Optimal Electric Field Research Articles

Electrotransformation of Saccharomyces cerevisiae protoplast by a plasmid of Escherichia coli

The experiments reported here are aimed at obtaining optimum conditions for gene transformation after electric field pulses. Saccharomyces cerevisiae DBY746 is used as the recipient strain for shuttle plasmid (YRp group). From the relationship between the optimum electric field conditions and the transformation efficiency it is discovered that the maximum transformation efficiency appears at a wide pulse length of 400 μs with an electric field strength of 4 kV/cm, yielding up to 273 transformants/μg DNA. The electroporation unit used in the experiment is a home-made set featuring simplicity, readiness and practicality.

Electrotransformation of Saccharomyces cerevisiae protoplast by a plasmid of Escherichia coli

The experiments reported here are aimed at obtaining optimum conditions for gene transformation after electric field pulses. Saccharomyces cerevisiae DBY746 is used as the recipient strain for shuttle plasmid (YRp group). From the relationship between the optimum electric field conditions and the transformation efficiency it is discovered that the maximum transformation efficiency appears at a wide pulse length of 400 μs with an electric field strength of 4 kV/cm, yielding up to 273 transformants/μg DNA. The electroporation unit used in the experiment is a home-made set featuring simplicity, readiness and practicality.

High Concentration Tritium Gas Measurement with Small Volume Ionization Chambers for Fusion Fuel Gas Monitors.

To apply ionization chambers to fusion fuel gas processing systems, high concentration tritium gas was experimentally measured with small volume 0.16 and 21.6 cm3 ionization chambers. From plateau curves, the optimum electric field strength was obtained as 100–200 V/cm. Detection efficiency was confirmed as dependent on the ionization ability of the filled gas, and moreover on its stopping power, because when the range of the β-rays was shortened, the probability of energy loss by collisions with the electrode and chamber wall increased. Loss of ions by recombination was prevented by using a small volume ionization chamber. For example the 0.16 cm3 ionization chamber gave measurements with linearity to above 40% tritium gas. After the tritium gas measurements, the concentration levels inside the chamber were estimated from their memory currents. Although more than 1/4,000 of the maximum current was observed as a memory effect, the smaller ionization chamber gave a smaller memory effect.

Expression of an exogenous gene in Trypanosoma cruzi epimastigotes

We have constructed a plasmid vector system into which a segment of the Trypanosoma cruzi gene encoding the spliced leader (SL) has been inserted to drive expression of a downstream gene encoding bacterial chloramphenicol acetyltransferase (CAT). We used this construct to establish conditions that permit reproducible transfection of cultured T. cruzi epimastigotes where transfection was mediated by electroporation and measured by assaying expression of CAT. CAT activity was detected only in T. cruzi lysates from cells transfected with constructs containing a properly oriented SL gene; constructs in which the gene was inserted in reverse orientation did not express CAT. The optimal electric field strength, cell density and DNA concentration for efficient transfection were established. This and similar systems will permit genetic dissection of this parasite.

High-efficiency gene transfection by in situ electroporation of cultured cells

It is demonstrated in this study that high-efficiency gene transfection can be obtained by directly electroporating cultured mammalian cells in their attached state using a pulsed radio-frequency (RF) electric field. A plasmid DNA containing the reporter gene β- gal was introduced into COS-M6 cells and CV-1 cells using this in situ electroporation method. At the optimal electric field strength (1.2 kV/cm), we found that over 80% of the M6 cells took up and expressed the β- gal gene with a cell survival rate of about 50%. In contrast, the transfection efficiency was less than 20% when the M6 cells were electroporated in suspension. It was shown that CV-1 cells could also be electroporated highly efficiency using the in situ method. Furthermore, we have measured the time required to express the β- gal gene after the plasmid DNA was introduced. We found that the percentage of cells expressing β- gal reached a peak value about 10 h after electroporation. This time-course was the same for both attached and suspended cells, suggesting that the observed difference in transfection efficiency was mainly the result of effects of the detachment treatment on the electroporation process rather than on the gene expression.

A nonuniform electrical field electroporation chamber design

We show an inexpensive design for an electroporation chamber which subjects electroporated cells to a nonuniform electrical field. Our design, which we call an electroporation cylinder, improved transfection efficiency over that of a uniform field design (electroporation cuvettes) by about sixfold when tested in five mouse cell lines with a transient gene expression assay. Electroporation cylinders subjected cells to electrical field strengths at least as powerful as those of electroporation cuvettes, as judged by comparing the percentages of cells killed by electroporation. Cylinder and cuvette designs were similar in their effect on the variability of transfection efficiency. Electroporation cylinders may be particularly useful when the optimal electrical field strength for a cell line is not known or is unattainable with a given power supply.

Transvenous and Subcutaneous Electrode System for an Implantable Defibrillator, Improved on Large Pigs

The currently required surgical procedure for implantable defibrillator implantation is a limiting factor and several groups are therefore investigating transvenous approaches. Our electrode system consists of a nondistal right ventricular catheter electrode and two or three subcutaneously (SC) placed electrodes. An optimal location for these SC electrodes is important to obtain the lowest possible defibrillation threshold (DFT) by allowing a more homogeneous current distribution within the thorax. An empirical approach consists of placing randomly the SC electrodes to find out the lowest possible DFT. A mathematical approach is to calculate the SC electrode locations for an optimal electric field distribution by using magnetic resonance images of thorax cross-sections and a specially designed computer program. Our recent experimental results are based on a series of 15 pigs weighing between 60 and 102 Kg. DFT ranged between 10 and 26 joules. We conclude that an electrode system with a right ventricular electrode and two or three subcutaneous electrodes can be optimized to reach a DFT for pigs with human-near body weights which is compatible with the energy capabilities of our implantable device.

Capillary zone electrophoresis : Quantitative study of the effects of some dispersive processes on the separation efficiency

The contributions of diffusion, Joule heating, sampling and electroosmotic flow to the dispersion of zones separated by capillary zone electrophoresis were evaluated theoretically and verified experimentally. The theoretical model predicts that an optimum electric field strength, E, exists at which the number of theoretical plates, N, is a maximum. Both open and closed capillaries were considered and a simple formula was derived for the calculation of a maximumm with a given experimental arrangement. The theoretical efficiency curves, N vs. E, were calculated and compared with those obtained experimentally in capillaries of various I.D.'s and filled with background electrolytes of various conductivities. The possibilities of minimization of some dispersion factors are discussed.

Bovine gallbladder muscularis: source of a myogenic receptor for cholecystokinin.

Despite being a classic target for the gastrointestinal peptide hormone, cholecystokinin (CCK), the gallbladder CCK receptor is not well characterized. Pharmacological studies of small species suggest that CCK action can be mediated by direct myogenic or by both myogenic and neurogenic receptors. To prepare for the biochemical characterization of a gallbladder CCK receptor and to define the subtype of the receptor being studied, we have performed autoradiographic localization and pharmacological characterization of CCK receptors on bovine gallbladder. Autoradiography demonstrated high-affinity specific CCK-binding sites only on the muscularis. CCK-8 stimulated tonic contraction of longitudinal strips of gallbladder muscularis in a concentration-dependent manner, with an ED50 of 0.2 nM. Antagonism at the cholinergic receptor with 1 microM atropine or axonal transmission with 1 microM tetrodotoxin did not modify CCK-induced contraction, supporting a direct myogenic effect of this hormone. Optimal electrical field stimulation (10 V, 10 Hz, 500 microseconds) to elicit a neuronal response resulted in muscle strip relaxation, which was abolished with adrenergic blockade (1 microM phentolamine, 1 microM propranolol). Although acetylcholine administration stimulated contraction, electrical field stimulation did not, even in the presence of phentolamine, propranolol, and/or CCK. Thus, in bovine gallbladder muscularis, there is evidence for a functional CCK receptor only on smooth muscle cells. Demonstration of a single, high-affinity specific CCK-binding site on an enriched plasma membrane preparation of bovine gallbladder muscularis is consistent with this representing a myogenic CCK receptor.

The effect of some factors on the inhibitory nervous systems of airway smooth muscle

To understand better the physiological role of the inhibitory nervous systems (i.e. adrenergic and nonadrenergic) operating in airway smooth muscle, the roles of different physiological conditions were studied using segments of trachea isolated from male guinea pigs (300–400 g BW; Camm-Hartley stain). Laryngeal, cervical and thoracic segments of trachea were used from each animal. Platinum plate electrodes were employed to generate an optimal electrical field stimulation of 40 V, 1.5 msec, 40 PPS of 12 sec duration. Biphasic contractile response was reported as change in the isometric force (g). Adrenergic and nonadrenergic components of airway smooth muscle relaxation were assessed using, D,L-propranolol (1 × 10 −6 M), a dose which did not influence the developed isometric force. We investigated the effect of the following experimental conditions on developed active relaxation: (a) different temperatures (29, 37 and 40°C), (b) pH=7.000, 7.38 and 7.56 (all ±0.05), and (c) moderate hypoxia (PO 2=50±5 Torr). It was found that: (1) No significant differences were detected in the active relaxation in three regions of guinea pig trachea. In guinea pig trechea nonadrenergic inhibition was responsible for approximately 75–80% of total relaxation; (2) A decrease in temperature to 29°C inhibited ( P<0.05) and an increase in temperature to 40°C potentiated ( P<0.01) relaxation in both adrenergic and nonadrenergic components (3) A low pH of 7.10±0.05 inhibited ( P<0.05) and a high pH of 7.56±0.05 potentiated ( P<0.05) relaxation as induced by electrical field stimulation; (4) Acute hypoxia, P O 2 = 50 ± 5 Torr, significantly inhibited ( P < 0.01) active relaxation of tracheal preparations. This inhibition, however, was reversible. These data suggest that in the condition of acidosis and hypoxia, such as is present in patients with chronic obstructive airway disease, the alteration of the inhibitory nervous systems of airway smooth muscle (both adrenergic and nonadrenergic) may contribute to the development of airway obstruction.

In vitro actin of progesterone on myometrium: I. Reversible modulation of the resistance of rabbit uterus to excitation-contraction uncoupling.

Active isometric tension provoked in isolated uterine strips by optimal electric field stimulation is an index of sarcoplasmic Ca++ concentration or the degree of excitation-contraction coupling in the myometrium. Removal of Ca++ from the bathing medium, while maintaining electric stimulation, results in a progressive uncoupling and a consequent loss of tension. The rate at which tension is lost by the uterine strips can be influenced by progesterone. When uterine strips from rabbits at Day 26 of pregnancy are incubated in tissue culture medium containing no added progesterone, there is a time dependent reduction in resistance to uncoupling. This response can be prevented in a log-dose manner by incubation in medium containing near physiological concentrations of progesterone while nonprogestagenic C21 steroids are ineffective. Incubation without steroid for 6 h followed by addition of progesterone results in a reversal of the initial loss of resistance to uncoupling. This reversal occurs after a latent period of ∼ 4 h. Because resistance to uncoupling is related to the availability of Ca++ in the vicinity of the sarcolemma, this study demonstrates that the association between the sarcolemma and Ca++ can be altered by progesterone in vitro.

Optimal confinement of collisionless plasmas by localized time-varying electric fields†

The problem of optimal confinement of collisionless plasmas by means of external amplitude-limited time-varying electric fields localized to an outer layer enclosing the confinement region (a specified closed bounded spatial domain) is considered. To confine the plasma, the external electric field (control) establishes a finite potential barrier for the escaping particles. The field direction for confinement depends on the sign of the electrical charge of the escaping particles inside the layer. The paper begins with a formal development of the plasma equations. Then, the existence and uniqueness of solutions of these equations are established. This is followed by the formulation of the optimal confinement problem along with a discussion of the existence of an optimal electric field (control). By introducing additional assumptions to simplify the problem, sub optimal feedback controls are obtained. Finally, the effectiveness of the derived feedback controls is determined numerically using a computer-simulated plasma.