Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.
Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.
Zein is renewable plant protein with valuable film-forming properties that can be used as a packaging material. It is known that the addition of natural cross-linkers can enhance a film's tensile properties. In this study, we aimed to prepare antimicrobial zein-based films enriched with monolaurin, eugenol, oregano, and thyme essential oil. Films were prepared using the solvent casting technique from ethanol solution. Their physicochemical properties were investigated using structural, morphological, and thermal techniques. Polar and dispersive components were analyzed using two models to evaluate the effects on the surface free energy values. The antimicrobial activity was proven using a disk diffusion method and the suppression of bacterial growth was confirmed via a growth kinetics study with the Gompertz function. The films' morphological characteristics led to systems with uniform distribution of essential oils or eugenol droplets combined with a flat-plated structure of monolaurin. A unique combination of polyphenolic eugenol and amphiphilic monoglyceride provided highly stretchable films with enhanced barrier properties and efficiency against Gram-positive and Gram-negative bacteria, yeasts, and molds. The prepared zein-based films with tunable surface properties represent an alternative to non-renewable resources with a potential application as active packaging materials.
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Antifungal Agents pharmacology MeSH
- Biomechanical Phenomena drug effects MeSH
- Calorimetry, Differential Scanning MeSH
- Escherichia coli drug effects MeSH
- Eugenol pharmacology MeSH
- Laurates pharmacology MeSH
- Microscopy, Atomic Force MeSH
- Monoglycerides pharmacology MeSH
- Food Packaging * MeSH
- Oils, Volatile pharmacology MeSH
- Steam MeSH
- Permeability MeSH
- Surface Properties MeSH
- Wettability MeSH
- Spectroscopy, Fourier Transform Infrared MeSH
- Staphylococcus aureus drug effects MeSH
- Zein pharmacology MeSH
- Publication type
- Journal Article MeSH
Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquid⁻crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquid⁻crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.
- MeSH
- 1,2-Dipalmitoylphosphatidylcholine chemistry MeSH
- 2-Naphthylamine analogs & derivatives chemistry MeSH
- Models, Chemical * MeSH
- Fluorescence MeSH
- Phosphatidylcholines chemistry MeSH
- Quantum Theory MeSH
- Laurates chemistry MeSH
- Lipid Bilayers chemistry MeSH
- Molecular Dynamics Simulation * MeSH
- Publication type
- Journal Article MeSH
Procyanidins, contained in many products abundant in human diet, exhibit high biological activity. However, this activity has not been fully explained at cellular and molecular levels. In this study, we determine the mechanism of interaction of procyanidin B3 with model lipid membrane. This mechanism was established on the basis of changes induced by B3 in the physical properties of lipid bilayer. The changes were investigated using steady state and time-resolved fluorescence, DSC, and FTIR. We show that procyanidin B3 causes changes in the arrangement of the polar heads of lipids, order of their acyl chains and the main lipid phase transition temperature. Furthermore, its presence in the membrane leads to a reduction in membrane dipole potential. Procyanidin B3 is anchored to membrane via hydrogen bonds formed between its OH groups and the PO2- and CO groups of lipids, causing changes in both hydrophilic and hydrophobic regions of the membrane.
- MeSH
- 2-Naphthylamine analogs & derivatives chemistry MeSH
- Biflavonoids chemistry MeSH
- Calorimetry, Differential Scanning MeSH
- Dihydropyridines chemistry MeSH
- Dimyristoylphosphatidylcholine chemistry MeSH
- Spectrometry, Fluorescence MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Catechin chemistry MeSH
- Laurates chemistry MeSH
- Lipid Bilayers chemistry MeSH
- Proanthocyanidins chemistry MeSH
- Spectroscopy, Fourier Transform Infrared MeSH
- Temperature MeSH
- Thermodynamics MeSH
- Hydrogen Bonding MeSH
- Phase Transition MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water-membrane interface. The implications of these findings for considerations of protein-cholesterol interactions are briefly discussed.
- MeSH
- 2-Naphthylamine analogs & derivatives chemistry MeSH
- Cholesterol chemistry MeSH
- Dihydropyridines chemistry MeSH
- Cholesterol Esters chemistry MeSH
- Phosphatidylcholines chemistry MeSH
- Laurates chemistry MeSH
- Lipid Bilayers chemistry MeSH
- Liposomes chemistry MeSH
- Protons * MeSH
- Molecular Dynamics Simulation MeSH
- Water chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Fluorescence methods are widely used in studies of biological and model membranes. The dynamics of membrane fluorescent markers in their ground and excited electronic states and correlations with their molecular surrounding within the fully hydrated phospholipid bilayer are still not well understood. In the present work, Quantum Mechanical (QM) calculations and Molecular Dynamics (MD) simulations are used to characterize location and interactions of two membrane polarity probes (Prodan; 6-propionyl-2-dimethylaminonaphthalene and its derivative Laurdan; 2-dimethylamino-6-lauroylnaphthalene) with the dioleoylphosphatidylcholine (DOPC) lipid bilayer model. MD simulations with fluorophores in ground and excited states are found to be a useful tool to analyze the fluorescent dye dynamics and their immediate vicinity. The results of QM calculations and MD simulations are in excellent agreement with available experimental data. The calculation shows that the two amphiphilic dyes initially placed in bulk water diffuse within 10 ns towards their final location in the lipid bilayer. Analysis of solvent relaxation process in the aqueous phase occurs on the picoseconds timescale whereas it takes nanoseconds at the lipid/water interface. Four different relaxation time constants, corresponding to different relaxation processes, where observed when the dyes were embedded into the membrane.
The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to "wobble-in-cone" model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.
- MeSH
- 2-Naphthylamine analogs & derivatives MeSH
- Cell Membrane metabolism drug effects MeSH
- Cetomacrogol pharmacology MeSH
- Diphenylhexatriene MeSH
- Diffusion MeSH
- Financing, Organized MeSH
- Fluorescent Dyes MeSH
- Spectrometry, Fluorescence MeSH
- Rats MeSH
- Laurates MeSH
- Brain metabolism drug effects MeSH
- Octoxynol pharmacology MeSH
- GTP-Binding Proteins metabolism MeSH
- Receptors, GABA-B metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
The need for detailed biophysical description of cationic lipid membranes, which are commonly used as gene transfection vectors, led us to study the properties of mixed cationic/zwitterionic lipid bilayers. Fluorescence solvent relaxation measurements of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) incorporated in a membrane consisting of cationic dimyristoyltrimethylammoniumpropane (DMTAP) and zwitterionic dimyristoylphosphatidylcholine (DMPC) were performed. The obtained results are compared with a recently measured system consisting of dioleoyltrimethylammoniumpropane (DOTAP) and dioleoylphosphatidylcholine (DOPC) (Jurkiewicz et al. Langmuir 22:8741-8749, 2006). The similar nonmonotonic dependence of the relaxation kinetics on cationic lipid content in the membrane was present for both systems. While the slowest solvent relaxation have been observed for 30 mol% of DOTAP in the DOPC bilayer (Jurkiewicz et al. Langmuir 22:8741-8749, 2006), for DMPC/DMTAP system it was found at 45 mol% of DMTAP, which agrees with the literature. Both membranes increased their hydration upon increased cationic lipid content.
- MeSH
- 2-Naphthylamine analogs & derivatives chemistry MeSH
- Dimyristoylphosphatidylcholine chemistry MeSH
- Financing, Organized MeSH
- Fluorescence MeSH
- Fluorescent Dyes chemistry MeSH
- Cations chemistry MeSH
- Quaternary Ammonium Compounds chemistry MeSH
- Laurates chemistry MeSH
- Lipid Bilayers chemistry MeSH
- Lipids chemistry MeSH
- Membranes chemistry MeSH
- Molecular Structure MeSH
- Myristates chemistry MeSH
- Solvents chemistry MeSH
- Water chemistry MeSH
New androstane brassinosteroids with 17beta-ester groups - butyrates, heptafluorobutyrates, and laurates (4-18) - were prepared. Brassinolide activity was evaluated using both the bean second internode bioassay and the rice lamina inclination test. Brassinosteroid 16 was found to be the most active by the bean second internode bioassay. This activity in the bean second internode bioassay corresponded with the field yield, while the RLIT bioassay does not.
- MeSH
- Androstanes pharmacology chemical synthesis chemistry MeSH
- Biological Assay MeSH
- Butyrates chemistry MeSH
- Cholestanols pharmacology chemical synthesis chemistry MeSH
- Esterification MeSH
- Phaseolus growth & development drug effects MeSH
- Financing, Organized MeSH
- Fluorocarbons chemistry MeSH
- Quantitative Structure-Activity Relationship MeSH
- Laurates chemistry MeSH
- Magnetic Resonance Spectroscopy MeSH
- Molecular Structure MeSH
- Oryza growth & development drug effects MeSH
- Steroids, Heterocyclic pharmacology chemical synthesis chemistry MeSH
