ABSTRACT
We measured temporal oscillations in thermodynamic variables such as temperature, heat flux, and cellular volume in suspensions of non-dividing yeast cells which exhibit temporal glycolytic oscillations. Oscillations in these variables have the same frequency as oscillations in the activity of intracellular metabolites, suggesting strong coupling between them. These results can be interpreted in light of a recently proposed theoretical formalism in which isentropic thermodynamic systems can display coupled oscillations in all extensive and intensive variables, reminiscent of adiabatic waves. This interpretation suggests that oscillations may be a consequence of the requirement of living cells for a constant low-entropy state while simultaneously performing biochemical transformations, i.e., remaining metabolically active. This hypothesis, which is in line with the view of the cellular interior as a highly structured and near equilibrium system where energy inputs can be low and sustain regular oscillatory regimes, calls into question the notion that metabolic processes are essentially dissipative.
Subject(s)
Entropy , Glycolysis , Models, Biological , Saccharomyces cerevisiae/physiology , Hot Temperature , ThermodynamicsABSTRACT
In this study, a simple purification protocol is developed to reduce the bovine serum albumin (BSA) content in commercially available bovine submaxillary mucin (BSM). This involved purification of the BSM by one-column anion-exchange chromatography protocol resulting in BSM with greatly reduced BSA content and homogeneously distributed size, and in a high yield of â¼43% from BSM as received from the manufacturer. The purity and composition of commercially acquired BSM were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry, which verified that BSA is the most abundant nonmucinous protein component. The purification effect was evident from a significantly altered circular dichroism (CD) spectrum of BSM after anion-exchange chromatography.
Subject(s)
Chromatography, Ion Exchange/methods , Mucins/isolation & purification , Animals , Cattle , Chromatography, Thin Layer , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Mucins/chemistry , Serum Albumin, Bovine/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We have in this study investigated the composition, structure and spectroscopical properties of multilamellar vesicles composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and up to 10mol% of triolein (TO), a triglyceride. We found in agreement with previous results that the mixtures with 10mol% TO spontaneously separate into two distinct phases, heavy (HF) and light (LF), with different densities and found this also to be the case for 2 and 5mol% TO. The compositions of the two phases were investigated by quantitative lipid mass spectrometric analysis, and with this method we found that TO had a solubility maximum of about 4mol% in the HF, whereas it was markedly up-concentrated in the LF. Electron paramagnetic resonance spectroscopy indicated POPC membranes of all tested concentrations of TO in both phases to be almost unperturbed by the presence of TO and to exist as vesicular structures containing entrapped water. Bilayer structure of the membranes was supported by small angle X-ray scattering that showed the membranes to form a lamellar phase. Fluorescence spectroscopy with the polarity sensitive dye Nile red revealed, that the LF samples with more than 5mol% TO contained pure TO domains. These observations are consistent with an earlier MD simulation study by us and our co-workers suggesting triglycerides to be located in lens shaped, blister-like domains between the two lipid bilayer leaflets (Khandelia et al. (2010) [26]).
Subject(s)
Lipid Bilayers/chemistry , Phosphatidylcholines/chemistry , Triglycerides/chemistry , Triolein/chemistry , Electron Spin Resonance Spectroscopy , Lipid Bilayers/metabolism , Magnetic Resonance Spectroscopy , Phosphatidylcholines/metabolism , Scattering, Small Angle , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Triglycerides/metabolism , Triolein/metabolismABSTRACT
In biophysical and biochemical studies of lipid bilayers the influence of the used buffer is often ignored or assumed to be negligible on membrane structure, elasticity, or physical properties. However, we here present experimental evidence, through bending rigidity measurements performed on giant vesicles, of a more complex behavior, where the buffering molecules may considerably affect the bending rigidity of phosphatidylcholine bilayers. Furthermore, a synergistic effect on the bending modulus is observed in the presence of both salt and buffer molecules, which serves as a warning to experimentalists in the data interpretation of their studies, since typical lipid bilayer studies contain buffer and ion molecules.
Subject(s)
Buffers , Lipid Bilayers/chemistry , Models, Chemical , Phosphatidylcholines/chemistryABSTRACT
Coffee brewed on light, and very light-roast coffee beans have emerged as a recent trend among specialty coffee drinkers. The acidity of such light-roast coffee, and coffee in general, is an important sensory characteristic, as there is demonstrated a clear correlation between the roast level and perceived acidity in brewed coffee. The acidity is believed to be strongly linked to the content and composition of organic acids in coffee. Still, there is limited literature on acid content in brewed coffee and on the relevance of specific acid concentrations to sensory perception. In this study, we determined concentrations of acids and sugars in French-press brewed specialty coffee. We used varying roast degrees in the light to very light range using five coffees from different geographical locations (Brazil, Bolivia, and Kenya) and determined the sensory detection threshold and recognition for selected acids. The concentration of all individual acids except one (formic) either significantly decreased (citric, malic, and chlorogenic acid) or increased (acetic, lactic, phosphoric, quinic, and glycolic acid) systematically with an increasing roast degree, while no systematic trends were found between the different coffee samples. The sugar content decreased with an increasing roast degree. The sensory detection threshold for malic, acetic, and lactic acid was determined to be above the actual concentration of said acids in the coffee and just below for phosphoric acid, indicating that these compounds are unlikely to individually be perceived in coffee. Only citric acid can be clearly detected in the threshold test (not identified by experts in coffee) in concentrations above the measured concentrations, as the detection threshold was below (<0.16 g/L) the concentration found in the investigated coffees (0.23-0.60 g/L). The measured citric acid concentration was found to be much higher for the Brazil coffees (0.49 ± 0.08 g/L) compared to the Bolivia coffee (0.40 ± 0.11 g/L), and the Kenya coffees (0.30 ± 0.07 g/L). Furthermore, none of the acids added to the coffee were correctly recognized by coffee experts when spiked with measured average concentrations. Combined, the results question the direct relation between individual organic acids and acidity in coffee and point towards a more complex understanding of perceived acidity.
ABSTRACT
We demonstrate here that triolein alters the mechanical properties of phospholipid membranes and induces extraordinary conformational dynamics. Triolein containing membranes exhibit fluctuations up to size range of 100µm and with the help of these are e.g. able to squeeze through narrow passages between neighbouring structures. Triolein-phosphatidylcholine membranes were found to have bending rigidity significantly lower than that of corresponding pure phosphatidylcholine membrane. Moreover, the triolein containing membranes were found to be reluctant to fuse, which is in good accordance with larger lamellar distances observed in the TOPOPC membranes. These findings suggest repulsion between adjacent membranes. We provide a comprehensive discussion on the possible explanations for the observed mechanics and dynamics in the TOPOPC system and on their potential cellular implications.
Subject(s)
Cell Membrane/chemistry , Membranes, Artificial , Phosphatidylcholines/chemistry , Triolein/chemistry , Calorimetry, Differential Scanning , Cell Membrane/ultrastructure , Elasticity , Fluorescence Resonance Energy Transfer , Membrane Fluidity , Membrane Fusion , Microscopy, Confocal , Microscopy, Electron, Transmission , Microscopy, Phase-Contrast , Molecular Conformation , Scattering, Small AngleABSTRACT
The leaves of the annual plant Perilla frutescens are used widely as a spice and a preservative in Asian food as well as in traditional medicine. The active compounds in the leaves are the cyclic monoterpene limonene (1) and its bio-oxidation products, perillaldehyde (2), perillyl alcohol (3), and perillic acid (4). These compounds are known to be biologically active and exhibit antimicrobial, anticancer, and anti-inflammatory effects that could all be membrane mediated. In order to assess the possible biophysical effects of these compounds on membranes quantitatively, the influence of limonene and its bio-oxidation products has been investigated on a membrane model composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) using differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC), and electron paramagnetic resonance spectroscopy (EPR). It was found that limonene (1), perillyl alcohol (2), and perillaldehyde (3) partitioned into the DMPC membrane, whereas perillic acid (4) did not. The DSC results demonstrated that all the partitioning compounds strongly perturbed the phase transition of DMPC, whereas no perturbation of the local membrane order was detected by EPR spectroscopy. The results of the study showed that limonene (1) and its bio-oxidation products affect membranes in rather subtle ways.
Subject(s)
Cell Membrane/physiology , Membrane Lipids/physiology , Perilla frutescens/chemistry , Plants, Medicinal/chemistry , Cyclohexenes/analysis , Cyclohexenes/chemistry , Cyclohexenes/pharmacology , Dimyristoylphosphatidylcholine/chemistry , Limonene , Models, Molecular , Molecular Structure , Monoterpenes/analysis , Monoterpenes/chemistry , Monoterpenes/pharmacology , Plant Leaves/chemistry , Terpenes/analysis , Terpenes/chemistry , Terpenes/pharmacologyABSTRACT
Secretory phospholipase A(2) (sPLA(2)) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (L(α)/L(ß)) lipid domains. In this work, we explore whether lipid domains can also direct the activity of the enzyme by inducing hydrolysis of certain lipid components due to preferential activity of the enzyme toward lipid domains susceptible to sPLA(2). Specifically, we show that the presence of L(α)/L(ß) and L(α)/P(ß') phase coexistence in a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) system results in the preferential hydrolysis of the shorter-chained lipid component in the mixture, leading to an enrichment in the longer-chained component. The restructuring process is monitored by atomic force microscopy on supported single and double bilayers formed by vesicle fusion. We observe that during preferential hydrolysis of the DMPC-rich L(α) regions, the L(ß) and P(ß') regions grow and reseal, maintaining membrane integrity. This result indicates that a sharp reorganization of the membrane structure can occur during sPLA(2) hydrolysis without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary. Differential scanning calorimetry results show that this preferential hydrolysis in the presence of lipid domains leads to a membrane system with a higher-temperature melting profile due to enrichment in DSPC. Together, these results show that the presence of lipid domains can induce specificity in the hydrolytic activity of the enzyme, resulting in marked differences in the physical properties of the membrane end-product.
Subject(s)
Lipids/chemistry , Membranes, Artificial , Phospholipases A2, Secretory/metabolism , Animals , Calorimetry, Differential Scanning , Dimyristoylphosphatidylcholine/chemistry , Hydrolysis , Lipid Bilayers/chemistry , Microscopy, Atomic Force , Phase Transition , Snake Venoms/enzymologyABSTRACT
Lysolipids and fatty acids are the natural products formed by the hydrolysis of phospholipids. Lysolipids and fatty acids form micelles in solution and acts as detergents in the presence of lipid membranes. In this study, we investigate the detergent strength of a homologous series of lyso-phosphatidylcholine lipids (LPCs) on 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphatidylcholine (POPC) lipid membranes by use of isothermal titration calorimetry and vesicle fluctuation analysis. The membrane partition coefficient (K) and critical micelle concentration (cmc) are determined by isothermal titration calorimetry and found to obey an inverse proportionality relation (cmc.K approximately 0.05-0.3). The partition coefficient and critical micelle concentration are used for the analysis of the effect of LPCs on the membrane bending rigidity. The dependency of the bending rigidity on LPC membrane coverage has been analyzed in terms of a phenomenological model based on continuum elastic theory, which yields information about the curvature-inducing properties of the LPC molecule. The results reveal: 1), an increase in the partition coefficient with increasing LPC acyl-chain length; and 2), that the degree of acyl-chain mismatch between LPC and POPC determines the magnitude of the membrane mechanical perturbation per LPC molecule in the membrane. Finally, the three-stage model describing detergent membrane interaction has been extended by a parameter D(MCI), which governs the membrane curvature stability in the detergent concentration range below the cmc-value of the LPC molecule.
Subject(s)
Detergents/chemistry , Lipid Bilayers/chemistry , Membrane Lipids/chemistry , Phosphatidylcholines/chemistry , Calorimetry , Detergents/pharmacology , Membranes/drug effects , MicellesABSTRACT
In mammalian cells, the organic osmolyte taurine is accumulated by the Na-dependent taurine transporter TauT and released though the volume- and DIDS-sensitive organic anion channel. Incubating Ehrlich Lettré tumor cells with methyl-ß-cyclodextrin (5 mM, 1 h) reduces the total cholesterol pool to 60±5% of the control value. Electron spin resonance data indicate a concomitant disruption of cholesterol-rich micro-domains. Active taurine uptake, cellular taurine content, and cell volume are reduced by 50, 20 and 20% compared to control values, respectively, whereas the passive taurine release is increased 4.5-fold under isotonic conditions following cholesterol depletion. However, taurine release under isotonic conditions is insensitive to DIDS and inhibitors of the volume-regulated anion channel. Uptake and release of meAIB are similarly affected following cholesterol depletion. Kinetic analysis reveals that cholesterol depletion increases TauT's affinity toward taurine but reduces its maximal transport capacity. Cholesterol depletion has no impact on TauT regulation by protein kinases A and C. Phospholipase A2 activity, which is required for the activation of volume-sensitive organic anion channel (VSOAC), is increased under isotonic and hypotonic conditions following cholesterol depletion, whereas taurine release under hypotonic conditions is reduced following cholesterol depletion. Hence, acute cholesterol depletion of Ehrlich Lettré cells leads to reduced TauT and VSOAC activities and at the same time increases the release of organic osmolytes via a leak pathway different from the volume-sensitive pathways for amino acids and anions.
Subject(s)
Carcinoma, Ehrlich Tumor/metabolism , Carcinoma, Ehrlich Tumor/pathology , Cholesterol/deficiency , Cholesterol/metabolism , Taurine/metabolism , Animals , Ion Channels/metabolism , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/metabolism , Mice , Osmosis , Phospholipases A2/metabolism , Tumor Cells, Cultured , beta-Cyclodextrins/pharmacologyABSTRACT
Imaging the microstructure of opaque composite foodstuffs and extracting quantitative chemical information about specific localized components is challenging. Herein, a method has been developed to determine spatially resolved concentrations of aqueous salt and applied to measure salt concentrations of water droplets in butter samples. This was done using Coherent Anti-Stokes Raman Scattering (CARS) microscopy which achieves non-invasive label free imaging based on visualization of specific chemical-bond vibrations. The concentration of salt in the dispersed water droplets in butter was determined based on the relative change in intensity of the CARS-signal at two distinct wavenumbers, which have been shown to be dependent on the inter-molecular coupling of water molecules and salt. The results provide the size and salt concentration distribution of the droplets in the samples. It is further shown that the average salt concentration in the whole sample can correctly be inferred from the concentration measured within the water droplets.
Subject(s)
Butter/analysis , Sodium Chloride/analysis , Spectrum Analysis, Raman/methods , Food Analysis , Microscopy , Oils/chemistry , Water/chemistryABSTRACT
Adequate protein intake is necessary to maintain muscle mass and function. Nutritionally dense(r) products are increasingly sought after by consumers that need a heightened protein intake, such as active young and elderly individuals. This paper focuses on functional snack sausages enriched with umami-tasting meat protein hydrolysates (MPH), developed by systematically varying in recipe, meat type and MPH content. A consumer study (Nâ¯=â¯100) was conducted where young and elderly consumers evaluated perceived acceptability and sensory quality of samples. Additionally, textural (Warner-Braztler shear test) and chemical (amino acids) analyses on the same samples were conducted to give a complete characterization of the functionality of the hydrolysates. Both the consumer test and the instrumental analyses consistently indicated that the enrichment with MPH had minor or no influence on the perceptual quality of the sausages, suggesting that this ingredient can be added to increase the nutritional density of a reference meat product without negatively affecting consumer acceptability.
Subject(s)
Consumer Behavior , Meat Products/analysis , Protein Hydrolysates , Taste , Adult , Aged , Aged, 80 and over , Animals , Cattle , Denmark , Female , Food Preferences , Humans , Male , Meat Proteins , Shear Strength , Sus scrofaABSTRACT
We have studied 15 different cultivars of chillies (Capsicum var.) grown in temperate climate Denmark and determined the contents of the four major capsaicinoids: capsaicin, dihydrocapsaicin, nordihydrocapsaicin, and homocapsaicin. From these contents we have, as commonly done for chillies, calculated the so-called pungency in Scoville heat units in order to compare with previous studies from other climatical zones. For three of the investigated cultivars, Serrano, Habanero and BIH Jolokia, for which reliable pungencies has previously been reported, we have found pungencies of 34,000±1400, 247,000±24,000 and 665,000±4000, respectively, which are all in the same ranges as found earlier for chillies grown in more traditional chilli growing areas. Furthermore we have found that the relative distribution of the four capsaicinoids in the 15 different cultivars is highly variable, with the content of capsaicin ranging from 31% to 71% of the total capsaicinoid content.
Subject(s)
Capsaicin/analogs & derivatives , Capsicum/chemistry , Capsaicin/analysis , Climate , DenmarkABSTRACT
Higher sterols are universally present in large amounts (20-30%) in the plasma membranes of all eukaryotes whereas they are universally absent in prokaryotes. It is remarkable that each kingdom of the eukaryotes has chosen, during the course of evolution, its preferred sterol: cholesterol in animals, ergosterol in fungi and yeast, phytosterols in higher plants, and e.g., fucosterol and desmosterol in algae. The question arises as to which specific properties do sterols impart to membranes and to which extent do these properties differ among the different sterols. Using a range of biophysical techniques, including calorimetry, fluorescence microscopy, vesicle-fluctuation analysis, and atomic force microscopy, we have found that fucosterol and desmosterol, found in red and brown macroalgae (seaweeds), similar to cholesterol support liquid-ordered membrane phases and induce coexistence between liquid-ordered and liquid-disordered domains in lipid bilayers. Fucosterol and desmosterol induce acyl-chain order in liquid membranes, but less effectively than cholesterol and ergosterol in the order: cholesterol>ergosterol>desmosterol>fucosterol, possibly reflecting the different molecular structure of the sterols at the hydrocarbon tail.
Subject(s)
Desmosterol/chemistry , Lipid Bilayers/chemistry , Seaweed/chemistry , Stigmasterol/analogs & derivatives , Calorimetry, Differential Scanning , Cell Membrane , Mechanical Phenomena , Microscopy, Atomic Force , Microscopy, Fluorescence , Molecular Structure , Stigmasterol/chemistryABSTRACT
The data in this paper are additional information to the research article entiltled "Inhibition of cholesterol transport in an intestine cell model by pine-derived phytosterols" (Yi et al.,2016) [1]. The data derived from the measurement on six liquid formulations of commercial pine-derived phytosterol (CPP) by dynamic light scattering. The data cover micelle size and the zeta-potential for formulations with cholesterol including monoglyceride, oleic acid, and bile salt. The data demonstrate the critical effect of the bile salt concentration on the size of cholesterol-digested fat micelles.
ABSTRACT
We have quantified the inhibition of intestinal cholesterol transport by pine-derived phytosterols using an HT29-MTX intestine cell model that forms a mucus layer similar to that in the intestine. An artificial intestinal fluid consisting of digested fat, bile salt, cholesterol, and phytosterols was formulated in order to mimic the conditions in the intestine. The apparent permeability coefficient (Papp) of the positive control, i.e., 0.1mM of cholesterol solubilized in the artificial intestine fluid, was found to be 0.33 (±0.17)×10-6cm/s. When 0.1mM ß-sitosterol was solubilized alongside, Papp was effectively zero, corresponding to a total inhibition of cholesterol transport. A similar strong inhibition was found when commercial pine-derived phytosterols, PinVita™ FSP DuPont, were co-solubilized with cholesterol in the dietary model micelles, leading to Papp=0.06 (±0.06)×10-6cm/s, i.e., 5.5 times lower than the cholesterol positive control. Additionally, the effect of potential oral administration formulations generated by the pine-derived phytosterols was also characterized. The formulations were produced as a liquid formulation of the cholesterol-containing artificial intestine fluid. Six liquid formulations were tested of which four displayed a Papp in the range of 0-0.09×10-6cm/s. The remaining two formulations did not show any inhibition effect on cholesterol transport and even enhanced cholesterol transport. It was furthermore observed that the phytosterols were found in the collected intestine cells but not transported to the basolateral region in the intestinal cell model system.
Subject(s)
Cholesterol/metabolism , Intestines/cytology , Intestines/drug effects , Models, Biological , Phytosterols/pharmacology , Pinus/chemistry , Absorption, Physiological , Biological Transport/drug effects , Cells, Cultured , Cholesterol/analysis , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , HT29 Cells , Humans , Intestinal Mucosa/metabolism , Particle Size , Phytosterols/chemistry , Structure-Activity Relationship , Surface PropertiesABSTRACT
Animal studies suggest a role of vitamin D in fetal lung development although not studied in preterm animals. We tested the hypothesis that vitamin D depletion aggravates respiratory insufficiency in preterm rat offspring. Furthermore, the effects of vitamin D depletion on growth and lung surfactant were investigated. Female Sprague-Dawley rats were randomly assigned low vitamin D (VDL) or control diet before mating and followed with serum 25-hydroxyvitamin D (s-25(OH)D) determinations. After cesarean section at gestational day 19 (E19) or day 22 (E22), placental weight, birth weight, crown-rump-length (CRL), oxygenation (SaO2) at 30 min and survival time were recorded. The pup lungs were analyzed for phospholipid levels, surfactant protein A-D mRNA and the expression of the vitamin D receptor (VDR). S-25(OH)D was significantly lower in the VDL group at cesarean section (12 vs. 30nmol/L, p<0.0001). Compared to the controls, E19 VDL pups had lower birth weight (2.13 vs. 2.29g, p<0.001), lung weight (0.09 vs. 0.10g, p = 0.002), SaO2 (54% vs. 69%, p = 0.002) as well as reduced survival time (0.50 vs. 1.25h, p<0.0001). At E22, the VDL-induced pulmonary differences were leveled out, but VDL pups had lower CRL (4.0 vs. 4.5cm, p<0.0001). The phospholipid levels and the surfactant protein mRNA expression did not differ between the dietary groups. In conclusion, Vitamin D depletion led to lower oxygenation and reduced survival time in the preterm offspring, associated with reduced lung weight and birth weight. Further studies of vitamin D depletion in respiratory insufficiency in preterm neonates are warranted.
Subject(s)
Lung/metabolism , Lung/physiology , Organ Size/physiology , Oxygen/metabolism , Premature Birth/blood , Premature Birth/physiopathology , Vitamin D Deficiency/blood , Vitamin D Deficiency/physiopathology , Vitamin D/blood , Animals , Body Weight/physiology , Female , Male , Pregnancy , Rats , Rats, Sprague-Dawley , Vitamin D/analogs & derivativesABSTRACT
Polyaromatic hydrocarbons (PAHs) is a group of compounds, many of which are toxic, formed by incomplete combustion or thermal processing of organic material. They are highly lipophilic and thus present in some seed oils used for human consumption as well as being increasingly common in aquaculture diets due to inclusion of vegetable oils. Cytotoxic effects of PAHs have been thought to be partly due to a membrane perturbing effect of these compounds. A series of studies were here performed to examine the effects of three different PAHs (naphthalene, phenanthrene and benzo[a]pyrene) with different molecular sizes (two, three and five rings, respectively) and fat solubility (Kow 3.29, 4.53 and 6.04, respectively) on membrane models. The effects of PAHs on liquid-liquid phase coexistence in solid-supported lipid bilayers (dioleoylphosphocholine:dipalmitoylphosphatidylcholine:cholesterol) were assessed using fluorescence microscopy. Benzo[a]pyrene had a slight affinity for the liquid-ordered phase, but there were no effects of adding any of the other PAHs on the number or size of the liquid domains (liquid-ordered and liquid-disordered). Benzo[a]pyrene and phenanthrene, but not naphthalene, lowered the transition temperature (Tm) and the enthalpy (ΔH) characterising the transition from the solid to the liquid-crystalline phase in DPPC vesicles. The membrane effects of the PAH molecules are likely related to size, with bigger and more fat-soluble molecules having a fluidising effect when embedded in the membrane, possibly causing some of the observed toxic effects in fish exposed to these contaminants.
Subject(s)
Lipid Bilayers/chemistry , Polycyclic Aromatic Hydrocarbons/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Benzo(a)pyrene/chemistry , Calorimetry, Differential Scanning , Cholesterol/chemistry , Microscopy, Fluorescence , Naphthalenes/chemistry , Phase Transition , Phenanthrenes/chemistry , Phosphatidylcholines/chemistry , Thermodynamics , Transition TemperatureABSTRACT
The compound 2,6-diisopropylphenol (Propofol, PRF) is widely used for inducing general anesthesia, but the mechanism of PRF action remains relatively poorly understood at the molecular level. This work examines the possibility that a potential mode of action of PRF is to modulate the lipid order in target membranes. The effect on monolayers and bilayers of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) was probed using Langmuir monolayer isotherms, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations. Increasing amounts of PRF in a DPPC monolayer causes a decrease in isothermal compressibility modulus at the phase transition. A partition constant for PRF in DPPC liposomes on the order of K≈1500 M(-1) was found, and the partitioning was found to be enthalpy-driven above the melting temperature (Tm). A decrease in Tm with PRF content was found whereas the bilayer melting enthalpy ΔHm remains almost constant. The last finding indicates that PRF incorporates into the membrane at a depth near the phosphatidylcholine headgroup, in agreement with our MD-simulations. The simulations also reveal that PRF partitions into the membrane on a timescale of 0.5 µs and has a cholesterol-like ordering effect on DPPC in the fluid phase. The vertical location of the PRF binding site in a bacterial ligand-gated ion channel coincides with the location found in our MD-simulations. Our results suggest that multiple physicochemical mechanisms may determine anesthetic potency of PRF, including effects on proteins that are mediated through the bilayer.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/metabolism , Anesthetics, Intravenous/pharmacology , Lipid Bilayers/metabolism , Phase Transition/drug effects , Propofol/pharmacology , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Anesthetics, Intravenous/analysis , Animals , Calorimetry, Differential Scanning , Lipid Bilayers/chemistry , Mice , Molecular Dynamics Simulation , Propofol/analysisABSTRACT
The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.