Electric field effects in systems of associating amphiphilic electrolytes

The thermodynamic stability and the structure of selfassociated amphiphilic electrolytes have been studied as a function of electric fields, the strength of which is of the order of 10⁴–10⁵ V/cm, thus corresponding to the conditions in the lipidic part of biological membranes. The experiments have been done with cetylpyridinium iodide and pinacyanol chloride, because these systems show association dependent changes of their optical absorption. The results which have been obtained in a field jump apparatus with short square wave pulses and optical detection can be classified as chemical and physical effects. Three chemical effects can be resolved in the cetylpyridinium iodide system. The concentration dependent relaxation times are of the order of 50 nsec, 1 μsec and 50 μsec. The nature of these effects is discussed with regard to the “Second Wien Effect”, the dipole field effect, and entropic events in the water structure. At higher concentrations of cetylpyridinium iodide physical field effects predominate in the optical observation. These electrodichroitic processes are attributed to the orientation or rearrangement of large anisometric micelles. The electric field effects in the pinacyanol system resemble those of the cetylpyridinium iodide: chemical reequilibration at lower concentrations and a superposition of chemical and physical effects in solutions with large aggregates.     

Differences in the electric birefringence of spectrin dimers and tetramers as shown by the fast reversing electric pulse method

The electric birefringence of purified Spectrin has been examined in medium of low ionic strength ai 20°C and for electric fields smaller than 4 × 10⁴ V m^?1. using the reversing electric pulse method. This technique allows study of the permanent and induced dipole electric moment of macromolecules more easily than in measurements using only rectangular pulses. We show that spectrin heterodimers and heterotetramers have different electro-optical properties. The relaxation time of the tetramer (7 μs) is significantly longer than that of the dimer (4.5 μs). Tetramers and dimers have also different polarizability parameters.     

Interactions of nucleic acid double helices induced by electric field pulses

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 × 10¹⁰ M^?1 helices s^?1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants k_d decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(k_d) and E² suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing sail concentration (measured in the range 1–5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 μs, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.     

Kinetic properties and the electric field effect of the helix-coil transition of poly(gamma-benzyl L-glutamate) determined from dielectric relaxation measurements

Dielectric relaxation of poly(γ-benzyl L -glutamate) in solution has been studied in the 5 kcps-10 Mcps range for various values of the helix content. The results give first experimental evidence for three effects of major significance. (1) The system exhibits dielectric relaxation due to a chemical rate process (namely helix formation). This confirms recent theoretical predictions. (2) The mean relaxation time τ* of the helix–coil transition could be evaluated as a function of the degree of transition. The results are in excellent agreement with a previously developed theory. At the midpoint of transition it is found τ*_max = 5 × 10^?7 sec. The elementary process of helical growth turns out to be practically diffusion-controlled (with a rate constant of hydrogen bond formation of 1.3 × 10¹⁰ sec^?1). (3) There is a considerable electric field effect of the helix–coil transition. This indicates that conformation changes in biological systems could be potentially caused by direct action of an electric field.     

Structural changes in bacteriorhodopsin induced by electric impulses

Electric impulses of high field intensity (2 × 10⁵ to 3 × 10⁶ Vm^?1, 1 to 20 μs duration) cause transient changes in the optical absorbance of suspended purple membranes of Halobacterium halobium. The electric dichroism at 1 mm NaCL, pH ≈ 6 and at 293K is dependent on field strength, pulse duration and wavelength of the monitoring, plane-polarized light in the range 400 to 650 nm. The optically detected processes are, however, independent of bacteriorhodopsin concentration, of ionic strenght and of the intensity of the monitoring light. These data together with the analysis of time course ands steady state of the reduced dichroism, suggest electric field-sensitive, intramemembraneous structural changes which lead to restricted orientation changes of the chromophore. A thoretical analysis of restricted orientation is developed and applied to the electro-optic data. As a result, it is found that the electric dichroism of purple membrane is associated with a large polarizability anisotropy of 2.4 × 10^?30 Fm² (2.2 × 10^?14 cm³); the electric permanent dipole moment which is involved amounts to 4.7 × 10^?28 Cm(140 Debye). The kinetic data suggest a cyclic reaction scheme with at least five different conformations. The high polarizability is probably due to displaceable ionic groups within the cooperative lattice of bacteriorhodopsin molecules in purple membranes.     

Flexibility of nucleosomal DNA

In this study transient electric birefringence (TEB) has been used to investigate the molecular flexibility of short fragments of DNA. Nucleosomal DNA always exhibits negative birefringence and Kerr behavior was observed up to high field strengths (6 KV/cm). The value of the Kerr constant is 3.5 10^?2 e.s.u.. Birefringence decays were single exponentials and a field dependence of the molecular orientational relaxation time τ was found: it is explained by an inherent flexibility of the DNA molecule. A 20 % decrease in the calculated length was observed with fields applied as low as 2 KV/cm. The results obtained at very low fields establish TEB as a method well suited to calculate accurate values for the length of small fragments of DNA: the τ value of 4.3 μsec corresponds to a DNA length of 660 Å.     

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.     

Electro-optic scattering studies on deoxyribonucleic acid

Measurements have been made of the intensity of light scattered from aqueous solutions of calf thymus DNA with and without the application of electric fields. For fields approaching 150 V/cm and frequencies below 2.5 KHz, changes (ΔI) of up to 10% in the residual scattered intensity were observed. In agreement with previous dielectric and electric birefringence measurements, a low frequency dispersion of ΔI was observed, from which a rotary diffusion constant (D) of 1200 s^?1 was determined. Interpreting the electric field data in terms of the classical dipolar orientation theory led to values of 2.4 × 10^?25 cm (7.4 × 10^?14 esu) and 4.3 × 10^?25 cm (13 × 10^?14 esu) for the permanent dipole moment and the anisotropy of the electric polarisabilities respectively. Furthermore the permanent dipole moment was along the major molecular axis and the particles orientated in the field as rigid entities. The zero field data indicated a molecular shape which was not rodlike but corresponded to the Kratky-Porod “stiffness” parameter of x = 24 for the wormlike coil model. Although curved, the molecules appeared to orientate in low-intensity electric fields as rigid, but not rodlike molecules. The implications of this on recent discrepancies in D determined by two or more dynamic relaxation methods is briefly discussed.     

Effects of Applied Electric Fields on Bile Salt Secretion by in Situ Rat Liver

The effects of weak external electric fields (1.5–3 V, 30–60 μA) on bile salt secretion by jji situ rat liver were studied after interruption (experiment A) and restoration (experiment B) of the enterohepatic bile salt cycle. In experiment A, bile salt tracer was injected in bolus 3 hr after application of the electric field (¹⁴C-TC, ¹⁴C-CA, or ¹⁴C-DCA). In experiment B, 36 μmoles/h of TC or CA were infused 150 min after application of the electric field until the end of the experiment. A bolus (¹⁴C-TC and ¹⁴C-CA) was injected 170 min after application of the electric field. The hepatic taurine pool was prelabeled in both experiments.

Application of electric fields delayed secretion of ¹⁴C-TC into the bile. This delay was longer at 60 μA than at 30 μA Neither field had any effect on ³H-tauroconjugation. Electric fields also led to the formation of osmiophilic globules in hepatocytes and, especially in experiment B, collagen fibers in intracellular spaces. Thus electric fields may provoke changes in plasmic membranes probably in connection with those in the extracellular matrix.     

Birefringence changes in vertebrate striated muscle

The changes in birefringence in the rigor to relax transition of single Triton-extracted rabbit psoas muscle fibers have been investigated. The total birefringence of rigor muscle fibers was dependent on sarcomere length and ranged from (1.46 ± 0.08) × 10⁻³ to (1.60 ± 0.06) ± 10⁻³ at sarcomere lengths from 2.70 μm to 3.40 μm. An increase in total birefringence was measured dependent on sarcomere length when 55 single fibers were relaxed from the rigor state with Mg-ATP. Pyrophosphate relaxation produced a smaller increase in retardation when compared to Mg-ATP. The expected change in intrinsic birefringence during the rigor to relax transition was calculated assuming a hinge function of the subfragment 2 moiety of myosin. The changes in birefringence during isometric contraction and relaxation have been discussed in relation to possible structural changes.     

Electric birefringence of bacterial flagellar protein filaments: evidence for field-induced interactions.

The time course for the build-up and decay of birefringence induced by a rectangular voltage pulse was measured on solutions of flagellar filaments from Salmonella equi-abortus (strain SJ25). These filaments are tubular polymers of protein (degree of polymerization ≈ 10³) constituted by non-covalent linkage of flagellin monomers of molecular weight 4 × 10⁴. The effect on electro-optical properties of solutions of filaments due to variations in temperature, concentration and mean length of protein filaments, and the duration and intensity of the applied electric field is described. Analysis of the field intensity dependence of the birefringence and comparison of the build-up and decay processes indicate that orientation in the field is due primarily to the existence of a permanent dipole moment in the filaments. At 18 °C the following values were obtained for a solution of filaments with mean length and standard deviation of 0.39 and 0.30 μm: specific Kerr constant (K_sp) = 6.14 × 10⁻³ electrostatic units; optical anisotropy factor (g₁ — g₂) = 5.66 × 10⁻³; dipole moment (μ) = 1.01 × 10⁵ Debye units; and mean relaxation time ($\text{}\text{̄}$gt) = 9.20 ms. At temperatures below 20 °C there is a marked increase in the optical anisotropy factor of the filaments which may be due to a change in their flexibility. The large values of K_sp obtained indicate the highly responsive nature of these filaments to an electric field. The birefringence decay curves were decomposed by computer into a specified number of exponential terms from which both the mean length and the size distribution of these polydisperse filaments were calculated. The results obtained were in substantial agreement with the values of these parameters observed by electron microscopy. A cumulative field effect dependent on field intensity and filament concentration was observed. Repeated pulsing of electric field, above threshold values of field intensity and filament concentration, produced decreases in the birefringence near 60% of its initial value. The effect was reversible with a time constant greater than two minutes. No appreciable change in the relaxation time for decay of birefringence was observed on multiple pulsing of these solutions. These results are interpreted consistently to arise from the sidewise aggregation of filaments induced by electrical impulses of sufficient intensity and duration. These properties appear relevant to bacterial motility: variations in electric potential along the membrane of the bacterium might serve first to orient these organelles and then to induce their coalescence to “bundles” of filaments. The latter structures are commonly observed in vivo. In this way the activity of flagella might be co-ordinated.     

The dichroism of DNA in electric fields

We have studied the dichroism of various samples of calf thymus DNA (of molecular weight from 3 × 10⁵ to 7 × 10⁶) in pulsed electric fields. The results may be summarized as follows:

• 1 We find that calf thymus DNA behaves in electrical orientation as if it possessed a large permanent dipole moment. This apparent moment is sensitive to such effects as Mg⁺⁺ binding which lower the net charge on DNA.
• 2 The limiting dichroism at infinite field corresponds to an angle of at least 80% between the transition moments at 265 nm and the helix axis, and could be consistant with a number of known forms of DNA. This result is independent of DNA molecular weight. There is evidence that the conformation may be different in 80% ethanol.
• 3 The dichroism relaxation curves contain a component with a relaxation time of about 8 μsec, which is nearly independent of molecular weight, and a longest component which behaves either according to the Broersma theory for low-molecular-weight samples, or the Zimm-Rouse theory at high molecular weights.

     

Kinetic studies of the helix–coil transition in aqueous solutions of poly(L-lysine)

The rate of conformational change of aqueous poly(α-L -lysine) solutions was measured using the electric field pulse relaxation method with conductivity detection. The relaxation time as a function of pH exhibits two maxima. One is assigned to a proton transfer reaction and the other to the helix–coil conformational transition. The helix nucleation parameter and the maximum relaxation time yield the rate constant of helix growth process (k_F) according to Schwarz's kinetic theory as k_F = 2 × 10⁷ sec^?1, which is comparable to that of the poly(glutamic acid) solution. The thermodynamic parameters of the helix growth process are compared with those of poly(glutamic acid).     

Optical Kerr effect in biopolymer solutions

The electric field in a single mode, YAG laser beam has been used to induce orientational birefringence in solutions of tobacco rattle virus, DNA, heparin, and hyaluronic acid. Using this laser in its “fixed-Q” mode, laser pulses were generated which persisted for up to 200 μsec in which the effective electric field vector rose to 5 kV cm^?1. The birefringence amplitudes so produced had a quadratic dependence on the effective field strength and thus obeyed Kerr's law. From the birefringence decay rates, relaxation times were determined which, by comparison with the birefringence induced by pulsed static electric fields revealed the biopolymer orientational origins of the effects. This indicated how these experiments can lead to the evaluation of particle geometry, the electronic contribution to electrical polarizabilities, and the optical polarizability of biopolymers in solution.     

Effect of the K channel openers NIP-121 and cromakalim on melittin-induced relaxation of guinea-pig isolated trachea

We have investigated the effect o f the K⁺ channel openers (+)-7,8- dihydro-6,6-dimethyl-7-hydroxy-8-(2-oxo-piperidin-1-yl)-6H-pyrano[2,3f]benz-2, 1,3-oxadiazole (NIP-121) and cromakalim on the relaxation induced by the phospholipase A2 activator melittin in guinea-pig isolated trachea. Melittin (0.1 to 3.0 μg/ml caused concentration-dependent relaxation of tracheal spirals precontracted with LTD₄. The magnitude of relaxation was about 40% of that obtained by 1 mM aminophylline. Melittin-induced relaxation was also observed in tracheas precontracted with histamine or the thromboxane A₂ mimetic U46619. The relaxation to melittin was prevented by the cyclooxygenase inhibitor indomethacin (5 μM) or by removal of the tracheal epithelium, suggesting that cyclooxygenase products, possibly dependent on the epithelium, may be implicated in the response to melittin. Pretreatment of tracheas with NIP-121 (0.03 μM) or cromakalim (0.4 μM) did not affect the contraction to LTD₄ but enhanced the relaxation to melittin. This enhancement to melittin was completely inhibited by the ATP-dependent K⁺ channel blocker glibenclamide (1 μM). Higher concentrations of NIP-121 (0.1 μM) or cromakalim (1 μM) did not enhance the response to melittin. In the presence of indomethacin, NIP-121 (0.03 μM) or cromakalim (0.4 μM) enhanced PGE₂ induced relaxation of tracheas precontracted with LTD₄. These results suggest that cyclooxygenase products, possibly dependent on the epithelium, may be involved in melittin-induced relaxation. The enhancement of relaxation to melittin by IK⁺ channel activation might be due, at least in part, to an increased.     

In Vivo Study of Transepithelial Potential Difference (TEPD) in Proximal Convoluted Tubules of Rat Kidney by Synchronization Modulation Electric Field

Synchronization modulation (SM) electric field has been shown to effectively activate function of Na⁺/K⁺ pumps in various cells and tissues, including skeletal muscle cells, cardiomyocyte, monolayer of cultured cell line, and peripheral blood vessels. We are now reporting the in vivo studies in application of the SM electric field to kidney of living rats. The field-induced changes in the transepithelial potential difference (TEPD) or the lumen potential from the proximal convoluted tubules were monitored. The results showed that a short time (20 s) application of the SM electric field can significantly increase the magnitude of TEPD from 1–2 mV to about 20 mV. The TEPD is an active potential representing the transport current of the Na/K pumps in epithelial wall of renal tubules. This study showed that SM electric field can increase TEPD by activation of the pump molecules. Considering renal tubules, many active transporters are driven by the Na⁺ concentration gradient built by the Na⁺/K⁺ pumps, activation of the pump functions and increase in the magnitude of TEPD imply that the SM electric field may improve reabsorption functions of the renal tubules.     

Transient electric birefringence studies of T2 bacteriophage and T2 ghost

The transient electric birefringence behavior of bacteriophage T2 and the T2 ghost or protein coal was studied. The field free relaxation measurements show both the intact virus and its ghost to have two rotary diffusion coefficients. These coefficients have values of 555 ± 54 and 111 ± 22 sec.^?1 for the intact virus and 688 ± 89 and 161 ± 29 sec.^?l for the ghost. The equivalent ellipsoids for the fast and slow relaxation coefficients were obtained by use of Perrin's equation and were related to the bacteriophage structure in terms of a possible extension of the tail fibers or an enlargement of the head structure. The saturation of the specific birefringence of the phage and the ghost when compared with the specific birefringence of the free nucleic acid gave an average optical orientation of 10 to 18% of the nucleic acid parallel to the main axis of the phage. The analysis of the birefringence versus applied field strength in the Kerr region gave the following values for the anisotropy of the polarixability. α_e,33 – α_e,11 and intrinsic dipole, μ, of both phage and ghost : for T2 phage α_e,33 – α_e,11 = 5.0 × 10^?14 cm.³ and μ = 64,400 Debyes; for T2 ghost α_e,33 – α_e,11 = 7.9 × 10^?14cm.³ and μ = 57,200 Debyes. The high intrinsic dipole for phage and ghost is interpreted as to be associated with the mechanisms of the virus for attachment, to the host cell wall.     

β-ADRENOCEPTOR-MEDIATED RELAXATION INHIBITED BY TETRAPENTYLAMMONIUM IONS IN RAT MESENTERIC ARTERY

The aim of this study was to examine the contribution of K⁺ channel activation to β-adrenoceptor-mediated relaxation in rat mesenteric arteries. Isoprenaline and fenoterol concentration-dependently relaxed the phenylephrine-preconstricted endothelium-intact arteries of the rat with EC₅₀ values of 0.26 ± 0.03 μM and 0.87 ± 0.12 μM, respectively. β-Adrenoceptor-mediated relaxation was significantly attenuated upon removal of endothelium. Tetrapentylammonium ions (TPA⁺) at low concentrations (1–5 μM) inhibited relaxations induced by β-adrenoceptor agonists in arteries with and without endothelium, while glibenclamide (3 μM) had no effect. TPA⁺ (5 μM) inhibited isoprenaline-induced relaxation in the presence of either iberiotoxin (100 nM) or glibenclamide (3 μM). TPA⁺ did not alter forskolin-induced relaxation. In the presence of 60 mM extracellular K⁺, the relaxations induced by two agonists were reduced in endothelium-intact arteries and abolished in endothelium-denuded arteries. The present results suggest that the activation of TPA⁺-sensitive K⁺ channels contributes toward the relaxations mediated through β- and β₂-adrenoceptor stimulation in rat mesenteric arteries. © 1998 Elsevier Science Inc.     

Sinusoidal 60 Hz electromagnetic fields failed to induce changes in protein synthesis in Escherichia coli

Escherichia coli JM83 {F^? ara Δ(lac-proAB) rpsL [?80dΔ(lacZ)M15]} in midlog growth phase at 30 °C were exposed to 60 Hz sinusoidal magnetic field of 3 mT of nonuniform diverging flux, inducing a nonuniform electric field with a maximum intensity of 32 μV/cm using an inductor coil. Exposed and unexposed control cells were maintained at 30.8 ± 0.1 °C and 30.5 ± 0.1 °C, respectively. Quadruplicate samples of exposed and unexposed E. coli cells were simultaneously radiolabeled with ³⁵S-L-methionine at 10 min intervals over 2 hr. Radiochemical incorporation into proteins was analyzed via liquid scintillation counting and by denaturing 12.5% polyacrylamide gel electrophoresis. The results showed that E. coli exposed to a 60 Hz magnetic field of 3 mT exhibited no qualitative or quantitative changes in protein synthesis compared to unexposed cells. Thus small prokaryotic cells (less than 2 μm × 0.5 μm) under constant-temperature conditions do not alter their protein synthesis following exposure to 60 Hz magnetic fields at levels at 3 mT. © 1994 Wiley-Liss, Inc.     

Magneto-electro-fusion of human erythrocytes

In inhomogeneous (static) magnetic fields close contact between ‘magnetic’ human erythrocytes was established. The cells were made magnetic by incubating them in a medium containing small Fe₃O₄-particles which adsorbed to the outer membrane surface. Fusion was induced by applying two electric field pulses (field strength: 8.5 kV · cm^?1; duration: 60 μs) to the magnetically collected cells. This procedure allowed the use of electrically conductive media (3 · 10^?1 Ω^?1 · cm^?1). Fusion of red blood cells occured very often. If cell suspensions of high density were used fusion resulted in the formation of giant red blood cells with osmotically intact membranes.