Electrical characterizations of biomimetic molecular layers on gold and silicon substrates.

Electrical impedance technology was used to characterize DNA recognition in a monolayer containing single-stranded DNA probes immobilized on a gold substrate using thiol self-assembly chemistry. Recognition of targeted complementary DNA was principally correlated with an eight-fold increase in the conductance of the monolayer and attributed to electron conduction through double helices formed upon the binding of the DNA targets to the probes. The high recognitive sensitivity was possible without the use of the redox labels or large bias voltages required for recognition using cyclic and Osteryoung square wave voltammetry. The impedance technology also provided atomic resolution of a hybrid bimolecular lipid membrane formed by deposition of a phospholipid:cholesterol monolayer onto a hydrophobic alkyl monolayer covalently attached to a silicon substrate via silicon-carbon bonds. Atomic resolution was achieved through preparation of membranes on surfaces approaching atomic flatness and the performance of impedance measurements over precisely defined areas of the surface in contact with solutions. Principally capacitive properties distinguished between the immobilized (octadecyl) and more fluidic (lipid:cholesterol) leaflets of the hybrid membrane. The lipid:cholesterol leaflets were structurally similar to those leaflets in free-standing bimolecular lipid membranes. The hybrid membrane therefore provides a highly stable and physiologically relevant surface for studying biomolecular interactions with membrane surfaces.

Adaptation of cell lines to serum-free culture medium.

The optimum conditions to allow proliferation of cells for the secretion of some growth factors and cytokines and the proliferation of cells in different media that do not contain proteins or serum from animals (serum-free media) were investigated. The culture of cell lines for the commercial production of products involves optimisation of cell proliferation and secretion in media from which the requisite proteins can be economically extracted. Some of these problems were addressed in this study. We used two different clones from a human myeloma cell line for adaptation to serum free medium in order to characterize long-term effects of the new medium. We gradually decreased serum content of medium and the results showed that cell mortality increased with serum reduction, antibody production lost by survived clones, and secretion of cytokines were always retained.

The physics of cell membranes.

An overview is given of the fundamental physics underlying the self-assembly, molecular organisation and electrical properties of the membranes that envelop living cells. These ultra thin (∼ 6 nm) membranes act as a diffusion barrier between the cell interior (cytoplasm) and the external medium. They consist basically of a bi-molecular film of lipid molecules in which are embedded functional proteins that perform a variety of functions, including energy transduction, signalling, transport of ions (and othermolecules), etc. Some examples are also presented of the fascinating and socially and commercially important applications of membrane biophysics.

Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae.

Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane. The predominant oxidation product accumulated was identified as 5alpha, 6alpha-epoxy-(22E)-ergosta-8,22-dien-3beta,7a lpha-diol (8-DED). 9(11)-dehydroergosterol (DHE) was identified as a minor oxidation product. In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE. Disrupted cell preparations of S. cerevisiae convert EEP to 8-DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity. Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation. The following reaction pathway is proposed: singlet oxygen-mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8-DED. Ergosterol 7-hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE. Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives. Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation.

Electrical impedance tomography study of biological processes in a single cell.

An in vivo electrical impedance tomography (EIT) study of single plant cells of Chara corallina is reported. When these aquatic cells grow in alkaline conditions, proton-translocating ATP synthases in the plasma membrane operate in reverse, utilizing ATP to translocate protons against an electrochemical gradient to the periplasm and creating localized acidic regions along the cell's cylindrical surface. These acidic regions, which appear as radial bands, approximately 5 mm long, between narrower alkaline bands, facilitate the uptake of bicarbonate, the plant's source of inorganic carbon for photosynthesis in the carbon dioxide-depleted alkaline conditions. Our EIT study of cell ultrastructure in the acidic and alkaline regions provides evidence that the plasma membrane is folded in localized regions (e.g., charasomes) in the acidic bands. The very low frequency capacitance dispersions were very similar to those of double fixed-charge structures. Such charge distributions are known to be present in the membrane-bound F0 portion of the ATP synthase. The theoretical dependence of the fixed-charge concentrations on pH in the proteins is shown to broadly account for the observed correlations between pH, membrane potential, conductance, and capacitance in these regions. In synthetically formed double fixed-charge membranes, electric field-induced dissociation of water into H+ and OH- occurs. This leads to the speculation that H+/OH- fluxes in ATP synthases located in the alkaline regions of Chara cells might also involve the electric field-induced dissociation of water.

Differential effects of cholesterol and oxidised-cholesterol in egg lecithin bilayers.

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.

Forces on biological cells due to applied alternating (AC) electric fields. II. Electro-rotation.

Measurements are presented of angular velocities of rotation of mammalian cells of K562 (human) and SP2 (mouse) in external alternating electric fields over a frequency range of 0.5 kHz to 12 MHz. Electro-rotation of the cells was observed for the case of "two cells in contact' using two parallel, cylindrical electrodes; only one cell was located on the electrode. A theoretical analysis is also presented which shows that the cell rotation arises from a torque produced by the interaction between the primary electric dipole moment induced in the spinning cell and the secondary electric fields, generated by the primary dipole induced in the adjacent cell. These secondary fields are out of phase with the applied electric field. The results show that (a) only the cell not located on the electrode rotates, (b) maximal electro-rotation occurs at two different excitation field frequency domains for the frequency range employed here, (c) the spin speed of the rotating cell at each frequency domain is much less than the excitation frequency, (d) the rotation direction of the cell depends on the angle (theta) between the external electric field and the line joining the centres of the two cells and (e) for a given angle theta, the rotation direction is the same for both excitation frequency domains. The experimental measurements allowed us to estimate the conductivities of the cytoplasms and membrane capacitances of the cells of K562 and SP2. The conductivities of the cytoplasms of the cells of K562 and SP2 were estimated to be 0.2 and 0.3 Sm-(1), respectively, whereas the membrane capacitances of these cells were found to be 2.7 +/- 0.8 and 9.8 +/- 0.6 mFm-(2), respectively.

Forces on biological cells due to applied alternating (AC) electric fields. I. Dielectrophoresis.

Measurements are presented of dielectrophoretic forces for SP2 (mouse) and K562 (human) cells in external alternating electric fields over a frequency range of 10 kHz to 2 MHz. Using a spherical shell model of the cell, the dielectrophoretic force is derived from the interaction between the induced electric dipole moment in the cell and the external electric field. The frequency dependence of the force has its origin in the dispersion with frequency of the impedances of the cell membrane, the cytoplasm and the external medium (a Maxwell-Wagner dispersion). The predicted tri-phasic form of the variation of the dielectrophoretic force is in good agreement with the experimental results presented. Using the theoretical model, the experimental measurements also provided an estimation of 0.18 +/- 0.03 S m-1 and 0.12 +/- 0.04 S m-1 for the conductivities of the cytoplasm of cells of SP2 and K562, respectively, and 6.0 +/- 2.0 mF m-2 and 2.0 +/- 1.0 mF m-2 for the capacitances of the plasma membrane of these cells.

The effect of benzyl alcohol and cholesterol on the acyl chain order and alkane solubility of bimolecular phosphatidylcholine membranes.

An investigation was made of the effects of cholesterol and benzyl alcohol on the partitioning of n-alkanes between lipid bilayer membranes and bulk lipid/alkane solutions (in the torus). Bilayers were formed from solutions containing alkanes of different chain lengths, together with phosphatidylcholine and cholesterol in varying proportions. The partitioning of the alkanes was determined from measurements of the very low frequency (1 Hz) capacitance of the membranes. Perturbation of the internal membrane structure by the inclusion of cholesterol and benzyl alcohol produced very significant changes in the n-alkane partition coefficient, cholesterol causing a decrease and benzyl alcohol an increase in the alkane partitioning into the bilayer. A correlation exists between the effects of these compounds on the alkane partitioning and their effect on the segmental chain order of the acyl chains in the bilayer and this correlation is consistent with a statistical-mechanical model of the lipid/alkane bilayers in the liquid crystalline state. The perturbation by cholesterol and benzyl alcohol of the internal structure of membranes bears on the conflicting reports of the effects of these substances on artificial lipid bilayers and could also be relevant to their known physiological effects.

The effect of temperature on lipid-n-alkane interactions in lipid bilayers.

The partitioning of n-alkanes between an egg-phosphatidylcholine bilayer and its torus was studied for alkanes with ten to sixteen carbon atoms using measurements of membrane capacitance. The partition coefficient was found to decrease with increasing alkane chainlength and to increase with increasing temperature. This is consistent with a well-known statistical model of lipid alkane bilayers in the liquid crystalline state. It was found that n-decane was unsuitable as a solvent in these experiments because significant partitioning of n-decane into the aqueous phase and atmosphere occurred and this could not be adequately controlled. Egg-phosphatidylcholine bilayers containing negligible amounts of solvent could be produced using a method similar to the 'freeze-out' method of White (Biochim. Biophys. Acta 356 (1974) 8-16). Bilayers formed using n-hexadecane were found to be virtually solvent-free at temperatures below 30 degrees C. The partition coefficient of n-alkanes in the bilayer was also found to depend on the alkane mole fraction. Thus it was concluded that the assumption of ideal mixing between acyl and alkane chains in the bilayer was not valid when the alkane mole fraction exceeded 40% (with respect to the acyl chains of the lipid). The variation of the standard chemical potential with temperature was measured for alkanes of different chainlengths and it was concluded that the enthalpy and entropy of the alkanes in the bilayer are in themselves temperature-dependent. This indicates that the state of the hydrophobic interior of lipid bilayers varies with temperature.

Effect of 2H2O/H2O replacement on the dielectric structure of lipid bilayer membranes.

Dielectric dispersion measurements were made on planar egg phosphatidylcholine and egg phosphatidylcholine/cholesterol bilayer membranes in H2O and 2H2O electrolyte solutions. The properties of the dielectrically distinct substructural layers of these membranes, which can be characterised by this technique, were found to be insensitive to replacement of H2O by 2H2O.

The molecular organisation of bimolecular lipid membranes. The dielectric structure of the hydrophilic/hydrophobic interface.

Improvements to a previously described very low-frequency impedance-measuring technique have now allowed the characterisation of a third, electrically distinct, type of substructural region in phosphatidylcholine biomolecular lipid membranes. This region was found to have properties intermediate to those of the hydrophobic (hydrocarbon) layer and the regions containing the polar heads of the phosphatidylcholine molecules. Its properties are consistent with it being associated with the oxygen-rich carboxyl ester portions of the phosphatidylcholine molecules which lie at the hydrophilic/hydrophobic interface. We will refer to these regions in the membrane as the acetyl regions. The individual properties of the three distinct types of region in the phosphatidylcholine membranes were determined at KCl electrolyte concentrations of 1, 10, 100 and 1000 mM. It was found that with increasing KCl concentration: (a) The capacitance, CH, of the hydrophobic region increased slightly, indicating a decrease in the thickness of this region. (b) The conductance, GH, of this hydrophobic region increased by a factor of 20 in going from 1 to 1000 mM electrolyte. (c) The capacitance of the acetyl region was independent of KCl concentration although its conductance increased 5-fold over the range 1-1000 mM KCl. (d) The volume-specific electrical properties of the region containing the polar heads appeared to be essentially independent of KCl concentration. However, a change in thickness of these regions was observed which was consistent with the cholinephosphate dipole being oriented normal to the bilayer surface in 1 mM KCl and parallel to the surface in 1000 mM KCl external solutions.

Location and effect of procaine on lecithin/cholesterol membranes using X-ray diffraction methods.

X-ray diffraction studies were made on lecithin/cholesterol multilayers with very high water content and containing the local anaesthetic procaine. Narrow-angle diffraction experiments show that the procaine molecules are located with the uncharged aromatic amine group approx. 10 A from the centre of the bilayer. The polar tertiary amine group of these molecules is almost certainly located in the polar head-group region of the membrane. Wide-angle diffraction experiments show that the incorporation of procaine molecules into such lipid membranes produces an approx. 30% increase in the spread of acyl chain separation, although the average spacing between the chains is slightly reduced.

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.

The effect of pressure on the electrical breakdown in the membranes of Valonia utricularis.

The interpretation of electrical breakdown in terms of electro-mechanical instabilities, predicts that the breakdown potential should decrease with increasing cell turgor pressure. Experiments were conducted to test this hypothesis on cells of Valonia utricularis over a turgor pressure range of 0.5-10(5)-5.0-10(5) N/m2. Electrical breakdown was measured using intracellular electrodes and 500 mus current pulses. The pressure was monitored by an intracellular micropipette pressure transducer. The results obtained show a linear decrease in the critical breakdown potential with pressure. The effective compressive modulus of the cell membrane, gamma, is calculated from the slope of this line to 69+/-10-10(5) N/m2 (average value of seven measurements). This is consistent with the theoretical prediction of the electromechanical model using our previously determined values of the elastic modulus of the membrane. A theoretical analysis is given of the effects of pressure on the breakdown, This includes also considerations of the indirect effect of pressure on the membrane via stretching of the cell wall with a possible coupling of such strains to the cell membrane. The results and analysis presented allow us to conclude on the basis of the experimentally determined breakdown P.K. of 959 mV that the region of membrane where electrical breakdown occurs is a dielectric with one of the following combinations of parameters: (A) a thickness delta=7-9 nm with a dielectric constant epsilon=greater than 10, e.g. a hydrated protein spanning the whole membrane. (B) delta=4-5 nm with epsilon=3-8, e.g. a lipoprotein of lipid bilayer dimensions. (C) delta approximately 2 nm with epsilon=2-3, e.g. a half lipid bilayer. If we assume that the breakdown P.D. of the tonoplast and plasmalemma are identical, that is 480 mV, then there is only one reasonable choice for the membrane thickness and the dielectric constant: delta=2 nm, epsilon=3-8, e.g. a (lipo) proteinaceous module facing a half life lipid bilayer.

Turgor pressure sensing in plant cell membranes.

Experimental evidence is reviewed which shows that the cell membrane is compressible by both mechanical and electrical forces. Calculations are given which show that significant changes in the thickness of cell membranes can occur as a result of (a) direct compression due to the turgor pressure; (b) indirect effects due to the stretching of the cell wall; and (c) the stresses induced by the electric field in the membrane.Such changes in the membrane thickness may provide the pressure-transducing mechanism required for osmoregulation and growth. An important feature of the model is that this pressure transduction can occur not only in the plasmalemma (where there is a pressure gradient), but also in the tonoplast.