New electric field methods in chemical relaxation spectrometry.

New stationary relaxation methods for the investigation of ionic and dipolar equilibria are presented. The methods are based on the measurement of non-linearities in conductance and permittivity under high electric field conditions. The chemical contributions to the nonlinear effects are discussed in their static as well as their dynamic behavior. A sampling of experimental results shows the potential and range of possible applications of the new techniques. It is shown that these methods will become useful in the study of nonlinear responses to perturbation, in view of the general applicability of the experimental principles involved.     

The effects of external electric field on the electron transport system of spinach chloroplasts

Since the thylakoid membranes of an active chloroplast are constantly exposed to the electric fields generated by the electron transport system inside the membranes, we have studied the effects of pretreating chloroplasts of spinach ( Spinacia oleracea L.) leaves with an external AC (alternating current) electric field on their electron transport system. It was found that a few minutes electric field pretreatment (333 V cm^-1 across chloroplast samples), especially at low frequency, irreversibly inhibited the activity of photosystem II (PSII), but under certain conditions, stimulated that of photosystem I (PSI). From the measurements of fluorescence from PSII, we ascribe the inhibition to a lesion close to its reaction center P680, leading to increased dissipation of excitation energy to heat. The effect on PSI was investigated by the reduction of its reaction center, P700 by various artificial donors. We suggest that the stimulative effect can be attributed to a positive shift of the surface charge density of thylakoid membranes that brings about an increase in the accessibility of exogenous electronegative donors.     

External electric field effects on prompt and delayed fluorescence in chloroplasts

An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233–240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulated luminescence supported the interpretation given above.Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.     

Exposure scheme separates effects of electric shock and electric field for honey bees, Apis mellifera L

Mechanisms to explain disturbance of honey bee colonies under a 765-kV, 60-Hz transmission line [electric (E) field = 7 kV/m] fall into two categories: direct bee perception of enhanced in-hive E fields, and perception of shock from induced currents. The same adverse biological effects previously observed in honey bee colonies exposed under a 765-kV transmission line can be reproduced by exposing worker bees to shock or E field within elongated hive entranceways (= tunnels). Exposure to intense E field caused disturbance only if bees were in contact with a conductive substrate. E-field and shock exposure can be separated and precisely defined within tunnels, eliminating dosimetric vagaries that occur when entire hives are exposed to E field.     

Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells

We have conducted experiments quantitatively investigating electroporative uptake kinetics of a fluorescent plasma membrane integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 μs pulsed electric fields (PEFs). These experiments were possible through the use of calibrated, real-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell cultures to intense PEFs while carrying out real-time microscopy. A finite-element electrostatic simulation was carried out to assess the degree of electric field heterogeneity between the microcuvette's electrodes allowing us to correlate trends in electroporative response to electric field distribution. Analysis of experimental data identified two distinctive electroporative uptake signatures: one characterized by low-level, decelerating uptake beginning immediately after PEF exposure and the other by high-level, accelerating fluorescence that is manifested sometimes hundreds of seconds after PEF exposure. The qualitative nature of these fluorescence signatures was used to isolate the conditions required to induce exclusively transient electroporation and to discuss electropore stability and persistence. A range of electric field strengths resulting in transient electroporation was identified for HL60s under our experimental conditions existing between 1.6 and 2 kV/cm. Quantitative analysis was used to determine that HL60s experiencing transient electroporation internalized between 50 and 125 million nucleic acid-bound PI molecules per cell. Finally, we show that electric field heterogeneity may be used to elicit asymmetric electroporative PI uptake within cell cultures and within individual cells.     

Determination of the induced ELF electric field distribution in a two layer in vitro system simulating biological cells in nutrient solution

In-vitro studies of biological effects of electromagnetic fields are often conducted with cultured cells either in suspension or grown in a monolayer. In the former case, the exposed medium can be assumed to be homogeneous; however, eventually the cells settle to the bottom of the container forming a two layer system with different dielectric and conductive properties. In the present work the effect of this separation on the electric field distribution is calculated and experimentally measured at selected positions for a commonly used exposure configuration. The settled cell suspension is modeled by a well-defined two layer system placed in a rectangular container with the base of the container parallel to the direction of the magnetic field. Theoretical calculations based on numerical techniques are done for various two layer systems with different conductivities in each layer. The agreement between the theoretical calculations and the experimental measurements is within ± 1.5 mV/m, or 10% of the maximum induced field when the conductivity of the lower layer is ten times that of the upper layer. This result is well within experimental error. When the thickness of one of the layers is small compared to the thickness of the other layer, it is found that the electric field distribution is essentially that of the homogeneous case. The latter situation corresponds to a typical cell exposure condition. © 1993 Wiley-Liss, Inc.     

Polyelectrolyte-based enzymatic diagnosticum with precipitation detection system for urea assay in solution

A polyelectrolyte-based enzymatic diagnosticum with a precipitation detection system that can be used as a biosensor was created. The detection method was based on the change in polyelectrolyte microcapsule weight with respect to the urea content. The possibility of biosensor reutilization was demonstrated. The appropriate ionic precipitator causing precipitation of insoluble carbonate within the microcapsules and the optimal microcapsule titre were found. In the solution of monovalent anions (chlorides), the activity of encapsulated urease was shown to increase monotonically as the square root of the ionic strength depending on the elevation of the salt content. The activity drastically increased in a narrow concentration interval (0.6–0.8 mM) of divalent anions (sulfates) and reached the level of the native enzyme activity.     

Superweak effects of chemical compounds and physical factors on biological systems

The results of extensive investigations of the responses of living systems to superweak influences are summarized. The results of the effect of ultralow concentrations of various biologically active substances and ultraweak physical fields (primarily electromagnetic) on biological systems of different level of organization, from molecular to population, were considered. Several single-type regularities peculiar to all systems were revealed: polymodal dose dependences, the efficiency of influences at doses below the background level and its dependence on the state of the system, the modification of the sensitivity of a system to subsequent influences, etc. Several hypotheses of the mechanisms of superweak influences and the role of water as a universal intermediate in these processes are presented. Possible consequences of the phenomenon and its applications in practice are discussed.     

Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells

Nanosecond pulsed electric fields (nsPEFs) are ultrashort pulses with high electric field intensity (kV/cm) and high power (megawatts), but low energy density (mJ/cc). To determine roles for p53 in response to nsPEFs, HCT116 cells (p53+/+ and p53-/-) were exposed to nsPEF and analyzed for membrane integrity, phosphatidylserine externalization, caspase activation, and cell survival. Decreasing plasma membrane effects were observed in both HCT116p53+/+ and p53-/- cells with decreasing pulse durations and/or decreasing electric fields. However, addition of ethidium homodimer-1 and Annexin-V-FITC post-pulse demonstrated greater fluorescence in p53-/- versus p53+/+ cells, suggesting a postpulse p53-dependent biological effect at the plasma membrane. Caspase activity was significantly higher than nonpulsed cells only in the p53-/- cells. HCT116 cells exhibited greater survival in response to nsPEFs than HL-60 and Jurkat cells, but survival was more evident for HCT116p53+/+ cells than for HCT116p53-/- cells. These results indicate that nsPEF effects on HCT116 cells include (1) apparent direct electric field effects, (2) biological effects that are p53-dependent and p53-independent, (3) actions on mechanisms that originate at the plasma membranes and at intracellular structures, and (4) an apparent p53 protective effect. NsPEF applications provide a means to explore intracellular structures and functions that can reveal mechanisms in health and disease.     

Oxidative effects of nanosecond pulsed electric field exposure in cells and cell-free media

Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1-13kV/cm) was assessed by oxidation of 2',7'-dichlorodihydrofluoresein (H(2)DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H(2)DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2',7'-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H(2)DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H(2)O(2) in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H(2)O(2) and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.     

A spectrophotometric method for detection of 5'-fluorouracil in plasma

A quantitative method using spectrophotometry was devised for the detection of 5'-Fluorouracil (FUra) in plasma. Plasma samples obtained at different time intervals from drug treated rabbits were deproteinised using phenol. The aqueous phase was lyophilised and dissolved in 0.01 N NaOH and absorption was monitored at 305 nm. Levels of FUra in the plasma were calculated by comparing with a standard graph generated from known concentrations of FUra. As low as 1 microgram of the drug per ml of plasma could be detected by this method.     

A comparison between the coupled spectrophotometric and uncoupled radiometric assays for RuBP carboxylase

The coupled spectrophotometric assay for RuBP carboxylase was compared with the conventional radiometric assay to assess the validity of its use in the measurement of initial and total activities in crude leaf extracts. At high magnesium concentrations both assays gave the same absolute values of initial and total activities, and resolved similarly the changes of total activity and activation state (ratio of initial to total activity) which occurred when the water status and light environment of leaves was altered prior to sampling. Although the magnesium concentration supporting the maximum rate of initial activity in soybean extracts was similar in the two assays, substantial differences of initial activity were observed at sub-optimal concentrations of magnesium. At low magnesium concentrations reaction rates in the spectrophotometric assay exhibited an initial phase of non-linearity which subsequently gave way to a linear rate. In contrast, reaction rates at low magnesium were linear from the time of initiation in the radiometric assay. Inclusion of EDTA in the reaction medium did, however, induce non-linear rates in the radiometric assay. The pre-addition of RUBP to extract immediately prior to dilution into the reaction medium did not eliminate the non-linearity in either assay system. The significance of these observations is discussed briefly in relation to the use of the spectrophotometric assay.     

Alternating current electric field effects on neural stem cell viability and differentiation

Methods utilizing stem cells hold tremendous promise for tissue engineering applications; however, many issues must be worked out before these therapies can be routinely applied. Utilization of external cues for preimplantation expansion and differentiation offers a potentially viable approach to the use of stem cells in tissue engineering. The studies reported here focus on the response of murine neural stem cells encapsulated in alginate hydrogel beads to alternating current electric fields. Cell viability and differentiation was studied as a function of electric field magnitude and frequency. We applied fields of frequency (0.1–10) Hz, and found a marked peak in neural stem cell viability under oscillatory electric fields with a frequency of 1 Hz. We also found an enhanced propensity for astrocyte differentiation over neuronal differentiation in the 1 Hz cultures, as compared to the other field frequencies we studied. Published 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Determination of hydrogen sulfide in a yeast culture solution by flow analysis utilising methylene blue spectrophotometric detection

Gas diffusion and flow injection analysis with a spectrophotometric detector has been developed for the determination of sulfide in a yeast culture solution. A detection limit (signal-to-noise ratio = 3) of 0.2 M sulfide was achieved. A relative standard deviation of 3.4% (n=12) was also achieved for 0.5 M sulfide. The technique is highly sensitive, accurate and precise, with low susceptibility to interferences, and allows significant reagent and instrument economy.     

Frequency-dependent effects in an alternating electric field on an isolated frog heart

An isolated frog heart placed in a special chamber with Ringer solution (pH 7.2) was stimulated by electric field at a frequency of 0.3-4.0 Hz with graphite electrodes immersed in the solution. Unusual resonance phenomena were observed during a progressive increase or decrease of the stimulation frequency: the amplitude of the mechanical responses of the preparation rose at multiple frequencies of stimulation, whereas the frequency of the responses remained unchanged.