative effects of halofantrine are observed to the same magnitude in uninfected and plasmodial-infected erythrocyte membranes, and may not be relevant to the antimalarial action of the drug. In contrast, the condensing effect of lumefantrine is significantly greater in infected erythrocyte membranes and may contribute to its antimalarial action.

Variations in the lipid profile of patients with chronic renal failure treated with pyridoxine

BackgroundHyperhomocysteinemia and lipid abnormalities are commonly found in patients with chronic renal failure; both are recognized as risk factors for atherosclerosis. The homocysteine-lowering effect of pyridoxine is controversial. This study was performed to determine the effect of a high dose of pyridoxine (300 mg i.v. three times a week) on plasma and red blood cell lipid profile and plasma homocysteine concentration in twelve chronic renal failure patients on regular hemodialysis. Fasting blood samples were taken at the beginning of the study (basal 1), after 30 and 60 days of treatment and 4 months after withdrawal (basal 2).ResultsPyridoxine supplementation induced a significant decrease in total plasma homocysteine level and also a lowering effect in plasma total cholesterol and triglycerides. These biochemical data increased when the samples were taken at basal 2, reaching the levels obtained at the beginning of the experiment. LDL cholesterol increased whereas HDL cholesterol was reduced during the treatment. In erythrocyte membranes vitamin B6 therapy enhanced the cholesterol/phospholipid ratio as well as the fluorescence anisotropy of diphenyl-hexatriene.ConclusionsWe conclude that high doses of pyridoxine represent an effective strategy to ameliorate both plasma homocysteine levels and lipid profiles in chronic renal failure patients, protecting them from atherosclerosis. Further research using a long-term treatment would be necessary in an attempt to restore the fatty acid pattern and the fluidity of red cell membranes.

Surface Charge Density Determines the Efficiency of Cationic Gemini Surfactant Based Lipofection

The efficiencies of the binary liposomes composed of 1,2-dimyristoyl- sn-glycero-3-phosphocholine and cationic gemini surfactant, (2S,3R)-2,3-dimethoxy-1,4-bis( N-hexadecyl- N, N-dimethylammonium)butane dibromide as transfection vectors, were measured using the enhanced green fluorescent protein coding plasmid and COS-1 cells. Strong correlation between the transfection efficiency and lipid stoichiometry was observed. Accordingly, liposomes with X SR−1 ≥ 0.50 conveyed the enhanced green fluorescent protein coding plasmid effectively into cells. The condensation of DNA by liposomes with X SR−1 > 0.50 was indicated by static light scattering and ethidium bromide intercalation assay, whereas differential scanning calorimetry and fluorescence anisotropy of diphenylhexatriene revealed stoichiometry dependent reorganization in the headgroup region of the liposome bilayer, in alignment with our previous Langmuir-balance study. Surface charge density and the organization of positive charges appear to determine the mode of interaction of DNA with (2S,3R)-2,3-dimethoxy-1,4-bis( N-hexadecyl- N, N-dimethylammonium)butane dibromide/1,2-dimyristoyl- sn-glycero-3-phosphocholine liposomes, only resulting in DNA condensation when X SR−1 > 0.50. Condensation of DNA in turn seems to be required for efficient transfection.

Changes in membrane microenvironment and signal transduction in platelets from NIDDM patients—a pilot study

Background: Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by the presence of abnormally active platelets in the circulation, leading to increased incidence of thrombotic complications. In this study, we have attempted to understand the pathophysiology of the platelets in NIDDM. Methods: Platelet aggregation was induced by thrombin receptor-activating peptide or epinephrine. Membrane fluidity was derived from the steady-state fluorescence anisotropy of diphenylhexatriene incorporated in the membrane. The phosphotyrosine content of the platelet proteins was probed using specific monoclonal antibodies. The extent of calpain activity was assessed from the proteolysis of calpain substrates. Results: Aggregation was significantly enhanced ( p<0.001) in the platelets obtained from the cases of NIDDM. Anisotropy measurements reflected a significant increase in the microviscosity of platelet membranes from 3.315 (±0.103) in the control to 4.153 (±0.119) in NIDDM. Proteins of relative mobilities of 131, 100, 47 and 38 kDa were found to remain phosphorylated on tyrosine in the resting platelets obtained from NIDDM patients, while they were not phosphorylated in the control counterparts. This was associated with heightened activity of the calcium-dependent thiol protease, calpain, in NIDDM. Conclusions: Taken together, these data indicated significant changes in the signaling mechanism in the platelets obtained from NIDDM, which could lead to platelet hyperactivity in this disease.

Stress-induced decrement in the plasticity of the physical properties of chick brain membranes.

The molecular basis underlying the stress-induced increment in the density of central benzodiazepine receptor from chick forebrain, observed previously at 4 degrees C, was studied from a biophysical perspective. The thermal dependence of [3H]flunitrazepam binding to the central benzodiazepine receptor and the supramolecular organization were studied in forebrain membranes from chicks submitted to partial water immersion. The equilibrium dissociation constants increased with temperature in membrane from both control and stressed chicks. The heat capacity values in control samples (deltaC(p, CON)) were significantly less negative than deltaC(p STR). Changes in deltaH and deltaS between 4-37 degrees C were greater in stressed chicks compared to control; however, the binding was exothermic and driven by enthalpy in both conditions. At 4 degrees C, the receptor density (B(max)) was higher in stressed chicks compared to control. Such a difference was lost irreversibly upon temperature elevation, possibly due to the hysteresis between the heating and cooling behaviour of B(max, CON) and the constancy in B(max, STR). The fluorescence anisotropy of diphenylhexatriene was higher in control samples with respect to stressed chicks below 10 degrees C. A temperature-induced increment in protein intrinsic-fluorescence was observed only in control, and was quenched by acrylamide more easily at 4 degrees C than at 25 degrees C. A higher microviscosity at 4 degrees C in control favoured more external localizations of integral proteins; at higher temperatures, tryptophan residues moved to hydrophobic membrane-regions. Changes in the membrane-organization towards more fluid states favoured the accessibility of benzodiazepine to the central benzodiazepine receptor, expressed by the higher values of B(max) found in stressed samples at low temperatures with respect to control samples.

Characterization of ginseng saponin ginsenoside-Rg3 inhibition of catecholamine secretion in bovine adrenal chromaffin cells

Since ginsenoside-Rg3, one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg3 inhibition were investigated. Although ginsenoside-Rg3 inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K+ or veratridine, an activator of the voltage-sensitive Ca2+ or Na+ channels, only slightly. The ACh-induced Na+ and Ca2+ influxes into the cells were also reduced by ginsenoside-Rg3. The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca2+. The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 μM ginsenoside-Rg3 and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 μM of this compound. Above 30 μM ginsenoside-Rg3 increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg3 at 30 μM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 μM. These results strongly suggest that ginsenoside-Rg3 blocks the nicotinic ACh receptor-operated cation channels, inhibits Na+ influx through the channels, and consequently reduces both Ca2+ influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.

Lipid fatty acid and protein pattern of equine prostasome-like vesicles

The semen of several mammals contains vesicles of different composition and origin. We have recently reported on the presence of lipoprotein vesicles in stallion semen. To a certain extent, these resemble human prostasomes, but differ from them in amount and composition. These horse-semen prostasome-like vesicles may be important, not only in horse reproductive physiology, but also in view of stallion semen cryopreservation. In this paper, we have studied horse-semen prostasome-like vesicles and found that they possess less saturated fatty acid than human prostasomes. Moreover, their protein pattern (SDS-PAGE electrophoresis) shows that the 30–50-kDa fraction is less abundant in stallion vesicles. In addition, fluidity (measured as fluorescence anisotropy of diphenylhexatriene) is higher in horse prostasome-like vesicles than in human prostasomes, albeit being much lower than that of most membranes. These findings may be connected to some species-related differences in reproductive physiology: the vaginal milieu of the mare is not acidic and the deposition of semen is intrauterine in the horse but vaginal in humans.

Chronic changes in plasma triglyceride levels do modify platelet membrane microviscosity in rats

Lipid metabolism disorders were proposed to mediate numerous cell membrane alterations in various forms of hypertension. Elevated plasma triglycerides were found to be associated with changes in membrane structure and function related to altered microviscosity in particular domains of the cell membrane. The aim of our study was to determine if an abnormal triglyceride metabolism might play a causal role in these alterations of membrane dynamics. Using genetically hypertensive rats of the Prague hereditary hypertriglyceridemic (HTG) strain we investigated whether the elevation of circulating triglycerides induced by high fructose intake and/or their lowering by chronic gemfibrozil treatment (for 10 weeks starting at the age of 6 weeks) are followed by reciprocal changes in membrane microviscosity. Two different fluorescent probes exploring either the outer membrane leaflet (TMA-DPH anisotropy) or the membrane lipid core (DPH anisotropy) were used in platelets of HTG rats. DPH (diphenylhexatriene) fluorescence anisotropy was decreased in platelets of fructose-treated HTG animals with highly elevated plasma triglyceride levels, whereas it was increased in gemfibrozil-treated HTG rats in which triglyceride levels were almost normalized. On the contrary, TMA-DPH (trimethylamino-diphenylhexatriene) anisotropy was not substantially altered in platelets from HTG rats by the above modifications of circulating triglycerides. No changes of plasma cholesterol or blood pressure were associated with the triglyceride-dependent modifications of membrane core microviscosity. Our interventional study demonstrates a major causal role of circulating triglycerides in the control of the microviscosity of membrane lipid core.

Effect of organochlorine insecticides on nicotinic acetylcholine receptor-rich membranes

The so-called generalized polarization (GP) of the fluorescent probe Laurdan and the steady-state fluorescence anisotropy of the probe diphenylhexatriene (DPH) and its phenylpropionic derivative (PA-DPH) were used to study the effects of several organochlorine insecticides of the chlorophenylethane, chlorinated cyclohexane and chlorinated cyclodiene families on the Torpedo nicotinic acetylcholine receptor (AChR)-rich native membrane. All insecticides, with the exception of Lindane, augmented Laurdan GP both in the native membrane and in model lipid systems. Most organochlorine compounds produced a concentration-dependent decrease of DPH and PA-DPH anisotropy in the AChR-rich membrane. These compounds exhibited a dual behavior vis-à-vis the native AChR-rich membrane, exerting disordering effects at the bilayer core while ordering and/or excluding water molecules from the lipid–protein interface region, as sensed by DPH anisotropy and Laurdan GP, respectively. Furthermore, all insecticides decreased the efficiency of fluorescence resonance energy transfer between the intrinsic protein and Laurdan, albeit to different extents. On the basis of all these observations, the existence of potential target sites for insecticides in the protein–lipid interface region is postulated.

Thyroid hormones modulate zinc transport activity of rat intestinal and renal brush-border membrane.

Thyroid hormone status influences the Zn2+ and metallothionein levels in intestine, liver, and kidney. To evaluate the impact of thyroid hormones on Zn2+ metabolism, Zn2+ uptake studies were carried out in intestinal and renal brush-border membrane vesicles (BBMV). Steady-state Zn2+ transport in intestinal and renal cortical BBMV was increased in hyperthyroid (Hyper-T) rats and decreased in the hypothyroid (Hypo-T) rats relative to euthyroid (Eu-T) rats. In both the intestinal and renal BBMV, Hyper-T rats showed a significant increase in maximal velocity compared with Eu-T and Hypo-T rats. Apparent Michaelis constant was unaltered in intestinal and renal BBMV prepared from the three groups. Fluorescence anisotropy of diphenyl hexatriene was decreased significantly in intestinal and renal brush-border membrane (BBM) isolated from Hyper-T rats compared with Hypo-T and Eu-T rats. A significant reduction in the microviscosity and transition temperature for Zn2+ uptake in intestinal and renal BBM from Hyper-T rats is in accordance with the increased fluidity of these BBMs. These findings suggest that the increased rate of Zn2+ transport in response to thyroid hormone status could be associated with either an increase in the number of Zn2+ transporters or an increase in the active transporters due to alteration in the membrane fluidity. Thus the thyroid hormone-mediated change in membrane fluidity might play an important role in modulating Zn2+ transport activity of intestinal and renal BBM.

Phase behavior of phosphatidylglycerol bilayers as a function of buffer composition: fluorescence studies using Laurdan probe

The influence of solution composition in lipid vesicles composed of different charged and zwitterionic phospholipids was studied using a Laurdan fluorescent probe. Particular results were found for dipalmitoylphosphatidylglycerol (DPPG) bilayers, in which quantitation of phase state domains as well as the main phase transition temperature are strongly dependent on ionic composition. The Laurdan generalised polarisation (GP) parameter decreases significantly in alkaline cation solutions in the concentration range 1–20 mM, in the order K +>Na +>Li +. Gel phase domains at 44°C change from 77% in vesicles prepared in pure milliQ water to 8% in 10 mM KCl solutions. Simultaneously, the steady-state fluorescence anisotropy of diphenylhexatriene (DPH) placed in the alkyl chain region of the bilayer decreases. No effect of alkaline ions on Laurdan relaxation was noticed in other negatively charged phospholipids such as dimyristoylphosphatidic acid (DMPA) or brain phosphatidylserine (PS). The addition of Ca 2+ to phosphatidylglycerol membranes was also detected even at micromolar concentrations. For example, 50 μM calcium concentration increases Laurdan GP from 0.17 to 0.477 at 51.6°C. The generation of a gel like phase was dependent on temperature and the presence of other ions in the medium. These results stress the particular phase behaviour of dipalmitoylphosphatidylglycerol (DPPG) membranes. Regarding these features, a structural mechanism related to phosphatidylglycerol arrangement is discussed.

Brain and heart membrane fatty acid composition in miniature swine fed diets containing corn and menhaden oils

Thirty-six female minipigs, from 4.7–10.6 years old, were fed corn oil (CO) and/or menhaden oil (MO) in diets containing 0, 0.75, or 15% MO and sufficient CO to provide 15% total oil for 6 months. Half the animals were made hypercholesterolemic by feeding them a 13% lard, 2% cholesterol diet for 2 months prior to the experimental feeding period. Samples were taken from the same location in each animal from forebrain, caudate nucleus, cerebellum, and heart to isolate brain synaptosomes and heart plasma membranes. Pretreatment of the minipigs with cholesterol had no effect on the fatty acid composition of the fractions isolated and their membrane fluidity. Despite MO feeding, the long chain n-3 fatty acids were low in the heart plasma membranes compared to heart tissue homogenates from the same animals. Minipigs fed 15% MO showed significant increases in synaptosomal eicosapentaenoate (EPA) and docosapentaenoate (DPA). Docosahexaenoate (DHA) was incorporated into synaptosomes in animals fed 0.75% MO, at concentrations significantly higher than in the CO-fed pigs. Pigs fed 15% MO showed significantly lower brain DHA than those fed CO. The synaptosomal data for the pigs fed the various diets suggest that metabolic synthesis from linolenate, 18:3(n-3), may be a more important source of brain DHA than diet, but that dietary n-6 n-3 ratios and levels of 18:3(n-3), EPA, DPA and DHA may require careful attention. The fatty acid changes did not affect fluidity determined by fluorescence anisotropy of diphenylhexatriene or its polar derivatives.