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Biomedical subjects

R D Astumian

Publications and source records attributed to R D Astumian.

At least 19 recordsLinked to original sources
PubMed1996

Protein kinase C activity is altered in HL60 cells exposed to 60 Hz AC electric fields.

We examined the effects of electric fields (EFs) on the activity and subcellular distribution of protein kinase C (PKC) of living HL60 cells. Sixty Hertz AC sinusoidal EFs (1.5-1,000 mV/cm p-p) were applied for 1 h to cells (10(7)/ml) in Teflon chambers at 37 degrees C in the presence or absence of 2 microM phorbol 12-myristate 13-acetate (PMA). PMA stimulation alone evoked intracellular translocation of PKC from the cytosolic to particulate fractions. In cells that were exposed to EFs (100-1,000 mV/cm) without PMA, a loss of PKC activity from the cytosol, but no concomitant rise in particulate PKC activity, was observed. In the presence of PMA, EFs (33-330 mV/cm) also accentuated the expected loss of PKC activity from the cytosol and augmented the rise in PKC activity in the particulate fraction. These data show that EFs alone or combined with PMA promote down-regulation of cytosolic PKC activity similar to that evoked by mitogens and tumor promoters but that it does not elicit the concomitant rise in particulate activity seen with those agents.

Carcinogens↗
PubMed1996

The physicochemical basis for thermal and non-thermal 'burn' injuries.

One of the most basic problems of burn science may well be the confusing nomenclature we use. The word 'burn' is used to identify several different mechanisms of tissue injury. This article describes the problem of accurately characterizing and defining the various burn injuries on the basis of molecular events. The most important objective is to distinguish between the various physicochemical injuries on the basis of differences in their fundamental physicochemical mechanisms and physiological consequences. Also, pathophysiologically important biophysical processes such as the central importance of cell membrane permeabilization in acute cellular necrosis, which different types of burn injury have in common, are emphasized. The biophysics of membrane formation and permeabilization is presented to clarify the conditions for membrane damage as well as to discuss the potential for therapeutic intervention. Where feasible, plausible new strategies to reverse the molecular alterations caused by injury are hypothesized.

Biophysical Phenomena↗
PubMed1996

Mechanochemical coupling of the motion of molecular motors to ATP hydrolysis.

The typical biochemical paradigm for coupling between hydrolysis of ATP and the performance of chemical or mechanical work involves a well-defined sequence of events (a kinetic mechanism) with a fixed stoichiometry between the number of ATP molecules hydrolyzed and the turnover of the output reaction. Recent experiments show, however, that such a deterministic picture of coupling may not be adequate to explain observed behavior of molecular motor proteins in the presence of applied forces. Here we present a general model in which the binding of ATP and release of ADP serve to modulate the binding energy of a motor protein as it travels along a biopolymer backbone. The mechanism is loosely coupled--the average number of ATPs hydrolyzed to cause a single step from one binding site to the next depends strongly on the magnitude of an applied force and on the effective viscous drag force. The statistical mechanical perspective described here offers insight into how local anisotrophy along the "track" for a molecular motor, combined with an energy-releasing chemical reaction to provide a source of nonequilibrium fluctuations, can lead to macroscopic motion.

Adenosine Triphosphate↗
PubMed1995

Rectification and signal averaging of weak electric fields by biological cells.

Oscillating electric fields can be rectified by proteins in cell membranes to give rise to a dc transport of a substance across the membrane or a net conversion of a substrate to a product. This provides a basis for signal averaging and may be important for understanding the effects of weak extremely low frequency (ELF) electric fields on cellular systems. We consider the limits imposed by thermal and "excess" biological noise on the magnitude and exposure duration of such electric field-induced membrane activity. Under certain circumstances, the excess noise leads to an increase in the signal-to-noise ratio in a manner similar to processes labeled "stochastic resonance." Numerical results indicate that it is difficult to reconcile biological effects with low field strengths.

Animals↗
PubMed1994

Selective and asymmetric molecular transport across electroporated cell membranes.

Transport of a divalent cation (Ca2+) and three DNA indicators [ethidium bromide (EB), propidium iodide (PI), and ethidium homodimer (EthD-1)] across electroporated membranes of several mammalian cell lines was found to be selective and asymmetrical. In low salt medium, Ca2+ and EB were preferentially transported across the anodefacing cell membrane while PI and EthD-1 predominately entered at the site facing the cathode. In high salt medium, the entry site for Ca2+ and EB was reversed to the cathode-facing hemisphere while it remained unchanged for PI and EthD-1. In all these experiments, the observed transport patterns remained unaffected whether the dyes (or ion) were present during or added after the electroporating pulse. The data suggest that asymmetric pores are created on both sides of the membrane facing the electrodes, with smaller pore size (but greater in number) on the anode side and larger pores (with a lower population) on the cathode side. Furthermore, the rate of resealing of the membrane pores is significantly enhanced in high ionic strength medium, thus affecting the entry site. The asymmetric transport pattern is neither caused by electrophoresis induced by the externally applied electric field nor due to one-sided membrane breakdown as previously believed.

3T3 Cells↗
PubMed1992

Interpretation of the effect of an oscillating electric field on membrane enzymes.

Theoretical expressions for the frequency and amplitude dependence of the rate of a catalyzed reaction are fitted to the data of Graziana et al. (1990) [Graziana, A., Ranjeva, R., & Teissié, J. (1990) Biochemistry 29, 8313-8318] for Ca2+ uptake by carrot protoplasts in an oscillating electric field. This uptake is a direct (linear) measure of the rate of increase of ATP caused by a plasma membrane enzyme in the oscillating field. The fit gives 20 ms and 33 microseconds for the relaxation times of the enzyme and roughly 3 for the effective number of elementary changes displaced across the membrane by a conformational change of the enzyme in its catalytic cycle. Additional experiments are suggested to define further the mechanism of the enzymatic reaction.

Adenosine Triphosphate↗
PubMed1992

Estimates for ELF effects: noise-based thresholds and the number of experimental conditions required for empirical searches.

Interactions between physical fields and biological systems present difficult conceptual problems. Complete biological systems, even isolated cells, are exceedingly complex. This argues against the pursuit of theoretical models, with the possible consequence that only experimental studies should be considered. In contrast, electromagnetic fields are well understood. Further, some subsystems of cells (viz. cell membranes) can be reasonably represented by physical models. This argues for the pursuit of theoretical models which quantitatively describe interactions of electromagnetic fields with that subsystem. Here we consider the hypothesis that electric fields, not magnetic fields, are the source of interactions, From this it follows that the cell membrane is a relevant subsystem, as the membrane is much more resistive than the intra- or extracellular regions. A general class of interactions is considered: electroconformational changes associated with the membrane. Expected results of such as approach include the dependence of the interaction on key parameters (e.g., cell size, field magnitude, frequency, and exposure time), constraints on threshold exposure conditions, and insight into how experiments might be designed. Further, because it is well established that strong and moderate electric fields interact significantly with cells, estimates of the extrapolated interaction for weaker fields can be sought. By employing signal-to-noise (S/N) ratio criteria, theoretical models can also be used to estimate threshold magnitudes. These estimates are particularly relevant to in vitro conditions, for which most biologically generated background fields are absent. Finally, we argue that if theoretical model predictions are unavailable to guide the selection of experimental conditions, an overwhelmingly large number of different conditions will be needed to find, establish, and characterize bioelectromagnetic effects in an empirical search. This is contrasted with well-established chemical dosimetry, which is much simpler. Because of the large number of possible electromagnetic field conditions, we also conclude that in vitro studies, rather than in vivo studies, should be emphasized in studies aimed at discovering and characterizing mechanisms for bioelectromagnetic effects.

Cell Membrane↗
PubMed1991

Electroporation by using bipolar oscillating electric field: an improved method for DNA transfection of NIH 3T3 cells.

Using the plasmid DNA pSV2-neo (which, when integrated into the cellular genome confers resistance to the antibiotic G418 for selection), we examined and compared the transfection efficiency on NIH 3T3 cells electropermeabilized by applying a sequence of high-frequency unipolar or bipolar square waves or a single square pulse. Results show that a bipolar square wave is, at least, 1.7- and 5.5-fold more efficient than the unipolar square wave and single square pulse, respectively. In the range of electric field strength used for optimum transfection, the survivability of electropermeabilized cells was comparable between the unipolar and bipolar square waves but fell considerably with the single square pulse. Qualitative comparison of cell permeabilization induced by the three types of wave forms and monitored by ethidium bromide uptake revealed that only the bipolar square wave permeabilizes the cell membrane symmetrically at the two hemispheres facing the electrodes. With unipolar square wave or single square pulse, the membrane is permeabilized either on one side or asymmetrically. Taken together, our result suggests that permeabilization of the membrane at multiple sites without affecting cell survivability may account for the improvements in transfection efficiency observed with bipolar oscillating electric fields.

Cell Line↗
PubMed1990

Electro-permeabilization of cell membranes: effect of the resting membrane potential.

Electric field induced permeabilization of cell membranes is an important technique for gene transfection and cell hybridization. Mechanistic studies of this process revealed that the uptake of fluorescent indicator by plant protoplasts occurs predominantly on the hemisphere facing the positive electrode, while in erythrocyte ghosts the probes exit through the hemisphere facing the negative electrode. To reconcile these observations symmetrical pore formation and a mechanism of molecular exchange by electroosmosis has been proposed. In light of these controversial observations, we conducted a systematic study of electroporation of NIH3T3 cells with varying electric field strength, waveform and frequency. Our data revealed that (i) symmetrical permeabilization of the cell membrane occurs only with bipolar a.c. fields. (ii) When a critical membrane breakdown potential, Vc, is applied using either an unipolar a.c. fields or a single d.c. square pulse, the cell membrane becomes permeabilized only at the hemisphere facing the positive electrode. (iii) When the pulse-induced membrane potential, Vm, is approximately equal to or larger than the intrinsic membrane potential (i.e. using d.c. or unipolar a.c. field), asymmetric permeabilization was observed with the hemisphere facing the positive electrode being most permeable. (iv) The rate of fluorescent indicator uptake is dependent on the concentration of the indicator. These results indicate that electro-permeabilization of cell membranes is affected by its resting potential and that electroosmosis is not the dominant mechanism for the cellular uptake of foreign molecules in electroporation.

Animals↗
PubMed1990

Activation of Na+ and K+ pumping modes of (Na,K)-ATPase by an oscillating electric field.

Serpersu and Tsong (Sepersu, E. H., and Tsong, T. Y. (1983) J. Membr. Biol. 74, 191-201; (1984) J. Biol. Chem. 259, 7155-7162) reported activation of a K+ pumping mode of (Na,K)-ATPase by an oscillating electric field (20 V/cm, 1.0 kHz). Their attempts to activate Na+ pumping at the same frequency were unsuccessful. We report here activation of a Na+ pumping mode with an oscillating electric field of the same strength as used previously (20 V/cm) but at a much higher frequency (1.0 MHz). At 3.5 degrees C and the optimal amplitude and frequency, the field-induced, ouabain-sensitive (0.2 mM ouabain incubated for 30 min) Rb+ influx ranged between 10 and 20 amol/red blood cell/h, and the corresponding Na+ efflux ranged between 15 and 30 amol/red blood cell/h, varying with the source of the erythrocytes. No Rb+ efflux nor Na+ influx was stimulated by the applied field in the frequency range 1 Hz to 10 MHz. These results indicate that only those transport modes that require ATP splitting under the physiological condition were affected by the applied electric fields, although the field-stimulated Rb+ influx and Na+ efflux did not depend on the cellular ATP concentration in the range 5 to 800 microM. Computer simulation of a four-state enzyme electroconformationally coupled to an alternating electric field (Tsong, T. Y., and Astumian, R. D. (1986) Bioelectrochem. Bioenerg. 15, 457-476; Tsong, T. Y. (1990) Annu. Rev. Biophys. Biophys. Chem. 19, 83-106) reproduced the main features of the above results.

Adenosine Triphosphate↗
PubMed1990

The response of living cells to very weak electric fields: the thermal noise limit.

A physical model in which cells are considered as possible detectors of very weak periodic electric fields yields a general relation between cell size and both thermally induced fluctuations in membrane potential and the maximum change in membrane potential caused by an applied field. The simplest version of the model provides a broad-band estimate of the smallest applied electric field to which membrane macromolecules can directly respond (about 10(-3) volt per centimeter). Much smaller fields (10(-6) volt per centimeter) can be detected if there is a response in only a narrow band of frequencies or if signal averaging occurs through field-induced variation in the catalytic activity of membrane-associated enzymes. Both extensions of the simplest version remove the apparent violation of the thermal noise limit found in some experiments.

Cell Physiological Phenomena↗
PubMed1990

Michaelis-Menten equation for an enzyme in an oscillating electric field.

The electric charges on an enzyme may move concomitantly with a conformational change. Such an enzyme will absorb energy from an oscillating electric field. If in addition the enzyme has a larger association constant for substrate than for product, as is often true, it can use this energy to drive the catalyzed reaction away from equilibrium. Approximate analytical expressions are given for the field-driven flux, electrical power absorbed, free-energy produced per unit time, thermodynamic efficiency, and zero-flux concentrations. The field-driven flux is written as a generalized Michaelis-Menten equation.

Biological Transport, Active↗
PubMed1990

Kinetics of a multistate enzyme in a large oscillating field.

A simple, general, and efficient method for calculating the response of a set of coupled first-order (or pseudo-first-order) chemical reactions to an arbitrarily large periodic field is described. The method is applied to a four-state membrane transport enzyme that is electroconformationally coupled to an ac field, i.e., the enzyme has electric charges that move concomitantly with a conformational transition. The calculation is done both for enzymes in a planar membrane and for enzymes in the spherical membrane of a cell or vesicle in suspension.

Enzymes↗