Leakage Of Carboxyfluorescein Research Articles

Functional interplay between short antimicrobial peptides and model lipid membranes

Antimicrobial peptides (AMPs) are considered an attractive generation of novel antibiotics due to their advantageous properties such as a broad spectrum of antimicrobial activity against pathogens, low cytotoxicity, and drug resistance. Although they have common structural features and it has been widely demonstrated that bacterial membranes represent the main target of the peptide activity, the exact mechanism underlying the membrane perturbation by AMPs is not fully understood. Nevertheless, all the proposed modes of action implicate the preliminary interaction of AMPs with the negatively charged lipids in bacterial membranes. Recently, the structural and functional characterization of two AMPs, RiLK1 and RiLK3, was reported. Specifically, both peptides were revealed to be multitalented compounds capable of binding Gram-positive and Gram-negative liposome models with high affinity, but their mechanism of action remains elusive. In this paper, the effects of RiLK1 and RiLK3 on vesicles mimicking prokaryotic and eukaryotic cell membranes were further examined by using different approaches. Fluorescence and quenching assays either by acrylamide or lipophilic probes suggested that the peptides were mainly located at the interface of the negatively charged membranes that mimicked those of Salmonella Typhimurium and Staphylococcus aureus, possibly oriented in a parallel manner. Furthermore, RiLK1 and RiLK3 caused a significant leakage of carboxyfluorescein from bacterial liposomes, demonstrating that they can permeabilize the target membranes at high doses. Conversely, both peptides appear to behave like cell penetrating peptides (CPPs) at concentrations near their MIC values evaluated against the bacterial targets. Moreover, Dynamic Light Scattering provided further insights on the mechanisms of antimicrobial peptide against the bacterial liposomes. Conclusively, in vitro experiments indicated that RiLK1 and RiLK3 displayed potent bacteriostatic efficacy at low micromolar concentrations against an antibiotic-resistant ESKAPE pathogen, making them a valuable tool in preventing and treating infections caused by such bacteria.

Effects of Conjugation of Ferrocene and Gallic Acid On desCys11/Lys12/Lys13-(p-BthTX-I)2K Peptide: Structure, Permeabilization and Antibacterial Activity.

Antimicrobial resistance is an emerging global health challenge that has led researchers to study alternatives to conventional antibiotics. A promising alternative is antimicrobial peptides (AMPs), produced as the first line of defense by almost all living organisms. To improve its biological activity, the conjugation of AMPs is a promising approach. In this study, we evaluated the N-terminal conjugation of p-Bt (a peptide derived from Bothrops Jararacuçu`s venom) with ferrocene (Fc) and gallic acid (GA). Acetylated and linear versions of p-Bt were also synthesized to evaluate the importance of N-terminal charge and dimeric structure. The compounds were obtained using solid-phase peptide synthesis. Circular dichroism, vesicle permeabilization, antimicrobial activity, and cytotoxicity studies were conducted. No increase in antibacterial activity against Escherichia coli was observed by adding either Fc or GA to p-Bt. However, Fc-p-Bt and GA-p-Bt exhibited improved activity against Staphylococcus aureus. No cytotoxicity upon fibroblast was observed for GA-p-Bt. On the other hand, conjugation with Fc increased cytotoxicity. This toxicity may be related to the membrane permeabilization capacity of this bioconjugate, which showed the highest carboxyfluorescein leakage in vesicle permeabilization experiments. Considering these observations, our findings highlight the importance of adding bioactive organic compounds in the N-terminal position as a tool to modulate the activity of AMPs.

Highly Stable Liposomes Based on Tetraether Lipids as a Promising and Versatile Drug Delivery System.

Conventional liposomes often lack stability, limiting their applicability and usage apart from intravenous routes. Nevertheless, their advantages in drug encapsulation and physicochemical properties might be helpful in oral and pulmonary drug delivery. This study investigated the feasibility and stability of liposomes containing tetraether lipids (TEL) from Thermoplasma acidophilum. Liposomes composed of different molar ratios of TEL:Phospholipon 100H (Ph) were produced and exposed to various temperature and pH conditions. The effects on size, polydispersity index, and zeta potential were examined by dynamic and electrophoretic light scattering. Autoclaving, which was considered an additional process step after fabrication, could minimize contamination and prolong shelf life, and the stability after autoclaving was tested. Moreover, 5(6)-carboxyfluorescein leakage was measured after incubation in the presence of fetal calf serum (FCS) and lung surfactant (Alveofact). The incorporation of TEL into the liposomes significantly impacted the stability against low pH, higher temperatures, and even sterilization by autoclaving. The stability of liposomes containing TEL was confirmed by atomic force microscopy as images revealed similar sizes and morphology before and after incubation with FCS. It could be concluded that increasing the molar ratio in the TEL:Ph liposome formulations improved the structural stability against high temperature, low pH, sterilization via autoclaving, and the presence of FCS and lung surfactant.

What different physical techniques can disclose about disruptions on membrane structure caused by the antimicrobial peptide Hylin a1 and a more positively charged analogue

The present work monitors structural changes in anionic membranes (DPPG; 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol)) caused by the native antimicrobial peptide (AMP) Hylin a1 (Hya1; IFGAILPLALGALKNLIK-NH2) and its synthetic analogue K0Hya1 (KIFGAILPLALGALKNLIK-NH2), with an extra positive residue of lysine at the N-terminus of the peptide chain. Anionic membranes were used to mimic anionic lipids in bacteria membranes. Differential scanning calorimetry (DSC) evinced that both peptides strongly disrupt the lipid bilayers. However, whereas the native peptide (+3) induces a space-average and/or time-average disruption on DPPG bilayers, the more charged, K0Hya1 (+4), appears to be strongly attached to the membrane, clearly giving rise to the coexistence of two different lipid regions, one depleted of peptide and another one peptide-disrupted. The membrane fluorescent probe Laurdan indicates that, in average, the peptides increase the bilayer packing of fluid DPPG (above the lipid gel-fluid transition temperature) and/or decrease its polarity. Spin labels, incorporated into DPPG membrane, confirm, and extend the results obtained with Laurdan, indicating that the peptides increase the lipid packing both in gel and fluid DPPG bilayers. Therefore, our results confirm that Laurdan is often unable to monitor structural modifications induced on gel membranes by exogenous molecules. Through the measurement of the leakage of entrapped carboxyfluorescein (CF), a fluorescent dye, in DPPG large unilamellar vesicles it was possible to show that both peptides induce pore formation in DPPG bilayers. Furthermore, CF experiments show that Hylin peptides are strongly bound to DPPG bilayers in the gel phase, not being able to migrate to other DPPG vesicles. Here we discuss the complementarity of different techniques in monitoring structural alterations caused on lipid bilayers by Hylin peptides, and how it could be used to help in the understanding of the action of other exogenous molecules on biological membranes.

Avoiding artifacts in liposome leakage measurements via cuvette- and liposome-surface modifications

The barrier properties of lipid membranes are often determined by investigating their solute permeability with the help of spectroscopic methods and the use of liposome-encapsulated self-quenching fluorescent dyes, for example, Carboxyfluorescein (CF). It was shown previously that liposome–surface interactions, and thus the choice of cuvette material, influence the result of such spectroscopic permeability/leakage experiments. In this work, we explore different methods to minimize the artifacts observed in spontaneous leakage measurements performed with cholesterol-containing liposomes. The spontaneous leakage of CF from liposomes with different composition and surface properties is monitored in cuvettes composed of quartz, polystyrene (PS), and Poly(methyl methacrylate) (PMMA). Our results show that significantly different leakage profiles are recorded for the exact same liposome batch depending on the cuvette material used. Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) experiments indicate that these discrepancies likely arise from side processes occurring at the solution-cuvette interface, mainly, the attaching and spreading of liposomes. Further, we show that in some cases it is possible to minimize liposome–cuvette interactions, and reduce the experimental artifacts, by supplementing the liposomes with polyethylene glycol (PEG)-grafted lipids or gangliosides, and/or by pre-adsorbing free PEG to the cuvette walls. The collected data suggest that quartz cuvettes modified by adsorption of PEG8000 are suitable for spontaneous leakage experiments with POPC:cholesterol-based liposomes, while other cuvette materials perform poorly in the same experiments.

Trialkyl(vinyl)phosphonium Chlorophenol Derivatives as Potent Mitochondrial Uncouplers and Antibacterial Agents.

Trialkyl phosphonium derivatives of vinyl-substituted p-chlorophenol were synthesized here by a recently developed method of preparing quaternary phosphonium salts from phosphine oxides using Grignard reagents. All the derivatives with a number (n) of carbon atoms in phosphonium alkyl substituents varying from 4 to 7 showed pronounced uncoupling activity in isolated rat liver mitochondria at micromolar concentrations, with a tripentyl derivative being the most effective both in accelerating respiration and causing membrane potential collapse, as well as in provoking mitochondrial swelling in a potassium-acetate medium. Remarkably, the trialkyl phosphonium derivatives with n from 4 to 7 also proved to be rather potent antibacterial agents. Methylation of the chlorophenol hydroxyl group suppressed the effects of P555 and P444 on the respiration and membrane potential of mitochondria but not those of P666, thereby suggesting a mechanistic difference in the mitochondrial uncoupling by these derivatives, which was predominantly protonophoric (carrier-like) in the case of P555 and P444 but detergent-like with P666. The latter was confirmed by the carboxyfluorescein leakage assay on model liposomal membranes.

Unraveling the mechanism of octenidine and chlorhexidine on membranes: Does electrostatics matter?

The increasing problem of antibiotic resistance in bacteria requires the development of new antimicrobial candidates. There are several well-known substances with commercial use, but their molecular mode of action is not fully understood. In this work, we focus on two commonly used antimicrobial agents from the detergent family—octenidine dichloride (OCT) and chlorhexidine digluconate (CHX). Both of them are reported to be agents selectively attacking the cell membrane through interaction inducing membrane disruption by emulsification. They are believed to present electrostatic selectivity toward charged lipids. In this study, we tested this hypothesis and revised previously proposed molecular mechanisms of action. Employing a variety of techniques such as molecular dynamics, ζ potential with dynamic light scattering, vesicle fluctuation spectroscopy, carboxyfluorescein leakage measurement, and fluorescence trimethylammonium-diphenylhexatriene- and diphenylhexatriene-based studies for determination of OCT and CHX membrane location, we performed experimental studies using two model membrane systems—zwitterionic PC and negatively charged PG (18:1/18:1):PC (16:0/18:1) 3:7, respectively. These studies were extended by molecular dynamics simulations performed on a three-component bacterial membrane model system to further test interactions with another negatively charged lipid, cardiolipin. In summary, our study demonstrated that detergent selectivity is far more complicated than supposed simple electrostatic interactions. Although OCT does disrupt the membrane, our results suggest that its primary selectivity was more linked to mechanical properties of the membrane. On the other hand, CHX did not disrupt membranes as a primary activity, nor did it show any sign of electrostatic selectivity toward negatively charged membranes at any stage of interactions, which suggests membrane disruption by influencing more discrete membrane properties.

Tuning stable noble metal nanoparticles dispersions to moderate their interaction with model membranes

HypothesisThe properties of stable gold (Au) nanoparticle dispersions can be tuned to alter their activity towards biomembrane models. ExperimentsAu nanoparticle coating techniques together with rapid electrochemical screens of a phospholipid layer on fabricated mercury (Hg) on platinum (Pt) electrode have been used to moderate the phospholipid layer activity of Au nanoparticle dispersions. Screening results for Au nanoparticle dispersions were intercalibrated with phospholipid large unilamellar vesicle (LUV) interactions using a carboxyfluorescein (CF) leakage assay. All nanoparticle dispersions were characterised for size, by dynamic light scattering (DLS) and transmission electron microscopy (TEM). FindingsCommercial and high quality home synthesised Au nanoparticle dispersions are phospholipid monolayer active whereas Ag nanoparticle dispersions are not. If Au nanoparticles are coated with a thin layer of Ag then the particle/lipid interaction is suppressed. The electrochemical assays of the lipid layer activity of Au nanoparticle dispersions align with LUV leakage assays of the same. Au nanoparticles of decreasing size and increasing dispersion concentration showed a stronger phospholipid monolayer/bilayer interaction. Treating Au nanoparticles with cell culture medium and incubation of Au nanoparticle dispersions in phosphate buffered saline (PBS) solutions removes their phospholipid layer interaction.

Liposome Fusion Mediated by Hydrophobic Magnetic Nanoparticles Stabilized with Oleic Acid and Modulated by an External Magnetic Field.

Membrane fusion is considered relevant in countless scientific areas and biotechnological processes, ranging from vital life events to biomedicine, pharmaceuticals, and materials engineering, among others. In this study, we employed hydrophobic oleic acid (OA)-coated magnetite (Fe3O4) nanoparticles (MNP-OA) as a platform to induce the fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes [large unilamellar vesicles (LUVs)] in a colloidal dispersion. This fusion was monitored through dynamic light scattering, turbidimetry, and fluorescence assay using the well-known Tb/dipicolinic acid (DPA) complex formation assay. MNP-OA have shown to be able to induce fusion with the mixing of liposomal inner content with direct dependence on the nanoparticle concentration added to the LUVs. Moreover, changes in the permeability of the liposome bilayer, upon the addition of MNP-OA to liposomes, were evaluated by studying the leakage of carboxyfluorescein and of the co-encapsulated Tb/DPA complex. These assays allowed us to determine that MNP-OA did not significantly modify liposome permeability during the fusion process. Transmission electron microscopy and confocal microscopy revealed that MNP-OA remained embedded in the lipid bilayer without producing membrane rupture, liposome deformation, or destruction. In addition, we evaluated the effect of applying a low-intensity magnetic field to the LUVs/MNP-OA system and observed that the nanoparticles considerably increased their fusogenic activity under this external stimulus, as well as they are capable of responding to low magnetic fields of around 0.45 mT. These results revealed the potential of hydrophobic magnetic nanoparticles, stabilized with OA, to act as a fusogen, thus representing a valuable tool for biotechnological applications.

Interaction of synthetic antimicrobial peptides of the Hylin a1 family with models of eukaryotic structures: Zwitterionic membranes and DNA

Antimicrobial peptides (AMPs) have been appointed as a possible alternative to traditional antibiotics in face of pathogens increasing resistance to conventional drugs. Hylin a1 (IFGAILPLALGALKNLIK), an AMP extracted from the skin secretion of a South American frog, Hypsiboas albopunctatus, was found to show a strong cytotoxicity against bacteria and fungus, but also a considerable hemolytic action. Considering the toxicity of the peptide in eukaryotic cells, this work focuses on investigating the effects of the interaction of the Hylin a1 analogues W6Hya1, D0W6Hya1 and K0W6Hya1 with models of eukaryotic structures, namely zwitterionic liposomes of dipalmitoyl phosphatidylcholine (DPPC) and calf-thymus DNA (CT DNA). Through intrinsic Trp fluorescence we determined that the peptide affinity for fluid DPPC bilayers follows the decreasing order: D0W6Hya1 (+2) > W6Hya1 (+3) » K0W6Hya1 (+4). Fluorescence data also indicate that the Trp residue in the more positively charged peptide, K0W6Hya1, is less deep in the bilayer than the residue in the other two peptides. This finding is supported by differential scanning calorimetry (DSC) data, which shows that both D0W6Hya1 and W6Hya1 disturb DPPC gel-fluid transition slightly more effectively than K0W6Hya1. DPPC DSC profiles are homogeneously disturbed by the three peptides, probably related to peptide-membrane diffusion. Surprisingly, the peptide that displays the lowest affinity for PC membranes and is located at the more superficial position in the bilayer, K0W6Hya1, is the most efficient in causing formation of pores on the membrane, as attested by carboxyfluorescein leakage assays. The three peptides were found to interact with CT DNA, with a deep penetration of the Trp residue into hydrophobic pockets of the double helix, as indicated by the significant blue shift on the Trp fluorescence, and the displacement of DNA-bound ethidium bromide by the peptides. The experiments of DNA electrophoresis confirm that Hylin peptides bind DNA in a concentration-dependent manner, inducing complete DNA retardation at the relative AMP/plasmid DNA weight ratio of ~17. These findings could help to better understand the AMPs toxic effects on eukaryotic cells, thus contributing to the design of healthier therapeutic agents.

Revealing the Mode of Action of Halictine Antimicrobial Peptides: A Comprehensive Study with Model Membranes.

Antimicrobial peptides are innate host defense molecules with the ability to kill pathogens. They have been widely studied for their membrane lytic activity and their potential to overcome the ever-increasing threat of antimicrobial resistance against conventional antibiotics. Here, we focus on two halictines, antimicrobial peptides first obtained from the venom of the eusocial bee Halictus sexcinctus. The peptides, HAL-1 and HAL-2, are cationic (with +3 and +4 charges, respectively) and amphipathic, have 12 amino acid residues, and exhibit high biological activity. For this study, the mechanism of action of HAL-1 and HAL-2 was studied in detail using large and giant unilamellar vesicles composed of pure palmitoyl oleoyl phosphatidyl choline (POPC) and a mixture of POPC and the anionic lipid palmitoyl oleoyl phosphatidyl glycerol (POPG) as biomimetic models of the membranes of eukaryotes and microorganisms, respectively. A set of complementary techniques was put forward: carboxyfluorescein leakage assay, phase contrast optical microscopy, ζ-potential, static and dynamic light scattering, fluorescence and circular dichroism spectroscopies, and isothermal titration calorimetry. The results show that both halictines are able to interact strongly with anionic membranes: The interaction is exothermic and accompanied by structuring of the peptides as an α-helix and deep insertion into the membrane causing substantial membrane permeabilization at very low peptide/lipid molar ratios. Extensive vesicle aggregation was detected only at a high peptide concentration. On the other hand, the interaction of the halictines with POPC is significantly milder. Yet, the peptides were able to permeabilize the POPC membranes to some extent. Comparing both peptides, HAL-1 showed a somewhat stronger effect on model membranes. Fits to the data revealed apparent binding constants on the order of 103-104 M-1 for anionic membranes and 1 order of magnitude lower for zwitterionic bilayers. When lytic activity results were compared at the same bound peptide/lipid ratio, the halictines exhibited a higher activity toward zwitterionic membranes. As novel peptides, small and with powerful activity, these halictines are potential candidates for becoming antimicrobial agents.

Identification of the potential biological target of N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline compounds active against gram-positive and gram-negative bacteria

The development of new antibiotics with activity towards a broad spectrum of bacteria, including multiresistant strains, is a very important topic for global public health. As part of previous works, N-benzenesulfonyl-1,2,3,4-tetrahydroquinoline (BSTHQ) derivatives were described as antimicrobial agents active against gram-positive and gram-negative pathogens. In this work, experimental and molecular modelling studies were performed in order to identify their potential biological target in the light of structure-based design efforts towards further BSTHQ derivatives. First, a carboxyfluorescein leakage assay was performed using liposomes to mimic bacterial membranes, which found no significative membrane disruption effects with respect to control samples. These results support a non-surfactant antimicrobial activity of the tested compounds. In a second stage, the inhibition of potential antimicrobial targets was screened using molecular modelling methods, taking into account previously reported druggable targets deposited in the ChEMBL database for Escherichia coli and Staphylococcus aureus. Two enzymes, namely D-glutamic acid-adding enzyme (MurD) and N-acetylglucosamine-1-phophate-uridyltransferase (GlmU), both involved in bacterial membrane synthesis, were identified as potential targets. Pharmacodynamic interaction models were developed using known MurD and GlmU inhibitors by applying state-of-the-art chemoinformatic methods (molecular docking, molecular dynamics and free energy of interaction analyses). These models were further extended to the analysis of the studied BSTHQ derivatives. Overall, our results demonstrated that the studied BSTHQ derivatives elicit their antibacterial activity by interacting with a specific molecular target, GlmU being the highly feasible one. Based on the presented results, further structure-aided design efforts towards the obtaining of novel BSTHQ derivatives are envisioned.Communicated by Ramaswamy H. Sarma

Differences in plasma membrane integrity may explain collapse of cardiac function in Antarctic notothenioids varying in thermal tolerance limits

Antarctic fishes of the suborder Notothenioidei are noted for their narrow temperature tolerance, yet the physiological factors underlying their limited capacity to endure elevated temperatures are unknown. Previous work by our group indicates that cardiac function may, in fact, delineate thermal tolerance. For example, the hemoglobinless (i.e., icefish) Chaenocephalus aceratus experiences persistent ventricular asystole at 14.1°C while the red‐blooded Notothenia coriiceps experiences asystole at 16.7°C, corresponding with established CTmax (associated with loss of right response) temperatures of 13.9°C and 17.1°C, respectively. In the current study, we tested the hypothesis that failure of membrane integrity is associated with collapse of cardiac function. Plasma membranes were isolated from ventricle tissue in both fish species and liposomes were prepared by rehydrating extracted lipids with the self‐quenching fluorophore 5(6)‐carboxyfluorescein (CF). Membrane structural integrity was assessed by quantifying percent of CF leakage from liposomes warmed in 1°C increments over a range of 0°C to 20°C, compared with CF leakage upon complete liposome dissolution. At physiological temperature (0°C), liposomes prepared from membranes of C. aceratus were significantly more permeable with initial leakage of 31% extending to 42% at 20°C, while membrane leakage was 1.5‐times less in N. coriiceps, beginning at 21% and increasing to only 27% over the same temperature range (P = 0.015). Liposomes prepared from membranes of the icefish also demonstrated increased thermal sensitivity compared with liposomes prepared from the red‐blooded species. Membrane leak increased most significantly between 0°C and 5°C in the icefish (P < 0.001), while liposomes from the red‐blooded species experienced the greatest change between 0°C and 9°C (P < 0.001), reflecting a reduced resilience to thermal change for the membrane integrity of C. aceratus compared to N. coriiceps (P < 0.001). Our data indicate that physical properties of the ventricular plasma membranes may indeed play a role in setting thermal tolerance limits in Antarctic notothenioid fishes.Support or Funding InformationSupported by NSF ANT 1341602.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH2). Two glycotriazole-peptides, namely [p-Glc-trz-G1]HSP1-NH2 and [p-GlcNAc-trz-G1]HSP1-NH2, were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO4·5H2O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G1]HSP1-NH2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH2 presents relatively low and [trz-G1]HSP1-NH2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

Cryopreservation of lipid bilayers by LEA proteins from Artemia franciscana and trehalose

The capacity of Late Embryogenesis Abundant (LEA) proteins and trehalose to protect liposomes against freezing-induced damage was examined by measuring the leakage of 5(6)-carboxyfluorescein (CF). Liposomes were prepared to simulate the lipid compositions of the inner leaflet of the plasma membrane, outer mitochondrial membrane (OMM), and inner mitochondrial membrane (IMM). Two recombinant LEA proteins belonging to Group 3 (AfrLEA2 and AfrLEA3m) were expressed and purified from embryos of Artemia franciscana. Only OMM-like liposomes were significantly protected by AfrLEA2 and AfrLEA3m against freeze-thaw damage; at the highest protein:lipid mass ratio tested, leakage of CF was 56.3% of control with AfrLEA3m and 29.3% with AfrLEA2. By comparison, trehalose provided protection to all compositional types. The greatest stabilization during freezing occurred when trehalose was present on both sides of the bilayer. When mitochondria isolated from rat liver were freeze-thawed in trehalose solution, the OMM remained intact based on the absence of increased oxygen consumption when cytochrome c was added during oxidative phosphorylation (OXPHOS). Respiratory control ratios (OXPHOS/LEAK) were depressed by only 30% after freeze-thawing in trehalose compared to non-frozen controls, which indicated some retention of OXPHOS capacity by the IMM. Trehalose then was loaded into the matrix (0.24 μmol/mg mitochondrial protein) by transient opening of the permeability transition pore, a procedure optimized for retention of OMM integrity. Surprisingly, respiratory control ratios were not improved after freeze-thawing with external plus matrix trehalose, when compared to external trehalose alone. This result could perhaps be explained by insufficient accumulation of matrix trehalose.

Fullerenol C60(OH)24 increases ion permeability of lipid membranes in a pH-dependent manner

Fullerenols are water-soluble analogs of fullerene exhibiting both antioxidant and prooxidant activities in vitro and in vivo. Here we report, for the first time, that fullerenol C60(OH)24 can induce ion permeability of a planar lipid bilayer membrane via the formation of ion pores or conductive defects with a preference for cations over anions. The fullerenol-mediated electrical current displayed non-linear concentration dependence and was reversibly enhanced by alkalinization. Calcium and magnesium ions decreased the fullerenol-induced potassium ion permeability. Voltage dependence of the current was sensitive to membrane composition, with the conductance being well pronounced in fully saturated diphytanoylphosphatidylcholine. Fullerenol did not induce carboxyfluorescein leakage from liposomes, suggesting a small size of fullerenol-induced pores. In contrast to ion permeability, the binding of C60(OH)24 to liposomes increased at acidic pH, as measured by fluorescence quenching of pyrene-labeled lipid. In line with this, the photodynamic action of fullerenol on the peptide gramicidin A also increased at low pH. It is hypothesized that aggregates of fullerenol may stabilize transient conductive lipid defects or pores formed under a variety of stress conditions.

Liposomes with diverse compositions are protected during desiccation by LEA proteins from Artemia franciscana and trehalose

Intracellular accumulation of Late Embryogenesis Abundant (LEA) proteins and the disaccharide trehalose is associated with cellular desiccation tolerance in a number of animal species. Two LEA proteins from anhydrobiotic embryos of the brine shrimp Artemia franciscana were tested for the ability to protect liposomes of various compositions against desiccation-induced damage in the presence and absence of trehalose. Damage was assessed by carboxyfluorescein leakage after drying and rehydration. Further, using a cytoplasmic-localized (AfrLEA2) and a mitochondrial-targeted (AfrLEA3m) LEA protein allowed us to evaluate whether each may preferentially stabilize membranes of a particular lipid composition based on the protein's subcellular location. Both LEA proteins were able to offset damage during drying of liposomes that mimicked the lipid compositions of the inner mitochondrial membrane (with cardiolipin), outer mitochondrial membrane, and the inner leaflet of the plasma membrane. Thus liposome stabilization by AfrLEA3m or AfrLEA2 was not dependent on lipid composition, provided physiological amounts of bilayer and non-bilayer-forming lipids were present (liposomes with a non-biological composition of 100% phosphatidylcholine were not protected by either protein). Additive protection by LEA proteins plus trehalose was dependent on the lipid composition of the target membrane. Minimal additional damage occurred to liposomes stored at room temperature in the dried state for one week compared to liposomes rehydrated after 24h. Consistent with the ability to stabilize lipid bilayers, molecular modeling of the secondary structures for AfrLEA2 and AfrLEA3m revealed bands of charged amino acids similar to other amphipathic proteins that interact directly with membranes.

Ubiquinone-10 alters mechanical properties and increases stability of phospholipid membranes

Ubiquinone-10 is mostly known for its role as an electron and proton carrier in aerobic cellular respiration and its function as a powerful antioxidant. Accumulating evidence suggest, however, that this well studied membrane component could have several other important functions in living cells. The current study reports on a previously undocumented ability of ubiquinone-10 to modulate the mechanical strength and permeability of lipid membranes. Investigations of DPH fluorescence anisotropy, spontaneous and surfactant induced leakage of carboxyfluorescein, and interactions with hydrophobic and hydrophilic surfaces were used to probe the effects caused by inclusion of ubiquinone-10 in the membrane of phospholipid liposomes. The results show that ubiquinone in concentrations as low as 2mol% increases the lipid packing order and condenses the membrane. The altered physicochemical properties result in a slower rate of release of hydrophilic components, and render the membrane more resistant towards rupture. As judged from comparative experiments using the polyisoprenoid alcohol solanesol, the quinone moiety is essential for the membrane stabilizing effects to occur. Our findings imply that the influence of ubiquinone-10 on the permeability and mechanical properties of phospholipid membranes is similar to that of cholesterol. The reported data indicate, however, that the molecular mechanisms are different in the two cases.

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.

Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules

Lucifer Yellow (LYCH) and carboxyfluorescein (CF) served in Medicago truncatula roots and root nodules as the markers of apoplastic and symplastic transport, respectively. The aim of this study was to understand better the water and photoassimilate translocation pathways to and within nodules. The present study shows that in damaged roots LYCH moves apoplastically through the vascular elements but it was not detected within the nodule vascular bundles. In intact roots, the outer cortex was strongly labeled but the dye was not present in the interior of intact root nodules. The inwards movement of LYCH was halted in the endodermis. When the dye was introduced into a damaged nodule by infiltration, it spread only in the cell walls and the intercellular spaces up to the inner cortex. Our research showed that in addition to the outer cortex, the inner tissue containing bacteroid-infected cells is also an apoplastic domain. Our results are consistent with the hypothesis that nodules do not receive water from the xylem but get it and photoassimilates from phloem. A comparison between using LYCH and LYCH followed by glutaraldehyde fixation indicates that glutaraldehyde is responsible for fluorescence of some organelles within root nodule cells. The influence of the fixation on nodule fluorescence has not been reported before but must be taken into consideration to avoid errors. An attempt was made to follow carboxyfluorescein (6(5) CF) translocation from leaflets into roots and root nodules. In root nodules, CF was present in all or a couple of vascular bundles (VB), vascular endodermis and some adjacent cells. The leakage of CF from the VBs was observed, which suggests symplastic continuity between the VBs and the nodule parenchyma. The lack of CF in inner tissue was observed. Therefore, photoassimilate entry to the infected region of nodule must involve an apoplastic pathway.