Dipalmitoylphosphatidycholine Research Articles

Cholesterol Translocation in a Phospholipid Membrane

Cholesterol (CHOL) molecules play a key role in modulating the rigidity of cell membranes and controlling intracellular transport and signal transduction. Using an all-atom molecular dynamics approach, we study the process of CHOL interleaflet transport (flip-flop) in a dipalmitoylphosphatidycholine (DPPC)-CHOL bilayer over a time period of 15 μs. We investigate the effect of the flip-flop process on mechanical stress across the bilayer and the role of CHOL in inducing molecular order in bilayer leaflets. The simulations are carried out at physiologically relevant CHOL concentration (30%), temperature (323 K), and pressure (1 bar). CHOL flip-flop events are observed with a rate constant of 3 × 104 s−1. Once a flip-flop event is triggered, a CHOL molecule takes an average of 73 nanoseconds to migrate from one bilayer leaflet to the other.

Cholesterol Flip-Flop Dynamics in a Phospholipid Bilayer: All Atom Molecular Dynamics Simulations

Presence of cholesterol (CHOL) molecules in cell membranes plays a key role in the structural properties of cell membranes. Also the dynamics of CHOL molecules in the cell membrane is an important biological process. Using all-atom molecular dynamics (MD) and parallel replica approach, we study the mechanism of CHOL flip-flop in a dipalmitoylphosphatidycholine (DPPC)-CHOL bilayer. The simulations are carried out at physiologically relevant CHOL concentration (30%), temperature 323 K and pressure 1 bar.

Small Interfering RNA Transfection Barriers Across a Lipid Membrane

Small interfering RNA (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference (RNAi) mechanism. We examine the effect of a bare siRNA on the structure and mechanical properties of a dipalmitoylphosphatidycholine (DPPC) bilayer. Our investigations are based on all-atom molecular dynamics (MD) simulations reaching 0.5 microseconds for systems containing 733,000 atoms. These simulations reveal that the bare siRNA transforms the liquid disordered (Lα) phase of the lipid bilayer at temperature T = 323 K into a phase of frustrated lipid gel domains which consist of lipid molecules with interdigitated and non-interdigitated hydrocarbon chains. We have also performed all-atom MD simulations to examine the interaction between the DPPC bilayer and a complex consisting of a siRNA with an oleic acid (OA) molecule connected to each 3′ end. In this case, the bilayer transforms into the liquid-ordered(Lo) phase. Steered MD simulations are also performed to study the transfection of a bare siRNA and siRNA/OA complex across the DPPC bilayer. Results for the transfection force and stress profiles across the lipid membrane will be presented.View Large Image | View Hi-Res Image | Download PowerPoint Slide

NUMERICAL SIMULATION OF NANOINDENTATION AND PATCH CLAMP EXPERIMENTS ON MECHANOSENSITIVE CHANNELS OF LARGE CONDUCTANCE IN ESCHERICHIA COLI.

A hierarchical simulation framework that integrates information from all-atom simulations into a finite element model at the continuum level is established to study the mechanical response of a mechanosensitive channel of large conductance (MscL) in bacteria Escherichia Coli (E.coli) embedded in a vesicle formed by the dipalmitoylphosphatidycholine (DPPC) lipid bilayer. Sufficient structural details of the protein are built into the continuum model, with key parameters and material properties derived from molecular mechanics simulations. The multi-scale framework is used to analyze the gating of MscL when the lipid vesicle is subjective to nanoindentation and patch clamp experiments, and the detailed structural transitions of the protein are obtained explicitly as a function of external load; it is currently impossible to derive such information based solely on all-atom simulations. The gating pathways of E.coli-MscL qualitatively agree with results from previous patch clamp experiments. The gating mechanisms under complex indentation-induced deformation are also predicted. This versatile hierarchical multi-scale framework may be further extended to study the mechanical behaviors of cells and biomolecules, as well as to guide and stimulate biomechanics experiments.

‘Free’ surfactant in gastric aspirates and bronchoalveolar lavage in children with and without reflux oesophagitis

Dipalmitoylphosphatidycholine (DPPC) is the characteristic and main constituent of surfactant. Adsorption of surfactant to epithelial surfaces may be important in the masking of receptors. The aims of the study were to (i) compare the quantity of free DPPC in the airways and gastric aspirates of children with gastroesophageal reflux disease (GORD) to those without and (ii) describe the association between free DPPC levels with airway cellular profile and capsaicin cough sensitivity. Children aged <14 years were defined as 'coughers' if a history of cough in association with their GORD symptoms was elicited before gastric aspirates and nonbronchoscopic bronchoalveolar lavage (BAL) were obtained during elective flexible upper gastrointestinal endoscopy. GORD was defined as histological presence of reflux oesophagitis. Spirometry and capsaicin cough-sensitivity test was carried out in children aged >6 years before the endoscopy. Median age of the 68 children was 9 years (interquartile range (IQR) 7.2). Median DPPC level in BAL of children with cough (72.7 microg/mL) was similar to noncoughers (88.5). There was also no significant difference in DPPC levels in both BAL and gastric aspirates of children classified according to presence of GORD. There was no correlation between DPPC levels and cellular counts or capsaicin cough-sensitivity outcome measures. We conclude that free DPPC levels in the airways and gastric aspirate is not influenced by presence of cough or GORD defined by histological presence of reflux oesophagitis. Whether quantification of adsorbed surfactant differs in these groups remain unknown. Free DPPC is unlikely to have a role in masking of airway receptors.

The Relationship Between Inflammation and Dipalmitoyl Phosphatidycholine in Induced Sputum of Children with Asthma

Background. Animal studies have shown elevated surfactant production in response to lung injury. In human airways, the contribution of surfactant to the airway epithelial barrier and importance of eosinophilic inflammation is increasingly appreciated. The relationship between blood and sputum inflammatory indices of childhood asthma to surfactant levels is unknown. In this study we hypothesized that the degree of inflammation influences the level of dipalmitoyl phosphatidycholine (DPPC) in airways of children with asthma. Methods. Sixteen children with asthma (ages 5.5–16 years) underwent venipuncture, skin prick test, spirometry, hypertonic saline challenge, and induced sputum during a nonacute phase. Sputum (sp) and blood (se) markers of inflammation (eosinophils, neutrophils, eosinophilic cationic protein [ECP]), were related to sputum DPPC levels and several markers of asthma severity (airway hyperresponsiveness, quality of life, FEV1). Results. On multiple regression, sp-DPPC significantly correlated to sp-ECP (r = 0.53, P = 0.0048). Se-ECP, se-Eo, sp-eosinophils, sp-neutrophils, se-neutrophils, and inhaled steroids dose did not significantly influence sp-DPPC. Exposure to smoke did not influence inflammatory markers. FEV1 and quality of life data did not relate to any blood or sputum variable. A significant association between AHR and se-eosinophils, but not between AHR and se-ECP, sp-eosinophils, or sp-ECP was found. Conclusion. Elevated DPPC levels occur in the presence of chronic eosinophilic inflammation in airways of children with stable asthma. Whether this represents an inherent lung mechanism for epithelial protection remains to be elucidated.

Interaction of GM 1 glycolipid in phospholipid monolayers with wheat germ agglutinin: effect of phospholipidic environment and subphase

Mixed monolayers of GM 1 glycolipid and stearoyl-oleoyl-phosphatidylcholine (SOPC) or dipalmitoyl-phosphatidycholine (DPPC) phospholipids were studied by surface pressure measurements. The effects induced by GM 1 on the mean molecular areas of mixtures and DPPC phase transition were followed for GM 1 concentrations ranging from 1 to 20 mol.%. Under our experimental conditions, one main parameter influencing the behavior of phospholipid–GM 1 monolayers is the ionic strength of the subphase. Mixed monolayers are in a more expanded state on buffer than on pure water. This could be due to a change of GM 1 orientation at the interface. The interaction of wheat germ agglutinin (WGA), a lectin recognizing specifically GM 1, with these monolayers was quantified in terms of the Gibbs equation. Specific WGA–GM 1 interactions are clearly reduced in the presence of DPPC as compared with SOPC, probably because of the higher packing density of these monolayers. Phospholipid–GM 1 monolayers could also undergo some rearrangements induced by WGA binding.

Characterization of the lipid-binding properties and lipoprotein lipase inhibition of a novel apolipoprotein C-III variant Ala23Thr

We have identified a G-to-A transition in exon 3 of the APOC3 gene resulting in a novel Ala23Thr apolipoprotein (apo) C-III variant, associated with apoC-III deficiency in three unrelated Yucatan Indians. The Ala23Thr substitution modifies the hydrophobic/hydrophilic repartition of the helical N-terminal peptide and hence could disturb the lipid association. In vitro expression in Escherichia coli of wild-type and mutant apoC-III enabled the characterization of the variant. Compared with wild-type apoC-III-Ala23, the mutant apoC-III-Thr23 showed reduced affinity for dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles with higher amounts of free apoC-III. Displacement of apoE from discoidal apoE:dipalmitoylphosphatidycholine (DPPC) complex by apoC-III-Thr23 was comparable to wild type but the less efficient binding of the apoC-III-Thr23 to the discoidal complex resulted in a higher apoE/apoC-III (mol/mol) ratio (34%) than with wild-type/apoE:DPPC mixtures. The inhibition of lipoprotein lipase (LPL) by apoC-III-Thr23 was comparable to that of wild type, and therefore effects on LPL activity could not explain the lower triglyceride (Tg) levels in Thr-23 carriers. Thus, these in vitro results suggest that in vivo the less efficient lipid binding of apoC-III-Thr23 might lead to a faster catabolism of free apoC-III, reflected in the reduced plasma apoC-III levels identified in Thr-23 carriers, and poorer competition with apoE, which might enhance clearance of Tg-rich lipoproteins and lower plasma Tg levels seen in Thr-23 carriers.—Liu, H., C. Labeur, C-F. Xu, R. Ferrell, L. Lins, R. Brasseur, M. Rosseneu, K. M. Weiss, S. E. Humphries, and P. J. Talmud. Characterization of the lipid-binding properties and lipoprotein lipase inhibition of a novel apolipoprotein C-III variant Ala23Thr. J. Lipid Res. 2000. 41: 1760–1771.

The structure of mixed monolayer films of DPPC and hexadecanol

Although dipalmitoyl phosphatidycholine (DPPC) is the primary surface active component of natural lung surfactant (NLS), under physiological conditions it does not adsorb rapidly to the interface or respread efficiently or rapidly after compression beyond film collapse. Other materials must be present to enhance these functions and one synthetic exogenous surfactant used in replacement therapy mixes DPPC with hexadecanol and tyloxapol. In the present study the interactions of DPPC and hexadecanol in spread monolayers have been investigated by film balance measurements, and Brewster angle microscopy. Two phases can be observed when the surface pressure is low and the mole fraction of DPPC is high: a condensed phase of hexadecanol with some DPPC and an expanded phase. Increasing surface pressure generates a second condensed phase that is DPPC rich. On further compression the expanded phase disappears and the two condensed phases merge, but miscibility rules indicate that the expanded phases do not mix even though there is no evidence for two phases in the experimental data.

Susceptibility of exogenous surfactant to phospholipase Α2 degradation

The inhibition of surfactant biophysical activity in vivo is potentially mediated by many factors, including serum proteins, particularly enzymatic proteins such as phospholipases. In the present study, we investigated the susceptibility of the phosphatidylcholine component of two exogenous surfactants, Exosurf and Survanta, to secretory-type phospholipase A2 (PLA2) deacylation in vitro. Lyophilized Exosurf and Survanta preparations were incubated at 37 degrees C for 120 min in the presence of bovine pancreatic PLA2, and the production of lysophosphatidylcholine was determined as a measure of the magnitude of phosphatidylcholine deacylation. The phosphatidylcholine component of Survanta was readily deacylated by PLA2, whereas the dipalmitoylphosphatidycholine (DPPC) component of Exosurf was resistant over the entire duration of the assay. To further evaluate this observed resistance the individual and combined effects of tyloxapol and hexadecanol, components of Exosurf, upon PLA2 deacylation of Survanta and DPPC were investigated. In both Survanta and DPPC preparations, PLA2-mediated deacylation was significantly inhibited in the presence of tyloxapol. We conclude that the presence of tyloxapol in the Exosurf preparation inhibits secretory type PLA2 mediated DPPC deacylation. This unique feature of Exosurf may be of clinical significance when this preparation is utilized in the treatment of surfactant-deficient infants.

Effect of lanthanide ions on the phase behavior of dipalmitoylphosphatidylcholine multilamellar liposomes

The effect of lanthanide ions (Ln 3+) and their coordination compounds of diethylenetriamine pentaacetic acid (DTPA) on the phase behavior of dipalmitoylphosphatidycholine (DPPC) multilamellar liposomes has been studied by differential scanning calorimetry (DSC), Raman spectroscopy, and freeze-fracture electron microscopic techniques. The displacement of Ca 2+ binding on DPPC liposomes by lanthanide ions was also studied. The results show that the binding degree of four kinds of chloride salts with DPPC liposomes is: YbCl 3 > GdCl 3 > LaCl 3 > CaCl 3. Lanthanide ions increase the phase transition temperature of DPPC liposomes and decrease the membrane fluidity. Freeze-fracture electron microscopic results show that La 3+ enhances the order of DPPC membrane. The effect of coordination compounds of lanthanides with DTPA on the phase behavior of DPPC liposomes is smaller than that of their chlorides. La 3+, Gd 3+, and Yb 3+, can displace Ca 2+ binding on DPPC liposomes, but there coordination compounds of DTPA can hardly displace Ca 2+. Raman spectroscopic results show that a very slight effect in lateral packing order of DPPC liposomes was observed at various concentrations of lanthanides.

Effect of Drug substances on the microviscosity of lipid bilayer of liposomal membrane

The microviscosities of the lipid bilayers of liposomal membranes of phospholipids were measured by the intermolecular excimer, formation method employing pyrene as a fluorescence probe, and the effects ofn-alkanols and other local anesthetics on the microviscosity were investigated. The results showed that then-alkanols and the other local anesthetics effectively lowered the microviscosity of the lipid bilayer of the dipalmitoyl phosphatidycholine liposomal membrane in proportion to the concentration of the additives. Moreover, there was a fairly good correlation between the local anesthetic activities and the microviscosity-lowering activities of these drugs. This results suggests that the nerveblocking activity of local anesthetics might have some relation with their activity fluidizing the lipid bilayer of biomembrane.

Enhanced luminol-dependent chemiluminescence of stimulated rat alveolar macrophages by pretreatment with alveolar lining material.

Previous reports indicate that the in vitro bactericidal activity of rat alveolar macrophages (AM) is dependent on the lipid fraction (ALM-L) of the alveolar lining material (ALM). The present study demonstrates that luminol-dependent chemiluminescence of stimulated rat AM is increased when rat AM are preincubated in the ALM or in the ALM-L. Evidence is presented that oxidation of the unsaturated lipids is responsible for the increase. In addition to pretreatment with the ALM, cells were also pretreated in commercial preparations of several lipids found to be present in the ALM. Preincubation in these lipids produced a significant increase in the luminol-dependent chemiluminescence response. However, when a saturated lipid, dipalmitoyl phosphatidylcholine, was used no increase was found. Pretreatment in ALM did not increase the nitro blue tetrazolium dye reduction by the AM, nor was the phagocytosis of latex beads by the AM altered by the addition of the ALM.

Influence of membrane viscosity on the lateral and transverse mobility of carboxylic ionophores

The rate of 45Ca or 22Na exchange-diffusion in multilamellar liposomes formed of dipalmitoyl-phosphatidycholine (DPPC) and cholesterol and containing the ionophore A23187 or Br-X537A was dramatically increased when the temperature and, hence, fluidity of the lipid bilayer were increased. In the case of 45Ca transport, i.e. when each Ca 2+ ion binds to two molecules of ionophore, the relative increment in transport velocity in response to a given increase in temperature or fluidity was much more marked in the high range of temperature (30–40°C) than in the low range of temperature (22–28°C). In the case of 22Na transport, however, i.e. when each Na + ion binds to only one ionophoretic molecule, the temperature-dependency of the transport process followed a single pattern throughout the entire range of temperature. In the latter case, the slope of the temperature-dependent line was the same as that seen for 45Ca transport by the same ionophore at high temperatures. A decrease in the ionophore content of the liposomes shifted to a higher temperature the transition point between the flat and steep lines characterizing the temperature dependency of 45Ca transport. It is concluded that the membrane viscosity affects both the lateral mobility of the ionophoretic molecules and the transverse mobility of the cation-ionophore complex.

Effect of a phase transition on the binding of 1-anilino-8-naphthalenesulfonate to phospholipid membranes

The binding of 1-anilino-8-naphthalenesulfonate (ANS) to dipalmitoyl-phosphatidycholine (DPPC)-sonicated vesicles was measured by a fluorimetric method in the vicinity of the gel-to-liquid crystalline phase transition temperature (Tm). A similar measurement was performed on large multimellar DPPC vesicles through equilibrium dialysis. Both measurements demonstrated anomalous dye binding in the temperature region of the Tm and slightly above (prefreezing region). The amount of ANS bound at this temperature region was in excess of what would be expected based on extrapolation of the high temperature binding data; just below the Tm, the amount of bound dye decreased abruptly. The fluorimetric studies on vesicles also indicated that inner monolayer binding of ANS was markedly inhibited below the Tm. The possibility that the increase in bound dye in the prefreezing region was caused by enhanced lateral compressibility, density fluctuations, or additional binding sites at the boundary of transient gel-like clusters is discussed and the general topic of anomalous increases in various membrane processes in the vicinity of a phase transition is briefly reviewed.

Measurement and interpretation of fluorescence polarisations in phospholipid dispersions

An instrument that measures the temperature dependence of fluorescence polarisation and intensity directly and continuously is described. The behaviour of four fluorescent probes bound to a number of well characterised model systems was then examined. The motional properties of the probes were determined from the polarisation and intensity data and were found to be sensitive to the crystallineliquid crystalline phase transitions in phospholipid vesicles of dimyristoyl and dipalmitoyl phosphatidylcholine. Binary mixture of dilauroyl and dipalmitoyl phosphatidycholine show lateral phase separation and in this system the probes partition preferentially into the more ‘fluid’ phase. In systems that have been reported to contain ‘short range order’ or ‘liquid clustering’, such as dioleoyl phosphatidylcholine and liquid paraffin, the motion of the probes was found to have anomalous Arrhenius behaviour consistent with the idea that homogeneous phases were not being sampled. The significance of these findings for the interpretation of the behaviour of fluorescent probes bound to natural membranes is discussed.