Semiconductor-controlled contour-clamped homogeneous electric field apparatus

The design and construction of a transistor-driven hexagonal contour-clamped homogeneous electric field (CHEF) apparatus is discussed in detail. The addition of computer control of pulsed-field timings and experiment duration gives rise to an efficient electrophoresis tool designed to achieve separation of DNA molecules in different size groupings. In particular, pulse time regimes which lead to the monotonic separation of DNA molecules ranging from 90 kbp to over a megabase pair are demonstrated. Theoretical treatment of electric field clamping with transistor-driven multiple electrodes is supported by measurements and by the actual performance of electrophoretic separation of yeast chromosomes. The large sample capacity of gels run in this apparatus coupled with the modest power requirements necessary to provide a homogeneous electric field offer significant advantages over earlier CHEF designs.     

Separation of large DNA by a variable-angle contour-clamped homogeneous electric field apparatus.

A device for separating large DNA molecules by pulsed field electrophoresis is described. Based on the principles of contour-clamped homogeneous electric fields (CHEF), it uses feedback to clamp voltages in a square electrode array, which is compact and inexpensive to construct, adaptable to computer control, and reorients the electric field by arbitrary angles. To illustrate its capabilities, pulsed fields with reorientation angles ranging from 90 to 140 degrees were used to separate DNAs of 4.7 and 5.7 megabases by up to four band-widths in 20 h. The combination of accessible technology and complete control of the electric field should facilitate the search for ways to resolve even larger DNA.     

Postimplantation development of tetraploid mouse embryos produced by electrofusion

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell 'fused' embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5-14 days p.c.     

Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and digested with a restriction endonuclease. Adjustment of the electrophoresis conditions enables the separation of DNA fragments with lengths from 10 kilobases up to 9 megabases in a size-dependent manner in agarose gels. The banding patterns can be used for epidemiological typing, the separated DNA can be immobilized onto a membrane and used for genetic mapping, or individual fragments can be extracted and used for downstream genetic manipulations. The protocol requires specialized equipment and can be completed in a maximum of 7 days.     

Measurement of the electric field of the heart in a homogeneous volume conductor]

The paper describes a technique and some results of experimental measurements of electrical potentials generated by an isolated dog heart in homogeneous conductor, drawing equipotential maps of the field, and calculating the characteristics of the dipole equivalent generator of the heart. The form of potential distribution on a spherical surface around the heart and its ideal orthogonal vectorcardiograms are discussed.     

Multiple Vibrio vulnificus strains in oysters as demonstrated by clamped homogeneous electric field gel electrophoresis.

Clamped homogeneous electric field gel electrophoresis and a computer program for managing electrophoresis banding patterns (ELBAMAP) were used to analyze genomic DNA of 118 Vibrio vulnificus strains, isolated from three oysters by direct plating. Analysis with SfiI resulted in 60 restriction endonuclease digestion profiles (REDP), while analysis with SrfI produced 53 different REDP. Similarities between REDP ranged from 7 to 93%. Principal-component analysis showed that the strains were heterogeneous.     

Interspecific somatic hybrids between Solanum khasianum and S. aculeatissimum produced by electrofusion

Summary Mesophyll derived protoplasts of Solanum khasianum Clarke and S. aculeatissimum Jacq. were fused by electrofusion and resulted in interspecific hybrids. In total, 249 calluses were obtained from which more than 65% produced shoots. From the regenerated plantlets 45% could be identified as hybrids by chromosome counts (4x = 48) and esterase isozyme pattern. Most hybrids showing intermediate phenotypic characteristics, had a normal flower morphology with fertile pollen and produced fertile berries. More than 80% of the seeds germinated.Abbreviations MS Murashige and Skoog medium - BAP 6-Benzylammopurine - NAA 1-Naphthaleneacetic acid - PAGE Poly-Acrylamide Gel Electrophoresis - AC alternating current - DC direct current     

Homokaryons from animal and plant cells generated by electrofusion

A new apparatus was constructed which enables the use of the electrofusion method to obtain polynuclear cells of various mammalian cell lines, erythrocytes and plant protoplasts. This technique was applied to both suspensions and monolayers. Electrical and other physical parameters were monitored to find optimal conditions for mutual contact of cells (dielectrophoresis) and subsequent fusion. In the suspension technique, dielectrophoresis of mouse erythrocytes occurred at a field frequency of 20 kHz and a strength of 500 V.cm-1, whereas cultured mammalian cells and plant protoplasts required a frequency of 1-1.4 MHz and a strength of 250-800 V.cm-1. Fusion of cells was induced after the application of 1 to 10 high-voltage pulses of 1-5 kV.cm-1, 10-36 microseconds duration. After these high-voltage pulses were to the monolayer of mouse L cells, about 12% viable homokaryons were obtained.     

Determination of electric field threshold for electrofusion of erythrocyte ghosts. Comparison of pulse-first and contact-first protocols.

Rabbit erythrocyte ghosts were fused by means of electric pulses to determine the electrofusion thresholds for these membranes. Two protocols were used to investigate fusion events: contact-first, and pulse-first. Electrical capacitance discharge (CD) pulses were used to induce fusion. Plots of fusion yield vs peak field strength yielded curves that intersected the field strength axis at positive values (pseudothresholds) which depended on the protocol and decay half time of the pulses. It was found that plots of pseudothreshold vs reciprocal half time were linear for each protocol; when extrapolated to reciprocal half time = 0 (i.e., t----infinity), these lines intersected the ordinate at values of the field strength considered to be the true electrofusion thresholds. In this fashion, the contact-first protocol gave an electrofusion threshold of 46.5 +/- 11.5 V/mm for hemoglobin-free ghosts (white ghosts) and 40.9 +/- 8.8 V/mm for ghosts with fractional hemoglobin (pink ghosts), while the threshold for the pulse-first protocol applied to pink ghosts was determined to be 93.4 +/- 11.0 V/mm. Although the thresholds depended on the electrofusion protocol, plots of critical field strength vs reciprocal time had the same slopes, i.e., approximately 24 Vs/mm. The results suggest that the fusogenic state induced by an electric pulse in either the contact-first protocol or the pulse-first protocol (long-lived fusogenic state) may in fact share a common mechanism, if the two states are not actually identical.     

Development and functional characterization of insulin-releasing human pancreatic beta cell lines produced by electrofusion

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.     

Electromechanical stresses produced in the plasma membranes of suspended cells by applied electric fields

Summary We analyze the electrical and mechanical stress in the bounding membrane of a cell (or vesicle) in suspension which is deformed by an external applied field. The membrane is treated as a thin, elastic, initially spherical, dielectric shell and the analysis is valid for frequencies less than the reciprocal of the charging time (i.e. less than MHz), or for constant fields. A complete analytic solution is obtained, and expressions are given which relate the deformation, the surface tension and the transmembrane potential difference to the applied field. We show that mechanical tensions in the range which lyse membranes are induced at values of the external field which are of the same order as those which are reported to lyse the plasma membranes of cells in suspension.     

Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts

Mesophyll derived protoplasts ofCatharanthus roseus cv. Little pinkie were fused with protoplasts derived from an habituated cell line ofC. roseus. Polyethylene glycol was used as agglutinating agent while fusions were induced by square pulses. Best results were obtained by fusing protoplasts from primary leaves with those from three-day-old cell cultures. Adding calcium ions considerably enhanced heterofusion rate. Good cell viabilities indicated that this fusion process was not cytotoxic. The heterofusion frequency was up to 10% or more. Most of the heterokaryons were able to regenerate their cell walls and underwent division. Communicated by J. TUPY     

Selective electrofusion of conjugated cells in flow.

Using a modified flow cytometer we have induced electrofusion of K562 and L1210 cells in flow. The two cell types are stained with two different fluorescent membrane probes, DiO and DiI, to facilitate optical recognition, and then coupled through an avidin-biotin bridge. In the flow cytometer, the hydrodynamically focused cells and cell pairs are first optically analyzed in a normal flow channel and then forced to flow through a Coulter orifice. If the optical analysis indicates that a cell pair is present, an electric pulse is applied across the orifice to induce fusion. The pulsed cell pairs were subsequently analyzed using normal and confocal microscopy to evaluate fusion induction. It appears that fusion can be induced in about 10% of pulsed cell pairs when one electric pulse with a duration of 10-15 microseconds and an effective electric field strength of 4-8 10(5) V/m is used.     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

Direct experimental evidence of the vectorial character of the interaction between electric pulses and cells in cell electrofusion

Fusion is induced between cultured mammalian cells growing on culture flasks by electric pulses. The yield of fusion and the level of polynucleation are dependent on the relative direction of the applied pulses. The effect of the pulse on the cells is thus proved to be vectorial and not scalar.     

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

Summary Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.     

Oscillations of electric spatial patterns emerging from the homogeneous state in characean cells

Electric spatial patterns of bands formed along the cell wall of the characean internode were studied using a multi-electrode measuring system. The electric potential near the surface of the cell was measured by arranging about 25 electrodes along the cell at approximately 1.6 mm intervals. Since the time required for one scan over the cell length is only 1 s, the temporal change in the spatial pattern of surface electric potential can be readily observed. Oscillations were sometimes found as the electric pattern started to appear after the cell was illuminated. Fourier analysis shows that a single spatial mode arises gradually and then becomes stabilized in an oscillatory manner. A simple electric circuit model comprising three variables, i.e., a membrane potential, an electric current across the membrane and an electromotive force, can simulate well the oscillatory rise of bands. These results imply that the electric spatial pattern observed in characean internodes is a self-organized structure emerging far from equilibrium, known as a dissipative structure. Biophysical mechanisms of band formation are discussed.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

Modeling the electric image produced by objects with complex impedance in weakly electric fish

Weakly electric fish generate an electric field around their body by electric organ discharge (EOD). By measuring the modulation of the electric field produced by an object in the field these fish are able to accurately locate an object. Theoretical and experimental studies have focused on the amplitude modulations of EODs produced by resistive objects. However, little is known about the phase modulations produced by objects with complex impedance. The fish must be able to detect changes in object impedance to discriminate between food and nonfood objects. To investigate the features of electric images produced by objects with complex impedance, we developed a model that can be used to map the electric field around the fish body. The present model allows us to calculate the spatial distribution of the amplitude and phase shift in an electric image. This is the first study to investigate the changes in amplitude and phase shift of electric images induced by objects with complex impedance in wave-type fish. Using the model, we show that the amplitude of the electric image exhibits a sigmoidal change as the capacitance and resistance of an object are increased. Similarly, the phase shift exhibits a significant change within the object capacitance range of 0.1–100 nF. We also show that the spatial distribution of the amplitude and phase shifts of the electric image resembles a “Mexican hat” in shape for varying object distances and sizes. The spatial distribution of the phase shift and the amplitude was dependent on the object distance and size. Changes in the skin capacitance were associated with a tradeoff relationship between the magnitude of the amplitude and phase shift of the electric image. The specific range of skin capacitance (1–100 nF) allows the receptor afferents to extract object features that are relevant to electrolocation. These results provide a useful basis for the study of the neural mechanisms by which weakly electric fish recognize object features such as distance, size, and impedance.     

Nonresonant excitation of surface waves by a normal electric field

A study is made of nonresonant parametric excitation of surface waves by a spatially uniform, time-dependent electric pump field directed perpendicular to a plane plasma-dielectric interface. A set of equations is derived that describes the dynamics of surface wave excitation. Expression for the growth rate in the linear stage of instability is obtained, and the threshold amplitude of the external electric field above which the parametric instability can occur is found. The spectrum of the excited waves is analyzed. Published in Russian in Fizika Plazmy, 2006, Vol. 32, No. 11, pp. 994–998. The article was translated by the author.