Fluorescence Anisotropy Of Diphenylhexatriene Research Articles

Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients.

This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar–nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes.

Differential interactions of bacterial lipopolysaccharides with lipid membranes: implications for TRPA1-mediated chemosensation

Bacterial lipopolysaccharides (LPS) activate the TRPA1 cation channels in sensory neurons, leading to acute pain and inflammation in mice and to aversive behaviors in fruit flies. However, the precise mechanisms underlying this effect remain elusive. Here we assessed the hypothesis that TRPA1 is activated by mechanical perturbations induced upon LPS insertion in the plasma membrane. We asked whether the effects of different LPS on TRPA1 relate to their ability to induce mechanical alterations in artificial and cellular membranes. We found that LPS from E. coli, but not from S. minnesota, activates TRPA1. We then assessed the effects of these LPS on lipid membranes using dyes whose fluorescence properties change upon alteration of the local lipid environment. E. coli LPS was more effective than S. minnesota LPS in shifting Laurdan’s emission spectrum towards lower wavelengths, increasing the fluorescence anisotropy of diphenylhexatriene and reducing the fluorescence intensity of merocyanine 540. These data indicate that E. coli LPS induces stronger changes in the local lipid environment than S. minnesota LPS, paralleling its distinct ability to activate TRPA1. Our findings indicate that LPS activate TRPA1 by producing mechanical perturbations in the plasma membrane and suggest that TRPA1-mediated chemosensation may result from primary mechanosensory mechanisms.

Rifampin- or Capreomycin-Induced Remodeling of the Mycobacterium smegmatis Mycolic Acid Layer Is Mitigated in Synergistic Combinations with Cationic Antimicrobial Peptides.

The mycobacterial cell wall affords natural resistance to antibiotics. Antimicrobial peptides (AMPs) modify the surface properties of mycobacteria and can act synergistically with antibiotics from differing classes. Here, we investigate the response of Mycobacterium smegmatis to the presence of rifampin or capreomycin, either alone or in combination with two synthetic, cationic, α-helical AMPs that are distinguished by the presence (D-LAK120-HP13) or absence (D-LAK120-A) of a kink-inducing proline. Using a combination of high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) metabolomics, diphenylhexatriene (DPH) fluorescence anisotropy measurements, and laurdan emission spectroscopy, we show that M.smegmatis responds to challenge with rifampin or capreomycin by substantially altering its metabolism and, in particular, by remodeling the cell envelope. Overall, the changes are consistent with a reduction of trehalose dimycolate and an increase of trehalose monomycolate and are associated with increased rigidity of the mycolic acid layer observed following challenge by capreomycin but not rifampin. Challenge with D-LAK120-A or D-LAK120-HP13 induced no or modest changes, respectively, in mycomembrane metabolites and did not induce a significant increase in the rigidity of the mycolic acid layer. Furthermore, the response to rifampin or capreomycin was significantly reduced when these were combined with D-LAK120-HP13 and D-LAK120-A, respectively, suggesting a possible mechanism for the synergy of these combinations. The remodeling of the mycomembrane in M.smegmatis is therefore identified as an important countermeasure deployed against rifampin or capreomycin, but this can be mitigated and the efficacy of rifampin or capreomycin potentiated by combining the drug with AMPs.IMPORTANCE We have used a combined NMR metabolomics/biophysical approach to better understand differences in the mechanisms of two closely related antimicrobial peptides, as well as the response of the model organism Mycobacterium smegmatis to challenge with first- or second-line antibiotics used against mycobacterial pathogens. We show that, in addition to membrane damage, the triggering of oxidative stress may be an important part of the mechanism of action of one AMP. The metabolic shift that accompanied rifampin and, particularly, capreomycin challenge was associated with modest and more dramatic changes, respectively, in the mycomembrane, providing a rationale for how the response to one antibiotic may affect bacterial penetration and, hence, the action of another. This study presents the first insights into how antimicrobial peptides may operate synergistically with existing antibiotics whose efficacy is waning or sensitize MDR mycobacteria and/or latent mycobacterial infections to them, prolonging the useful life of these drugs.

Role of Membrane Cholesterol in the Apoptosis Induced by Ginsenoside Rh2, A Steroid Saponin

Some saponins (digitonin, alpha-hederin, ginsenoside) exhibit several biological and pharmacological activities such as antibacterial and anticancer effect. Many studies attributed the activity of saponins to their interactions with membrane cholesterol. In this study, we focus on the ginsenoside Rh2, the main metabolite of ginseng. Our goal is to explore whether and how cholesterol could play a role in the molecular mechanisms involved in apoptosis induced by Rh2. We first characterized the Rh2 cytotoxicity using U937 cells depleted or not in cholesterol. We observed higher cellular uptake of Rh2 upon cholesterol depletion explaining why the Rh2-induced apoptosis is observed earlier in cholesterol-depleted cells compared to non-depleted cells. Mechanism includes the inactivation of Akt/PI3K cell survival pathways, known to be associated with cholesterol-enriched membrane domains. We therefore developed biophysical approaches and produced artificial membranes composed of cholesterol or not to assess its potential role in the Rh2-induced cytotoxicity. Using large unilamellar vesicles (LUVs), we investigated whether Rh2 could interact with membrane and examined its effect on different biophysical parameters: fluidity (fluorescence anisotropy of diphenylhexatriene and generalized polarization of laurdan), permeability (calcein release) and fusion (R18 dequenching). Rh2 increases membrane rigidity, induces the release of calcein and provokes hemifusion of membranes. These effects appear higher and/or earlier in liposomes without cholesterol as compared to liposomes containing cholesterol. Altogether, our data suggest that Rh2 insertion within the bilayer could be enhanced upon low cholesterol content, thereby explaining earlier and greater Rh2-induced apoptosis in cholesterol-depleted cells as compared to non-depleted cells. Moreover, regarding earlier and/or higher changes of biophysical liposomal membrane properties in the absence of cholesterol, cholesterol could not be considered as the specific target of Rh2. On the contrary, cholesterol seems to depress the sensitivity of membrane to Rh2.

Docosahexaenoic Acid (DHA), but not Eicosapentaenoic Acid (EPA), Increases Both Membrane Fluidity and Cholesterol Crystalline Domain Formation in Lipid Vesicles

Elevated free cholesterol levels lead to atherosclerotic-like changes in membrane structure, including cholesterol crystalline domain formation. Omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may differentially affect cholesterol domain formation as a result of their distinct biophysical properties. In this study, we tested the comparative effects of EPA and DHA on lipid oxidation, membrane fluidity, and lipid structural organization. The lipid oxidation effects of each agent (10 μM) were measured in vesicles prepared as binary mixtures of dilinoleoylphosphatidylcholine and cholesterol and exposed to autoxidative conditions for 72 hr. Changes in membrane fluidity and lipid structural organization were measured using diphenylhexatriene (DPH) fluorescence anisotropy and small angle x-ray scattering approaches, respectively, in palmitoyloleoylphosphatidylcholine-enriched membrane vesicles prepared at 50 mol% cholesterol in the absence or presence of each agent at 5-10 mol% and tested over a broad range of temperatures. EPA and DHA were found to reduce lipid oxidation to a similar extent. However, only DHA affected membrane lipid fluidity, reducing DPH apparent rotational correlation values by 20% (19.4 to 15.6 nsec). Notably, DHA also induced cholesterol domain formation and reduced the lipid bilayer unit cell periodicity (d-space) from 57A to 55A with increasing temperature (15-30°C). By contrast, EPA had no effect on cholesterol domain formation and produced larger d-space values (60A to 57A, with increasing temperature) as compared to DHA (p<0.05). These data indicate that EPA and DHA, despite similar antioxidant benefits, had different effects on membrane bilayer width, lipid fluidity, and cholesterol crystalline domain formation. These results suggest that omega-3 fatty acids may differentially affect membrane lipid dynamics and structural organization as a result of their unique phospholipid/sterol interactions.

Formation of Gel-like Nanodomains in Cholesterol-Containing Sphingomyelin or Phosphatidylcholine Binary Membrane As Examined by Fluorescence Lifetimes and (2)H NMR Spectra.

In this study, we measured the time-resolved fluorescence of trans-parinaric acid (tPA), steady-state fluorescence anisotropy of diphenylhexatriene (DPH), and (2)H NMR of 10,10-d2-stearoyl lipids in stearoyl sphingomyelin with cholesterol (SSM/Chol) and l-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine with Chol (PSPC/Chol) binary membranes. The results suggest that the membrane order obtained from the fluorescence experiments shows a similar temperature dependency as those of the (2)H NMR data. More importantly, the time-resolved fluorescence data implied the presence of at least two types of domains, cholesterol-poor gel-like domains (CPGLD) and cholesterol-enriched liquid-ordered (Lo) domains. These domains appear on a nano-to-micro second time scale for both SSM-Chol and PSPC-Chol membranes. The relative size of the gel-like domain was also estimated from the temperature-dependent lifetime measurements and (2)H NMR spectral changes. The results imply that the size of the gel-like domains is very small, probably on the nanometer scale, and smaller in SSM-Chol membrane than those in PSPC-Chol bilayers, which could account for the higher thermal stability of SM-Chol membranes. The present study demonstrates that gel-like nanodomains occur in SM-Chol binary membrane even with Chol content of over 33 mol %, which has been thought to consist exclusively of Lo phase, implying that not only Lo domains but also gel-like nanodomains are important for formation of lipid-ordered phase in SM-Chol and PC-Chol membranes.

Flavonoids in Microheterogeneous Media, Relationship between Their Relative Location and Their Reactivity towards Singlet Oxygen.

In this work, the relationship between the molecular structure of three flavonoids (kaempferol, quercetin and morin), their relative location in microheterogeneous media (liposomes and erythrocyte membranes) and their reactivity against singlet oxygen was studied. The changes observed in membrane fluidity induced by the presence of these flavonoids and the influence of their lipophilicity/hydrophilicity on the antioxidant activity in lipid membranes were evaluated by means of fluorescent probes such as Laurdan and diphenylhexatriene (DPH). The small differences observed for the value of generalized polarization of Laurdan (GP) curves in function of the concentration of flavonoids, indicate that these three compounds promote similar alterations in liposomes and erythrocyte membranes. In addition, these compounds do not produce changes in fluorescence anisotropy of DPH, discarding their location in deeper regions of the lipid bilayer. The determined chemical reactivity sequence is similar in all the studied media (kaempferol < quercetin < morin). Morin is approximately 10 times more reactive than quercetin and 20 to 30 times greater than kaempferol, depending on the medium.

Effect of amyloid β-peptide on the fluidity of phosphatidylcholine membranes: Uses and limitations of diphenylhexatriene fluorescence anisotropy

There is accumulating evidence that peptide-induced perturbations in the order and dynamics of cellular membranes may play a role in the neurotoxicity of amyloid β-peptide (Aβ). Several studies have reported that Aβ decreases fluidity of membranes based on an Aβ-induced increase in the fluorescence anisotropy of diphenylhexatriene (DPH). However, the effect of Aβ on the membrane fluidity is still a subject of controversy, because other studies that employed pyrene as a fluorescent probe have shown that Aβ has the opposite effect. To reveal the reason for this discrepancy, we have examined the effect of Aβ on the fluidity of phosphatidylcholine membranes using spectroscopic methods. The fluorescence anisotropy of DPH is dramatically increased on addition of Aβ to DPH-containing phosphatidylcholine membranes. However, Aβ does not affect the Raman spectrum of the membrane, which is sensitive to the packing order of the hydrocarbon chains of lipids. We have also found that circular dichroism (CD) bands of DPH appear during incubation of DPH-containing membranes with Aβ, whereas DPH is an achiral molecule. The observed CD bands of DPH are induced by a chiral environment of Aβ but not by that of the lipids, because positive CD bands appear regardless of the d/l-chirality of phosphatidylcholine. The findings obtained from CD measurements provide evidence that DPH molecules translocate from the membrane to Aβ. The peptide-mediated extraction of DPH from the membrane may cause changes in the fluorescence anisotropy of DPH, even though Aβ does not affect the fluidity of membranes.

Surfactant-modified fatty acid composition of Citrobacter sp. SA01 and its effect on phenanthrene transmembrane transport

The effects of the surfactants, Tween 80 and sodium dodecyl benzene sulfonate (SDBS) on a membrane’s fatty acid composition and the transmembrane transport of phenanthrene were investigated. The results indicated that both surfactants could modify the composition of fatty acids of Citrobacter sp. Strain SA01 cells, 50mgL−1 of both surfactants changed the composition of the fatty acids the most, increasing the amount of unsaturated fatty acids. The comparison of fatty acid profiles with diphenylhexatriene fluorescence anisotropy, a probe for plasma membrane fluidity, suggested that an increased amount of unsaturated fatty acids corresponded to greater membrane fluidity. In addition, increased unsaturated fatty acids promoted phenanthrene to partition from the extracellular matrix to cell debris, which increased reverse partitioning from the cell debris to the cytochylema. The results of this study were expected in that the addition of a surfactant is a simple and effective method for accelerating the rate-limiting step of transmembrane transport of hydrophobic organic compounds (HOCs) in bioremediation.

Fluorescence anisotropy of diphenylhexatriene and its cationic Trimethylamino derivative in liquid dipalmitoylphosphatidylcholine liposomes: opposing responses to isoflurane

BackgroundThe mechanism of action of volatile general anesthetics has not yet been resolved. In order to identify the effects of isoflurane on the membrane, we measured the steady-state anisotropy of two fluorescent probes that reside at different depths. Incorporation of anesthetic was confirmed by shifting of the main phase transition temperature.ResultsIn liquid crystalline dipalmitoylphosphatidylcholine liposomes, isoflurane (7-25 mM in the bath) increases trimethylammonium-diphenylhexatriene fluorescence anisotropy by ~0.02 units and decreases diphenylhexatriene anisotropy by the same amount.ConclusionsThe anisotropy data suggest that isoflurane decreases non-axial dye mobility in the headgroup region, while increasing it in the tail region. We propose that these results reflect changes in the lateral pressure profile of the membrane.

The Action of Alpha Hederin, a Triperpenoid Saponin, on Membranes

α-hederin is a triterpenoid saponin which has shown hemolytic and apoptotic properties. (Chwalek et al. 2006, Gauthier et al. 2009). Many saponins are known to interact with biological membranes on a cholesterol dependent or independent manner (Francis et al. 2002) but the underlying mechanisms need to be further explored. We have studied the effects of α-hederin on size, membrane permeability and fluidity in a LUV model composed of DMPC including 25% (mol/mol) of cholesterol or not. Size was determined by quasi-elastic light scattering spectroscopy. Permeability was evaluated by the release of calcein, entrapped at self-quenching concentrations in liposomes. Membrane dynamics at the hydrophobic core was evaluated by fluorescence anisotropy of diphenylhexatriene, upon increasing temperatures.α-hederin increased the size of cholesterol-rich liposomes and induced release of calcein entrapped within liposomes, on a time- and concentration-dependent manner. It was without effect when cholesterol was absent. α-hederin reduced the transition temperature on a concentration-dependent manner only in liposomes containing cholesterol but increased anisotropy beyond the transition temperature in both types of liposomes.Our data suggest that cholesterol is required for α-hederin's effects on liposome's size, permeability and transition temperature but probably not for the effect on membrane fluidity, when lipids are in fluid phase. The role of these effects for apoptosis induced by α-hederin is under investigation.

Sphingolipid-Enriched Microdomains in the Plasma Membrane of Saccharomyces Cerevisiae: Ergosterol-Free «Lipid Rafts» in the Gel Phase

The plasma membrane (PM) of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid (t-PnA) and diphenylhexatriene (DPH), providing the first direct evidence for the presence of gel domains in living cells and showing that the PM is mostly constituted by ordered domains(1). The fluorescence lifetimes of t-PnA are particularly sensitive to the presence and nature of ordered domains, and here the first measurements in yeast cells are reported. A long fluorescence lifetime typical of the gel phase (> 30 ns) was found in mid-exponential phase wild-type (wt) cells from two different genetic backgrounds, at both 24 and 30°C. The confirmation of the gel nature was achieved by comparing t-PnA and DPH fluorescence anisotropy, and studying the thermotropic transitions of liposomes reconstituted from PM lipid-extracts. To address the location and composition of the domains spheroplasts, the isolated PM, liposomes from total lipid-extracts, and living cells of several mutant strains with deletions in genes coding for enzymes involved in ergosterol-, sphingolipid- and GPI-anchor biosynthesis were also studied. It was shown that the gel domains are not ergosterol-enriched lipid rafts, but mainly composed of sphingolipids and GPI-anchored proteins, suggesting important roles in membrane traffic, signaling, and interactions with the cell wall. The findings provide a biophysical mechanism for the targeting of some proteins to the PM, which is sphingolipid-dependent but sterol-independent and for the formation of membrane compartments in the yeast PM (MCP and MCC)(2). Acknowledgements: PTDC/QUI-BIQ/104311/2008, Ciência2007 and PEst-OE/QUI/UI0612/2011 (FCT, Portugal). (1) F. Aresta-Branco, A.M. Cordeiro, H.S. Marinho, L. Cyrne, F. Antunes, R.F.M. de Almeida (2011) J. Biol. Chem. 286:5043-5054. (2) Poolman, B. (2011) Evaluation of: [Aresta-Branco F et al. Gel domains in the plasma membrane ...]. Faculty of 1000, 26 Apr 2011. F1000.com/9922956.

Stereoselective effects of monoterpenes on the microviscosity and curvature of model membranes assessed by DPH steady‐state fluorescence anisotropy and light scattering analysis

Here, we evaluated stereoselectivity in monoterpenes (MTs) ability to disturb membrane dynamics. Correlations between molecular structure and physicochemical properties of pinenes, menthols, and carvones enantiomers were investigated through cluster and principal component analysis. Therefore, MTs' concentration-dependent changes in light scattering and diphenylhexatriene (DPH) fluorescence polarization induced by MTs were measured on large unilamellar vesicles (LUVs) of dipalmitoylphosphatidylcholine. The behavior of the less polar compounds (hydrocarbons) was characterized by a membrane expansion (increase in light scattering), detectable within the low-concentration range. They remained in the membrane up to the highest concentrations tested exhibiting a concentration-dependent anisotropy decrease. Within the more polar terpenes (alcohols) prevailed a budding phenomenon with the production of small LUVs with roughly constant curvature (more evident at medium and high concentrations), which explains the slight change in microviscosity (DPH fluorescence anisotropy). These behaviors were compatible with the deeper localization within the membrane core of the formers compared with the latters as predicted from the corresponding polar charge distribution in their molecular structures. The enantioselectivity was expressed by neomenthol at low concentration and carvone at medium concentration. Inhibition and potentiation were evidenced, within the low-concentration range, by the racemic mixtures in neomenthol and β-pinenes, respectively.

Abstract B65: Molecular basis of mitigated alkaline phosphatase activity in renal cell carcinoma

Abstract The present study was conducted to find out the molecular basis of aberrant expression/activity of ALP in BBM from RCC in comparison to normal renal BBM. Thirty histopathologically confirmed cases of RCC were included in the present investigation. A significant decrease was observed in the activity of alkaline phosphatase in BBM isolated from RCC as compared to BBM isolated from normal renal parenchyma. Kinetic studies revealed that diminished activity of alkaline phosphatase in BBM isolated from RCC was fraternized with decrease in maximal velocity (Vmax) and increase in affinity constant (Km) of the enzyme. SDS-PAGE analysis illustrated the less expression of higher molecular weight BBM proteins in RCC. Incubation of native polyacrylamide gel with ALP specific BCIP/NBT dye showed reduced expression of ALP in BBM from RCC as compared to BBM from normal kidney tissue. In addition, Western blot analysis using anti-ALP antibody also confirmed the reduced expression of ALP protein in BBM of RCC. This finding was corroborated by immunofluorescence and immunohistochemical study. Further, semiquantitative mRNA expressions of ALP demonstrated a markedly reduced expression of ALP gene in RCC as compared to normal kidney tissue. No mutation in C-terminal end of ALP gene was detected. Therefore the C-terminal end of the ALP gene might not play a role in decreased activity of ALP in RCC. Further, to elucidate the role of secondary messengers viz. cAMP and intracellular Ca2+ in the reduction of ALP activity/expression. cAMP and intracellular Ca2+ levels were measured in RCC. The cAMP level was found to be significantly increased while the intracellular Ca2+ was significantly decreased in RCC as comparison to normal renal tissue indicating their possible role in reduced activity of ALP in RCC. Antecedent studies have suggested that ALP is directly correlated with membrane lipid composition and fluidity. In view of this, we determined the role of membrane lipid composition and fluidity of BBM in decreased activity of ALP in BBM from RCC. The fluorescence anisotropy (rDPH) and polarization (pDPH) of diphenyl hexatriene were significantly decreased. These parameters are inversely proportional to membrane fluidity. Therefore, this finding was suggestive of increased membrane fluidity in BBM isolated from RCC. Cholesterol has condensing effect on phospholipid and significantly reduced membrane fluidity. The molar ratio of cholesterol to phospholipid in BBM was of utmost importance to determine and compare the membrane fluidity. BBM lipid composition analysis revealed that phospholipids and glycolipids were increased whereas, cholesterol was decreased in BBM of RCC as compared to that in normal renal BBM. Cholesterol to phospholipid ratio was found to be decreased in BBM from RCC. The differential membrane phospholipid was also found altered in BBM from RCC. Therefore, the alteration in membrane lipids, cholesterol to phospholipid molar ratio and membrane fluidity might be playing an important role in decreased activity of ALP in RCC as compared to normal renal tissue. Results from the present study revealed those molecular events that might play a collaborative role in decreased activity of ALP in RCC. Our data suggest that the reduced activity of ALP in BBM isolated from RCC is due to decreased expression of ALP gene at transcription level. Besides this membrane fluidity and lipid composition might also contribute to the decrease activity of ALP. In the above mechanisms secondary messengers such as cAMP and intracellular Ca2+ may be having contributory role. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B65.

Bilayer heating in magnetite nanoparticle–liposome dispersions via fluorescence anisotropy

Temperature measurements have been made within magnetite (Fe 3O 4) nanoparticle–liposome dispersions subjected to electromagnetic field at radiofrequency (RF) heating based on the fluorescence anisotropy of diphenylhexatriene (DPH) embedded within the bilayer. Incorporating cholesterol within dipalmitoylphosphatidylcholine (DPPC) bilayers broadened the anisotropy window associated with lipid melting. Cryogenic transmission electron microscopy showed that the dispersions contained magnetoliposomes with nanoparticle aggregates at both low and high encapsulation densities. RF heating results demonstrated the ability to measure the temperature of the ML bilayer with on/off RF cycles using DPH anisotropy. These measurements reflected the temperature of the bulk aqueous phase, which is consistent with previous work showing rapid heat dissipation from a nanoparticle surface during RF heating and a negligible difference between surface and bulk temperature.

Differences in Cold Adaptation of Bacillus subtilis under Anaerobic and Aerobic Conditions

Bacillus subtilis, which grows under aerobic conditions, employs fatty acid desaturase (Des) to fluidize its membrane when subjected to temperature downshift. Des requires molecular oxygen for its activity, and its expression is regulated by DesK-DesR, a two-component system. Transcription of des is induced by the temperature downshift and is decreased when membrane fluidity is restored. B. subtilis is also capable of anaerobic growth by nitrate or nitrite respiration. We studied the mechanism of cold adaptation in B. subtilis under anaerobic conditions that were predicted to inhibit Des activity. We found that in anaerobiosis, in contrast to aerobic growth, the induction of des expression after temperature downshift (from 37 degrees C to 25 degrees C) was not downregulated. However, the transfer from anaerobic to aerobic conditions rapidly restored the downregulation. Under both aerobic and anaerobic conditions, the induction of des expression was substantially reduced by the addition of external fluidizing oleic acid and was fully dependent on the DesK-DesR two-component regulatory system. Fatty acid analysis proved that there was no desaturation after des induction under anaerobic conditions despite the presence of high levels of the des protein product, which was shown by immunoblot analysis. The cold adaptation of B. subtilis in anaerobiosis is therefore mediated exclusively by the increased anteiso/iso ratio of branched-chain fatty acids and not by the temporarily increased level of unsaturated fatty acids that is typical under aerobic conditions. The degrees of membrane fluidization, as measured by diphenylhexatriene fluorescence anisotropy, were found to be similar under both aerobic and anaerobic conditions.

Differential Susceptibility of Normal and Transformed Human Leukocytes to Hydrolytic Attack by Secretory Phospholipase A2

Previous experiments with cultured lymphoma cells demonstrated that secretory phospholipase A2 (sPLA2) distinguishes healthy cells from those that are dying by apoptosis or necrosis. This distinction depends on cell membrane properties including the amount of negative charge at the bilayer surface and the strength of interactions among neighboring phospholipids. These results raised two important questions. 1) Does the enzyme's ability to distinguish healthy and dying cells apply to normal human leukocytes? 2) Does sPLA2 differentiate between normal and tumor cells? These questions were addressed by comparing membrane properties and susceptibility to hydrolysis among cultured transformed leukocytes and freshly-isolated human neutrophils and lymphocytes. Membrane properties were assessed by flow cytometry, merocyanine 540 fluorescence spectra, trimethylammonium diphenylhexatriene fluorescence anisotropy, and two-photon scanning microscopy with laurdan. Similar to the behavior of transformed cells, normal human leukocytes resisted hydrolysis by sPLA2. Upon addition of a calcium ionophore, ionomycin, the cells became vulnerable to hydrolysis, again analogous to the results observed with tumor cells. However, several important quantitative distinctions were observed. First, the various types of normal leukocytes responded differently to the enzyme; lymphocytes exhibited significantly greater rates of hydrolysis by sPLA2 compared to granulocytes. Second, hydrolysis was substantially slower in normal cells compared to transformed cells. Third, the time required for ionomycin to induce cells to be attacked by sPLA2 was greater in normal compared to transformed cells. Likewise, changes in membrane physical properties following ionomycin treatment were more subtle in normal cells than they were in transformed cells. These results suggest the possibility that sPLA2 could function as a therapeutic ally during cancer chemotherapy to assist with the demise of tumor cells.

Toxicity of a serotonin-derived neuromelanin

Postoperative Cognitive Dysfunction (POCD) is associated with increased mortality in the elderly and may occur from lipid peroxidation in aging. We previously showed that sevoflurane sequesters acrolein, which promotes the formation of a novel species of a putative neuromelanin. The current study examined the properties of this serotonin-derived melanoid (SDM). The interaction of SDM with unilamellar vesicles (ULVs) was examined using lipid membrane probes. Vesicle disruption was investigated by leakage of dye from calcein-loaded ULVs. We observed that SDM decreased diphenyl-hexatriene fluorescence anisotropy and increased the temperature-dependent change in anisotropy. SDM changed the absorbance of merocyanin-bound ULVs. SDM increased detergent-mediated calcein leakage. SDM structure was dramatically altered upon interaction with ULVs. We also observed that SDM enhanced detergent-mediated leakage of loaded ULVs, suggesting that SDM may be neurotoxic. We propose that inhalational agents, which sequester acrolein, may promote the production of certain species of neuromelanin that depletes local serotonin and enhances neuronal vulnerability.

Regulation of Membrane Proteins by Dietary Lipids: Effects of Cholesterol and Docosahexaenoic Acid Acyl Chain-Containing Phospholipids on Rhodopsin Stability and Function

Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol % cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature ( T m) of rhodopsin was scan rate-dependent, demonstrating the presence of a rate-limited component of denaturation. The activation energy of this kinetically controlled process ( E a) was determined from DSC thermograms by four separate methods. Both T m and E a varied with bilayer composition. Cholesterol increased the T m both the presence and absence of docosahexaenoic acid acyl chains (DHA). In contrast, cholesterol lowered E a in the absence of DHA, but raised E a in the presence of 20 mol % DHA-containing phospholipid. The relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. The T m for thermal unfolding was inversely related to acyl chain packing order. Rhodopsin kinetic stability ( E a) was reduced in highly ordered or disordered membranes. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing, such as DHA and cholesterol, may be required for maximum protein stability.

Effect of sodium deoxycholate and sodium cholate on DPPC vesicles: A fluorescence anisotropy study with diphenylhexatriene

Effects of two bile salts, namely sodium deoxycholate (NaDC) and sodium cholate (NaC), on DPPC small unilamellar vesicles have been investigated using the steady-state fluorescence anisotropy (r ss ) of diphenylhexatriene (DPH) as a tool. It was found that the variation of r ss is sensitive enough to monitor different stages of interaction of bile salts with DPPC vesicles. NaDC induced significant changes in the membrane well below its CMC (6 mM). Even at 4 mM, which is still lower than the CMC, the phospholipids were completely solubilised by the NaDC micelles. The effect of NaC on DPPC vesicles, however, was much less significant, especially in the sub-micellar concentration regime. Being more hydrophilic NaC does not interact with the membrane efficiently. Complete solubilisation of phospholipids took place only when the concentration of NaC was above its CMC (16 mM). The experiments also showed that the bile salt-induced changes of vesicle structure were strongly dependent on the concentration of the bile salt and not on the molar ratio of lipid and bile salt.