Separation of submicron bioparticles by dielectrophoresis.

Submicron particles such as latex spheres and viruses can be manipulated and characterized using dielectrophoresis. By the use of appropriate microelectrode arrays, particles can be trapped or moved between regions of high or low electric fields. The magnitude and direction of the dielectrophoretic force on the particle depends on its dielectric properties, so that a heterogeneous mixture of particles can be separated to produce a more homogeneous population. In this paper the controlled separation of submicron bioparticles is demonstrated. With electrode arrays fabricated using direct write electron beam lithography, it is shown that different types of submicron latex spheres can be spatially separated. The separation occurs as a result of differences in magnitude and/or direction of the dielectrophoretic force on different populations of particles. These differences arise mainly because the surface properties of submicron particles dominate their dielectrophoretic behavior. It is also demonstrated that tobacco mosaic virus and herpes simplex virus can be manipulated and spatially separated in a microelectrode array.     

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion.     

The electric unit size of thylakoid membranes.

The size of the function unit of electrical events in thylakoid membranes was estimated by the minimum amount of gramicidin needed to discharge the flash light generated electrical potential difference. Early flash spectroscopic measurements have indicated that a single gramicidin dimer operates on an electrical function unit containing at least 2 x 10(5) chlorophyll molecules. In this study we present gramicidin titrations with more intact thylakoid preparations which revealed a more than hundred-fold greater lower limit for the electric unit size, namely 5 x 10(7) chlorophyll molecules. It is conceivable that the whole complicated thylakoid structure inside a chloroplast constitutes a single electric unit. It comprises more than 2 x 10(8) chlorophyll molecules in an area of more than 400 microns 2.     

High electric field effects on the cell membranes of Halicystis parvula

The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically induced cell-to-cell fusion. Using the charge pulse technique, the membranes were charged at different pulse lengths to the maximum voltage V_c. Because of a reversible, high-conductance state of the membrane (electrical breakdown), it was not possible to exceed the critical membrane breakdown potential. The breakdown voltage exhibited a strong dependence on the charging time (pulse length) between 10 s and 100 s. Below 10 s the breakdown voltage of the two membranes, tonoplast and plasmalemma, assumed a constant value of about 1.9 V, whereas above a pulse length of about 100 s the breakdown voltage was nearly constant with a value of about 0.6 V. The extreme values for the breakdown voltage at very short and at very long charging times agree fairly well with results which have been obtained on cells of Valonia utricularis and planar lipid bilayer membranes. However, the pulse length dependence of the breakdown voltage was found to be quite different in H. parvula. In addition, the membrane conductance increase during breakdown in H. parvula cells is much more pronounced than in membranes of V. utricularis, but similar to lipid bilayer membranes. From this result it is suggested that the membrane structure of H. parvula is quite different from V. utricularis (larger lipid domains).     

60 Hz electric field parameters associated with the perturbation of a eukaryotic cell system

Summary Roots ofPisum sativum were exposed for seven days to 60 Hz electric fields ranging from 70–430 V/m in an aqueous medium whose conductivity was approximately 0.07 mho/m. (Corresponding current densities in the exposure medium associated with these field strengths ranged from 0.5–3.0 mA/cm²). Control and exposed roots were grown concomitantly in the same tank whose growth medium was continuously circulated. Temperature in the exposure medium was held at a constant 19° C. All experiments were conducted double blind. Root growth rates were determined daily. No perturbations in root growth were observed with electric fields of 150 V/m; there was a slight effect at 360 V/m, and a pronounced decrease in growth rate occurred at 430 V/m. Root conductivities are comparable to that of the growth medium. Under conditions in which growth inhibition occurs, it is estimated that induced 60 Hz cell membrane potentials would be of the order of 3–8 mV.     

Liposomal membranes. XIV. Fusion of liposomal membranes induced by polyisoprenoids as monitored by fluorescence quenching method

Fusion of the single-walled liposomes of egg phosphatidylcholine as induced by the polyisoprenoids such as solanesol, trans-ethyl decaprenoate (EDP), coenzyme Q10, and dolichol has been investigated adopting the fluorescence quenching method. Relative efficiency of the polyisoprenoids employed on the induced fusion of liposomes was a sequence of solanesol less than or equal to EDP much less than CoQ10, dolichol, which was consistent with the result previously obtained by the dye-release method.     

Electroporation of cell membranes.

Electric pulses of intensity in kilovolts per centimeter and of duration in microseconds to milliseconds cause a temporary loss of the semipermeability of cell membranes, thus leading to ion leakage, escape of metabolites, and increased uptake by cells of drugs, molecular probes, and DNA. A generally accepted term describing this phenomenon is "electroporation." Other effects of a high-intensity electric field on cell membranes include membrane fusions, bleb formation, cell lysis... etc. Electroporation and its related phenomena reflect the basic bioelectrochemistry of cell membranes and are thus important for the study of membrane structure and function. These phenomena also occur in such events as electric injury, electrocution, and cardiac procedures involving electric shocks. Electroporation has found applications in: (a) introduction of plasmids or foreign DNA into living cells for gene transfections, (b) fusion of cells to prepare heterokaryons, hybridoma, hybrid embryos... etc., (c) insertion of proteins into cell membranes, (d) improving drug delivery and hence effectiveness in chemotherapy of cancerous cells, (e) constructing animal model by fusing human cells with animal tissues, (f) activation of membrane transporters and enzymes, and (g) alteration of genetic expression in living cells. A brief review of mechanistic studies of electroporation is given.     

Interfacial electric polarizability of purple membranes in solution.

Chloroplasts in higher magnetic fields align with their equatorial plane perpendicular to the field. Because of the nonrandom orientation of the chromophores in the membrane the fluorescence radiation will be partially polarized. The chloroplast concentration, magnetic field, and temperature dependence of the fluorescence polarization has been investigated. The results are compared with a simplified model calculation. It is shown that the concentration dependence can be related to the linear dichroism of the fluorescence radiation and self-adsorption. Taking these effects into account results in the calculation of a higher fluorescence polarization (FP) ratio and higher inclination of chlorophyll dipoles to the membrane plane. Analyzing the magnetic field dependence of the FP ratio, we conclude that in a magnetic field not only will be chloroplasts be aligned, but the thylakoid stacks as well. A decrease in the FP ratio was observed around 20 degrees C. It is suggested that this decrease reflects a phase transition in the photosynthetic membrane.     

Determination of the DNA-synthesizing cell count by the immunofluorescence method

In DNA-synthesizing cells DNA is partially single-stranded. Anti-thymidine antibodies, while specifically reacting with this DNA, form a complex which may be revealed using indirect immunofluorescent technique. A comparative determination of DNA-synthesizing cell number in tumor tissue (larynx squamous cell carcinoma) was performed using immunofluorescent technique and radioautography. The former method showed the labeling index (LI) to vary from 1.2 to 9.9%, while the latter showed it to vary from 1.0 to 8.2%. The correlation ratio between the LI values obtained by the two techniques was 0.79. To eliminate a possible reaction of anti-thymidine antibodies with cellular RNA, specimens were preincubated in solutions with RNAase. No more than 6 hours were required to stain specimens using this LI estimation technique. This investigation allows to reveal DNA synthesizing cells not only in the periphery of a histological section, as does routinely radioautography, but also in its centre.     

An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.     

Permeability changes induced by electric impulses in vesicular membranes

Summary Electric impulses were found to cause transient permeability changes in the membranes of vesicles storing biogenic amines. Release of catecholamines induced by electric fields (of the order of 20 kV/cm and decaying exponentially with a decay time of about 150 sec) was studied, using the chromaffin granules of bovine adrenomedullary cells as a vesicular model system. Far-UV-absorption spectroscopy was applied to determine the amount of catecholamines released from suspended vesicles. A polarization mechanism is suggested for the induction of short-lived permeability changes caused by electric fields. Such transient changes in permeability may possibly represent a part of the sequence of events leading to stimulated neurohumoral secretion.     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current.

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.     

Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.     

Inhibition of specific effector T cell-target cell conjugates by cell plasma membranes.

Conjugate formation between effector T cells raised in vivo in a BALB/c hose had fluorescein diacetate-labeled allogeneic EL4 tumor target cells was observed and scored by using a fluorescence mirroscope. This conjugate formation was found to be specifically inhibited by plasma membrane vesicles isolated from the EL4 cells.     

Effect of electric field gradients on lipid monolayer membranes.

Externally applied nonuniform electric fields can strongly affect thermodynamic phases in a lipid monolayer when applied under conditions of temperature, pressure, and composition that are near phase boundaries. Under such conditions nonuniform applied fields can produce or suppress phase separations. Field-induced phase-separated domains have sizes that are in good agreement with calculations. Field gradients can also produce large concentration gradients in binary mixtures just above their critical points. The present work elaborates our earlier studies of these field effects using thermodynamic models of the phase behavior of two-component liquid mixtures. The calculations are of interest in connection with biological membranes that, at the growth temperature, are in a liquid state close to a phase boundary.     

Studies of electric capacitance of membranes

Summary The electric capacitance and conductance of a model membrane composed of a hydrophobic filter paper and a synthetic lipid analogue, i.e., dioleylphosphate, immersed in an electrolyte solution were observed with various frequencies ranging from 20 to 3×10⁶ Hz. With successive increase of salt concentration in the external solution, the capacitance and conductance of the membrane increased discontinuously at a certain critical value of the external salt concentration. This variation of the capacitance and conductance of the membrane with the salt concentration was found to be reversible, and the critical value of salt concentration was independent of the adsorbed quantity of the lipid, and of the pore size of the filter paper as far as the adsorbed quantity of the dioleylphosphate was large.A theoretical analysis based on the membrane model for the filter paper-phospholipid system proposed in Part I of this series revealed that the dioleylphosphate impregnated in the filter paper changed its conformation from oil droplets or globular micelles to a number of bilayer membranes when the salt concentration reached the critical value for a given pair of electrolyte species and the membrane. The conformational change of the lipid analogue in the filter paper is discussed in connection with the ability of formation and stability of a black bilayer membrane of the dioleylphosphate.