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R C MacDonald

Publications and source records attributed to R C MacDonald.

At least 37 records · Page 2Linked to original sources
PubMed1996

Delivery of membrane-impermeant fluorescent probes into living neural cell populations by lipotransfer.

Use of fluorescent probes to monitor f-actin in living cells typically relies on difficult microinjection procedures. The current work has developed cationic lipotransfer of membrane-impermeant probes as an alternative to microinjection. BODIPY FL-phallacidin, a fluorescent f-actin probe, was packaged into 40-50 nm cationic liposomes. Packaging, verified by gel filtration, enabled delivery of the probe into living nerve cells and provided an image of f-actin that was identical to that seen in fixed, permeabilized cells. Phallacidin alone did not enter living cells, nor was its uptake stimulated by the presence of empty liposomes. All predicted f-actin structures were fluorescent in living cells, indicating a high efficacy of delivery. Cationic lipotransfer of fluorescent probes was rapid, not disruptive to cells, and delivered a probe en masse to a large sample population. Lipotransfer appears to be a promising alternative to microinjection for introducing membrane-impermeant probes and reagents into living cells.

Actins↗
PubMed1996

A tethered adhesive particle model of two-dimensional elasticity and its application to the erythrocyte membrane.

A new model of two-dimensional elasticity with application to the erythrocyte membrane is proposed. The system consists of a planar array of self-adhesive particles attached to nearest neighbors with flexible tethers. Stretching from the equilibrium dimension is resisted because force is required to dissociate the particle clusters and to decrease the distribution entropy. Release of the external force is accompanied by a contraction as thermal diffusion randomizes the particles and allows interparticle attachments to form again. Analysis of membrane thermodynamics and mechanics under the two-state particle assumption results in a shear softening stress-strain relation. The shear modulus is found proportional to the square root of the surface density of particles, the interparticle adhesive energy, and is inversely proportional to the tether length. Applied to the erythrocyte membrane under the assumption that band 3 tetramer represents the particle and spectrin the tether, the shear modulus predicted corresponds to the measured value when the interparticle adhesive energy is approximately 4.0-5.9 kT, where kT is the Boltzmann constant multiplied by the temperature. This model suggests a mechanism wherein erythrocyte membrane deformability depends on integral protein homomultimeric interactions and can be modulated from the external surface.

Adhesiveness↗
PubMed1995

Neurotransmitter release evoked by nerve impulses without Ca2+ entry through Ca2+ channels in frog motor nerve endings.

1. The requirement for extracellular Ca2+ in the process of evoked acetylcholine (ACh) release by nerve impulses was tested at endplates in frog skeletal muscle. Ca(2+)-containing lipid vesicles (Ca2+ liposomes) were used to elevate cytoplasmic Ca2+ concentrations under conditions in which Ca2+ entry from the extracellular fluid was prevented. 2. In an extracellular solution containing no added Ca2+ and 1 mM Mg2+ ('Ca(2+)-free' solution), Ca2+ liposomes promoted the synchronous release of ACh quanta, reflected electrophysiologically as endplate potentials (EPPs), in response to temporally isolated nerve impulses. 3. Motor nerve stimulation generated EPPs during superfusion with Ca2+ liposomes in Ca(2+)-free solutions containing the Ca2+ channel blocker Co2+ (1 mM), and the Ca2+ chelator EGTA (2 mM). As a physiological control for Ca2+ leakage from the liposomes to the extracellular fluid, the effect of Ca2+ liposomes on asynchronous evoked ACh release mediated by Ba2+ was examined. In contrast to the effects of 0.2-0.3 mM extracellular Ca2+, which generated EPPs but antagonized Ba(2+)-mediated asynchronous ACh release, Ca2+ liposomes generated EPPs but did not reduce asynchronous release mediated by Ba2+. The effects of Ca2+ liposomes were thus not due to leakage of Ca2+ from the liposome to the extracellular fluid. 4. Morphological studies using fluorescently labelled liposomes in conjunction with a confocal microscope demonstrate that lipid is transferred from the liposomes to nerve endings and liposomal contents are delivered to the nerve terminal cytoplasm. 5. The results suggest that when intracellular Ca2+ is elevated using liposomes as a vehicle, evoked ACh release can occur in the absence of Ca2+ entry via Ca2+ channels.

Acetylcholine↗
PubMed1995

Effects of chain unsaturation on the equation of state for lipid monolayers at the air-water interface.

An equation of state for lipid monolayers at the air-water interface is presented, which takes into account the effects of the conformation and the number and position of double bonds of the hydrocarbon chains. The total Hamiltonian of the monolayer is assumed to consist of three terms. Two of them are calculated exactly within the limitations of the formulation. These are the two-dimensional entropy of mixing of the lipid and water molecules at the surface and the conformational entropy of the chains using a model available from the literature. These two terms give rise to positive surface pressure. The third term, which includes all energies that are not amenable to calculation, was obtained as the difference between the sum of the two calculated terms and experimental data and is found to represent an approximately area-independent tension. The effects of chain unsaturation on the equation of state were modeled in the present theory in two ways; the chain bend caused by cis double bonds increases the minimal molecular area, and the double bond linkage on a chain decreases the degrees of freedom of the chain. Calculations revealed that the former is highly significant whereas the latter is negligible. The deduced equation of state reproduces experimental data with appropriate values for three parameters, which represent the collapse area, the overlap of adjacent chains, and the combined effects of the intra- and intermolecular interactions other than the surface mixing entropy and the chain conformational energy.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

A metastable state of high surface activity produced by sonication of phospholipids.

Sonication of phosphatidylcholine dispersions generates a metastable high energy assembly of molecules, the existence of which is revealed by its conspicuous surface activity. Freshly sonicated liposome dispersions release molecules to the air/water interface at rates sufficient to produce a close-packed monolayer within minutes. In contrast, monolayers at the surface of multilamellar and extruded vesicles take hours to form. The highly surface active species appears within the first few minutes of sonication, long before a major reduction in turbidity occurs, and accumulates over the course of a few hours of sonication. It disappears upon exhaustive sonication, extrusion, addition of extruded vesicles, or, more slowly, simply on standing. Tests for extraneous substances in the lipids before as well as after sonication revealed amounts of degradation products too small to represent the observed surfactant. Direct evidence that the metastable aggregate releases intact phospholipids was provided by a novel procedure to characterize monolayer composition by comparing surface tension with surface potential, both as a function of surface density. Centrifugation and gel filtration chromatography indicate that the surface activity is associated with a particle of diameter larger than a lysophosphatidylcholine micelle but not larger than limit sonicated vesicles. The metastable material appears to be lipid molecules in other than the normal stable vesicular state, perhaps an incompletely closed vesicle, one in which the inner and outer monolayers have not equilibrated, or possibly a micellar form.

Lipid Bilayers↗
PubMed1994

Fragmentation into small vesicles of dioleoylphosphatidylcholine bilayers during freezing and thawing.

Multilayered liposomes of some phosphatidylcholines progressively fragment into small vesicles when the electrolyte solution in which they are suspended is subjected to successive cycles of freezing and thawing. The fragmentation process, routinely monitored by absorbance measurements and verified by electron microscopy and dynamic light scattering, involves bilayer breakage and resealing. After 10 cycles of freezing and thawing in 0.1 M electrolyte solution, the result is a population of vesicles smaller than 200 nm diameter. Sucrose, a common cryoprotectant, completely inhibits fragmentation. Fragmentation is absolutely dependent upon the presence of an electrolyte. Those electrolytes most effective in promoting liposome fragmentation have large freezing point depressions and corresponding high solubilities at the freezing point. This, coupled with the observation that saturating concentrations of electrolyte are less effective than 0.1 M solutions indicates that an essential stage in the fragmentation process is osmotic extraction of water from the vesicles, i.e., ice formation in the external phase leads to a progressive increase in the electrolyte concentration of the residual external solution, which, in turn, dehydrates the vesicle. In addition, for maximal fragmentation, the minimum temperature must be at least as low as the solute eutectic temperature. Particular physical properties of the bilayer are also important, for dioleoyl and diphytanoyl derivatives are much more susceptible to fragmentation than are other phosphatidylcholines, and inclusion of 50 mol% cholesterol in dioleoylphosphatidylcholine completely inhibits membrane breakup. This system provides insight into mechanisms of freezing damage to membranes and may also offer a very simple and rapid assay for biological cryoprotectants.

Cholesterol↗
PubMed1994

Lipid unsaturation influences melittin-induced leakage of vesicles.

Investigation of vesicles composed of different phosphatidylcholines revealed that the extent of leakage of internal contents induced by the lytic agent melittin can range from practically none to essentially complete, depending upon the fatty acyl chain composition of the phospholipid. The extent of leakage increases with the number of double bonds in the series dioleoylphosphatidylcholine < dilinoleoylphosphatidylcholine < dilinolenoylphosphatidylcholine. It depends on the length of the saturated chain with 1-myristoyl-2-arachidonoylphosphatidylcholine vesicles being more sensitive to melittin induced leakage than 1-palmi-toyl-2-arachidonoylphosphatidylcholine, 1-stearoyl-2-arachidonoylphosphatidylcholine or 1-palmitoyl-2- docosahexaenoylphosphatidylcholine vesicles. The extent of leakage induced by melittin from vesicles composed of 1-palmitoyl-2-oleoylphosphatidylcholine, dioleoylphosphatidylcholine, 1-palmitoyl-2-arachidonoylphosphatidylcholine and 1-palmitoyl-2-docosahexaenoyl-phosphatidylcholine increases with the free volume parameter of these lipids for 1,6-diphenylhexatriene (Straume, M. and Litman, B.J. (1987) Biochemistry 26, 5113-5120). Among the lipid examined here, diphytanoylphosphatidylcholine vesicles were least susceptible to melittin induced leakage. The results indicate that lipid fatty acyl structure may be important in lipid-protein interactions of the kind simulated by melittin.

Kinetics↗
PubMed1994

Fluorescence studies of spectrin and its subunits.

To better understand the solution structure of spectrin, the environment of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its alpha and beta subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the beta subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated beta chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2-p-toluidinyl napthalene-6-sulfonic acid (TNS). It therefore appears that low ionic strength, alpha-beta association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solution does not involve exposure of significant numbers of hydrophobic sites.

Ethanol↗
PubMed1993

Band 4.1 enhances spectrin binding to phosphatidylserine vesicles.

Erythroid band 4.1 enhances the binding of erythroid spectrin to phosphatidylserine vesicles under conditions of ionic strength and at protein concentrations similar to those in the red cell. The extent of enhancement depends on the concentration of band 4.1; at 2 microM 4.1, spectrin binding increases approximately 10-fold (to 600 mumoles/mole lipid). The Kd is 0.5 microM, as measured by SDS-PAGE of protein-bound vesicles recovered by ficoll gradient centrifugation. The 4.1-enhanced binding of spectrin was also measured by a gel filtration assay. The electrostatic nature of the enhancement of spectrin binding is indicated by its dependence on the phosphatidylserine content of the vesicles.

Chromatography, Ion Exchange↗
PubMed1993

Experimental method to correct fluorescence intensities for the inner filter effect.

An empirical procedure for the correction of measured fluorescence intensity for the inner filter effect is described. The procedure is more reliable than those described previously as fluorophores with the fluorescence properties of interest are sequestered from the external aqueous phase. The inner filter effect can therefore be assessed in the absence of chemical reaction or Forster energy transfer between the fluorophore and chromophore. A correction curve for the inner filter effect is obtained by measuring the fluorescence intensity of the sequestered fluorophore after adding a chromophore to increase the absorbance of the solution. The procedure was tested with liposomes containing calcein and two commercial polymer bead preparations containing embedded fluorophores. The former have the advantage of a wider choice of fluorophores and the latter of stability and convenience. The inner filter effect was varied using small aliquots of potassium chromate or pyridinium chloride solution. It was shown that the magnitude of the inner filter effect must be experimentally determined for each instrument and whenever the instrumental configuration is altered if an accurate correction for the inner filter effect is to be obtained. The magnitude of the correction depends on the wavelength range and the pathlength but not on slit-width or sample turbidity for the instrument employed for most of the experiments reported here. This empirical procedure makes possible studies of protein fluorescence quenching by agents previously unsuitable because of high absorbances.

Fluorescence↗
PubMed1991

Characteristics of spectrin-induced leakage of extruded, phosphatidylserine vesicles.

At neutral pH spectrin induces modest leakage of trapped calcein from reverse-phase or extruded, but not sonicated, vesicles composed of phosphatidylserine, but not phosphatidylcholine. The extent of leakage from extruded vesicles is not or is only slightly affected by magnesium ions at a physiological concentration or calcium ions at a greater than physiological concentration, respectively. In addition to accounting for several previously discrepant observations on the lytic effects of spectrin, these findings indicate that some proteins like spectrin may destabilize vesicles with low curvature more readily than vesicles of high curvature, in contrast to certain amphiphilic peptides. 60% less leakage is induced from phosphatidylserine vesicles by heat-denatured than by native spectrin. In contrast, both trypsin- and subtilisin-treated spectrins, if sufficiently digested, induce several-fold more leakage than undigested spectrin. Since spectrin prepared either by 1 M Tris dissociation of Triton-extracted cytoskeletons or by low ionic strength extraction of ghosts released the same amounts of calcein from vesicles of various compositions, these effects are unlikely to reflect artifacts of spectrin preparation. Furthermore, spectrin is unlikely to promote leakage in vivo, since vesicles composed of phosphatidylserine, cholesterol and/or phosphatidylethanolamine, which constitute the lipid composition of the inner monolayer of the red cell membrane, did not leak on addition of spectrin, whereas vesicles composed of phosphatidylserine and phosphatidylcholine, did leak in the presence of spectrin.

Calcium↗
PubMed1991

Small-volume extrusion apparatus for preparation of large, unilamellar vesicles.

The design and performance of a filter holder which enables convenient preparation of volumes of up to a milliliter of large, unilamellar vesicles formed by extrusion (LUVETs) from multilamellar vesicles (MLVs) are described. The filter holder provides for back-and-forth passage of the sample between two syringes, a design that minimizes filter blockage, eliminates the need to change filters during LUVET preparation and reduces preparation time to a few minutes. Replicas of slam-frozen LUVETs in the electron microscope are unilamellar and reasonably homogeneous with an average diameter close to the pore size of the filters used to extrude them. Extrusion per se does not destabilize the vesicles, which trapped a fluorescent dye only when they were disrupted on freeze-thawing and during the first extrusion when most of the MLVs were apparently converted to LUVETs.

Biomarkers↗
PubMed1990

Spontaneous rupture of liver due to cholangitis.

Spontaneous rupture of the liver is rare in the Western world and most cases are associated with primary or metastatic tumours. Spontaneous rupture of the intrahepatic biliary tree is not well documented and only two reports have been found in the literature. The authors describe a further case due to ascending cholangitis but presenting with biliary peritonitis. The literature is reviewed and the diagnostic problems discussed.

Cholangitis↗
PubMed1990

Surface chemistry of phospholipid vesicles relevant to their aggregation and fusion.

A general description of those forces that are important in the stability of the lipid bilayer is presented. The analysis focuses on those that may change under conditions in which aggregation or fusion of vesicles or exchange of their molecules may occur. It is that class of membrane forces that is most likely to play a role in the process(es) that leads to the formation of cholesterol crystals in bile, which eventually grow into gallstones. A specific, hypothetical mechanism for cholesterol crystal nucleation based on vesicle fusion is also given. This mechanism is consistent with the principles of surface chemistry outlined.

Chemical Phenomena↗
PubMed1989

Interactions of liposomes with planar bilayer membranes.

The adhesion to horizontal, planar lipid membranes of lipid vesicles containing calcein in the aqueous compartment or fluorescent phospholipids in the membranes has been examined by phase contrast, differential interference contrast and fluorescence microscopy. With water-immersion lenses, it was possible to study the interactions of vesicles with planar bilayers at magnifications up to the useful limit of light microscopy. In the presence of 15 mM calcium chloride, vesicles composed of phosphatidylserine and either phosphatidylethanolamine or soybean lipids adhere to the torus, bilayer and lenses of planar bilayers of the same composition. Lenses of solvent appear at the site where vesicles attach to decane-based bilayers and lipid fluorophores move from the vesicles to the lenses. Because the calcein contained in such vesicles is not released, we interpret this as indicating fusion of only the outer monolayer (hemifusion) of the vesicles with the decane lenses. In the case of squalene-based black lipid membranes (BLMs), in contrast, vesicles do not nucleate lenses but they apparently do fuse with the torus at the bilayer boundary. Interactions leading to hemifusions between vesicles and planar membranes thus occur predominantly in regions where hydrocarbon solvent is present. Osmotic water flow, induced by addition of urea to the compartment containing vesicles, causes coalescence of lenses in decane-based BLMs as well as coalescence of the aqueous spaces of the vesicles that have undergone hemifusion with the lenses. We did not observe transfer of the aqueous phase of vesicles to the trans side of either decane- or squalene-based planar membranes; however, we cannot rule out the possibility particularly in the latter case, that rupture of the planar membrane may have been an immediate result of vesicle fusion and thus precluded its detection.

Fluoresceins↗
PubMed1989

A fluorometric assay for acetylcholine with picomole sensitivity.

A sensitive fluorogenic assay for acetylcholine is described. The assay is based upon the reactions: (1) hydrolysis of acetylcholine to choline and acetate, catalyzed by acetylcholinesterase. (2) Oxidation of choline to betaine and hydrogen peroxide by choline oxidase. (3) Oxidation of p-hydroxyphenylacetic acid by hydrogen peroxide to a fluorescent product, catalyzed by peroxidase. With a sensitivity comparable to chemiluminescence procedures, the assay should find application to those situations requiring the persistence of a fluorescence signal or the necessity of assaying small volumes.

Acetylcholine↗
PubMed1989

Aerobic ethanol production by leaves: Evidence for air pollution stress in trees of the Ohio River Valley, USA.

We measured the frequency with which leaves of trees in the Ohio River Valley produced ethanol aerobically, to determine if aerobic ethanol production might provide a viable field assay for air pollution stress. Leaves were collected from trees during the summers of 1985 and 1986 and ethanol production was determined using headspace GC. Frequency of ethanol production was compared with environmental factors, including air pollution concentrations. We found frequent foliar ethanol production and elevated alcohol dehydrogenase activity in the leaves of several species of trees in the Ohio River Valley, USA. The ethanol concentrations measured were often equivalent to those produced by anaerobic leaves. Ethanol production was associated with hot, hazy weather and elevated NO(2) concentrations. Ethanol production was more frequent in urban and industrialized areas. Ethanol production was not associated with natural stresses such as flooding and herbivory. We propose that aerobic ethanol production is the result of cell acidification due to the accumulation of acidic gases in the cytoplasm. The use of ethanol production as a diagnostic tool for detecting stress imposed by acidic gases is discussed.

Journal Article↗