Kinetics of homomeric GluR6 glutamate receptor channels.

We studied the kinetics of the unedited version of rat GluR6 glutamate (glu) receptor channels, GluR6Q, in outside-out patches using a system for submillisecond solution exchange. Half-maximum activation of the channels was reached with approximately 0.5 microM glu. The maximum slope of the double-logarithmic plot of the peak current versus glu was approximately 1.3, indicating that at least two binding steps are necessary to open the channels. Currents in response to a pulse of 10 microM glu had a short rise time (10-90% of peak current) of approximately 220 microseconds at approximately 20 degrees C. The rise time increased with falling glu concentration, reaching approximately 6.0 ms with 10 microM glu. In the continued presence of glu, the channels desensitized, and this desensitization can be described with a single time constant of approximately 7.0 ms for a pulse of 10 microM glu. The steady-state current in response to a long pulse of 10 microM glu was below 1/280th of the peak current. The time constant of desensitization was found to be independent of concentration between 30.0 and 0.3 microM glu, but to be increased for lower concentrations. After a short pulse of 1 ms duration and 10 or 0.3 microM glu, currents decayed with a time constant of approximately 2.5 ms. Recovery from desensitization after a pulse took approximately 5 s, and the half-time of recovery was approximately 2.2 s. Continuous application of low concentrations of glutamate reduced the peak currents in response to a pulse of 10 microM glu markedly. Fifty percent response reduction was observed in the continuous presence of approximately 0.3 microM glu. Our results for homomeric GluR6 agree with a cyclical reaction scheme developed for completely desensitizing, glu-activated channels on crayfish muscles.     

Dye exclusion artifact in flow cytometers

BACKGROUND: Cells exclude their own volume of dye solution in the sample flow which carries them through the flow chamber of the flow cytometer, thereby affecting the otherwise constant signal arising from the fluorescence of this solution. Under certain conditions, this phenomenon may significantly influence the fluorescence signal of the cells. MATERIALS AND METHODS: Using the slit scan technique, we studied this phenomenon as observed for monodisperse polystyrene particles in fluorescein solution. RESULTS: The measurements show that dye solution accumulates just in front of the particle and just behind it, with a relative void in between. This phenomenon is most likely caused by the rapid constriction of the flow as it enters the orifice of the nozzle or flow chamber, giving rise to a pulse of fluorescence which adds to that of the particle or cell itself. The magnitude of this artifact depends on the design and dimensions of the nozzle/flow chamber as well as on the rate of sample flow. CONCLUSIONS: The dye exclusion artifact may affect measurements of cells when they are in a dye solution having a fluorescence per unit volume which is significant compared to that of the cells, especially at low sample flow rates.     

Dynamic parameters of the dipole layer formed during pulsed photoemission discharging of a plane vacuum diode

The parameters of the current and dipole moment that arise during the discharging of a plane vacuum diode by a short photoemission pulse are studied analytically and numerically. Electron emission is induced by the ionizing radiation incident obliquely on the cathode. An approximate analytic model is developed that, under certain conditions, makes it possible to reduce the problem to that having a known solution for the normally incident ionizing radiation. In the case of a short pulse, scaling relationships for the parameters of the dipole layer are obtained as functions of the diode parameters (the interelectrode gap and applied voltage) and the angle of incidence of the ionizing radiation.     

Performance analysis of a dual-buffer architecture for digital flow cytometry.

BACKGROUND: Most current commercial flow cytometers employ analog circuitry to provide feature values describing the pulse waveforms produced from suspended cells and particles. This restricts the type of features that can be extracted (typically pulse height, width, and integral) and consequently places a limit on classification performance. In previous work, we described a first-generation digital data acquisition and processing system that was used to demonstrate the classification advantages provided by the extraction of additional waveform features. An improved version of the system is discussed in this paper, focusing on dual-buffering to ensure increased pulse capture. A mathematical model of the system is also presented for performance analysis. METHODS: The second-generation system incorporates fast digitization of analog pulse waveforms, instantaneous pulse detection hardware, and a novel dual-buffering scheme. A mathematical model of the system was developed to theoretically compute the capture-rate performance. RESULTS: The capture rate of the system was theoretically analyzed and empirically measured. Under typical conditions, a capture rate of 8,000 pulses/s was experimentally achieved. CONCLUSIONS: Based on these results, the dual-buffer architecture shows great potential for use in flow cytometry.     

Selective recognition of d-tryptophan from d/l-tryptophan mixtures in the presence of Cu(II) by electropolymerized l-lysine film

Selective recognition of d-tryptophan (d-Trp) in the presence of Cu(II) was investigated at poly-l-lysine (p-l-Lys) film using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). p-l-Lys film was immobilized on a glassy carbon electrode (GCE) by cyclic voltammetry between 0.0 and 1.9 V in 20 mM phosphate buffer solution (pH 8.6). After the p-l-Lys/GCE electrode was incubated with d-Trp solution containing Cu(II) ions, obvious enhancement of electron transfer resistance and decrease of voltammetric current could be observed. If d-Trp was replaced by l-tryptophan (l-Trp), there was no apparent resistance and current changes. Moreover, no resistance and current changes could be found in the absence of Cu(II). It may be due to the formation of Cu complex with l-lysine and d-tryptophan. Finally, this method was successfully applied to monitoring enantiomeric composition of the d-Trp and l-Trp mixtures.     

Numerical simulations of spark channels propagating along the ground surface: Comparison with high-current experiment

A numerical model of a spark discharge propagating along the ground surface from the point at which an ∼100-kA current pulse is input into the ground has been developed based on experiments in which the velocity of a long leader was measured as a function of the leader current. The results of numerical simulations are in good agreement with the measured characteristics of creeping discharges excited in field experiments by using a high-power explosive magnetic generator. The reason why the length of a spark discharge depends weakly on the number of simultaneously developing channels is found. Analysis of the influence of the temporal characteristics of the current pulse on the parameters of the creeping spark discharge shows that actual lighting may exhibit similar behavior.     

Revisiting excitation pattern design for magnetic resonance imaging through optimisation of the signal contrast efficiency

The design of excitation signals for Magnetic Resonance Imaging (MRI) is cast as an optimal control problem. Here, we demonstrate that signals other than pulse excitations, which are ubiquitous in MRI, can provide adequate excitation, thus challenging the optimality and ubiquity of pulsed signals. A class of on-resonance piecewise continuous amplitude modulated signals is introduced. It is shown that despite the bilinear nature of the Bloch equations, the spins system response is largely analytically tractable for this class of signals, using Galerkin approximation methods. To challenge the optimality of the pulse excitation, an appropriate cost criterion, the Signal Contrast Efficiency (SCE), is developed. It is to be optimised subject to dynamics expressed by the Bloch equations. To solve the problem the Bloch equation is transferred to the excitation dependent rotating frame of reference. The numerical solutions to the problem for different tissue types show that for a short period of time, pulse excitations provide the maximum signal contrast. However, the problem should be solved for longer periods of time which may result in a different answer than a pulse. For this purpose, the approximate analytic solution which is derived based on averaging the Bloch equation in the excitation dependent rotating frame of reference will be used to find the optimal excitation pattern. The solution to the optimisation problem is potentially useful for all forms of MRI including structural and functional imaging. The objective of this paper is to show that while classically transient response of pulses have been monitored so far, the optimal excitation pattern may be the steady state response of a non-pulse excitation.     

Synthesis of acylglucuronides of drugs using immobilized dog liver microsomes octadecylsilica particles coated with phospholipid

Immobilized dog liver microsome octadecylsilica (ODS) particles coated with phospholipid were developed for the synthesis of acylglucuronides of drugs. The phospholipid-coated ODS particles were readily prepared by stirring a solution containing L-alpha-dipalmitoylphosphatidylcholine with the ODS particles, in which the phospholipid was absorbed on the ODS surfaces by hydrophobic interaction between the acyl group of phospholipid and the otcadecyl group of the ODS particles. Similarly, the microsome-immobilized particles were readily prepared by stirring a buffer solution containing dog liver microsomes with the phospholipid-coated ODS particles, in which the microsomes were immobilized on the phospholipid-coated ODS particles by hydrophobic binding. The microsome-immobilized particles exhibited UDP-glucuronosyltransferase activity which catalyzed the glucuronidation of ketoprofen and a nonpeptide endothelin receptor antagonist, S-1255 ([R]-[+]-2-[benzo(1,3)dioxol-5-yl]-6-isopropyl-4-[4-methoxyphenyl]-2H-chromene-3-carboxylic acid), to the corresponding acylglucuronide in the presence of uridine 5(')-diphosphate (UDP)-glucuronic acid, and two acylglucuronides of ketoprofen and S-1255 were synthesized using the microsome-immobilized particles. These acylglucuronides were synthesized by simply shaking the microsome-immobilized particles adsorbed on the substrate in a buffer solution containing UDP-glucuronic acid with a thermostated mixer. The molecular weights and chemical structures of the synthesized acylglucuronides were identified by mass spectrometry and nuclear magnetic resonance, respectively. The productivity of S-1255 acylglucuronide using microsome-immobilized particles was approximately threefold higher than that observed with free microsomes, whereas the ketoprofen acylglucuronide productivity was slightly lower than that observed with free microsomes. The present method should be very useful for the synthesis of acylglucuronides of drugs, which are slightly soluble aqueous solutions in the drug development stage.     

Synchronization of Na/K pump molecules by a train of squared pulses

We experimentally studied the Na/K pump currents evoked by a train of squared pulses whose pulse-duration is about the time course of Na-extrusion at physiological conditions. The magnitude of the measured pump current can be as much as three-fold of that induced by the traditional single pulse measurement. The increase in the pump current is directly dependent on the number of pre-pulses. The larger the number of the pre-pulses is, the higher the current magnitude can be obtained. At a particular number of pre-pulses, the pump current becomes saturated. These results suggest that a large number of pre-pulses may synchronize the pump molecules to work at the same pace. As a result, the pump molecules may extrude Na ions at the same time corresponding to the stimulation pulses, and pump in K ions at the same time during the pulse intervals. Therefore, the measured pump current is three-fold of that measured by a single pulse where the outward and inward pump currents are canceled each other.     

Current noise around steady states in discrete transport systems

Subject of this paper is the transport noise in discrete systems. The transport systems are given by a number (n) of binding sites separated by energy barriers. These binding sites may be in contact with constant outer reservoirs. The state of the system is characterized by the occupation numbers of particles (current carriers) at these binding sites. The change in time of the occupation numbers is generated by individual “jumps” of particles over the energy barriers, building up the flux matter (for charged particles: the electric current). In the limit n → ∞ continuum processes as e.g. usual diffusion are included in the transport model. The fluctuations in occupation numbers and other quantities linearly coupled to the occupation numbers may be treated with the usual master equation approach. The treatment of the fluctuations in fluxes (current) makes necessary a different theoretical approach which is presented in this paper under the assumption of vanishing interactions between the particles. This approach may be applied to a number of different transport systems in biology and physics (ion transport through porous channels in membranes, carrier mediated ion transport through membranes, jump diffusion e.g. in superionic conductors). As in the master equation approach the calculation of correlations and noise spectra may be reduced to the solution of the macroscopic equations for the occupation numbers. This result may be regarded as a generalization to non-equilibrium current fluctuations of the usual Nyquist theorem relating the current (voltage) noise spectrum in thermal equilibrium to the macroscopic frequency dependent admittance.The validity of the general approach is demonstrated by the calculation of the autocorrelation function and spectrum of current noise for a number of special examples (e.g, pores in membrances, carrier mediated ion transport).     

Two-component structure of the current pulse of a ranaway electron beam generated during electric breakdown of elevated-pressure nitrogen

Conditions are investigated at which two current pulses of ranaway electron beams are generated in elevated-pressure nitrogen during one voltage pulse. It is shown that the regime with two runaway electron beam current pulses takes place at decreased values of the electric field strength E in the gap (or decreased values of the parameter E/p, where p is the gas pressure). The regime with two runaway electron beam current pulses is observed both at high (1500?C3000 Torr) and low (below 100 Torr) pressures. It is shown that, for the second runaway electron beam current pulse to form, the voltage across the gap should be partially reduced during the first pulse. At low nitrogen pressures (~10 Torr), the regime in which two runaway electron beams are generated can be implemented by increasing the breakdown strength of the gap and/or increasing the value of E/p. In experiments carried out in atmospheric-pressure air with a picosecond time resolution, a rather complicated structure of the beam current pulse is observed at a voltage rise time of ~300 ps.     

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

Measurement of fast dynamic intracellular pH in Saccharomyces cerevisiae using benzoic acid pulse

pH affects many processes on cell metabolism, such as enzyme kinetics. To enhance the understanding of the living cells, it is therefore indispensable to have a method to monitor the pH in living cells. To accomplish this, a dynamic intracellular pH measurement method applying low concentration benzoic acid pulse was developed. The method was thoroughly validated and successfully implemented for measuring fast dynamic intracellular pH of Saccharomyces cerevisiae in response to a glucose pulse perturbation performed in the BioSCOPE set-up. Fast drop in intracellular pH followed by partial alkalinization was observed following the pulse. The low concentration benzoic acid pulse which was implemented in the method avoids the undesirable effects that may be introduced by benzoic acid to cell metabolism.     

Detection of Charge-Neutral Near-Equilibrium Processes at Na-Metal Electrodes by Electrochemical Microcalorimetry

Investigations on electrochemical kinetics usually rely on the measurement of current or potential as a function of time. Charge-neutral process steps or side reactions are naturally disguised in the electrical signals and have only indirect impact. However, all processes will contribute to heat evolution. In this work, heat absorption/liberation is measured as a function of time for pulsed Na deposition/dissolution on a Na-electrode in a 1 m NaPF₆/diglyme solution, in addition to the standard electrochemical signals. While potential and current transients both exhibited sharp rectangular shapes, indicating instantaneous electrochemical Na deposition or dissolution on the time scale of the pulse (10 ms), heat absorption or liberation continued up to about 0.5 s after the pulse. Since heat evolution is to large extent reversible, this corresponded to entropy changes in the absence of external electric current flow, pointing to a reversible, charge-neutral chemical process accompanying Na deposition or dissolution. From the observed entropy changes, it is suggested that upon Na deposition solvated Na⁺ ions are instantaneously transferred into the outer layers of the solid electrolyte interphase, followed by slow desolvation.     

Physical nature of the electric current produced by the asymmetry of particle motion in toroidal magnetic confinement systems

A new type of longitudinal electric current is revealed by analyzing the drift trajectories of charged particles in a tokamak—the current that may be referred to as the asymmetry current because it is associated with the asymmetry of the boundary between trapped and transit particles in phase space. The generation of this current is explained by the fact that the motions of the particles that cross the magnetic surface at a given point in opposite directions are qualitatively different. The asymmetry current results from the toroidal variations of the magnetic field and is maintained by the radial momentum flux of transit particles. The contribution of the particles of different species to the asymmetry current density is proportional to their pressure, is independent of the gradients of the plasma parameters, is maximum at the magnetic axis, and decreases toward the plasma periphery. In contrast to standard neoclassical theory, the asymmetry current can be found only from exact particle trajectories. The asymmetry current is calculated for tokamaks with differently shaped magnetic surfaces and for a model stellarator. By exploiting the newly revealed asymmetry current, together with the bootstrap current, it may be possible to substantially simplify the problem of creating a tokamak reactor.     

Optocapacitive Generation of Action Potentials by Microsecond Laser Pulses of Nanojoule Energy

Millisecond pulses of laser light delivered to gold nanoparticles residing in close proximity to the surface membrane of neurons can induce membrane depolarization and initiate an action potential. An optocapacitance mechanism proposed as the basis of this effect posits that the membrane-interfaced particle photothermally induces a cell-depolarizing capacitive current, and predicts that delivering a given laser pulse energy within a shorter period should increase the pulse’s action-potential-generating effectiveness by increasing the magnitude of this capacitive current. Experiments on dorsal root ganglion cells show that, for each of a group of interfaced gold nanoparticles and microscale carbon particles, reducing pulse duration from milliseconds to microseconds markedly decreases the minimal pulse energy required for AP generation, providing strong support for the optocapacitance mechanism hypothesis.     

Dynamics of the optical emission from a high-voltage diffuse discharge in a rod-plane electrode system in atmospheric-pressure air

Results are presented from experimental investigations of the dynamics of optical emission from a nanosecond diffuse discharge in a rod-plane electrode system. A study was made of discharges in a 10-cm-long interelectrode gap in atmospheric-pressure air (the cathode being a 1-cm-diameter rod with a bullet-shaped end). The voltage across the discharge gap was 220 kV and the voltage pulse duration was 180 ns, the voltage rise time being 10 ns. In experiments, the discharges were observed to evolve through two stages: the bridging stage and the conduction stage. The bridging stage begins with intense optical emission from the cathode region, the onset of the emission being delayed with respect to the beginning of the voltage pulse. Simultaneously with the onset of optical emission, a displacement current corresponding to the motion of charged particles begins to be generated in the cathode region. The duration of this current corresponds to the time the emission front takes to bridge the gap. As the emission front reaches the anode region, the current increases abruptly, indicating the beginning of the conduction stage. It was found that the time delay of optical emission relative to the beginning of the voltage pulse largely governs the discharge parameters: as the time delay becomes longer, the emission front velocity in the bridging stage increases from 0.6 to 1.5 cm/ns, the probability of realizing a multichannel structure of the discharge becomes higher, and the discharge current and the intensity of X-ray emission from the discharge grow.     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.     

Flicker noise of ion-selective membranes and turbulent convection in the depleted layer

Flicker noise of electric currents through ion-selective membranes is explained. It is attributed to the depletion of salt on one side of the membrane, which creates a thin layer of high resistance. Joule heating in this depletion layer and the ensuing temperature gradient, as well as the concentration gradient, give rise to buoyant forces which may create a turbulent convection current. The turbulence mixes the depletion layer so that the electric resistance fluctuates, and consequently the current flickers.Experiments with ion-selective membranes support this conjecture. They show that 1) Noise is coincident with the increase of the electric resistance by the depletion process. 2) When the current density is reduced, it reaches a critical value, below which the convection current changes from turbulent to laminar, and the noise disappears. 3) Noise reduces with temperature, because the expansion coefficient of water decreases with temperature, and its viscosity increases. 4) A non-ionic water-soluble polymer added to the compartment on the side of the depletion layer reduces the noise, by increasing the bulk viscosity of the solution. 5) Noise depends on the membrane's orientation in the gravitational field. 6) The convection-current in the depletion layer can be observed directly, using a laser-beam, by adding latex particles which create optical noise as they drift with the convection current across the beam. The optical noise is observed only coincidently with the current noise.A. Katzir-Katchalsky Fellow     

A stochastic model for DNA translocation through an electropore

A 1D Fokker-Planck simulation of DNA translocation through an electropore under finite pulses is presented. This study is motivated by applications relevant to DNA electrotransfer into biological cells via electroporation. The results review important insights. The translocation may occur on two disparate time scales, the electrophoretic time (~ms), and the diffusive time (~s), depending on the pulse length. Furthermore, a power-law correlation is observed, F-PST~(V(m)t(p))(a)/N(b), where F-PST is the final probability of successful translocation, V(m) is the transmembrane potential, t(p) is the pulse length, and N is the DNA length in segments. The values for a and b are close to 1 and 1.5, respectively. The simulated results are compared with previous data to interpret the trends. In particular, the diffusive time scale is used to explain the frequency dependence observed in electroporation experiments with uni- and bi-polar pulse trains. The predictions from the current model can be harnessed to help design experiments for the further understanding and quantification of DNA electrotransfer.