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Biomedical subjects

R C MacDonald

Publications and source records attributed to R C MacDonald.

At least 55 records · Page 3Linked to original sources
PubMed1989

Fusion of synaptic vesicle membranes with planar bilayer membranes.

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.

Animals↗
PubMed1988

Membrane surface pressure can account for differential activities of membrane-penetrating molecules.

It is a common observation that cis-unsaturated and branched chain fatty acids, which are usually liquid, affect membrane function differently from saturated and trans-unsaturated fatty acids, which are usually solid. We also found that the former are much more potent than the latter in inhibiting viral hemolytic activity. A search for the origin of this difference revealed a correlation between inhibition and equilibrium surface pressure (the surface pressure at the air/water interface of a solution of the substance in question). Using a simple but rigorous thermodynamic analysis, we show that penetration of a lipid bilayer is correlated with equilibrium surface pressure of the penetrating molecule. We therefore conclude that an important reason for the difference in effects of liquid and solid fatty acids on membranes is the greater penetrability of the former relative to the latter. We suggest that attributing such effects to fluidity changes in the membrane should await demonstration of actual intramonolayer residence of the fatty acid in the membrane. The thermodynamic analysis is readily generalized and, in the absence of specific interactions between penetration and bilayer molecules, provides a convenient method for predicting membrane penetration by virtually any type of exogenous molecule.

Fatty Acids, Nonesterified↗
PubMed1987

Determination of reflection coefficients of liposomes for some non-electrolytes by osmotic pressure measurement.

The reflection coefficients of bilayer lipid vesicles (liposomes) of various compositions have been determined for a number of non-electrolytes. The solutes were the same and the method of measurement was essentially the same as those which have been used to estimate an equivalent pore radius for erythrocytes. The method involves matching the osmotic pressure of solutions of a permeant test solute with that of a known inpermeant solute. Reflection coefficients for cholesterol-containing liposomes and those of erythrocytes are, when account is taken of those solutes known to permeate the erythrocyte by specialized pathways, not greatly different. Lipid bilayers can thus account for most of the permeability characteristics of the cell originally interpreted as due to aqueous pores. Reflection coefficients are significantly higher for egg phosphatidylcholine membranes that contain cholesterol than those which do not. There is a strong correlation between relative permeabilities derived from reflection coefficients and oil-water partition coefficients. There is also good agreement between these permeabilities and permeabilities measured by others, which exhibit an inverse dependence on molecular size. It is suggested that this tendency of membranes to pass small molecules more readily than large molecules, other properties being equal, is a consequence of the surface pressure of the constituent monolayers of the membrane.

Diffusion↗
PubMed1987

Lipid monolayer states and their relationships to bilayers.

Uncommon methods of formation and analysis of lipid monolayers have enabled the recognition of several monolayer states and the identification of that in which molecular organization corresponds closely to that of the bilayer. Monolayers were formed by continuously adding a solution of phospholipid [dimyristoyl phosphatidylcholine in hexane/ethanol, 9:1 (vol/vol)] to the air/water interface of a constant-area trough. This procedure generates unconventional surface pressure (pi)-surface concentration (gamma) isotherms, which for liquid-crystalline monolayers consist of straight lines with three prominent intersections, two of which are not apparent in conventional pi-A isotherms. The regions of linear change of pi are explicable in terms of the area dependence of alkyl chain entropy. The two breaks at lower pi delimit states in which both chains lie parallel to the surface. The third occurs at collapse, which corresponds to a true equilibrium for unstressed liposomes. Mechanical and thermodynamic properties of bilayers, particularly phase-transition parameters, correspond closely to those of monolayers with which they are in equilibrium.

Cholesterol↗
PubMed1984

Inhibition of sendai virus-induced hemolysis by long chain fatty acids.

A number of fatty acids were found to inhibit Sendai virus-induced hemolysis. cis-Unsaturated fatty acids such as oleate, as well as the methyl-branched isostearate, completely inhibited viral hemolysis at concentrations as low as 5-10 micrograms/ml, whereas the saturated, normal acids such as palmitate and stearate were comparably inhibitory only at 2-5 times those concentrations. trans-Unsaturated acids, as well as several other amphiphilic compounds, were either not or only weakly inhibitory. In contrast to their disparate effects on viral hemolysis, cis- and trans-unsaturated acids lysed erythrocytes in the same concentration range, which is several times higher than that at which the former compounds inhibited viral hemolysis. The mechanism of inhibition of viral hemolysis by isostearate involves the inactivation of viral hemolytic activity per se, since isostearate neither inhibited viral hemagglutination nor rendered erythrocytes significantly less susceptible to hemolysis. Furthermore, the concentration dependence of hemolysis inhibition by isostearate was biphasic, increasing sharply at the critical micelle concentration from a linear relationship below that concentration. Finally, an inhibitory concentration of isostearate was well below that at which amphiphiles dissolved membranes and did not dissolve Sendai virus, as shown by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was concluded that low concentrations of fatty acids--particularly cis-unsaturated or fluid-phase types--could block the fusion, as opposed to agglutination, step of viral hemolysis by perturbing hydrophobic regions of the Sendai virus membrane.

Detergents↗
PubMed1983

Lipid mixing during freeze-thawing of liposomal membranes as monitored by fluorescence energy transfer.

A new pair of fluorescence-energy-transferring probes, dansylphosphatidylethanolamine and dioctadecylindocarbocyanine, were incorporated separately into phospholipid vesicles to monitor intervesicle lipid mixing under various conditions. The transfer efficiencies of mixtures of sonicated vesicles labeled with 2 wt% donor dansylphosphatidylethanolamine (DnsPE) or with 1 wt% acceptor dioctadecylindocarbocyanine (DiI-C18) were negligible, but increased to about 25% after the vesicles had been frozen in a solid CO2/ethanol bath, thawed and diluted. The freeze-thaw-induced mixing of lipids between vesicles, signified by energy transfer, was dependent on lipid concentration and was promoted by 0.5-1.5 M KCl, 0.5 M potassium trichloroacetate and 5 mM sodium acetate (pH 4) and inhibited by 0.5 M LiCl, 0.5 M glycerol, 0.5 M sucrose, 0.15 M KCl and 0.15-1.5 M NaCl. These results support and complement previously reported measurements of the trapped volumes, turbidities and population size distributions of similarly treated liposomes. Comparison of the responses of paucilamellar vesicles with those of multilamellar vesicles suggests that lipid mixing during freeze-thawing can occur either during interaction of the outermost bilayers of vesicles or during interaction of all bilayers, possibly as a result of breakdown and reformation of bilayer structure.

Dansyl Compounds↗
PubMed1983

Characterization of a fluorescence assay to monitor changes in the aqueous volume of lipid vesicles.

The fluorescent compound, 4',5'-bis[N,N-bis(carboxymethyl)aminomethyl] fluorescein (calcein) has been characterized for use in lipid vesicle studies. Particularly useful is its reaction with Co2+, which results in fluorescence quenching. This is accompanied by about a 10-nm blue shift in the uv absorbance bands and a small reduction in the visible absorbance band. For vesicle studies, Co2+ may be combined with citrate, which does not significantly hinder calcein quenching by Co2+. It does augment the absorbance of the metal ion. No significant interaction of citrate X Co2+ with phosphatidylserine vesicles was observed. Zn2+ is capable of displacing Co2+ and restoring calcein fluorescence. Fluorescence quenching due to formation of the calcein X Co2+ complex can also be reversed with EDTA. Thus, calcein is the basis of some simple reactions which can be used to assay changes in the aqueous volume of lipid vesicles.

Buffers↗
PubMed1983

Detergent effects on enzyme activity and solubilization of lipid bilayer membranes.

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturing them. Various concentrations each of detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents most effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.

Alkaline Phosphatase↗
PubMed1983

Solubilization of phospholipids by chaotropic ion solutions.

Phosphatidylcholine (PC) dissolves in solutions of concentrated neutral trichloroacetate and tribromoacetate, anions known to be extremely chaotropic. Solubilization of egg yolk PC occurs in Na-trichloroacetate between 2 and 3 M and in Na-tribromoacetate between 1.5 and 2 M. The resulting optically clear solutions were found by gel exclusion chromatography to consist of micelles containing of the order of 10(2) lipid molecules which, according to 31P NMR, are undergoing rapid, isotropic motion. Dipalmitoyl-PC is not solubilized below its normal phase transition temperature, although it remains in solution if first dissolved above that temperature; transition temperature and enthalpy change are both drastically depressed in 3 M Na-trichloroacetate. Na-trichloroacetate (3 M) also dissolves sphingomyelin but not phosphatidylethanolamine, acidic phospholipids, or egg PC-cholesterol mixtures of more than 33% cholesterol. Destabilization of the lamellar phase of egg PC relative to the micellar phase is promoted by trichloroacetate binding to and intercalating between the lipid molecules. Trichloroacetate was found to bind to egg PC liposomes with an affinity constant of about 1 M-1, so that nearly 1:1 association between egg PC and trichloroacetate is predicted at the concentration at which solubilization occurs. A surface area dilation by an amount (50+%) consistent with formation of micelles was confirmed by surface tension isotherms of egg PC on 3 M Na-trichloroacetate. The chaotropic effect may synergize binding by reducing the energy cost of the exposure of hydrophobic portions of lipid that is necessitated by the small radius of curvature of micelles. Several chaotropic agents less potent than trichloroacetate and tribromoacetate did not solubilize egg PC.

Calorimetry, Differential Scanning↗
PubMed1983

Sendai virus-mediated lysis of liposomes requires cholesterol.

Vesicles were constituted with glycophorin, the Sendai virus receptor of human erythrocytes, and loaded with calcein, a polar derivative of fluorescein, at self-quenching concentrations. On exposure to Sendai virus and mild hypo-osmotic stress, vesicles of the appropriate composition released a significant portion of their internal contents, as indicated by an increase in calcein fluorescence. Susceptible liposomes were not induced to leak by heat-inactivated virus or by trypsin-treated virus. The response of the vesicles to virus attachment is thus analogous to virus-induced hemolysis and presumably involves fusion of the vesicle and virus membranes. In addition to glycophorin and phosphatidylcholine, cholesterol was absolutely required for the lytic response to the virus. The need for cholesterol was not attributable to inactivation of the virus by liposomes without cholesterol. The presence of gangliosides increased the encapsulated volume of the liposomes, but gangliosides did not effectively substitute for glycophorin. Thin-layer chromatography of lipid extracted from incubated virus and liposomes containing a small amount of a fluorescent phosphatidylcholine indicated that phosphatidylcholine in the vesicle is not chemically altered by functional interaction with the virus.

Cell Aggregation↗
PubMed1982

A fluorescence assay to monitor vesicle fusion and lysis.

An assay based on the fluorescent compound 2',7'-([bis(carboxymethyl)amino]methyl) fluorescein (calcein) has been developed to investigate vesicle fusion and lysis. The assay involves encapsulating the nonfluorescent Co2+ complex of calcein in one set of vesicles and EDTA in a second set. If fusion occurs, EDTA chelates Co2+, releasing calcein which may be assayed by means of its intense fluorescence. Leakage of either component of the reaction can be directly quantitated by titrating the external medium with the other component. This assay was used to investigate the effects of calcium ion on small phosphatidylserine vesicles, a system reported to undergo fusion. It revealed immediate and extensive leakage of the encapsulated contents; membrane fusion must, therefore, be simultaneously with or subsequent to, loss of vesicle integrity. This assay is convenient and flexible; it should be useful in a variety of investigations of membrane-bounded compartments.

Animals↗
PubMed1982

Preparation of giant liposomes.

A method is described for the preparation of giant unilamellar lipid vesicles that are stable in electrolyte solution. In general, it involves dialysis of lipid and indifferent solute in a water-miscible organic solvent against an aqueous buffer. During dialysis the concentration of organic solvent decreases so that vesicles form under conditions where their internal contents are continuously hyperosmotic. Interlamellar attractive forces are neutralized, even between bilayer membranes with no net charge, and giant vesicles are generated in large numbers. The population is heterogeneous but most large vesicles have diameters between 10 and 100 micron. The method is simple. One procedure involves dialysis for a day or more of a methanol solution of phosphatidylcholine, supersaturated with methylglucoside, against an aqueous phase containing up to 1 M univalent electrolyte. The procedure is effective over a wide range of temperature and pH.

Dialysis↗
PubMed1980

The organization of n-alkanes in lipid bilayers.

The interaction of n-alkanes (C6--C16) with phosphatidylcholine has been studied by the combined use of differential scanning calorimetry, X-ray diffraction and monolayer techniques. It has been found that the thermal properties and ultrastructure of lipid-alkane vesicles are strongly dependent on the length of the n-alkanes. Long alkanes, such as tetradecane and hexadecane, increase the transition temperature of dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, while the X-ray data indicate that these long alkanes align parallel to the lipid acyl chains. In contrast, shorter alkanes, such as hexane and octane, decrease and broaden the thermal transition and electron density profiles show that these alkanes increase bilayer width by partitioning between the apposing monolayers of the bilayer. For lipids in the gel and liquid crystalline states, the short alkanes form an alkane region in the geometric center of the bilayer.

Alkanes↗
PubMed1979

Effect of benzyl alcohol on lipid bilayers. A comparisons of bilayer systems.

The effect of the small anesthetic molecule, benzyl alcohol, on the structure of various bilayer system has been studied by optical, electrical, and x-ray diffraction techniques. We find that the modifications in bilayer thickness caused by benzyl alcohol differ dramatically for planar (or black lipid) bilayers containing solvent, planar bilayers containing little or no solvent, and vesicular bilayers. Benzyl alcohol increases the thickness of planar bilayers containing n-alkane solvents, yet decreases the thickness of "solvent-free" planar bilayers. The effect of benzyl alcohol on vesicular bilayers below the phase transition temperature also depends on whether solvent is present in the bilayers. Without solvent, gel-state bilayers are reduced in thickness by benzyl alcohol, whereas in the presence of solvent, the thickness is unchanged. Above the phase transition temperature, benzyl alcohol has no measurable effect on vesicular bilayer thickness, whether solvent is present or not. These results indicate that different model membrane systems respond quite differently to a particular anesthetic.

Benzyl Alcohol↗
PubMed1977

Dry column chromatography of phospholipids.

Separation of common phospholipids can be effected by dry column chromatography on silica gel. The method involves packing the column with dry gel and developing it in solvent mixtures used for thin-layer chromatography of the same lipids. Solvent is allowed to migrate only to the end of the column; access to the bands of separated material is obtained by using columns with a removable glass front. RF values of lipids on development columns and those on thin-layer plates are nearly identical when the column is packed with thin-layer chromatography gel. Such columns, however, develop very slowly. Columns packed with fine silica gel designed for elution column chromatography develop very rapidly and yield separations that are still quite comparable to those obtainable from thin-layer plates. Such columns are convenient for the purification of phospholipids in amounts of 10 mg to about 10 g. Column design and construction are described in detail.

Chromatography, Gel↗