13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

Influence of a pulsed electric field on the spores and oxygen consumption of Aspergillus niger and its citric acid production

Spores of Aspergillus niger were exposed to a pulsed electric field. After treatment by the electric field, the viability of the conidia of A. niger varied depending on the field strength, pulse width and frequency. In all cases, these parameters reduced the viability rate of the conidia from 2.0 × 10⁷ to a range from 6.2 × 10⁶ to 8.5 × 10⁶ spores/ml (3.1 to 42.6%). After pulse treatment, the conidia were used as the inoculum for citric acid fermentation in shake flasks. The highest increase in citric acid yield (about 1.4-fold) was reached at a field strength of 2.85 kV/cm, a frequency of 1 Hz and a pulse width of 1 ms. When the parameters of the electric field increased there were important changes in the respiration rate of the Aspergillus niger mycelium (48-h-old) after electric shock treatment. The highest consumption of dissolved oxygen (22.9%) in the medium by Aspergillus niger mycelium was observed at an electric field strength of 2.85 kV/cm, a 1 Hz frequency, a pulse width of 1 ms and a 1-min exposure period. It seems that an electric-field stimulation of the conidia prior to inoculation may offer an important method of improving the efficiency of citric acid. The treatment of the conidia is both simple from the technical point of view and extremely rapid.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

Optimum Conditions for Electric Pulse-mediated Gene Transfer to Bacillus subtilis Cells

It was found that plasmid DNA (pUB 110) can be introduced into not only protoplasts but also intact cells of Bacillus subtilis by electric field pulses. The transformation of, B. subtilis using protoplasts results in an efficiency of 2.5 × 10⁴ transformants per μg of DNA, with a single pulse of 50 jisec with an initial electric field strength of 7kV/cm. Even transformation of intact B. subtilis cells results in a maximum efficiency of 1.5 × 10³ transformants per μg DNA, with a single pulse of 400 μsec with an initial electric field strength of 16kV/cm. The cell survival of protoplasts and intact cells was approximately 100% and 30%, respectively, under the conditions found to be optimal for the transformation process. Plasmid DNA isolated from pUB 110 containing transformants was indistinguishable from authentic preparations of pBU 110 on gel electrophoretic analysis.     

Relative stability of the α-helix of deuterated poly(γ-benzyl-L-glutamate)

The coil-to-helix transition temperatures of hydrogen bearing and deuterated poly(γ-benzyl-L -glutamate) in 1,3-dichlorotetrafluoroacetone/H₂O and/D₂O mixtures, respectively, have been determined. Together with previously obtained data for the conformational transition of this polypeptide in normal and deuterated dichloroacetic acid, these results have been used in an analysis of the effect of deuterium substitution on the intrinsic stability of the α-helical form of poly(γ-benzyl-L -glutamate). The findings, consistent for both solvent systems, showed that the deuterated polypeptide is some 5% more stable than the normal protonated poly(γ-benzyl-L -glutamate), while the polypeptide-active solvent interaction enthalpy is also slightly increased by deuterium substitution in the respective molecules. A consideration of available data for poly(β-benzyl-L -aspartate) reveals an anomaly with respect to the present analysis.     

Synthesis and conformational study of poly(N -benzyl-L-lysine) and its benzyloxycarbonyl derivative

The solvent-and pH-induced conformational changes are examined in order to investigate the influence of benzyl group. Polymer was prepared via N^?-benzyloxycarbonyl, N^?-benzyl-N^α-carboxy-L -lysine anhydride. The resulting poly (N^?-benzyloxycarbonyl, N^?-benzyl-L -lysine) was obtained in high yield and had a high molecular weight. The protected polymer was removed into poly (N^?-benzyl-L -lysine) by treating it with hydrogen bromide. From the results of the ORD and CD, the protected polymer has a righthanded α-helix, showing [m′]₂₃₃ = –10,300, [θ]₂₂₀ = –27,600 and [θ]₂₀₇ = –25,100 in dioxane. The breakdown of the helical conformation is found to occur at 8% dichloroacetic acid in chloroform-dichloroacetic acid mixture. In the pH range 3.35–6.90, poly (N^?-benzyl-L -lysine) is in a random coil structure. In the pH range 7.50–13.0, the polypeptide has a right-handed α-helix structure; [m′]₂₃₃ = –12,000, [0]₂₂₀ = –27,200, and [0]₂₀₇ = –27,000. In comparison with poly-L -lysine, the coil-to-helix transition is observed at lower pH range in 50% n-propanol. Above pH 8 by heating, the α ? β transition of poly (N^?-benzyl-L -lysine) is not observed in an aqueous media.     

Effects of pulsed electric fields on mouse spleen lymphocytes in vitro

The effects of pulsed electric fields on cell membranes were investigated. In vitro exposure of mouse splenocytes to a single high-voltage pulse resulted in an increase in membrane permeability that was dependent on both the electric field strength and the pulse duration. Exposure to a 2 μs, 3.0 kV/cm pulse resulted in the induction of a 1.26 V transmembrane potential, and elicited a 50% loss of intracellular K⁺. These results are in agreement with previous studies of the effects of pulsed electric fields on erythrocytes and microorganisms. The effect of pulsed electric fields on the functional integrity of lymphocytes was i vestigated by measuring [³H]thymidine incorporation by cells cultured in the presence and absence of various mitogens following exposure to an electrical pulse. No statistically significant effects on the response of mouse spleen lymphocytes to concanavalin A, phytohemagglutinin or lipopolysaccharide were observed following exposure to 2 μs electric pulses at amplitudes of up to 3.5 kV/cm. Exposure to a single 10 μs pulse of 2.4–3.5 kV/cm produced a statistically significant reduction in the response of lymphocytes to lipopolysaccharide stimulation that was attributed to cell death.     

Kinetics of permeability changes induced by electric impulses in chromaffin granules

Summary Electric field pulses, ranging in intensity from 20 to 50 kV/cm and in duration from 10 to 40 sec, caused a transient increase in the membrane permeability of chromaffin granules from the bovine adrenal medulla, that led to partial release of granule soluble constituents. This transient permeability change was long-lived, as compared to the pulse duration, and the main part of material efflux occurred after the termination of the pulse. During the latter phase the temporarily increased permeability decayed to its original value, in the absence of the electric field. This indicated that the structural perturbation induced in the membrane was transient and apparently reversible. The release event was characterized by a field-dependent permeability coefficient ranging from 2×10^–4 cm/sec at 30 kV/cm to 3×10^–3 cm/sec at 50 kV/cm. The resealing process of the membrane could be described by two relaxation times, both of which decreased with increasing field strength. ₁ varied from about 3.0 msec at 30 kV/cm to less than 2.0 msec at 50 kV/cm, while ₂ varied from about 100 to about 40 msec in the same interval of field strength. The distribution in the degree of filling of granules that had been partially depleted by an electric field pulse indicated that the population could be considered homogeneous with respect to release.     

Effect of concentration on the aggregation of poly(γ-benzyl-L-glutamate) in dioxane: Critical concentration and maximum length for linear head to tail aggregates

Dielectric relaxation studies in the frequency range 100 Hz to 2 MHz of poly(γ-benzyl-L -glutamate) in dioxane have been carried out over a range of concentration 10^?4–10^?2g/g. The structure of aggregates is analyzed in terms of dipole moment and relaxation time. A critical concentration (? 10^?3 g/g for the studied molecular weights) has been determined below which the aggregates are found to have linear head to tail type structure. Above the critical concentration a different structure of aggregates is apparent which could not be fully analyzed by these measurements alone. Possible forms of aggregation above the critical concentration are discussed. Formation of long range order which would lead to nematic liquid crystalline phase at higher concentrations has been discussed as one of the possible explanations for the observed behavior above the critical concentration. Maximum length of linear head to tail type aggregates for poly(γ-benzyl-L -glutamate) in dioxane as determined from these results correspond to an α-helix of molecular weight 210,000. A slight difference in the purity of dioxane has been shown to have an influence on the reproducibility of the state of aggregation as well as on the rate of disaggregation on dilution.     

Calculation of electric fields and currents induced in a millimeter-resolution human model at 60 Hz using the FDTD method

The finite-difference time-domain (FDTD) method has previously been used to calculate induced currents in anatomically based models of the human body at frequencies ranging from 20 to 915 MHz and resolutions down to about 1.25 cm. Calculations at lower frequencies and higher resolutions have been precluded by the huge number of time steps that would be needed in these simulations. This paper describes a method used to overcome this problem and efficiently calculate induced currents in an MRI-based, 6-mm-resolution model of the human under a high-voltage transmission line. This model is significantly higher resolution than the 1.31-cm-resolution model previously used; therefore, it can be used to pinpoint locations of peak current densities in the body. Proposed safety guidelines would allow external electric fields of 10 kV/m and 25 kV/m for exposure to 60 Hz fields of the general public and workers, respectively. For this external electric field exposure of 10 kV/m, local induced current densities as high as 20 mA/m² are found in the head and trunk with even higher values (above 150 mA/m²) in the legs. These currents are considerably higher than the 4 or even 10 mA/m² that have been suggested in the various safety guidelines, thus indicating an inconsistency in the proposed guidelines. In addition, several ratios of E/H typical of power line exposures were examined, and it was found that the vertical electric field couples strongly to the body, whereas the horizontal magnetic field does not. Bioelectromagnetics 19:293–299, 1998. © 1998 Wiley-Liss, Inc.     

Expression of voltage-gated calcium channels augments cell susceptibility to membrane disruption by nanosecond pulsed electric field

We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca²⁺. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (>80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC+ cells) was about twofold greater than in cells which did not (VGCC− cells). Among VGCC+ cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca²⁺ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC+ cells. However, it remains to be established whether larger transients reflected additional Ca²⁺ entry through VGCC, or were a result of more severe electropermeabilization of VGCC+ cells.     

Quantitative studies on the viability of yeast protoplasts following dielectrophoresis

Abstract Protoplasts from Saccharomyces cerevisiae and Saccharomyces diastaticus were collected in a non-homogeneous alternating electric field. The dependence of the viability of the protoplasts on different conditions of collection was tested by determining the regeneration rates in each case. The parameters varied in collection were the field strength (0.33 kV/cm–6.67 kV/cm), the frequency of the alternating field (1–2 MHz) and the collection time (2–10 min). The introduction of a new type of fusion chamber (meander chamber) permitted, for the first time, quantitative exposure of protoplasts to the electric field as well as their complete transference into the regeneration medium. The regeneration rates of yeast protoplasts collected under those conditions employed for electrofusion did not differ from those of protoplasts which had been maintained under the same experimental conditions but were not subject to the influence of an alternating electric field. The two yeast strains were fused together (collection 1 kV/cm; pulse 15 kV/cm; duration of pulse 40 μs) and the fusion products were introduced into a selection medium for regeneration. The fusion rate was about 4.8 × 10⁻⁴; on average 272 colonies grew on the selection medium for each chamber filling.     

Electric Field Pulses Can Induce Apoptosis

Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane. Here we demonstrate that application of supercritical field pulses between 4.5 and 8.1 kV/cm strength and 40 μsec duration induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage of the poly(ADP ribose) polymerase. Apoptosis induction did not depend on the presence of any particular electrolyte in the extracellular medium. However, no apoptosis was observed in solutions without a minimum amount of salt. Apoptotic DNA fragmentation was prevented by the caspase inhibitor zVAD. Received: 16 December 1998/Revised: 24 February 1999     

A conformation change of single stranded polyriboadenylate induced by an electric field

Absorbance measurements performed with high molecular weight poly A at pH 8 show that the degree of single strand stacking present at high ionic strength is reduced at low ionic strengths. The salt dependence of the poly A conformation is assigned to an electrostatic repulsion between subsequent turns of the single strand “helix” structure. - Electric fields of 5 to 80 kV/cm induce an increase in the poly A absorbance consistent with a decrease in the ion concentration in the environment of the polymer. The increase of the absorbance is a linear function of the field strength suggesting that the conformation change is caused by a dissociation field effect. At increasing ionic strength, threshold values of the electric field strength have to be exceeded in order to induce measurable absorbance changes. - The time required for the conformation change decreases from about 2 μsec at 10⁻⁴ M ionic strength to about 0.3 μsec at high ionic strengths. At low ionic strengths the ion equilibration may influence the rate limiting step, whereas the arrangement of the nucleotide residues into the ordered structure is rate limiting at high ionic strengths.     

Nanosecond pulse electric field (nanopulse): a novel non-ligand agonist for platelet activation

Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca²⁺) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca²⁺ ion concentration ([Ca²⁺]_i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300 ns pulse with an electric field of 30 kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7 kV/cm with maximum effects seen with approximately 30 kV/cm. The increases in intracellular Ca²⁺ release and Ca²⁺ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30 kV/cm. The increases in [Ca²⁺]_i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca²⁺ to leak out of intracellular Ca²⁺ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca²⁺ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca²⁺]_i, however by a different mechanism.     

Natural Electrotransformation of Lightning-Competent Pseudomonas sp. Strain N3 in Artificial Soil Microcosms

The lightning-competent Pseudomonas sp. strain N3, recently isolated from soil, has been used to study the extent of natural electrotransformation (NET) or lightning transformation as a horizontal gene transfer mechanism in soil. The variation of electrical fields applied to the soil with a laboratory-scale lightning system provides an estimate of the volume of soil affected by NET. Based on the range of the electric field that induces NET of Pseudomonas strain N3, the volume of soil, where NET could occur, ranges from 2 to 950 m³ per lightning strike. The influence of DNA parameters (amount, size, and purity) and DNA soil residence time were also investigated. NET frequencies (electrotransformants/recipient cells) ranged from 10⁻⁸ for cell lysate after 1 day of residence in soil to 4 × 10⁻⁷ with a purified plasmid added immediately before the lightning. The electrical field gradient (in kilovolts per cm) also played a role as NET frequencies ranging from 1 × 10⁻⁵ at 2.3 kV/cm to 1.7 × 10⁻⁴ at 6.5 kV/cm.     

Comparison of the coupling of grounded humans,swine and rats to vertical, 60-Hz electric fields

Published and new data for grounded humans, swine, and rats exposed to vertical, 60-Hz electric fields are used to determine field strengths at the surfaces of the bodies and average components of induced-current density along the axes of the bodies. At the tops of the bodies, surface electric fields are increased (enhanced) over the unperturbed field strength present before the subjects entered the field by factors of 17,7, and 4 for humans, swine, and rats, respectively. For an unperturbed field strength of 10 kV/m, average induced axial current densities in the neck, chest, abdomen, and feet are: 550, 190, 250, and 2000 nA/cm², respectively, for humans; 40, 13, 20, and 1100 nA/cm², respectively, for swine; and 28, 16, 2, and 1400 nA/cm², respectively, for rats. These data are used to show that the actual electric fields experienced by animals depend strongly on the shape of the body and its orientation relative to the electric field and ground plane. This fact must be taken into account if biological data obtained with laboratory animals are to be used for the assessment of possible hazards to humans exposed to 60-Hz electric fields.     

The effect of pulse field strength on electric field stimulated biosorption of uranium by Kluyveromyces marxianus IMB3

Summary Improved biosorption of uranium by Kluyveromyces marxianus IMB3 biomass was achieved by increasing the electric field strength of delivered pulses from 1.25kV/cm to 2.5kV/cm. Although this had little or no effect on the maximum biosorption capacity (q_max), at low concentrations of uranium the amount bound to the biomass increased from 70 to 140mg uranium/g biomass. Significant increases in the maximum biosorption capacities (119–180 mg uranium/g biomass) were observed when the pulse field strength was increased from 2.5kV/cm to 3.25kV/cm.     

Perception and the Characteristics of the Response of Honey Bees,Paper Wasps,and Red Wood Ants to a Low-Frequency Electric Field

Bees, wasps, and ants have no specialized receptors for the perception of an electric field. An appropriate response to naturally occurring electric fields in bees and ants is associated with atmospheric exposure, amplified by the approach of the front of a thunderstorm. The primary transducers of mechanoreceptors that respond to displacement are related to the perception of low-frequency electric fields of high intensity by insects. The non-specific mechanism of perception of electric fields is based on irritation by induced currents that flow in the locations of their contact with each other and/or conductive surfaces. The frequency dependence of the electric field sensitivity is determined mainly by the magnitude of the current induced by it and the intensity of its contact action. The magnitude of the current induced in the outer part of the insect body is non-linearly related to the frequency of the electric field. The region with the highest sensitivity to electric fields is close to 500 Hz, which is consistent with the maximum magnitude of the induced current. At the same time, the threshold of the sensitivity to an electric field in wasps is approximately 0.04 kV/m, while in bees it is 0.45 kV/m. Ants react to the action of an electric field of 7–10 kV/m by slowing their movement. Magnetic fields and ionization, which accompany the generation of an electric field whose intensity reaches 15–20 kV/m, do not stimulate changes in the behavior of insects.

     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.