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PubMed2002

Influence of voltage and extracellular Na(+) on amiloride block and transport kinetics of rat epithelial Na(+) channel expressed in Xenopus oocytes.

We expressed the three subunits of the epithelial amiloride-sensitive Na(+) channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na(+) accumulation we performed all experiments in low-Na(+) solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2 pi f(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V(m)) of -60 mV k(on) was 80.8 +/- 5.1 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (V(m)=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K(mic,ami) decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na(+)] ([Na(+)](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na(+)](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na(+) for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na(+)](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na(+)](o), indicating that Na(+) and amiloride do not compete for the same binding site at the channel.

Amiloride↗
PubMed2002

Rapid, non-genomic actions of progesterone and estradiol on steady-state calcium and resting calcium influx in lens epithelial cells.

The effects of steroids on the steady-state intracellular [Ca(2+)] ([Ca(2+)](i)) and resting Ca(2+) influx in Fura-2-loaded bovine lens epithelial cells were examined to identify potential rapid, non-genomic actions. When administered in the presence of 1-2 mM extracellular Ca(2+) ([Ca(2+)](o)), 100 micro M progesterone produced large (up to 12-fold) and transient (5 min) increases in [Ca(2+)](i). These effects were abolished in EGTA-containing solutions, and were associated with large increases in the rate at which extracellularly administered Mn(2+) quenched the intracellular Fura signal. Lower concentrations of progesterone (10-100 micro M) produced smaller increases in [Ca(2+)](i) that were concentration dependent, and 17beta-estradiol induced large, rapid and brief increases in [Ca(2+)](i) at 100 nM and smaller oscillations in [Ca(2+)](i) at 10 nM. In cells pretreated with thapsigargin, 100 micro M progesterone produced slower increases in [Ca(2+)](i) that were maintained for several minutes. These results demonstrate rapid non-genomic actions of progesterone and estradiol on resting Ca(2+) influx and [Ca(2+)](i) that may involve specific interactions with a recently discovered steroid-binding protein in the plasma membrane of lens epithelial cells.

Animals↗
PubMed2006

Parametric study of the biopotential equation for breast tumour identification using ANOVA and Taguchi method.

Extensive literatures have shown significant trend of progressive electrical changes according to the proliferative characteristics of breast epithelial cells. Physiologists also further postulated that malignant transformation resulted from sustained depolarization and a failure of the cell to repolarize after cell division, making the area where cancer develops relatively depolarized when compared to their non-dividing or resting counterparts. In this paper, we present a new approach, the Biofield Diagnostic System (BDS), which might have the potential to augment the process of diagnosing breast cancer. This technique was based on the efficacy of analysing skin surface electrical potentials for the differential diagnosis of breast abnormalities. We developed a female breast model, which was close to the actual, by considering the breast as a hemisphere in supine condition with various layers of unequal thickness. Isotropic homogeneous conductivity was assigned to each of these compartments and the volume conductor problem was solved using finite element method to determine the potential distribution developed due to a dipole source. Furthermore, four important parameters were identified and analysis of variance (ANOVA, Yates' method) was performed using design (n = number of parameters, 4). The effect and importance of these parameters were analysed. The Taguchi method was further used to optimise the parameters in order to ensure that the signal from the tumour is maximum as compared to the noise from other factors. The Taguchi method used proved that probes' source strength, tumour size and location of tumours have great effect on the surface potential field. For best results on the breast surface, while having the biggest possible tumour size, low amplitudes of current should be applied nearest to the breast surface.

Analysis of Variance↗
PubMed1995

Stable transfection of cloned murine T helper cells.

Here we describe a protocol for the stable transfection of murine T helper (Th) cells and long term culture of the resulting transfectants. The electroporation protocol was established for the murine Th2 clone L1/1 by testing different parameters determining the electric field (capacitance, voltage, single or twin pulse) as well as the activation status of the cells. The transfected T cells were genetically altered by stable integration of the neomycin resistance gene, encoded in the vector pM5neo, into the genome. For selection and long term culture of stable transfectants a scheme combining selection with the antibiotic neomycin (G-418, Geneticin) and repeated stimulation with antigen presenting cells (APC) and antigen was established. This protocol should also be applicable to other antigen reactive T cells. The resistance of the T cells to neomycin correlated directly with expression of the transferred neomycin resistance gene as demonstrated by mRNA analysis. Applying periodic reselection with neomycin the transfected Th2 cells were found to be stable for more than 18 months in culture and displayed an unaltered antigen recognition and lymphokine production pattern as compared with the untransfected L1/1 Th2 cells.

Animals↗
PubMed2005

Nanopowder molding method for creating implantable high-aspect-ratio electrodes on thin flexible substrates.

Metal nanoparticles and a nanopowder molding process were used to fabricate 2D and 3D patternable structures having a height-to-width ratio of up to 10:1. By means of this process, an entire neural stimulation circuit, including stimulating electrode, connection trace, and contact pad, can be fused into one continuous, integrated structure where different sections can have different heights, widths, and shapes. The technique is suitable for mass production, and the fabricated electrode is robust and very flexible. More importantly for biomedical applications, the entire fabricated structure can be packed at room temperature onto a biocompatible flexible substrate, such as polydimethylsiloxane, parylene, and polyimide as well as other temperature-sensitive or vacuum-sensitive materials. The electrodes and wires have about the same electrical resistivities as bulk materials and desirable electrochemical properties, including low impedance.

Electric Capacitance↗
PubMed2004

Understanding capture detection.

Automatic capture detection systems are currently available in several cardiac pacing devices. All current systems use low-polarization electrodes and no beat to beat detection system is available for all types of electrodes. In addition the success ratio for currently available systems is not always 100%. Failure to detect capture reliably is often related to the behaviour of the electrode-tissue interface under different circumstances. Pacemaker electrodes can be considered electrochemical cells with complicated characteristics depending on time, temperature and electrical charge. This electrochemical cell is disturbed when a charge is transferred across the electrode-tissue interface during pacing. Several measures can be taken in order to minimise this disturbance or pace polarization artefact (PPA) including the use of high active surface area electrodes and application of tri-phasic pacing pulses. Another factor influencing detection of evoked potentials is the input circuit of the pacemaker affecting the PPA and the evoked response. Positive PPAs can be falsely interpreted as evoked potentials due to the undershoot of the second order filters applied in modern cardiac pacemakers. This paper explains the behaviour of the interface between the electrode and the cardiac tissue in combination with the pacemaker output circuits and input amplifiers under different circumstances.

Cardiac Pacing, Artificial↗
PubMed2006

The microwave absorption of emulsions containing aqueous micro- and nanodroplets: a means to optimize microwave heating.

The microwave absorption at frequencies between 10 MHz and 4 GHz is measured for aqueous brine droplets dispersed in a dielectric medium (epsilon(')=2.0). By varying the size of the droplets, ion type and ion concentration, it is found that the microwave absorption goes through a maximum which depends on the type of ions and their concentration. The absorption process is attributed to the polarization of the microdroplets through surface charges. Means to optimize microwave heating in emulsions is discussed.

Electric Capacitance↗
PubMedDate not supplied

Mechanisms of thermal injury to the lower genital tract with radiofrequency resectoscopic surgery.

STUDY OBJECTIVE: Identification of mechanisms of thermal injury to the lower genital tract during radiofrequency (RF) resectoscopic surgery. DESIGN: Laboratory comparative study using uterovaginal tissue models. SETTING: University surgical laboratory. PATIENTS: No patients involved. INTERVENTIONS: A tissue model was created to simulate the uterus, contiguous posterior vaginal wall, and the introitus. Instrumentation included a RF electrosurgical generator; a continuous-flow resectoscope; and rollerball electrodes, both intact and with insulation defects created in a standardized fashion. The resectoscope was serially activated, varying electrode insulation defects and RF waveforms and wattage, both in open-circuit conditions, with or without cervical contact, and with variable amounts of the external sheath within the simulated cervical canal. The cervix was either overdilated or minimally dilated so that the surrogate cervical tissue was snug to the external sheath. After activation of the generator, the external sheath was brought into contact with the proximal vagina and perineum, any visible arcing was noted, and tissue effects were visually graded according to a zero-to-three scale. MEASUREMENTS AND MAIN RESULTS: When the resectoscope was in contact with the cervix, the prerequisites for vaginal injury included cervical overdilation, a proximal electrode insulation defect, and less than 2 cm of the external sheath in the canal. There was greater risk with larger electrode insulation defects located beside the telescope, and there was a greater degree of coupling and injury with high voltage outputs. CONCLUSION: The incidence of vaginal and perineal burns associated with unipolar RF resectoscopes can likely be minimized by careful attention to technique.

Animals↗
PubMed2006

Neuron firing in driven nonlinear integrate-and-fire models.

Statistical properties of neuron firing are studied in the framework of a nonlinear leaky integrate-and-fire model that is driven by a slow periodic subthreshold signal. The firing events are characterized by first passage time densities. The experimentally better accessible interspike interval density generally depends on the sojourn times in a refractory state of the neuron. This aspect is not part of the integrate-and-fire model and must be modelled additionally. For a large class of refractory dynamics, a general expression for the interspike interval density is given and further evaluated for the two cases with an instantaneous resetting (i.e. no refractory state) and a refractory state possessing a deterministic lifetime. First passage time densities and interspike interval densities following from the proposed theory compare favorably with precise numerical simulations.

Action Potentials↗
PubMed2006

Intrinsic coherence resonance in excitable membrane patches.

The influence of intrinsic channel noise on the spiking activity of excitable membrane patches is studied by use of a stochastic generalization of the Hodgkin-Huxley model. Internal noise stemming from the stochastic dynamics of individual ion channels does affect the electric properties of the cell-membrane patches. There exists an optimal size of the membrane patch for which the internal noise alone can cause a nearly regular spontaneous generation of action potentials. We consider the influence of intrinsic channel noise in presence of a constant and an oscillatory current driving for both, the mean interspike interval and the phenomenon of coherence resonance for neuronal spiking. Given small membrane patches, implying that channel noise dominates the excitable dynamics, we find the phenomenon of intrinsic coherence resonance. In this case, the relatively regular spiking behavior becomes essentially independent of an applied stimulus. We observed, however, the occurrence of a skipping of supra-threshold input events due to channel noise for intermediate patch sizes. This effect consequently reduces the overall coherence of the spiking.

Action Potentials↗
PubMed2006

Steady-state solutions in mathematical models of atrial cell electrophysiology and their stability.

The steady states of the Fenton-Karma, the Courtemanche and the Nygren cell models were studied by determining the fixed points of the dynamical system describing their cell kinetics. The linear stability of the fixed points was investigated, as well as their response to external stimuli. Symbolic calculations were carried out as far as possible in order to prove the existence of these fixed points. In the Fenton-Karma model, a unique stable fixed point was found, namely the resting state. In contrast, the Courtemanche model had an infinite number of fixed points. A bifurcation diagram was constructed by classifying these fixed points according to a conservation law. Initial conditions were identified, for which the dynamical behavior of the cell was auto-oscillatory. In its original formulation, the Nygren model had no fixed point. After having restored charge conservation, the system was found to have an infinite number of fixed points, resulting in a bifurcation diagram similar to that of the Courtemanche model. The approach proposed in this paper assists in the exploration of the high-dimensional parameter space of the cell models and the identification of the conditions leading to spontaneous pacemaker activity.

Algorithms↗
PubMed2005

The steroid-binding subunit of the Na/K-ATPase as a progesterone receptor on the amphibian oocyte plasma membrane.

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.

Animals↗
PubMed2004

Multi-frequency harmonic arrays: initial experience with a novel transducer concept for nonlinear contrast imaging.

Nonlinear contrast imaging modes such as second harmonic imaging (HI) and subharmonic imaging (SHI) are increasingly important for clinical applications. However, the performance of currently available transducers for HI and SHI is significantly constrained by their limited bandwidth. To bypass this constraint, a novel transducer concept termed multi-frequency harmonic transducer arrays (MFHA's) has been designed and a preliminary evaluation has been conducted. The MFHA may ultimately be used for broadband contrast enhanced HI and SHI with high dynamic range and consists of three multi-element piezo-composite sub-arrays (A-C) constructed so the center frequencies are 4f(A) = 2f(B) = f(C) (specifically 2.5/5.0/10.0 MHz and 1.75/3.5/7.0 MHz). In principle this enables SHI by transmitting on sub-array C receiving on B and, similarly, from B to A as well as HI by transmitting on A receiving on B and, likewise, from B to C. Initially transmit and receive pressure levels of the arrays were measured with the elements of each sub-array wired in parallel. Following contrast administration, preliminary in vitro HI and SHI signal-to-noise ratios of up to 40 dB were obtained. In conclusion, initial design and in vitro characterization of two MFHA's have been performed. They have an overall broad frequency bandwidth of at least two octaves. Due to the special design of the array assembly, the SNR for HI and SHI was comparable to that of regular B-mode and better than commercially available HI systems. However, further research on multi-element MFHA's is required before their potential for in vivo nonlinear contrast imaging can be assessed.

Acoustics↗
PubMed2005

Electro-acoustical characterization procedure for cMUTs.

A procedure for the electro-acoustical characterization in air of cMUTs is reported. First, the measured input electrical impedance of the transducer is used to calculate the transducer parameters at different bias voltages by fitting it with the Mason model. Then, the single membrane equivalent circuit can be calculated. Second, the cMUT impulse response is obtained through a send-and-receive experiment to be compared with the one predicted by the Mason model. In order to minimize the influence of the emitter in the obtained impulse response an ad hoc broadband piezoelectric transducer centered at the resonant frequency of the cMUT was fabricated. Using this transducer, no deconvolution of the impulse response of the emitter in the cMUT reception pulse is necessary. The procedure is tested for two cMUTs with silicon-rich nitride as structural layer and different membrane diameters (60 and 70 microm).

Electric Capacitance↗
PubMed2003

Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers.

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.

Calcium Channels↗
PubMed2001

Dielectric relaxation in a single tryptophan protein.

Although dielectric relaxation can significantly affect the intrinsic fluorescence properties of a protein, usually it is fast compared to fluorescence timescales and needs to be slowed down by adding viscogens or lowering temperature before its impact on fluorescence can be studied. We report here a remarkable blue shift in fluorescence upon bimolecular quenching in the single-tryptophan thermostable protein Bj2S, the 2S seed albumin from Brassica juncea, at ambient temperature and viscosity. The magnitude of the blue shift ( approximately 5 nm at 50% quenching by acrylamide) is striking in a single-tryptophan protein and is attributed to a slowly relaxing dielectric environment in Bj2S from red edge excitation, steady-state polarization and time-resolved fluorescence experiments. Our results have important implications on interpretation of fluorescence of proteins with highly constrained backbones and in designing model systems for studying slow protein solvation dynamics using Trp fluorescence as the reporter probe.

Albumins↗
PubMed2002

Transient absorption and photovoltage study of' self-assembled bacteriorhodopsin/polycation multilayer films.

A series of organized (PDAC/PM)(n) (poly(diallyldimethylammonium chloride)/purple membrane) multilayer films were prepared by alternate adsorptions of positively charged PDAC polyelectrolyte and negatively charged purple membrane (PM). The kinetics of the photocycle of bacteriorhodopsin (bR) in PM was studied by flash photolysis and transient photovoltage methods. Although the orientation of the adsorbed bR depends on the pH of the PM suspension, the kinetics of the photo-induced reaction cycle in dehydrated films is independent of the deposition pH. In dry (PDAC/PM)(n) films the decay of the M intermediate to the initial bR state is multiexponential and delayed to several minutes for both orientations. A simultaneous two-exponential decay in millisecond time domain was observed at red wavelengths. The source of the red-shifted absorption is suggested to be the C(610) intermediate of the cis photocycle of bR.

Bacteriorhodopsins↗