Decreased membrane deformability in Melanesian ovalocytes from Papua New Guinea.

We examined the ability of Melanesian ovalocytes from Papua New Guinea to be deformed in order to probe the resistance of these cells to invasion by several species of malaria parasite. We found ovalocytes were refractile to drug-induced endocytosis, that they formed abnormal rouleaux, showed reduced deformability when aspirated into 0.6-micron diameter pores in polycarbonate sieves, and failed to crenate when mounted under a glass coverslip. No substantial differences were found between normocytes and ovalocytes in their initial rate of filtration through 4.5-micron pore polycarbonate sieves, their membrane fluidity as measured by the rate of depolarization of fluorescent probes or the rate of extraction of cytoskeletal proteins in low ionic strength buffers. We conclude that the resistance of ovalocytes to undergo localized deformation might be significant in explaining the resistance of these cells to invasion by malarial merozoites.

Vasopressin analogs that antagonize antidiuretic responses by rats to the antidiuretic hormone.

Four new synthetic analogs of vasopressin (antidiuretic hormone) can antagonize the antidiuretic response to intravenous vasopressin in anesthetized, water-loaded rats. They also cause a diuresis resembling that of diabetes insipidus when given intraperitoneally to conscious rats. Such antagonists may prove to be useful both pharmacologically and therapeutically.

Cytosine arabinoside influx and nucleoside transport sites in acute leukemia.

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [(3)H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx (<0.4 pmol/10(7) cells per min) and NBMPR binding (<3,000 sites/cell) for the treated group. In summary, myeloblasts have both higher araC transport rates and more nucleoside transport sites than lymphoblasts and this factor may contribute to the greater sensitivity of AML to this drug. AraC transport varied >10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC.

Cytosine arabinoside transport and metabolism in acute leukemias and T cell lymphoblastic lymphoma.

Cytosine arabinoside (araC) has proven efficacy in acute myeloid leukemia (AML), but its place in the treatment of acute lymphoblastic leukemia (ALL) and T lymphoblastic lymphoma is uncertain. The therapeutic potential of araC has been assessed in patients with AML, ALL, and T lymphoblastic lymphoma by measuring the conversion of araC to its active metabolite, the 5'-triphosphate of araC (araCTP), in purified blasts from patients as well as in normal polymorphs and lymphocytes. In all leukemias, araCTP was the major intracellular metabolite of araC. The highest araCTP formation was in blasts from T lymphoblastic lymphoma, which formed threefold more nucleotide than myeloblasts, and in turn myeloblasts formed twofold more araCTP than lymphoblasts from ALL. The mean araCTP formation in myeloblasts was sixfold greater than polymorphs, but in contrast, lymphoblasts and lymphocytes formed low and similar amounts of this nucleotide. Reasons for the sixfold range in araCTP accumulation in the various leukemic blasts were studied. The mean size of myeloblasts was 35-70% larger than lymphoblasts when compared on the basis of protein or intracellular water content, but T lymphoblastic lymphoma blasts and lymphoblasts were the same size. Activities of deoxycytidine kinase, deoxycytidylate deaminase, and pyrimidine nucleoside monophosphate kinase were not different between any of the leukemic cell types. The number of nucleoside transport sites on blasts was estimated by measuring the equilibrium binding of [3H]nitrobenzylthioinosine (NBMPR), which binds with high affinity to the transporter. Scatchard analysis yielded mean values of 27,500 sites/cell for T lymphoblastic lymphoma blasts, 10,000 sites/cell for myeloblasts, and 2,300 sites/cell for lymphoblasts. Our previous work has shown that araC influx correlates with the maximum number of 3H-NBMPR binding sites in leukemic and normal white cells. A strong correlation was observed between the number of nucleoside transport sites per leukemic blast cell and the accumulation of intracellular araCTP from extracellular araC at 1 microM. Membrane transport of araC at the low concentrations (approximately 1 microM), which are achieved therapeutically, is a major rate-limiting step in its conversion to araCTP by leukemic blast cells. Myeloblasts form more araCTP than lymphoblasts because of both higher nucleoside transport capacity and larger cell size. The highest nucleoside transport capacity and largest conversion of araC to araCTP is in T lymphoblastic lymphoma, which suggests that araC may be effective in the treatment of this disease.

Oriented circular dichroism of a class A amphipathic helix in aligned phospholipid multilayers.

The effect of lipid phase state on the orientation and conformation of a class A alpha-helical peptide on aligned lipid multilayers was examined using oriented circular dichroism spectroscopy. A comparison of oriented spectra in aligned peptide-lipid multilayers with CD spectra of unaligned peptide lipid vesicle complexes is consistent with a preferential alignment of helices parallel to the membrane surface at temperatures above and below the main acyl-chain melting transition temperature of the phospholipid. Changes are observed in the oriented CD spectra with lipid phase state which are attributed to a subtle conformational change of the peptide on the lipid surface. The results are compared with available experimental data on membrane-active lytic and antimicrobial helical peptides.

Effect of cholesteryl ester on the distribution of fluorescent cholesterol analogues in triacylglycerol-rich emulsions.

Lipid emulsions consisting of a surface monolayer of phospholipid enclosing a core of neutral lipid (cholesteryl ester and/or triacylglycerol) are useful models of the lipid phase of lipoproteins. The physical state of the emulsion surface may determine the extent and nature of interaction of enzymes and lipid transfer proteins (e.g., lipoprotein lipase, cholesteryl ester transfer protein) with the particle. Unesterified cholesterol, which is a major determinant of the physical state of the surface phase, is able to partition between surface and core compartments. This report describes a fluorescence quenching method which determines the equilibrium distribution of a fluorescent cholesterol analogue (dehydroergosterol) between the surface and core compartments of triacylglycerol-rich emulsions. Quenching by iodide is used to distinguish a pool of unesterified cholesterol readily accessible to the aqueous phase. Quenching by 5-nitroxystearate identifies a pool of unesterified cholesterol in the phospholipid monolayer and the pool of unesterified cholesterol in the core compartment is found by difference. It is shown that the substitution of cholesteryl oleate for triolein in the core of the emulsion substantially increases the partition of unesterified cholesterol into the core compartment with a consequent depletion of unesterified cholesterol in the surface monolayer. The distribution of unesterified cholesterol between surface and core compartments is largely enthalpically driven.

Properties and the locations of a set of fluorescent probes sensitive to the fluidity gradient of the lipid bilayer.

The synthesis and properties of a set of four fluorescent probes (n-(9-anthroyloxy) fatty acids, n = 2, 6, 9, 12) sensitive to the fluidity gradient of the lipid bilayer are described. Fluorescent quenching experiments show that the probes locate at a graded series of depths in the bilayer. A fifth probe, methyl-9-anthroate, locates near the bilayer centre. As an example of their application, the probes are used to study the phase transitions of dipalmitoyl phosphatidyl-choline. Changes in the rotational relaxation times of the probes across the transitions are more pronounced at the centre of the bilayer than at the surface.

Depth-dependent fluorescent quenching of a tryptophan residue located at defined positions on a rigid 21-peptide helix in liposomes.

Five lipophilic 21-peptide analogs of the potential-dependent pore-former, alamethicin, were synthesized bearing tryptophan residues at the position 1, 6, 11, 16 and 21 on a long, conformationally rigid, alpha-helix. The alpha-helical conformation was induced and stabilized using the sequential oligomers (Ala-Aib-Ala-Aib-Ala)n as analyzed by CD and NMR. The partitioning of the N-t-butoxycarbonyl 21-peptide methyl esters and the N-terminally deprotected alpha-helices was followed by fluorescence enhancement in phospholipid bilayer vesicles. Quenching experiments were performed by titrating with n-doxyl stearic acids bearing the nitroxide label at positions 5, 7, 10, 12 and 16. This well-defined system revealed that the N- and C-terminal tryptophan residues become situated in the hydrophilic region. Tryptophan at position 11 was found in the lipophilic core, whereas the tryptophan at positions 6 and 16 were localized at intermediate depths of the lipid membrane. Therefore, the helices span the lipid bilayer with their long axis normal to the membrane surface.

The association of acrylamide with proteins. The interpretation of fluorescence quenching experiments.

The association properties of acrylamide with a number of proteins in aqueous solution have been investigated by a fluorescence-quenching method previously used in micelles and lipid bilayers (Blatt, E., Chatelier, R.C. and Sawyer, W.H. (1984) Chem. Phys. Lett. 108, 397-400). At pH 7.0, acrylamide partitions between the bulk aqueous phase and the proteins, human serum albumin, monellin and ovalbumin. Comparison with an earlier method of analysis (Sikaris, K.A., Thulborn, K.A. and Sawyer, W.H. (1981) Chem. Phys. Lipids 29, 23-36) confirms the data quantitatively. For human serum albumin at pH 2.2, acrylamide associates according to both partition and binding processes. Equilibrium dialysis experiments performed for the latter system verify that acrylamide associates with proteins.

Perturbation of phospholipid bilayers by DDT.

The localization of the effects of DDT (5-50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23 degrees C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54 degrees C or egg PC at 23 degrees C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.

Structural organisation of band 3 in Melanesian ovalocytes.

The diffusional freedom of human erythrocyte band 3 (anion exchanger 1) has been measured in membranes from normocytic and ovalocytic erythrocytes. A dramatic reorganisation of band 3 in the ovalocyte membranes is indicated by a markedly restricted rotational mobility. Extraction of spectrin from erythrocyte membranes had no effect on normocyte band 3 mobility, but partially relieved the restrictions on ovalocyte band 3 mobility. Further removal of ankyrin and band 4.2 resulted in an increase in the rotational mobility of both ovalocyte and normocyte band 3 to similar levels. The results suggest that the molecular basis of the unusual shape and decreased deformability of ovalocytes resides in an altered interaction of band 3 with one or more of the peripheral proteins. We present a model which illustrates a possible role for band 3 aggregation in controlling erythrocyte deformability.

Rotational dynamics of erythrocyte spectrin.

The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere and rigid rod. The data indicate that the ribbon-like spectrin molecule possesses considerable torsional and segmental flexibility. These motions are restricted, but not abolished, when spectrin is reconstituted into cross-linked cytoskeletal protein networks, or bound to spectrin-actin depleted erythrocyte membrane vesicles.

Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli.

Elongation factor Tu from Escherichia coli is known to polymerize at slightly acidic pH and low ionic strength. The structure and dynamics of these aggregates have been examined using imaging and spectroscopic methodologies. Electron microscopy provides evidence for two-dimensional sheets and bundled filaments of EF-Tu, whereas fluorescence microscopy of EF-Tu covalently labeled with tetramethylrhodamine isothiocyanate showed highly branched polymers of EF-Tu several microns in diameter. These polymers were studied using quasi-elastic light scattering to determine the evolution of the translational diffusion coefficient during the polymerization process. The rotational dynamics of the aggregate were investigated using phosphorescence anisotropy of EF-Tu covalently labeled with erythrosin isothiocyanate. A high infinite-time anisotropy was observed, suggesting a lack of motion or entanglement of EF-Tu polymers. A sub-microsecond motion which was slowed in the presence of glycerol may be due to local flexibility of the polymers. The possible relevance of polymeric EF-Tu to its function in vivo is discussed.

The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipid bilayers.

A set of n-(9-anthroyloxy) fatty acid probes (n = 2, 6, 9, 12) have been used to examine gradients in fluorescence polarization, lifetime (tau F), relative quantum yield (phi rel) and positions of emission maxima (lambda max) through bilayers composed of synthetic phospholipids. The fluorophores of these probes report the environment at a graded series of depths from the surface to the centre of the bilayer structure. 1. Polarizations decrease as the fluorophore is moved deeper into the bilayer indicating greater rotational motion of the fluorophore in the hydrocarbon core of the bilayer. 2. The different responses of the probe diphenylhexatriene and the anthroyloxy fatty acids to the action of cholesterol on lipid bilayers are discussed in terms of the orientation of these probes in the bilayer and the types of anisotropic rotational motions which result in depolarization of fluorescence. 3. Stearic acid derivatives which have the fluorophore in the 6-, 9- and 12-positions along the acyl chain have a similar response to solvent polarity as measured by values of lambda max and phi rel in a variety of organic solvents. 4. The position of the emission maximum has little dependence on solvent viscosity, but viscosity does change the degree of vibrational structure seen in the emission spectrum. The vibrational structure itself may be used as an indication of the 'mciroviscosity' gradient in the transverse plane of the bilayer. 5. Values of lambda max, tau F and phi rel indicate that a gradient of polarity exists from the surface to the centre of the bilayer. For dipalmitoyl phosphatidylcholine in the crystalline phase, cholesterol acts to make this polarity gradient shallower.

Interaction of immunologically-active lipopeptides with membranes.

Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators. In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells. Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent. In addition, capping and patching was observed. Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment. These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it. The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells.

Synthesis and characterisation of fluorescent oligonucleotides. Effect of internal labelling on protein recognition.

Fluorescently labelled 42 base pair DNA duplexes were synthesised to examine the interaction between the TyR repressor protein of Escherichia coli and its DNA recognition sequence. An Fmoc-protected 5-(3-aminoprop-l-yn-l-yl)-2'-deoxyuridine phosphoramidite was synthesised and incorporated into oligonucleotides using standard beta-cyanoethyl phosphoramidite chemistry. Oligonucleotides containing the 3-aminopropynyl nucleotide at internal positions were reacted with fluorescein isothiocyanate to generate fluorescent DNA molecules useful for characterising interactions between DNA and proteins. Short DNA duplexes were investigated with respect to their melting temperatures and their ability to bind TyrR. Oligonucleotides containing a TyrR binding site were labelled in the central region of the recognition sequence or near the 5' edge of the recognition sequence. Fluorescein-labelled oligonucleotides could hybridise to form duplex DNA, and gel retardation experiments showed that the presence of the dye did not alter the binding affinity for the TyrR protein significantly. Fluorescence anisotropy measurements were used to examine the binding equilibrium in low and high salt buffers. A dissociation constant of 200-500 nM was obtained for the interaction of the TyrR dimer with a 42 bp duplex containing a centrally located 22 bp TyrR binding site.

Response of rat heart membranes and associated ion-transporting ATPases to dietary lipid.

The effects of different dietary fat intake on the lipid composition and enzyme behaviour of sarcolemmal (Na+ + K+)ATPase and sarcoplasmic reticulum Ca2+-ATPase from rat heart were investigated. Rat diets were supplemented with either sunflower seed oil (unsatd./satd. 5.6) or sheep kidney fat (unsatd./satd. 0.8). Significant changes in the phospholipid fatty acid composition were observed in both membranes after 9 weeks dietary lipid treatment. For both membranes, the total saturated/unsaturated fatty acid levels were unaffected by the dietary lipid treatment, however the proportions of the major unsaturated fatty acids were altered. Animals fed the sunflower seed oil diet exhibited an increase in n-6 fatty acids, including linoleic (18:2(n-6] and arachidonic (20:4(n-6] while the sheep kidney fat dietary rats were higher in n-3 fatty acids, principally docosahexaenoic (22:6), with the net result being a higher n-6/n-3 ratio in the sunflower seed oil group compared to sheep kidney fat dietary animals. Fluorescence polarization indicated that the fluidity of sarcoplasmic reticular membrane was greater than that of sarcolemmal membrane, with a dietary lipid-induced decrease in fluidity being observed in the sarcoplasmic reticular membrane from sheep kidney fat dietary animals. Despite these significant changes in membrane composition and physical properties, neither the specific activity nor the temperature-activity relationship (Arrhenius profile) of the associated ATPases were altered. These results suggest that with regard to the parameters measured in this study, the two ion-transporting ATPases are not modulated by changes which occur in the membrane lipid composition as a result of the diet.

Perturbations to lipid bilayers by spectroscopic probes as determined by dielectric measurements.

Dielectric measurements on lecithin/cholesterol bimolecular lipid membranes have indicated that the series of extrinsic fluorescent probe molecules, the n-(9-anthroyloxy) fatty acids, cause significant perturbation to the bilayer structure at concentrations equivalent to those used in fluorescence experiments (0.1 mol%). Perturbations were observed in the capacitance and conductance of the electrically distinct substructural regions of the bilayer that were consistent with the putative location of the probe molecules. Inclusion of stearic acid decreased the thickness of the hydrocarbon region of the membrane, presumably by expanding the average surface area per unit membrane mass, and also significantly disrupted the surface regions. The attachment of the anthroyloxy moiety to position 2 of a fatty acid accentuated both these effects. Attachment at position 12 had the reverse effect by increasing the volume of the hydrocarbon region without further disturbance of the surface organisation. The 9-positioned probe had an intermediate effect. The degree of perturbation by the 2-positioned probe was dependent on the probe concentration within the range (probe:lipid) 1:1000 to 1:10 000. The technique therefore detects perturbation of structure at probe levels which are lower than those commonly used in fluorescence-labelling experiments.

A spectroscopic and equilibrium binding analysis of cationic detergent-protein interactions using soluble and insoluble recombinant porcine growth hormone.

Overexpression of cloned eukaryote genes in bacteria often leads to the formation of insoluble refractile bodies which require solubilization by harsh denaturants or detergents. We describe the conformational changes associated with the binding of a surfactant, cetyltrimethylammonium chloride (CTAC) to recombinant porcine growth hormone (PGH). The stoichiometry of binding by CTAC to the soluble and insoluble forms of recombinant PGH was also assessed. Optimum CTAC binding and protein solubilisation were obtained at 50 degrees C and at extreme pH. Increased ionic strength and changes in pH towards the isoelectric point of PGH (pH 6) decreased both the binding of CTAC and the efficiency of solubilising PGH from inclusion bodies. The positive charge on the quaternary ammonium head group of CTAC was found to be critical in the binding of CTAC to PGH and for the subsequent solubilisation of inclusion bodies. The binding of CTAC to the soluble form of PGH caused appreciable changes to the tertiary structure of the protein but did not significantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that urea, guanidine hydrochloride and CTAC solubilized recombinant PGH molecules behave differently during in vitro refolding (Puri, N.K., Crivelli, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Brandon, M.R. (1992) Biochem. J. 285, 871-879.).

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula.

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-3H]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin 1. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin 1 using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an alpha-helical secondary structure.