Biomedical subjects

Marjorie L Longo

Publications and source records attributed to Marjorie L Longo.

14 recordsLinked to original sources

PubMed2006

Long-term stability by lipid coating monodisperse microbubbles formed by a flow-focusing device.

In this letter, the long-term stabilization of monodisperse microbubbles produced by flow focusing is demonstrated using lipid encapsulation. Fluorescence microscopy, high-speed camera imaging, and particle size analysis were used to investigate the roles of lipid phase behavior, dissolution, Ostwald ripening, and coalescence in the stability of microbubbles formed by flow focusing. It was found that these behaviors were controlled through compositional changes with respect to lipid, emulsifier, and viscosity agents. Microbubbles coated with lipid and PEG emulsifier in a viscous solution were found to contain an extremely narrow size distribution (diameter(av) = 51 microm, standard deviation = 4 microm), which was maintained for up to several months.

Microbubbles↗

PubMed2006

Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry.

Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of approximately 100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.

Cell Membrane↗

PubMed2006

Karhunen-Loeve analysis for pattern description in phase separated lipid bilayer systems.

Karhunen-Loeve analysis, a special variant of principal component analysis, is used to describe and analyze the dynamics of self-assembled pattern formation in a mixed phospholipid bilayer. The dominant modes of the evolving heterogeneities in density and dynamics are elucidated. At low temperatures the evolution of patterns can be followed by the principal modes of the systems. We find that the higher modes only evolve after the dominant modes have been established. At high temperatures no such dominant modes are found. So, a clear descriptor of an evolving self-assembled pattern can be identified and its time evolution can be monitored. This analysis suggests, additionally, a new way of determining the equilibration decision in complex systems.

Computer Simulation↗

PubMed2006

Collapse and shedding transitions in binary lipid monolayers coating microbubbles.

We report on a fluorescence microscopy study of the monolayer collapse and shedding behavior due to shell compression during the dissolution of air-filled, lipid-coated microbubbles in degassed media. The monolayer shell was comprised of saturated diacyl phosphatidylcholine (C12:0 to C22:0) and an emulsifier, poly(ethylene glycol)-40 stearate. The morphologies of monolayer collapse structures and shed particles were monitored as a function of phospholipid acyl chain length (n) and temperature. The two components formed a single miscible phase when the phospholipid was near or above its main phase transition temperature, and collapse occurred via suboptical particles to vesicles (both were shed) and tubes as chain length increased. Conversely, two-phase coexistence was observed when the lipid was below its main phase transition temperature. For these bubbles, a transition from primary collapse to secondary collapse was observed. Primary collapse was observed as a loss of expanded phase due to vesiculation. Secondary collapse involved the rapid propagation of monolayer folds and simultaneous deformation. For very rigid monolayers, we observed substantial surface buckling with simultaneous nucleation and growth of folds. The folds merged at a single point or region, providing a conduit for the entire excess lipid to shed in a single event, and the bubble smoothed and became more spherical. These results are discussed in the context of general binary phospholipid collapse behavior, microbubble dissolution behavior, medical applications, and the dissolution behavior of natural microbubbles.

Hot Temperature↗

PubMed2006

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions.

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer-sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1, and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in supported lipid bilayers. Using "slow-cooled vesicle fusion" to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area/perimeter ratio (A(D)/P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), "quenched vesicle fusion", and find that the microstructures are remarkably similar above a cholesterol mol fraction of approximately 0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose-specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection.

Cholesterol↗

PubMed2005

Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study.

A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry; but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported lipid bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald ripening, the smaller domains in one leaflet erode away whereas the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

4-Chloro-7-nitrobenzofurazan↗

PubMed2005

Elasticity and phase behavior of DPPC membrane modulated by cholesterol, ergosterol, and ethanol.

Giant vesicles formed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and sterols (cholesterol or ergosterol) in water and water/ethanol solutions have been used to examine the effect of sterol composition and ethanol concentration on the area compressibility modulus (K(a)), overall mechanical behavior, vesicle morphology, and induction of lipid alkyl chain interdigitation. Our results from micropipette aspiration suggest that cholesterol and ergosterol impact the order and microstructure of the gel (L(beta)') phase DPPC membrane. At low concentration (10-15 mol%) these sterols disrupt the long-range lateral order and fluidize the membrane (K(a) approximately 300 mN/m). Then at 18 mol%, these sterols participate in the formation of a continuous cohesive liquid-ordered (L(o)) phase with a sterol-dependent membrane density (K(a) approximately 750 for DPPC/ergosterol and K(a) approximately 1100 mN/m for DPPC/cholesterol). Finally at approximately 40 mol% both cholesterol and ergosterol impart similar condensation to the membrane (K(a) approximately 1200 mN/m). Introduction of ethanol (5-25 vol%) results in drops in the magnitude of K(a), which can be substantial, and sometimes individual vesicles with lowered K(a) reveal two slopes of tension versus apparent area strain. We postulate that this behavior represents disruption of lipid-sterol intermolecular interactions and therefore the membrane becomes interdigitation prone. We find that for DPPC vesicles with sterol concentrations of 20-25 mol%, significantly more ethanol is required to induce interdigitation compared to pure DPPC vesicles; approximately 7 vol% more for ergosterol and approximately 10 vol% more for cholesterol. For lower sterol concentrations (10-15 mol%), interdigitation is offset, but by <5 vol%. These data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L(beta)I) phase bilayer is a major factor in the disruption of cellular activity, which typically occurs between approximately 12 and approximately 16 vol% ethanol in yeast fermentations. We summarize our findings by producing, for the first time, "elasticity/phase diagrams" over a wide range of sterol (cholesterol and ergosterol) and ethanol concentrations.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2005

Effect of microstructure on molecular oxygen permeation through condensed phospholipid monolayers.

A method is presented that allows novel measurement of the effect of microstructure on the oxygen permeability of highly condensed, polycrystalline phospholipid monolayers. Oxygen permeability of the polycrystalline shell coating a stationary microbubble is measured directly using an apposing microelectrode in the induced transfer mode and modeling oxygen flux through the shell and intervening aqueous medium. Varying cooling rate through the phospholipid main phase transition permits control of shell microstructure by manipulation of crystalline domain size and shape. Domain boundary density, defined as the ratio of the mean domain perimeter to the mean domain area, of the microbubble shell is determined by fluorescence microscopy. Oxygen permeability was shown to increase linearly with domain boundary density at a constant phospholipid acyl chain length and, accordingly, was shown to decrease exponentially with increasing chain length at a constant domain boundary density. Modification of the energy barrier theory to account for microstructural effects, in terms of the domain boundary density, provides a general equation to model passive transport through polycrystalline monolayer films. Results from this method show promise in determining the gas transport kinetics of medical microbubbles and the gas exchange characteristics of biological monolayers.

Cell Membrane Permeability↗

PubMed2004

Surface phase behavior and microstructure of lipid/PEG-emulsifier monolayer-coated microbubbles.

Langmuir trough methods and fluorescence microscopy were combined to investigate the phase behavior and microstructure of monolayer shells coating micron-scale bubbles (microbubbles) typically used in biomedical applications. The monolayer shell consisted of a homologous series of saturated acyl chain phospholipids and an emulsifier containing a single hydrophobic stearate chain and polyethylene glycol (PEG) head group. PEG-emulsifier was fully miscible with expanded phase lipids and phase separated from condensed phase lipids. Phase coexistence was observed in the form of dark condensed phase lipid domains surrounded by a sea of bright, emulsifier-rich expanded phase. A rich assortment of condensed phase area fractions and domain morphologies, including networks and other novel structures, were observed in each batch of microbubbles. Network domains were reproduced in Langmuir monolayers under conditions of heating-cooling followed by compression-expansion, as well as in microbubble shells that underwent surface flow with slight compression. Domain size decreased with increased cooling rate through the phase transition temperature, and domain branching increased with lipid acyl chain length at high cooling rates. Squeeze-out of the emulsifier at a surface pressure near 35 mN/m was indicated by a plateau in Langmuir isotherms and directly visualized with fluorescence microscopy, although collapse of the solid lipid domains occurred at much higher surface pressures. Compression of the monolayer past the PEG-emulsifier squeeze-out surface pressure resulted in a dark shell composed entirely of lipid. Under certain conditions, the PEG-emulsifier was reincorporated upon subsequent expansion. Factors that affect shell formation and evolution, as well as implications for the rational design of microbubbles in medical applications, are discussed.

Lipids↗

PubMed2004

Modeling amyloid beta-peptide insertion into lipid bilayers.

Inspired by recent suggestions that the Alzheimer's amyloid beta peptide (Abeta) can insert into cell membranes and form harmful ion channels, we model insertion of the 40- and 42-residue forms of the peptide into cell membranes using a Monte Carlo code which is specific at the amino acid level. We examine insertion of the regular Abeta peptide as well as mutants causing familial Alzheimer's disease, and find that all but one of the mutants change the insertion behavior by causing the peptide to spend more simulation steps in only one leaflet of the bilayer. We also find that Abeta42, because of the extra hydrophobic residues relative to Abeta40, is more likely to adopt this conformation than Abeta40 in both wild-type and mutant forms. We argue qualitatively why these effects happen. Here, we present our results and develop the hypothesis that this partial insertion increases the probability of harmful channel formation. This hypothesis can partly explain why these mutations are neurotoxic simply due to peptide insertion behavior. We further apply this model to various artificial Abeta mutants which have been examined experimentally, and offer testable experimental predictions contrasting the roles of aggregation and insertion with regard to toxicity of Abeta mutants. These can be used through further experiments to test our hypothesis.

Algorithms↗

PubMed2004

The influence of short-chain alcohols on interfacial tension, mechanical properties, area/molecule, and permeability of fluid lipid bilayers.

We used micropipette aspiration to directly measure the area compressibility modulus, bending modulus, lysis tension, lysis strain, and area expansion of fluid phase 1-stearoyl, 2-oleoyl phosphatidylcholine (SOPC) lipid bilayers exposed to aqueous solutions of short-chain alcohols at alcohol concentrations ranging from 0.1 to 9.8 M. The order of effectiveness in decreasing mechanical properties and increasing area per molecule was butanol>propanol>ethanol>methanol, although the lysis strain was invariant to alcohol chain-length. Quantitatively, the trend in area compressibility modulus follows Traube's rule of interfacial tension reduction, i.e., for each additional alcohol CH(2) group, the concentration required to reach the same area compressibility modulus was reduced roughly by a factor of 3. We convert our area compressibility data into interfacial tension values to: confirm that Traube's rule is followed for bilayers; show that alcohols decrease the interfacial tension of bilayer-water interfaces less effectively than oil-water interfaces; determine the partition coefficients and standard Gibbs adsorption energy per CH(2) group for adsorption of alcohol into the lipid headgroup region; and predict the increase in area per headgroup as well as the critical radius and line tension of a membrane pore for each concentration and chain-length of alcohol. The area expansion predictions were confirmed by direct measurements of the area expansion of vesicles exposed to flowing alcohol solutions. These measurements were fitted to a membrane kinetic model to find membrane permeability coefficients of short-chain alcohols. Taken together, the evidence presented here supports a view that alcohol partitioning into the bilayer headgroup region, with enhanced partitioning as the chain-length of the alcohol increases, results in chain-length-dependent interfacial tension reduction with concomitant chain-length-dependent reduction in mechanical moduli and membrane thickness.

Adsorption↗

PubMed2002

Conformational partitioning of the fusion peptide of HIV-1 gp41 and its structural analogs in bilayer membranes.

Experiments have shown that the ability of the HIV-1 virus to infect cells can be greatly diminished by deactivation of the N-terminal (fusion) peptide of its glycoprotein gp41. Deactivation can be achieved by the deletion of several amino acid residues, or replacement of a hydrophobic residue with a polar residue, to form mutant variants of the wild-type peptide. We report Monte Carlo simulation studies of a simplified peptide/membrane model, representing the interaction of an HIV-1 fusion peptide (FP) and four closely related mutagens with a lipid bilayer. In agreement with experimental results, we show that FP inserts deeply into the bilayer at approximately 40 degrees to the bilayer normal. We also show a previously unreported behavior of membrane peptides, namely their equilibrium partitioning between several distinct conformations within the bilayer. We quantify this partitioning behavior and characterize each conformation in terms of its geometry, energy, and entropy. The diminished ability of FP mutagens to hemolyse and aggregate red blood cells due to their partitioning into unfavorable conformations, is also discussed. Our analysis supports a negative curvature mechanism for red blood cell hemolysis by FP. We also suggest that the small repulsive forces between surface-adsorbed peptides in opposing membrane surfaces may block aggregation.

Amino Acid Sequence↗

PubMed2002

Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach.

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.

Cell Membrane↗

PubMed2002

A Monte Carlo study of peptide insertion into lipid bilayers: equilibrium conformations and insertion mechanisms.

The membrane insertion behavior of two peptides, Magainin2 and M2 delta, was investigated by applying the Monte Carlo simulation technique to a theoretical model. The model included many novel aspects, such as a new semi-empirical lipid bilayer model and a new set of semi-empirical transfer energies, which reproduced the experimental insertion behavior of Magainin2 and M2 delta without parameter fitting. Additionally, we have taken into account diminished internal (intramolecular) hydrogen bonding at the N- and C-termini of helical peptides. All simulations were carried out at 305 K, above the membrane thermal phase transition temperature, and at pH 7.0. The peptide equilibrium conformations are discussed for a range of bilayers with tail polarities varying from octanol-like to alkane-like. Probability distributions of the individual amino-acid-residue positions show the dynamic nature of these equilibrium conformations. Two different insertion mechanisms for M2 delta, and a translocation mechanism for Magainin2, are described. A study of the effect of bilayer thickness on M2 delta insertion suggests a critical thickness above which insertion is unfavorable. Additionally, we did not need to use an orientational potential or array of hard cylinders to persuade M2 delta to insert perpendicular to the membrane surface. Instead, we found that diminished internal hydrogen bonding in the helical conformation anchored the termini in the headgroups and resulted in a nearly perpendicular orientation.

Amino Acid Sequence↗