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Biomedical subjects

M J Hope

Publications and source records attributed to M J Hope.

At least 55 records · Page 3Linked to original sources
PubMed1986

Polymorphic phase behavior of unsaturated lysophosphatidylethanolamines: a 31P NMR and X-ray diffraction study.

The polymorphic phase behavior of aqueous dispersions of 1-oleoyl-, 1-linoleoyl-, and 1-linolenoyl-sn-3-glycerophosphoethanolamine (1-C18:1c-PE, 1-C18:2c-PE, and 1-C18:3c-PE, respectively) has been investigated by 31P NMR, small-angle and wide-angle X-ray diffraction, and freeze-fracture techniques in response to changes in temperature and pH. Between -20 and 0 degrees C at pH 7, NMR and X-ray data indicate that 1-C18:1c-PE adopts a lamellar phase. Above 20 degrees C, the X-ray diffraction from 1-C18:1c-PE reveals no long-range lattice order, whereas the NMR data indicate lamellar structure to 90 degrees C. Freeze-fracture electron microscopy shows that 1-C18:1c-PE at pH 8.2 forms closed multilamellar vesicles upon dispersion and also that large unilamellar vesicles produced by extrusion techniques (LUVETs) can be made from 1-C18:1c-PE at pH 7. Such LUVETs can trap [3H]inulin and support a K+ diffusion potential for up to 4 h. At pH 8.5 and above, 1-C18:1c-PE forms optically clear, fluid dispersions with NMR and X-ray characteristics consistent with a micellar (noninverted) phase structure. Attempts to prepare LUVETs from 1-C18:1c-PE at pH 9 result in structures that can neither trap [3H]inulin nor support a membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Fatty Acids, Unsaturated↗
PubMed1985

Protection of large unilamellar vesicles by trehalose during dehydration: retention of vesicle contents.

The ability of trehalose and other sugars to maintain the integrity of large unilamellar vesicles subjected to dehydration and rehydration has been investigated. It is shown, employing freeze-fracture techniques, that large unilamellar vesicles prepared in the presence of trehalose at 125 mM or higher concentration do not exhibit significant structural changes during the dehydration-rehydration cycle. Further, up to 90% of entrapped 22Na or [3H]inulin is retained during this process. Other sugars also exhibited similar protective effects where trehalose was most effective, followed by sucrose, maltose, glucose and lactose. It is demonstrated that proton or Na+/K+ electrochemical gradients can be maintained during the dehydration-rehydration process, which can subsequently be used to drive the uptake of lipophilic cationic drugs such as adriamycin. The implications for long-term storage of liposomal systems for use in drug-delivery protocols are discussed.

Desiccation↗
PubMed1985

Solute distributions and trapping efficiencies observed in freeze-thawed multilamellar vesicles.

It has recently been observed (Gruner, Lenk, Janoff and Ostro (1985) Biochemistry, in the press) that mechanical dispersion of dry lipid in an aqueous buffer to form multilamellar vesicle (MLV) systems does not result in equilibrium trans-membrane distributions of solute. In particular, the entrapped buffer exhibits reduced solute concentrations. Here we demonstrate that egg phosphatidylcholine MLV systems dispersed in the presence of Mn2+ also exhibit non-equilibrium solute distributions, and that repetitive freeze-thawing cycles can remove such solute heterogeneity. Further, the resulting freeze-thawed MLVs exhibit dramatically enhanced trapped volumes and trapping efficiencies. At 400 mg phospholipid per ml, for example, the trapping efficiencies can be as high as 90%. This is associated with a remarkable change in MLV morphology where large inter-bilayer separations are commonly observed.

Freeze Fracturing↗
PubMed1985

Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential.

Many drugs exhibit lipophilic and cationic (basic) characteristics. Previous studies have shown that lipophilic cations can be accumulated into model membrane 'liposomal' (vesicular) systems in response to establishing a membrane potential (inside negative) across the vesicle membrane. We demonstrate here that the anticancer drugs, adriamycin and vinblastine, can be rapidly accumulated into egg phosphatidylcholine large unilamellar vesicles in response to a valinomycin-dependent K+ diffusion potential (delta psi) to achieve high effective interior concentrations. Further, trapping efficiencies approaching 100% can be easily achieved. The influence of lipid composition and the requirement for valinomycin have been examined for adriamycin. Equimolar cholesterol levels inhibit the uptake process at 20 degrees C. However, incubation at higher temperature results in enhanced uptake. Similarly, the presence of egg phosphatidylserine or incubation at elevated temperatures results in significant adriamycin uptake in the absence of valinomycin. It is shown that the adriamycin retention time in the vesicles is enhanced by an order of magnitude or more when actively trapped by the presence of a membrane potential in comparison to passive trapping procedures. It is suggested that such active trapping procedures may be of use for loading liposomal systems for drug delivery applications, and may provide avenues for controlled release of encapsulated material.

Antineoplastic Agents↗
PubMed1985

Uptake of dibucaine into large unilamellar vesicles in response to a membrane potential.

Local amine anesthetics appear to exert their effects in the charged (protonated) form on the cytoplasmic side of excitable membranes. Two features of interest are the mechanism whereby these drugs move across the membrane to the inner monolayer and the actual membrane concentrations achieved. In this work, we have investigated the influence of a K+ diffusion potential, delta psi, on the transmembrane distribution and concentration of the local anesthetic dibucaine employing large unilamellar vesicle systems. It is demonstrated that egg phosphatidylcholine large unilamellar vesicles exhibiting a delta psi (interior negative) actively accumulate dibucaine to achieve high interior concentrations. 31P and 13C nuclear magnetic resonance studies show that the internalized drug is localized to the vesicle inner monolayer, and suggest that the protonated form of the anesthetic is the species that is actively transported. The inner monolayer anesthetic concentrations thus achieved can be an order of magnitude or more larger than predicted on the basis of anesthetic lipid-water partition coefficients. It is suggested that these effects may be related to the mechanisms whereby local anesthetics are localized and concentrated at their sites of action in nerve membranes.

Anesthetics, Local↗
PubMed1984

Phosphatidic acid as a calcium ionophore in large unilamellar vesicle systems.

The ionophoretic capabilities of dioleoylphosphatidic acid (DOPA) for transporting calcium across phospholipid bilayers have been investigated. Calcium uptake by large unilamellar vesicles is shown to depend on the presence of DOPA. This uptake is sensitive to the nature and concentration of calcium chelators in the vesicle interior, indicating that accumulation results from DOPA-mediated translocation of calcium across the membrane. Further, it is shown that characteristics of DOPA-mediated Ca2+ uptake are similar to those observed for the fungal calcium ionophore, A23187.

Biological Transport↗
PubMed1984

Influence of vesicle size and oxidase content on respiratory control in reconstituted cytochrome oxidase vesicles.

Previous work has shown that the respiratory control or coupling exhibited by reconstituted cytochrome oxidase systems can be markedly sensitive to the lipid composition, the lipid to protein ratio, and the vesicle size. In this work we have attempted to ascertain which, if any, of these factors plays a definitive role in determining the observed coupling. Vesicles prepared from dioleoylphosphatidylcholine-dioleoylphosphatidylethanolamine (1:4) were fractionated by DEAE chromatography to obtain a population homogeneous with respect to their lipid to protein ratio. This subpopulation was then applied to a Sepharose 4B-CL column to separate the vesicles by size. Fractions eluted from the column were assayed for enzyme activity in the presence and absence of carbonyl cyanide ( trifluoromethoxy )phenylhydrazone plus valinomycin. The coupling ratio was found to be dependent upon vesicle size; the smaller the vesicles, the higher the ratio. This suggests that lipid composition per se does not determine coupling characteristics. Reducing vesicle size for a given lipid to protein ratio has two effects. First, the radius of curvature is increased and, second, the average number of oxidase molecules per vesicle is reduced. In order to identify which of these factors was responsible for the observed tighter coupling, the oxidase was reconstituted with dioleoylphosphatidylcholine into vesicles of a defined size and the number of oxidase molecules per vesicle varied. The highest coupling ratios were observed for vesicles containing on average only one oxidase dimer. As the fraction of vesicles containing more than one protein was increased, the coupling ratio rapidly declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-Free System↗
PubMed1983

Lipid requirements for coupled cytochrome oxidase vesicles.

Cytochrome c oxidase has been reconstituted with two synthetic phospholipids, dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine. Vesicles prepared from either of these two lipids alone showed no stimulation of enzyme activity upon addition of carbonyl cyanide (trifluoromethoxy)phenylhydrazone and valinomycin, indicating that they were leaky to small ions. However, when mixtures of the two lipids were used for the reconstitution, tightly coupled vesicles could be obtained. The coupling ratio was dependent upon the ratio of dioleoylphosphatidylcholine to dioleoylphosphatidylethanolamine and also on the lipid-to-protein ratio. Maximal rates of enzyme activity were not significantly different with different lipid mixtures. The results are discussed in terms of both the size distribution of the reconstituted vesicles and the possible requirement for a variety of lipid species to ensure tight sealing at the lipid-protein interface.

Animals↗
PubMed1983

Polymorphic phase preferences of phosphatidic acid: A 31P and 2H NMR study.

31P NMR, 2H NMR and freeze fracture techniques have been employed to investigate the structural preferences of dioleoyl phosphatidic acid (DOPA) under various conditions of pH and divalent cation content. It is shown that DOPA increasingly prefers the HII organization below pH 5, and that low levels of Ca2+ and Mg2+ (Me2+/DOPA = 0.5) induce HII phase structure for pH less than 6.0. Higher Mg2+ and Ca2+ levels (Me2+/DOPA greater than 1.0) induce more complex structures at pH greater than 5.0 which may correspond to intermediates between lamellar and HII organization. This work illustrates the utility of 2H NMR techniques in conjunction with suitable 2H labelled lipids to provide structural information on lipid-water systems, and suggests that 31P NMR techniques for determinations of lipid organization can be applied to advantage in PA containing membranes.

Calcium↗
PubMed1983

Ca2+ and pH induced fusion of small unilamellar vesicles consisting of phosphatidylethanolamine and negatively charged phospholipids: a freeze fracture study.

Fusion processes in small (sonicated) unilamellar vesicle (SUV) systems composed of 80 mol % unsaturated phosphatidylethanolamine and 20 mol % of phosphatidylserine, phosphatidylinositol, phosphatidic acid or cardiolipin have been examined by freeze-fracture techniques. All these mixtures have the property that Ca2+ and, in some cases low pH, can trigger bilayer to hexagonal (HII) phase transitions in large multilamellar dispersions of these lipid mixtures. It is shown that when the SUV systems are subjected to similar protocols, the vesicles first fuse to form larger systems prior to HII phase formation and this fusion is accompanied by the appearance of lipidic particle structures, often localized to the fusion interface. We conclude that factors promoting HII phase structure initially result in fusion of unilamellar systems and that this fusion proceeds via intermediary formation of non-lamellar, possibly inverted micellar, structure.

Calcium↗
PubMed1983

Polymorphism of phosphatidylethanolamine-phosphatidylserine model systems: influence of cholesterol and Mg2+ on Ca2+-triggered bilayer to hexagonal (HII) transitions.

Previous work has shown that Ca2+ can trigger bilayer to hexagonal (HII) polymorphic phase transitions in (unsaturated phosphatidylserine (PS)-phosphatidylethanolamine (PE) model systems. In this work we examine the influence of cholesterol and Mg2+ on the phase preferences of PS-PE systems. Subsequently, the influence of cholesterol and Mg2+ on the levels of Ca2+ required to trigger bilayer-HII transitions in these mixed systems is studied. It is shown that at 30 degrees C the presence of equimolar (with respect to phospholipid) levels of cholesterol engenders formation of the HII phase for PE-PS systems containing 15 and 30 mol% PS, whereas bilayer structure is maintained for PE-PS-cholesterol (1:1:2) dispersions. However, the polymorphic phase preferences of the latter system are much more sensitive to the presence of monovalent and divalent cations. In the absence of cholesterol, Mg2+ and high salt concentrations do not affect the polymorphic phase preferences of PE-PS (1:1) systems. In contrast, 8 mM or higher Mg2+ levels or salt concentrations greater than 1.0 M induce HII-phase formation in PE-PS-cholesterol (1:1:2) systems. Further, lower Mg2+ concentrations (2 mM) act as a powerful adjunct to Ca2+ triggering of HII-phase structure in such systems, reducing the Ca2+ concentration required from 4 to 0.25 mM. These results are discussed in terms of Ca2+ concentrations required for fusion events and the influence of cholesterol on the structural preferences of the inner monolayer lipids of the erythrocyte membrane.

Calcium↗
PubMed1982

Phospholipids as adjuncts for calcium ion stimulated release of chromaffin granule contents: implications for mechanisms of exocytosis.

Structure-function relationships for the lipid component of chromaffin granules isolated from the bovine adrenal medulla have been investigated by employing 31P nuclear magnetic resonance (NMR), freeze-fracture, and spectrophotometric techniques. Two aspects have been studied in detail, namely, the structural preferences of lipids in the isolated granule membrane and derived liposomal model membrane systems as well as the influence of exogenous lipid (in the form of sonicated vesicle systems) on the Ca2+-stimulated release of granule contents. It is shown that at least 90% of endogenous granule membrane phospholipids assume a liquid-crystalline bilayer configuration at physiological temperatures. Liposomal dispersions of total granule lipid also exhibit bilayer structure, consistent with a structural role of phospholipids in vivo. Incubation of intact isolated granules in the presence of up to 10 mM Ca2+ does not induce significant release of contents above background levels. However, it is shown that incubation of granules in the presence of sonicated phospholipid systems which undergo structural transitions in the presence of Ca2+ can cause immediate and total release of granule contents at Ca2+ levels of 2 mM or more. This behavior is attributed to disruption of granule membrane integrity due to fusion of the vesicle systems with the chromaffin granules. Direct evidence for such fusion is obtained by freeze-fracture electron microscopy. On the basis of this information and with the assumption that the inner leaflet of the adrenal cell plasma membrane is composed predominantly of phosphatidylethanolamine and phosphatidylserine, a mechanism of Ca2+-stimulated exocytotic release of catecholamines in vivo is proposed.

Adrenal Medulla↗
PubMed1982

Structural preferences of phosphatidylinositol and phosphatidylinositol-phosphatidylethanolamine model membranes. Influence of Ca2+ and Mg2+.

The structural preferences of soya phosphatidylinositol in isolation and in mixtures with soya phosphatidylethanolamine, and the influence of Ca2+ and Mg2+ on these preferences, have been examined employing 31P-NMR and freeze-fracture techniques. It is shown that phosphatidylinositol assumes the bilayer organization on hydration both in the presence and absence of Ca2+ and Mg2+. In mixed systems with (HII phase) phosphatidylethanolamine, phosphatidylinositol induces lipidic particle structure at low (less than 10 mol%) concentrations and bilayer structure at higher levels. In systems containing 15 or 20 mol% phosphatidylinositol, Ca2+ (but not Mg2+) can induce HII phase structure. The results indicate that phosphatidylinositol is a more effective agent than other acidic phospholipids for stabilizing bilayer structure, particularly when high levels of divalent cations are present. These findings are discussed in terms of functional roles of phosphatidylinositol and mechanisms whereby Ca2+ induces structural reorganizations in mixed systems containing acidic phospholipids and phosphatidylethanolamine.

Calcium↗
PubMed1981

Effect of dimyristoyl phosphatidylcholine on intact erythrocytes. Release of spectrin-free vesicles without ATP depletion.

Incubation of human erythrocytes with suspensions of sonicated dimyristoyl phosphatidylcholine resulted in dramatic morphological changes of the cells and release of membrane vesicles. The shedding of membrane vesicles was not preceded by ATP depletion and only occurred at temperatures of incubation that were above the phase transition temperature of dimyristoyl phosphatidylcholine. Membrane vesicles were separated from intact erythrocytes and exogenous dimyristoyl phosphatidylcholine by a series of centrifugation steps. The lipid composition of the membrane vesicles was similar to that of the native erythrocyte, and the predominant membrane proteins were band 3, glycophorin and acetylcholinesterase. Spectrin was not detected. Freeze-fracture electron microscopy showed vesicles (150 nm in diameter) with protein particles embedded in the lipid bilayer.

Acetylcholinesterase↗
PubMed1981

The role of nonbilayer lipid structures in the fusion of human erythrocytes induced by lipid fusogens.

(1) A comparative study of the polymorphic phase behaviour of reconstituted erythrocyte phospholipase with 'fusogens' and chemically related 'non-fusogens' using 31P-NMR techniques has been made. We demonstrate that only fusogens can induce a conformational change in the membrane lipids from the bilayer of the hexagonal (HII) phase. (2) The tendency of erythrocyte (ghost) membranes to adopt nonbilayer structures is correlated with the extent of fusion in erythrocytes at various membrane concentrations of glycerol monooleate. Above a certain critical membrane concentration of glycerol monooleate, there is an increase in the tendency of ghost membranes to adopt the HII phase, which is paralleled by a marked increase in fusion processes in the intact erythrocytes. (3) These observations are consistent with a common mechanism of action for fusogenic lipids whereby inverted lipid structures such as long cylinders (HII configuration) or inverted micelles are induced, which serve as intermediates in the fusion event.

Cell Fusion↗
PubMed1980

Structural properties of phospholipids in the rat liver inner mitochondrial membrane.

1. The 31P-NMR characteristics of intact rat liver mitochondria, mitoplasts and isolated inner mitochondrial membranes, as well as mitochondrial phosphatidylethanolamine and phosphatidylcholine, have been examined. 2. Rat liver mitochondrial phosphatidylethanolamine hydrated in excess aqueous buffer undergoes a bilayer-to-hyexagonal (HII) polymorphic phase transition as the temperature is increased through 10 degrees C, and thus prefers the HII arrangement at 37 degrees C. Rat liver mitochondiral phosphatidylcholine, on the other hand, adopts the bilayer phase at 37 degrees C. 3. Total inner mitochondrial membrane lipids. dispersed in an excess of aqueous buffer, exhibit 31P-NMR spectra consistent with a bilayer arrangment for the majority of the endogeneous phospholipids; the remainer exhibit spectra consistent with structure allowing isotropic motional averaging. Addition of Ca2+ results in hexagonal (HII) phase formation for a portion of the phospholipids, as well as formation of 'lipidic particles' as detected by freeze-fracture techniques. 4. Preparations of inner mitochondrial membrane at 4 and 37 degrees C exhibit 31P-NMR spectra consistent with a bilayer arrangement of the large majority of the endogenous phospholipids which are detected. Approx. 10% of the signal intensity has characteristics indicating isotropic motional averaging processes. Addition of Ca2+ results in an increase in the size of this component, which can become the domiant spectral feature. 5. Intact mitochondria, at 4 degrees C, exhibit 31P-NMR spectra arising from both phospholid and small water-soluble molecules (ADP, Pi, etc.). The phospholipid spectrum is characteristic of a bilayer arrangement. At 37 degrees C the phospholipids again give spectra consistent with a bilayer; however, the labile nature of these systems is reflected by increased isotropic motion at longer (at least 30 min) incubation times. 6. It is suggested that the uncoupling action of high Ca2+ concentrations on intact mitochondria may be related to a Ca2+-induced disruption of the integrity of the inner mitochondrial phospholipid bilayer. Further, the possibility that non-bilayer lipid structures such as inverted micelles occur in the inner mitochondrial membrane cannot be excluded.

Animals↗