Biomedical subjects

K R Barber

Publications and source records attributed to K R Barber.

51 records · Page 3Linked to original sources

PubMed1990

Glycosphingolipid interdigitation in phospholipid bilayers examined by deuterium NMR and EPR.

Glycosphingolipid fatty acids commonly have up to eight methylene carbons more than do their surrounding phospholipid-attached counterparts. The resultant 'extra' segment may very well modulate glycosphingolipid function as receptor and structural element. As part of an investigation of this phenomenon, galactosylceramide was prepared with a deuterated 18-carbon fatty acid chain. Deuterium-labelled galactosylceramide was assembled at 10 mol% into unsonicated phosphatidylcholine bilayers having all 14-carbon or all 18-carbon saturated fatty acid chains (DMPC and DSPC, respectively). The systems were studied by 2H-NMR spectroscopy above and below the phase transition temperatures, Tm, of the host matrices. At comparable reduced temperatures in fluid membranes the degree of motional order exhibited by the glycolipid fatty acid was significantly higher in the phospholipid host matrix that was four carbons shorter. The fatty acid chain segment least affected by the change from long to short chain host matrix was the terminal (deutero)methyl group (an increase of 8% in quadrupolar splitting for the terminal methyl vs. 16% for deuterons at C17 and 23-28% for the remainder of the chain). Order parameter profiles for galactosylceramide were qualitatively very similar in the two host membranes, arguing against any major conformational difference between the arrangement of the 18-carbon glycolipid fatty acid in the 18-carbon vs. 14-carbon host matrices. Similarly a nitroxide spin probe covalently attached to carbon-12 of the galactosylceramide fatty acid gave clear indication of greater order in the fluid 14-carbon fatty acid phospholipid bilayer. These results are consistent with 'tethering' of the extra length of fatty acid via interdigitation into the opposing monolayer. There was no spectroscopic evidence of any intrinsic difference in glycolipid behaviour in the two fluid host matrices. 2H-NMR spectra of galactosylceramide at comparable reduced temperatures below Tm of the phospholipid bilayer were very different for 14-carbon vs. 18-carbon host matrices. The glycolipid fatty acid showed evidence of relatively reduced mobility in the shorter chain matrix.

Electron Spin Resonance Spectroscopy↗

PubMed1989

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment.

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Amphotericin B↗

PubMed1988

Globoside with spin-labelled fatty acid: bilayer lateral distribution and immune recognition.

We have critically addressed the question of lateral distribution of glycolipids in bilayer membranes, and the effect of glycolipid fatty acid chain length upon such distribution. For this purpose we synthesised the complex neutral glycosphingolipid, globoside, with spin-labelled fatty acid. Base hydrolysis to remove the natural fatty acid was found to deacetylate the GalNAc residue concomitantly, necessitating application of the synthetic route described for gangliosides by Neuenhofer et al. (Biochemistry 24, 525-532 (1985)). Globosides were produced with 18-carbon and 24-carbon fatty acids bearing a spin label at the C-16 position. Spin-labelled globosides were incorporated at 2 and 10 mol% into rigid, highly cooperative bilayer matrices of 1,2-dipalmitoylglycerophosphocholine (DPPC) and also into semi-fluid, non-cooperative membranes of DPPC/cholesterol. Recorded electron paramagnetic resonance (EPR) spectra were analysed by comparison with a library of standards representing samples of known composition. Spectra were manipulated using a computer program which permitted linear combination of standards to stimulate coexistence of laterally separated domains of different composition. The most important conclusions were as follows: (1) at least 80% of the globoside was definitely not confined to domains highly enriched in glycolipid, although there was evidence of binary-phase separation in the rigid DPPC/globoside matrix; (2) the presence of 33 mol% cholesterol reduced the evidence of globoside phase separation; (3) there was remarkably little difference in results whether the globoside fatty acid chain length was similar to that of the phospholipid host matrix or eight carbons longer. Temperature profiles derived over the phase-transition region of DPPC using spin-labelled globoside or an unattached amphiphilic spin label were consistent with these findings. The same systems lent themselves to consideration of the role of glycolipid fatty acid chan length and cholesterol in determining glycolipid crypticity in membranes: (1) polyclonal anti-globoside IgG bound to globoside in DPPC liposomes without inducing agglutination. (2) The same antibodies did agglutinate DPPC/cholesterol liposomes bearing globoside. (3) The effect of cholesterol probably was upon glycolipid dynamics or attitude in the membrane, rather than upon distribution. (4) These observations were basically unaffected by the choice of 18-carbon vs. 24-carbon glycolipid fatty acids.(ABSTRACT TRUNCATED AT 400 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

Evidence that trans-bilayer interdigitation of glycosphingolipid long chain fatty acids may be a general phenomenon.

'Interdigitation' is a term coined to describe the phenomenon whereby pure phosphatidylcholines with intramolecular fatty acid chain length heterogeneity when hydrated to form bilayers may insert the methyl ends of long fatty acids from one side across more than half of the membrane thickness to protrude amongst the acyl chains of the opposite side of the bilayer (Keough, K.M.W. and Davis, P.J. (1979) Biochemistry 18, 1453-1459; Huang, C. and Mason, J.T. (1986) Biochim. Biophys. Acta 864, 423-470). In this article we address the fate of long fatty acid chains of glycosphingolipids present as minor components in membranes of non-interdigitating phosphatidylcholines. In this pursuit, derivatives of galactosyl ceramide, lactosyl ceramide, globoside and GM1 were synthesized having either 18-carbon or 24-carbon fatty acid with a spin label covalently attached at C-16. Labelled glycolipids were incorporated at 1-2 mol% into bilayers of synthetic phosphatidylcholines, their mixtures with cholesterol, or natural egg phosphatidylcholine. In each case the C-16 carbon of the glycolipid long chain fatty acid showed considerably greater 'order' and immobility than did C-16 of the fatty acid which was similar in length to the host matrix phospholipids. We interpret this as strong evidence that the long chain fatty acid interdigitates across the mid point of the bilayer in the systems studied. Clearly this phenomenon did not require that the phospholipid host matrix have mixed chain lengths. Furthermore it was totally independent of glycolipid family: for a given host matrix and (glycolipid) fatty acid chain length the order parameter values found were the same amongst all four glycolipid families tested.

Fatty Acids↗

PubMed1987

Double label freeze-etch study of the relative topography of concanavalin A and wheat germ agglutinin receptors at the myoblast surface.

This report records for the first time double-label freeze-etch electron microscopy of cells in culture. On L6 rat myoblasts, receptors for wheat germ agglutinin and concanavalin A were found distributed together in irregular granular microclusters of cell surface material up to 60 nm in diameter. Simultaneous localization of two different receptor families to such small regions using colloidal gold and ferritin to differentiate between lectin markers proved difficult in our hands. We were able to achieve the desired result using native concanavalin A and ferritin-conjugated wheat germ agglutinin, whose shadowed diameters are measurably different.

Animals↗

PubMed1987

A long chain spin label for glycosphingolipid studies: transbilayer fatty acid interdigitation of lactosyl ceramide.

16-Carbon and 18-carbon fatty acids with covalently attached nitroxide free radicals have seen wide usage in membrane studies of phospholipid dynamics, orientation, and associations. However, they are inadequate for dealing with some very important questions that relate to glycosphingolipids. We report here the synthesis of a long chain (24-carbon) spin-labelled fatty acid designed for such problems. We have used both the new 24-carbon and the more conventional 18-carbon spin-labelled fatty acids to replace the natural fatty acid of lactosyl ceramide so that we may begin to compare short and long chain derivatives to analyse the molecular basis of their functional differences. Spectra seen are consistent with the view that in a bilayer host matrix the methyl end of the long fatty acid crosses the hydrophobic membrane center and interdigitates with fatty acids of phospholipids of the opposing monolayer.

Antigens, CD↗

PubMed1987

A phospholipid spin label used as a liposome-associated MRI contrast agent.

Given current clinical use of phospholipid bilayer structures (liposomes/vesicles) as nontoxic drug delivery vehicles, we have addressed the possibility of employing the phospholipids themselves as MRI contrast agents. To this end we have synthesized phosphatidylcholine with a nitroxide spin label replacing one methyl residue of the choline headgroup. This material was mixed with natural phosphatidylcholine in mole ratios from 1:50 to 1:1 and used to prepare sonicated unilamellar vesicles in saline. Expected structural features of these vesicles were verified by freeze-fracture electron microscopy. Proton T1 values of saline were readily decreased to less than 0.3 s by such preparations, yielding a net relaxivity of 0.6 M-1 s-1. The approach seems to be a realistic way of firmly associating a contrast agent of minimal toxicity with ordinary liposomes/vesicles in a manner that is not subject to leakage.

Contrast Media↗

PubMed1987

Mechanism of the selective toxicity of amphotericin B incorporated into liposomes.

Previously, it has been shown that incorporation of the membrane channel-forming polyene antibiotic, amphotericin B (AMB), into liposomes composed of dimyristoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol (7:3 ratio) results in reduced drug toxicity to animals with full retention of therapeutic activity against systemic fungal infections. In this report we explore the cellular and biochemical bases of the enhanced therapeutic index of liposomal amphotericin B (L-AMB). AMB and L-AMB are equally potent and both promptly induce rapid cation efflux from Candida albicans cells. By contrast, AMB, but not L-AMB, induces cation efflux and cell lysis in mammalian erythrocytes, demonstrating the selectivity of L-AMB at the cellular level. The characteristics of the lipid of the erythrocyte membrane seem to be the most important determinant of cellular sensitivity, since AMB, but not L-AMB, induces cation release from large unilamellar liposomes composed of red cell membrane lipids, thus paralleling the observations on intact cells. The ability of L-AMB to induce cation release and cause toxicity to erythrocytes, however, can be modulated by changing the lipid composition of the liposome carrier. Thus, AMB-containing liposomes composed of phospholipids with saturated acyl chains are nontoxic, whereas AMB liposomes composed of phospholipids containing unsaturated acyl chains are almost as toxic as AMB itself. The acyl chain composition rather than the head group composition seems most important, although substitution of anionic phosphatidylglycerols for phosphatidylcholines contributes somewhat to the protective effect. Analysis of several types of liposomes containing AMB at concentrations up to 5 mol %, using electron paramagnetic resonance and freeze fracture electron microscopy, shows that the drug is incorporated in the lipid bilayer but produces only modest disruptive effects on bilayer structure. Current results are interpreted in terms of a selective transfer of AMB from "donor" liposomes to "target" cell membranes. The transfer process probably occurs by diffusion of AMB through the solvent but is regulated by the physical properties of both donor and target membranes.

Amphotericin B↗

PubMed1986

Visualization of domains in rigid ganglioside/phosphatidylcholine bilayers: Ca2+ effects.

We have considered the extent to which details of lectin binding directly visualized by freeze-etch electron microscopy are consistent with current concepts of ganglioside arrangement in phosphatidylcholine bilayer membranes. Native lectins in general seem appropriate labels for this type of study. Wheat germ agglutinin, Ricinus communis agglutinin, and peanut agglutinin are adequately resolved on membrane surfaces as spherical particles of diameters 6 nm, 10 nm, and 13 nm, respectively (uncorrected for platinum shadow thickness). The finite areas covered by these markers correspond to some 56, 157, and 265 lipid molecules, respectively, on the surfaces of the shadowed rigid phosphatidylcholine matrices employed here; and this constitutes a basic limitation to the precision with which one can localize a given glycolipid receptor. Ricinus communis agglutinin provides a marker whose size permits adequate quantitation of bound material while minimally obscuring detail. Using it we estimated the size limits of GM1-enriched domains, since this is the ganglioside which has shown the greatest evidence of discontinuous distribution in our hands (Peters, M.W., Mehlhorn, I.E., Barber, K.R. and Grant, C.W.M. (1984) Biochim. Biophys. Acta 778, 419-428). Results of such analyses indicate the probable existence of phase separated domains selectively enriched in GM1 up to 60 nm in extent (5600 lipid molecules) for rigid dipalmitoylphosphatidylcholine membranes bearing up to 14 mol% GM1. Similar observations were true of rigid bilayers of dimyristoylphosphatidylcholine; however, if domains enriched in GM1 exist in fluid dimyristoylphosphatidylcholine, they are on the order of 6 nm or less in diameter (or are dispersed by lectin binding). Employing the small lectin, wheat germ agglutinin, which binds to all gangliosides, we then examined the effect of exposure to Ca2+ ions (while in the fluid state) on the ganglioside 'domain structure' referred to above in rigid dipalmitoylphosphatidylcholine host matrices. GM1, GD1a and GT1b were studied at 0, 2 and 10 mM Ca2+ concentrations. It was demonstrated by spin label measurements that the dipalmitoylphosphatidylcholine matrix retained its basic melting characteristics in the presence of added Ca2+ and ganglioside under these conditions. Within the technique's functional resolution limit of some 6 nm we were unable to identify any effect of Ca2+ in physiological concentration on ganglioside topography as reflected by bound lectin distribution.(ABSTRACT TRUNCATED AT 400 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1986

Freeze-etch visualization of the outer surface of myoblasts in culture.

The Pfenninger device is one of several types of specimen holders designed to permit freeze-fracture electron microscopy of cultured cells growing attached to solid substrates. It achieves this by directing a fracture plane horizontally through a monolayer of cells frozen with overlying medium (without need for prior disruption of cell attachments or relationships). The end result is a platinum-shadowed replica of the cell membrane hydrophobic interior. Here we describe the features seen when this traditional fracture step is followed by a lengthy etching step, making possible views of the cell membrane outer surface with a resolution 100 X better than that of fluorescence microscopy. Because of the technical difficulties involved, such views have in past been restricted to samples which may be handled in suspension, particularly blood cells and model membranes. Thus we have been able to examine extensive regions of the myoblast outer surface (the so-called etch face) at a magnification that permits visualization of details on the order of individual macromolecules. Prominent clumps of glycocalyx material occupying some 50% of the surface can be readily resolved as a closely spaced network of uniformly distributed 20- to 60-nm irregular granular patches. Receptors for wheat germ agglutinin were found to be associated almost exclusively with these surface prominences, so that bound lectin tended to exist in a uniform distribution of small clusters corresponding to the patches described above. When cells were not fixed until 15 min after lectin addition there was visibly more binding, but in a similar distribution. The details of cell surface architecture recorded here at a resolution of 2-4 nm are well below the limit of resolution of light microscopy and complement existing studies by fluorescence techniques. The presence of surface receptors in small patches reinforces the possibility that some literature observations of receptor interaction may be explained on the basis of direct receptor-receptor contact.

Animals↗

PubMed1984

Evidence of a distribution difference between two gangliosides in bilayer membranes.

Freeze-etch electron microscopy, a platinum shadowing technique, has been used to compare the lateral distribution of several gangliosides in bilayer model membranes by directly visualizing bound lectin molecules. In particular, GM1 and GD1a, major components of brain ganglioside, were studied in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholines exposed to Ricinus communis agglutinin and wheat germ agglutinin. The distribution of glycolipid showed evidence of microheterogeneity in that bound lectin tended to occur in clusters of several or more molecules. With GD1a as receptor such clusters were small and very uniformly distributed over the membrane surface. Somewhat larger, irregularly spaced clusters of up to a dozen lectin particles were more typical of membranes bearing GM1 and, in addition, there were occasional extensive patches of bound lectin coexisting with areas apparently devoid of glycolipid receptor in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholine. Gangliosides in the latter mixtures were not obviously influenced in their lateral distribution by the presence of coexisting fluid and rigid domains. These basic observations seem to extend to bilayer membranes containing mixtures of two gangliosides. The patterns of lectin binding were not grossly affected by incubation time or history of warming and cooling. This study was extended to bilayers of pure dipalmitoylphosphatidylcholine in expectation that the distinctive features characteristic of the P beta' phase of this lipid might accentuate any behavioural differences between GM1 and GD1a. GM1 was found to exist preferentially in the 'trough' regions between P beta' ripples, while GD1a showed no apparent preferential arrangement. Given that bound lectins adequately reflect glycolipid distribution in membranes, it would appear that structurally different glycolipids from the same host membrane can assume different distributions on the basis of interactions with defined lipid host matrices.

Freeze Fracturing↗

PubMed1984

Lateral distribution of gangliosides in bilayer membranes: lipid and ionic effects.

Native Ricinus communis agglutinin (RCA 60) has been used as a visual marker to localize the ganglioside GM1 under a variety of conditions in model membranes. By employing the technique of freeze-etch electron microscopy, it was possible to resolve membrane structural details down to some 3 nm. The most striking feature was frequent existence of the marker lectin in clusters--presumably reflecting the underlying presence of clustered receptor. This feature persisted at glycolipid concentrations from 0.5 to 7 mol %. It was visible in membranes of single pure phospholipids above and below their phase transition temperatures, in membranes of mixed phospholipids, and in membranes containing cholesterol. The presence of cations, including Ca2+, was not seen to alter the pattern of lectin binding at a resolution of 3 nm. In pure dipalmitoyl phosphatidylcholine, a lining-up of glycolipid in the "troughs" between ripples in rigid lipid was apparent, in agreement with a similar phenomenon reported by Tillack et al for a neutral glycosphingolipid in pure dimyristoyl phosphatidylcholine [1982: Biochim Biophys Acta 691:261-273]; but this feature was not evident in host matrices composed of several different lipids.

Biophysical Phenomena↗

PubMed1983

Glycolipid crypticity in membranes--not a simple shielding effect of macromolecules.

The potential of membrane-bound macromolecules for shielding glycolipids from involvement in specific binding events was considered in model membranes. Serum albumin and several Dextrans were covalently derivatized with oleic acid so that they adsorbed irreversibly to lipid bilayers. This provided a means of generating bilayer membranes with a considerable layer of attached material. Gangliosides dispersed in such membranes were subjected to attack by the enzyme, neuraminidase, in order to assess their "accessibility'. We were surprised to find that we could not demonstrate any significant reduction in ganglioside hydrolysis in phosphatidylcholine bilayers bearing extensive surface coats of protein or polysaccharide. We conclude that non-specific, physical shielding by macromolecules is an unlikely source of the often-observed "crypticity' of glycolipids at the cell surface. Consistent with this interpretation was a relative lack of headgroup motional restriction seen for spin-labelled ganglioside headgroups in the same bilayers and in cell membranes.

Electron Spin Resonance Spectroscopy↗

PubMed1982

Headgroup behaviour of an uncharged complex glycolipid.

A globoside spin labelled on the terminal sugar residue has been synthesized, and employed in model membranes to study headgroup behaviour of complex uncharged glycolipids. The labelled headgroup demonstrated a high degree of motional freedom limited to the aqueous region of the interface between lipid bilayer and surrounding medium. This observation was unaltered by the presence of a dense, tightly-bound surface layer of peripheral proteins or polysaccharide--which might be expected to reproduce conditions present at a cell surface. Headgroup dynamics were only very modestly correlated with the physical state (i.e., fluidity) of the membrane itself. In spite of the absence of charged sugar residues in globoside, the aspects of its headgroup behaviour monitored here we found to be similar to those of oligosaccharide chains on gangliosides and several sialic acid-rich glycoproteins.

Animals↗

PubMed1980

Ganglioside headgroup dynamics.

Gangliosides, spin-labelled specifically on N-acetylneuraminic acid residues or on random-headgroup sugars, have been used to extend previous studies of headgroup behaviour. Headgroup sugar mobility is seen to be homogeneous and relatively unrestricted in a range of systems including three lines of cultured cells. The effects of temperature and pH have been considered. Binding of small quantities of the lectin, wheat germ agglutinin, was found to increase average headgroup mobility for gangliosides in lipid bilayers, most likely as a result of a disordering effect on ganglioside clusters.

Animals↗