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R M Epand

Publications and source records attributed to R M Epand.

At least 145 records · Page 8Linked to original sources
PubMed1992

Structure-activity study and design of multidrug-resistant reversal compounds by a computer automated structure evaluation methodology.

We have studied the relation between the structure and the multidrug resistance-reversal activity of a set of diverse chemicals with the MULTICASE structure-activity program. A number of key structural features were identified as being related to multidrug resistance reversal activity. Using these key features, we identified seven new compounds predicted to have substantial activity. These were obtained and tested experimentally on a CHO/CHRC5 cell line derived from the AB1 Chinese hamster ovary line in the presence of vincristine and vinblastine. Of the seven compounds tested so far, four showed substantial reversal activity, the most potent of them exhibiting activity at par with verapamil.

Animals↗
PubMed1992

Zwitterionic amphiphiles that raise the bilayer to hexagonal phase transition temperature inhibit protein kinase C. The exception that proves the rule.

We synthesized the zwitterionic amphiphile cholesterylphosphorylethylpyridinium. This substance activated protein kinase C (PKC) in a micelle-based assay, but in a vesicle assay it was inhibitory. An analog of this compound, in which the pyridine ring is saturated and the nitrogen methylated, showed similar behaviour with PKC. Replacing cholesterol by an aliphatic alcohol lowered the extent of activation in the micelle assay. These results demonstrate that, with some membrane additives, the vesicle and micelle assays give opposite results. Results from the membrane-based vesicle assay for PKC are in accord with the generalization that zwitterionic amphiphiles that raise the bilayer to hexagonal phase transition temperature in model membranes are inhibitors of PKC.

Cholesterol Esters↗
PubMed1992

On the mechanism of inhibition of viral and vesicle membrane fusion by carbobenzoxy-D-phenylalanyl-L-phenylalanylglycine.

The mechanism by which the hydrophobic peptide Z-D-Phe-L-PheGly inhibits membrane fusion was investigated. Differential scanning calorimetry, 2H nuclear magnetic resonance (NMR), and 13C NMR of phosphatidylcholine bilayers in the presence of Z-D-Phe-L-PheGly indicate that this hydrophobic peptide penetrates the phospholipid bilayer but does not strongly perturb the properties of phosphatidylcholine bilayers with a large effective radius of curvature. However, Z-D-Phe-L-PheGly does affect the properties of highly curved phosphatidylcholine bilayers. Small, sonicated vesicles (SUV) of dipalmitoylphosphatidylcholine (DPPC) were made in the presence and absence of Z-D-Phe-L-PheGly. At pH 4.5, the presence of Z-D-Phe-L-PheGly inhibited the formation of SUV. At pH 6.7 and 7.4, SUV could form. Once sonication ceased and the vesicles were incubated at 23 degrees C, modest growth in vesicle size occurred for pure DPPC SUV. Rapid change to large sheetlike structures occurred in the presence of Z-D-Phe-L-PheGly. Z-D-Phe-L-PheGly appeared to favor the formation of phospholipid structures with large radii of curvature. In these experiments, Z-D-Phe-L-PheGly had access to both sides of the bilayer. If Z-D-Phe-L-PheGly was added to preformed DPPC SUV (and thus present initially only in the outer leaflet of the vesicle bilayer) and incubated at 23 degrees C, only modest growth in vesicle size was observed with little difference from control values at short to intermediate incubation times.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Hexagonal phase forming propensity detected in phospholipid bilayers with fluorescent probes.

The fluorescence emission spectrum of N epsilon-dansyl-L-Lys undergoes a marked blue shift when incorporated from aqueous solution into phospholipid bilayers. This shift is greater for membranes composed of dipalmitoleoylphosphatidylcholine than for membranes of dipalmitoleoylphosphatidylethanolamine. With the latter but not the former lipid, the fluorescence emission from N epsilon-dansyl-L-Lys is markedly temperature-dependent. The marked temperature dependence of N epsilon-dansyl-L-Lys fluorescence in bilayers of dipalmitoleoylphosphatidylethanolamine is greatest as the sample is heated close to the bilayer to hexagonal phase transition temperature. The fluorescence emission properties of another probe of membrane surface hydrophobicity, Laurdan, also exhibit marked changes at temperatures just below the bilayer to hexagonal phase transition temperature. At these temperatures, the generalized polarization begins to increase rather than decrease with temperature, and the emission intensity decreases markedly. Such effects are not observed over the same temperature range with phosphatidylcholine. Thus, both dansyl-L-lysine and Laurdan provide probes to measure changes in the physical properties of membrane bilayers which occur when these bilayers are heated close to the temperature required for transition to the hexagonal phase.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Hydrophobic lipid additives affect membrane stability and phase behavior of N-monomethyldioleoylphosphatidylethanolamine.

The rate of formation of high-curvature intermediates or disordered cubic phases in N-methyldioleoylphosphatidylethanolamine (N-methyl-DOPE) dispersions with or without additives was studied by 31P NMR spectroscopy. In N-methyl-DOPE dispersions, both the L alpha liquid-crystalline phase and the hexagonal HII phase convert into phases of high curvature giving rise to isotropic 31P NMR resonances. Addition of the bilayer destabilizers 1,2-diolein, 1,3-diolein, or eicosane lowers the threshold temperature of the isotropic phase. The isotropic threshold temperature is strongly correlated with the L alpha-HII phase transition temperature (TH). The addition of hexagonal phase promoters does not change the rate of formation of the isotropic phase at a temperature shifted by a fixed amount below TH. However, the formation of "isotropic" phases from the additive-stabilized hexagonal phase is slow compared to that observed in pure N-methyl-DOPE lipid dispersions. Membrane leakage and fusion are promoted by the dioleins and well as by eicosane, but changes in the rates of these processes do not correlate well with the extent of formation of isotropic phases. All three additives have similar effects on phase behavior and on vesicle leakage and fusion. These similarities occur despite the fact that eicosane is believed to partition differently into the membrane than diolein. In addition to the general similarities in the effects of the two diolein isomers, 1,2-diolein is somewhat more potent in promoting the hexagonal phase and in increasing rates of leakage and fusion than is 1,3-diolein.

Alkanes↗
PubMed1992

Peptide models for the membrane destabilizing actions of viral fusion proteins.

The fusion of enveloped viruses to target membranes is promoted by certain viral fusion proteins. However, many other proteins and peptides stabilize bilayer membranes and inhibit membrane fusion. We have evaluated some characteristics of the interaction of peptides that are models of segments of measles and influenza fusion proteins with membranes. Our results indicate that these models of the fusogenic domains of viral fusion proteins promote conversion of model membrane bilayers to nonbilayer phases. This is opposite to the effects of peptides and proteins that inhibit viral fusion. A peptide model for the fusion segment of the HA protein of influenza increased membrane leakage as well as promoted the formation of nonbilayer phases upon acidification from pH 7-5. We analyze the gross conformational features of the peptides, and speculate on how these conformational features relate to the structures of the intact proteins and to their role in promoting membrane fusion.

Amino Acid Sequence↗
PubMed1992

Formation of a new stable phase of phosphatidylglycerols.

Dilauroyl and dimyristoylphosphatidylglycerol (DMPG) form a more stable gel state when aqueous suspensions are incubated several days at low temperature (0-2 degrees C), pH 7.4 with 0.15 M NaCl. This gel state is characterized by a higher transition temperature and a higher transition enthalpy. The geometry of this gel state is distinguishable from the metastable gel state that forms rapidly upon hydration on the basis of its x-ray diffraction pattern. Infrared spectra in the CH2 scissoring region indicate that the stable gel phase of DMPG is also characterized by reduced reorientational fluctuations of acyl chains and increased interchain interactions. Analysis of vibrational bands due to ester carbonyl groups of DMPG suggests that the transition to a new gel phase is initiated by changes in the interfacial and/or headgroup region of the bilayer, most likely via formation of interlipid hydrogen bonds. The melting of the stable gel phase of DMPG is accompanied by a gross morphological change resulting in vesiculation.

Calorimetry, Differential Scanning↗
PubMed1992

Fatty-acid chain tilt angles and directions in dipalmitoyl phosphatidylcholine bilayers.

X-ray diffraction has been applied to determine the various tilt angles and directions (if any) which can be assumed by oriented gel phase multilayers of dipalmitoyl phosphatidylcholine (DPPC) as a function of hydration. We report for the first time that oriented DPPC multilayers with a repeat spacing (d-spacing) of 55.2A at 25 degrees C and 0% relative humidity (RH) have hydrocarbon chains tilted at an angle theta of 21.5 degrees with respect to the bilayer normal. In addition, the chains are tilted along one of the bisectors (omega = 0 degrees) of the hexagonal lattice (8 wide-angle maxima, 2 unique), a phase not previously reported in DPPC studies. At 100% RH, the chain tilt angle and d-spacing increased to approximately 29.0 degrees and 58.9A, respectively. Since at 100% RH only 4 wide-angle maxima are observed, we analyze the data on the assumption that the hydrocarbon chains may rotate independently of the hexagonal lattice (omega = 0-30 degrees), at a fixed chain tilt angle theta (Stamatoff, J.B., et al. 1979. Biophys. J. 25:253-262). The largest observed angle phi made by the wide-angle maxima with the equator is 29.5 degrees corresponding to a theta of approximately 32.6 degrees (omega avg. = 24 degrees) and the sample having a d-spacing of 64.0 A (excess water condition). Finally, theta remains relatively constant (approximately 21.5 degrees) up to a RH of approximately 45% and a d-spacing of 57.8A, after which, with increases in RH, theta increases to a maximum of 32.6 degrees.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

A comparison of membrane properties and composition between cell lines selected and transfected for multi-drug resistance.

Cell lines selected (CHRC5) and transfected (LR-73-1A) for multi-drug resistance have total lipid compositions which are indistinguishable between resistant and parental cells. Lipid composition was evaluated by 1H NMR and the total fatty acid content by GLC. No change in surface hydrophobicity, as measured with the fluorescent probe dansyl-PE, was observed as a result of transfection of CHO cells with the mdr1 gene. However, the selected cell line, CHRC5, showed a decreased surface hydrophobicity. This decreased surface hydrophobicity was indicated by an 8 nm increase in the fluorescence emission of dansyl-PE in the CHRC5 cell line compared with the AB1. Both resistant cell lines showed an increase in the polarisation of the fluorescent probe, TMA-DPH in the plasma membranes corresponding to a 14% and a 24% change in fluorescence polarisation for the selected and transfected cell lines, respectively. This is indicative of reduced mobility of the acyl chains in the resistant cell lines. Both the CHRC5 and the transfected cell lines showed almost a 2-fold increase in the initial rate of membrane cycling. The membrane cycling could be inhibited by a known bilayer stabiliser, the N-carbobenzoxy-D-Phe-L-Phe-Gly. These results indicate that the properties of the plasma membrane from resistant cells are altered compared with their parental cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Effects of the 'fusion peptide' from measles virus on the structure of N-methyl dioleoylphosphatidylethanolamine membranes and their fusion with Sendai virus.

31P nuclear magnetic resonance spectroscopy (31P-NMR) was used to study phospholipid organization in hydrated preparations of N-methyl dioleoylphosphatidylethanolamine and a 'fusion peptide' with the sequence: FAGV-VLAGAALGVAAAAQI, which corresponds to the amino terminus of the F1 subunit of the membrane fusion protein of measles virus. These amino acids are believed to mediate syncytia formation, host-cell penetration and hemolysis by infectious virus. The presence of the peptide at 0.5 mole percent significantly facilitated the formation of isotropic 31P resonances. The effects at 1 mole percent peptide were substantially enhanced over the effects observed at 0.5 mole percent, leading to a decrease in the onset temperature of the formation of the isotropic 31P-NMR resonances by about 30 degrees C. The formation of such isotropic 31P-NMR resonances has been previously associated with an increased rate of fusion of large unilamellar vesicles composed of N-methyl dioleoylphosphatidylethanolamine. Enhanced fusion of octadecyl rhodamine-labelled Sendai virus with N-methyl dioleoylphosphatidylethanolamine large unilamellar vesicles was observed when the 'fusion peptide' was incorporated into the large unilamellar vesicles.

Amino Acid Sequence↗
PubMed1991

Mimicking the membrane-mediated conformation of dynorphin A-(1-13)-peptide: circular dichroism and nuclear magnetic resonance studies in methanolic solution.

The structural requirements for the binding of dynorphin to the kappa-opioid receptor are of profound clinical interest in the search for a powerful nonaddictive analgesic. These requirements are thought to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13)-peptide, Tyr1-Gly2-Gly3-Phe4-Leu5-Arg6-Arg7-Ile8-Arg9-Pro10- Lys11-Leu12-Lys13. Schwyzer has proposed an essentially alpha-helical membrane-mediated conformation of the 13 amino acid peptide [Schwyzer, R. (1986) Biochemistry 25, 4281-4286]. In the present study, circular dichroism (CD) studies on dynorphin A-(1-13)-peptide bound to an anionic phospholipid signified negligible helical content of the peptide. CD studies also demonstrated that the aqueous-membraneous interphase may be mimicked by methanol. The 500- and 620-MHz 1H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13)-peptide in methanolic solution were sequence-specifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive COSY (DQF-COSY), relayed COSY (RELAY), and nuclear Overhauser enhancement spectroscopy (NOESY). 2-D CAMELSPIN/ROESY experiments indicated that at least the part of the molecule from Arg7 to Arg9 was in an extended or beta-strand conformation, which agreed with deuterium-exchange and temperature-dependence studies of the amide protons and analysis of the vicinal spin-spin coupling constants 3JHN alpha. The results clearly demonstrated the absence of extensive alpha-helix formation. chi 1 rotamer analysis of the 3J alpha beta demonstrated no preferred side-chain conformations.

Cell Membrane↗
PubMed1991

Action of insulin in rat adipocytes and membrane properties.

Several small peptides inhibit insulin-promoted glucose uptake in rat adipocytes. At 10 microM peptide concentration, the extent of their inhibition of the insulin effect is related to the ability of these peptides to raise the bilayer- to hexagonal-phase transition temperature in model membranes. Hexane and DL-threo-dihydrosphingosine lower this phase transition temperature in model membranes, and they promote glucose uptake in adipocytes. There is thus an empirical relationship between the action of membrane additives on glucose uptake in adipocytes and their effect on the hexagonal-phase-forming tendency in model membranes. The most potent of the bilayer-stabilizing peptides tested in this work is carbobenzoxy-D-Phe-L-Phe-Gly. This peptide also inhibits insulin-stimulated protein synthesis in adipocytes. In contrast, DL-threo-dihydrosphingosine stimulates protein synthesis. The uptake of [125I]iodoinsulin by adipocytes is inhibited by carbobenzoxy-D-Phe-L-Phe-Gly. The mechanism of action of the bilayer-stabilizing peptides includes inhibition of insulin-dependent protein phosphorylation in adipocytes. The peptides are not specific inhibitors of a single function but are suggested to cause their effects by altering the physical properties of the membrane in a nonspecific manner. These results demonstrate that insulin-dependent functions of rat adipocytes can be modified by membrane additives in a manner predictable from the properties of these additives in model membranes.

Adipose Tissue↗
PubMed1991

Evidence for the regulation of the activity of protein kinase C through changes in membrane properties.

We measured the effects of two branched-chain analogs of distearoyl-phosphatidylcholine, containing either a methyl or an n-butyl group at the 8 position, on the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine. The former compound raised the bilayer to hexagonal phase transition temperature while the latter compound lowered it. The opposite effects of these amphiphiles on protein kinase C activity (inhibition and activation, respectively) correlated with their effects on lipid polymorphism. Because of the similarity of the structures of these two compounds, it seems likely that their opposite effects on the activity of protein kinase C is a result of their alteration of the lipid environment of the membrane rather than to binding to a specific site on the protein. We also compared the effects of hexachlorophene on lipid polymorphism and protein kinase C activity at high and at low calcium concentrations. We also found that the effect of hexachlorophene forming a complex with Ca2+ is to increase both the hexagonal phase forming propensity of the membrane as well as to increase the activity of protein kinase C, again demonstrating the correlation between lipid phase propensity and effects on protein kinase C activity.

Animals↗
PubMed1991

Reversal of intrinsic multidrug resistance in Chinese hamster ovary cells by amiloride analogs.

A number of amiloride analogs can sensitise wild type Chinese Hamster ovary (CHO) cells to the cytotoxic action of vinblastine, daunomycin, puromycin or colchicine. Some of these analogs also have weak sensitising effects on the multidrug resistant CHO cell line, CHRC5. The unusual feature of most of the active amiloride analogs is that they are more potent in reversing the intrinsic multidrug resistance (MDR) phenotype of CHO cells than their acquired MDR characteristic. Human HeLa cells that do not exhibit intrinsic MDR are not affected by these agents. Several of the amiloride analogs have a greater effect in increasing adriamycin uptake in wild type CHO cells than they do with CHRC5 cells. The differential effect of amiloride analogs on intrinsic versus acquired MDR characteristics of Chinese hamster cells suggests some differences in the underlying resistance mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Autoradiographic localization of human calcitonin sensitive binding sites in rat brain.

Using 125I-salmon calcitonin (sCT) as a ligand, in vitro autoradiography of rat brain outlined specific anatomical localization of human calcitonin (hCT) sensitive binding sites. The results presented herein show that there are hCT sensitive binding sites in the ventral part of the lateral septum among the sCT specific binding sites distributed throughout the diencephalon.

Animals↗
PubMed1990

Thermodynamics of the binding of human apolipoprotein A-I to dimyristoylphosphatidylglycerol.

The interaction of human serum apolipoprotein A-I with dimyristoylphosphatidylglycerol was analyzed by isothermal titration calorimetry. Binding of the apolipoprotein A-I to large unilamellar vesicles of dimyristoylphosphatidylglycerol, a negatively charged phospholipid, is characterized by thermodynamic parameters which are invariant over the 30-40 degrees C temperature range. The enthalpy change resulting from the first additions of lipid are positive and decline in magnitude with subsequent additions of lipid. After several additions of lipid, the sign of the enthalpy changes to negative and then reaches a constant value/injection. This exothermic process is larger and opposite in sign to the heat of dilution. Similar behavior is also observed when the lipid is in the form of a dispersion in distilled water. Only a non-saturable exothermic process is observed at 30 degrees C with large unilamellar vesicles of the zwitterionic lipid, dimyristoylphosphatidylcholine. The beginning of an exothermic process can also be observed prior to the larger endotherm in the first injections of large unilamellar vesicles of dimyristoylphosphatidylglycerol into the protein. We analyze the enthalpy changes for the reaction of dimyristoylphosphatidylglycerol with the protein as arising from two distinct processes, one endothermic and the other exothermic. The binding isotherms for the high affinity binding of the apolipoprotein A-I to large unilammelar vesicles of dimyristoylphosphatidylglycerol, over the temperature range 30-40 degrees C, gave an enthalpy change of 1.43 +/- 0.07 kcal/mol of protein and a free energy change of -5.91 +/- 0.04 kcal/mol of protein for the binding of the protein to a cluster of 25 +/- 2 lipid molecules. Thus this reaction is entropically driven.

Apolipoprotein A-I↗
PubMed1990

Dimerization of the P-glycoprotein in membranes.

Plasma membranes from a CHO cell line, CHRC5, which exhibits multidrug resistance was studied using radiation inactivation analysis. The P-glycoprotein content of the membrane was determined by Western blots. Irradiation resulted in the loss of P-glycoprotein. The dependence of this loss on radiation dose corresponded to a target size of 250 kDa which is the molecular mass of a dimer of the P-glycoprotein. This is strong evidence to indicate that the P-glycoprotein self associates in the membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗