Concentration Jumps Research Articles

Biologically Active Molecules with a “Light Switch”

Biologically active compounds which are light-responsive offer experimental possibilities which are otherwise very difficult to achieve. Since light can be manipulated very precisely, for example, with lasers and microscopes rapid jumps in concentration of the active form of molecules are possible with exact control of the area, time, and dosage. The development of such strategies started in the 1970s. This review summarizes new developments of the last five years and deals with "small molecules", proteins, and nucleic acids which can either be irreversibly activated with light (these compounds are referred to as "caged compounds") or reversibly switched between an active and an inactive state.

Temperature, pressure, and concentration jumps for a binary mixture of vapors on a plane condensed phase: Numerical analysis of the linearized Boltzmann equation

The half-space problem of the temperature, pressure, and concentration jumps for a binary mixture of vapors is investigated on the basis of the linearized Boltzmann equation for hard-sphere molecules with the complete condensation condition. First, the problem is shown to be reduced to three elemental ones: the problem of the jumps caused by the net evaporation or condensation, that caused by the gradient of temperature, and that caused by the gradient of concentration. Then, the latter two are investigated numerically in the present contribution because the first problem has already been studied [Yasuda, Takata, and Aoki, Phys. Fluids 17, 047105 (2005)]. The numerical method is a finite-difference one, in which the complicated collision integrals are computed by the extension of the method proposed by Sone, Ohwada, and Aoki [Phys. Fluids A 1, 363 (1989)] to the case of a gas mixture. As a result, the behavior of the mixture is clarified not only at the level of the macroscopic quantities but also at the level of the velocity distribution function. In addition, accurate formulas of the temperature, pressure, and concentration jumps are constructed for arbitrary values of the concentration of the background reference state by the use of the Chebyshev polynomial approximation. The solution of the corresponding problem of a vapor-gas mixture and that of the temperature-jump problem on a simple solid wall are also obtained as special cases of the present problem.

The Minimal, Phase-Transition Model for the Cell-Number Maintenance by the Hyperplasia-Extended Homeorhesis

Oncogenic hyperplasia is the first and inevitable stage of formation of a (solid) tumor. This stage is also the core of many other proliferative diseases. The present work proposes the first minimal model that combines homeorhesis with oncogenic hyperplasia where the latter is regarded as a genotoxically activated homeorhetic dysfunction. This dysfunction is specified as the transitions of the fluid of cells from a fluid, homeorhetic state to a solid, hyperplastic-tumor state, and back. The key part of the model is a nonlinear reaction-diffusion equation (RDE) where the biochemical-reaction rate is generalized to the one in the well-known Schlögl physical theory of the non-equilibrium phase transitions. A rigorous analysis of the stability and qualitative aspects of the model, where possible, are presented in detail. This is related to the spatially homogeneous case, i.e. when the above RDE is reduced to a nonlinear ordinary differential equation. The mentioned genotoxic activation is treated as a prevention of the quiescent G0-stage of the cell cycle implemented with the threshold mechanism that employs the critical concentration of the cellular fluid and the nonquiescent-cell-duplication time. The continuous tumor morphogeny is described by a time-space-dependent cellular-fluid concentration. There are no sharp boundaries (i.e. no concentration jumps exist) between the domains of the homeorhesis- and tumor-cell populations. No presumption on the shape of a tumor is used. To estimate a tumor in specific quantities, the model provides the time-dependent tumor locus, volume, and boundary that also points out the tumor shape and size. The above features are indispensable in the quantitative development of antiproliferative drugs or therapies and strategies to prevent oncogenic hyperplasia in cancer and other proliferative diseases. The work proposes an analytical-numerical method for solving the aforementioned RDE. A few topics for future research are suggested.

The Glutamate Transporter Subtypes EAAT4 and EAATs 1-3 Transport Glutamate with Dramatically Different Kinetics and Voltage Dependence but Share a Common Uptake Mechanism

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-μs time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 μM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na+-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at −90 mV). Applying step changes to the transmembrane potential, V m, of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a τ of ∼15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<−40 mV) and activation at positive V m (>0 mV). A similar inhibitory effect at V m < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V m curve. Jumping the glutamate concentration to 100 μM generated biphasic, saturable transient transport and anion currents (K m ∼ 5 μM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1–3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.

Maximum likelihood fitting of single channel NMDA activity with a mechanism composed of independent dimers of subunits

Steady-state single channel activity from NMDA receptors was recorded at a range of concentrations of both glutamate and glycine. The results were fitted with several plausible mechanisms that describe both binding and gating. The mechanisms we have tested were based on our present understanding of receptor structure, or based on previously proposed mechanisms for these receptors. The steady-state channel properties appear to have virtually no dependence on the concentration of either ligand, other than the frequency of channel activations. This limited the ability to discriminate detail in the mechanism, and, along with the persistence of open-shut correlations in high agonist concentrations, suggests that NMDA channels, unlike other neurotransmitter receptors, cannot open unless all binding sites are occupied. As usual for analyses of NMDA channels, the applicability of our results to physiological observations is limited by uncertainties in synaptic zinc and hydrogen ion concentrations, both of these being known to affect the receptor. The mechanism that we propose, on the basis of steady-state single channel recordings, predicts with fair accuracy the apparent open and shut-time distributions in different concentrations of agonists, correlations between open and shut times, and both the rising and falling phases of the macroscopic response to concentration jumps, and can therefore account for the main features of synaptic currents.

Activation and inactivation kinetics of a Ca2+-activated Cl- current: photolytic Ca2+ concentration and voltage jump experiments

The activation kinetics of the endogenous Ca(2+)-activated Cl(-) current (I (Cl,Ca)) from Xenopus oocytes was investigated in excised "giant" membrane patches with voltage and Ca(2+) concentration jumps performed by the photolytic cleavage of the chelator DM-nitrophen. Currents generated by photolytic Ca(2+) concentration jumps begin with a lag phase followed by an exponential rising phase. Both phases show little voltage dependence but are Ca(2+)-dependent. The lag phase decreases from about 10 ms after a small Ca(2+) concentration jump (0.1 microM) to less than 1 ms after a saturating concentration jump (55 microM). The rate constant of the rising phase is half-maximal at about 5 microM. At saturating Ca(2+) concentrations, the rate constant is 400 to 500 s(-1). The Ca(2+) dependence of the stationary current can be described by the Hill equation with n=2.3 and K (0.5)=0.5 microM. The amplitude of the stationary current decreases after the excision of the membrane patch with t (1/2) approximately 5 min (run-down). The activation kinetics of the current elicited by a Ca(2+) concentration jump is not affected by the run-down phenomenon. At low Ca(2+) concentration (0.3 microM), voltage jumps induce a slowly activating current with voltage-independent time-course. Activation is preceded by an initial transient of about 1-ms duration. At saturating Ca(2+) levels (1 mM), the initial transient decays to a stationary current. The transient can be explained by a voltage-dependent inactivation process. The experimental data reported here can be described by a linear five-state reaction model with two sequential voltage-dependent Ca(2+)-binding steps, followed by a voltage-independent rate-limiting transition to the open and a voltage-dependent transition to a closed, inactivated state.

Evaporation and condensation of a binary mixture of vapors on a plane condensed phase: Numerical analysis of the linearized Boltzmann equation

Half-space problem of evaporation and condensation of a binary mixture of vapors is investigated on the basis of the linearized Boltzmann equation for hard-sphere molecules with the complete condensation condition. The problem is analyzed numerically by a finite-difference method, in which the complicated collision integrals are computed by the extension of the method proposed by Y. Sone, T. Ohwada, and K. Aoki [“Temperature jump and Knudsen layer in a rarefied gas over a plane wall: Numerical analysis of the linearized Boltzmann equation for hard-sphere molecules,” Phys. Fluids A 1, 363 (1989)] to the case of a gas mixture. As a result, the behavior of the mixture is clarified not only at the level of the macroscopic quantities but also at the level of the velocity distribution function. In addition, accurate formulas of the temperature, pressure, and concentration jumps caused by the evaporation and condensation are constructed for arbitrary values of the concentration of the background reference state by the use of the Chebyshev polynomial approximation.

Detection of channel proximity by nanoparticle-assisted delaying of toxin binding; a combined patch-clamp and flow cytometric energy transfer study.

Gold nanoparticles of 30 nm diameter bound to cell-surface receptor major histocompatibility complex glycoproteins (MHCI and MHCII), interleukin-2 receptor alpha subunit (IL-2Ralpha), very late antigen-4 (VLA-4) integrin, transferrin receptor, and the receptor-type protein tyrosin phosphatase CD45 are shown by the patch-clamp technique to selectively modulate binding characteristics of Pi(2) toxin, an efficient blocker of K(v)1.3 channels. After correlating the electrophysiological data with those on the underlying receptor clusters obtained by simultaneously conducted flow cytometric energy transfer measurements, the modulation was proved to be sensitive to the density and size of the receptor clusters, and to the locations of the receptors as well. Based on the observation that engagement of MHCII by a monoclonal antibody down-regulates channel current and based on the close nanometer-scale proximity of the MHCI and MHCII glycoproteins, an analogous experiment was carried out when gold nanoparticles bound to MHCI delayed down-regulation of the K(v)1.3 current initiated by ligation of MHCII. Localization of K(v)1.3 channels in the nanometer-scale vicinity of the MHC-containing lipid rafts is demonstrated for the first time. A method is proposed for detecting receptor-channel or receptor-receptor proximity by observing nanoparticle-induced increase in relaxation times following concentration jumps of ligands binding to channels or to receptors capable of regulating channel currents.

Response changes of some wells in the mainland subsurface fluid monitoring network of China, due to the September 21, 1999, Ms7.6 Chi-Chi Earthquake

About 60 hydrologic changes in response to the Chi-Chi earthquake with Ms7.6 on September 21, 1999, occurred in 52 wells, including groundwater level, temperature, discharge rate, well pressure and radon, etc., in the subsurface fluid monitoring network. These response changes were mainly co-seismic, but some pre- and post-earthquake changes occurred mainly within 5 days before and after the Chi-Chi earthquake. The response changes of different wells clustering in different tectonic areas showed different features. These changes are distributed in five areas named as A, B, C, D and E. The response changes in A area with short hypo-central distance (less than 550 km) were mainly pre-earthquake changes occurring more than 5 days before the event. Those in area B (in Huanan tectonic block) and C (in Huabei tectonic block) were mainly co-seismic changes. The hypo-central distance is about 1100–1280 and 800–1160 km, respectively. These changes were high-frequency water-level oscillations induced by seismic waves and accompanied by prominent and permanent water-level jumps and drops. There are also some post-seismic changes including discharge rate and water radon and well pressure changes in area C. Those in area D in the Yanshan tectonic block were mainly co-seismic and post-seismic changes including water level, water temperature, and water radon concentration, etc., showing prominent and permanent water-level jumps and drops and rising concentrations of water radon. The hypo-central distance is about 1750–2060 km. Those in Area E were mainly co-seismic changes showing prominent and permanent water-level jump. The hypo-central distance is about 1810–2120 km. Three moderate earthquakes occurred in area D and one strong earthquake occurred in area E 4 months after the Chi-Chi earthquake. The different features of the response changes might be caused by the changes of local hydrologic conditions (like permeability) induced by seismic waves. On the other hand, these response changes might indicate the near-critical conditions in the area where the response changes clustered. Such changes might be understood by the crustal buckling hypothesis. It is thought that the response changes might be a kind of precursor that implies elevated earthquake risk in the region.

Using Light to Induce Temperature Jumps (T-jumps) and Cofactor Concentration Jumps (C-jumps) in Cryoelectron Microscopy Specimens: Design Criteria

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Time-Resolved Charge Translocation by Sarcoplasmic Reticulum Ca-ATPase Measured on a Solid Supported Membrane

Sarcoplasmic reticulum vesicles were adsorbed on an octadecanethiol/phosphatidylcholine mixed bilayer anchored to a gold electrode, and the Ca-ATPase contained in the vesicles was activated by ATP concentration jumps both in the absence and in the presence of K + ions and at different pH values. Ca 2+ concentration jumps in the absence of ATP were also carried out. The resulting capacitive current transients were analyzed together with the charge under the transients. The relaxation time constants of the current transients were interpreted on the basis of an equivalent circuit. The current transient after ATP concentration jumps and the charge after Ca 2+ concentration jumps in the absence of ATP exhibit almost the same dependence upon the Ca 2+ concentration, with a half-saturating value of ∼1.5 μM. The pH dependence of the charge after Ca 2+ translocation demonstrates the occurrence of one H + per one Ca 2+ countertransport at pH 7 by direct charge-transfer measurements. The presence of K + decreases the magnitude of the current transients without altering their shape; this decrease is explained by K + binding to the cytoplasmic side of the pump in the E 1 conformation and being released to the same side during the E 1–E 2 transition.

Calcium transport by sarcoplasmic reticulum Ca-ATPase can be investigated on a solid-supported membrane

Sarcoplasmic reticulum (SR) native vesicles incorporating Ca-ATPase are adsorbed on a solid-supported lipid membrane (SSM). Upon adsorption, the ion pumps are chemically activated by concentration jumps of ATP and the capacitive current transients generated by SR Ca-ATPase are measured under potentiostatic conditions. The Michaelis–Menten constant, K M, for ATP is evaluated by varying the concentration of ATP in the activating solution. This preliminary result shows that ion transport by SR Ca-ATPase can be suitably investigated by a technique based on concentration jumps on an SSM.

平面凝縮相における2成分混合蒸気の蒸発・凝縮 : 線形化ボルツマン方程式の数値解析(S27-2 熱流体現象に対する分子気体力学および分子動力学法の応用(2),S27 熱流体現象に対する分子気体力学および分子動力学法の応用)

Half-space problem of evaporation and condensation of a binary mixture of vapors is investigated on the basis of the linearized Boltzmann equation for hard-sphere molecules with the complete condensation condition. The problem is analyzed numerically by a finite-difference method, in which the complicated collision integrals are computed by the extension of the method proposed by Sone, Ohwada, and Aoki [Phys. Fluids A 1, 363 (1989)] to the case of a gas mixture. As a result, the behavior of the mixture is clarified not only at the level of the macroscopic quantities but also at the level of the velocity distribution function. In addition, accurate formulas of the temperature, pressure, and concentration jumps caused by the evaporation or condensation are constructed for arbitrary values of the concentration of a component gas by the use of the Chebyshev polynomial approximation.

Biphasic excitation by leucine in Escherichia coli chemotaxis.

Leucine concentration jumps (applied by photolysis of inert derivatives) triggered swim or tumble responses in Escherichia coli mutants lacking Tsr or Tar, respectively. Wild-type E. coli bacteria were attracted in spatial assays when the initial leucine concentration difference was 5 to 120 micro M but were repulsed when it was over 0.5 mM. Their responses to concentration jumps confirmed earlier deductions regarding biphasic excitation.

Investigation of Na +,K +-ATPase on a solid supported membrane: the role of acylphosphatase on the ion transport mechanism

Charge translocation by Na +,K +-ATPase was investigated by adsorbing membrane fragments containing Na +,K +-ATPase from pig kidney on a solid supported membrane (SSM). Upon adsorption, the ion pumps were activated by performing ATP concentration jumps at the surface of the SSM, and the capacitive current transients generated by Na +,K +-ATPase were measured under potentiostatic conditions. To study the behavior of the ion pump under multiple turnover conditions, ATP concentration jump experiments were carried out in the presence of Na + and K + ions. Current transients induced by ATP concentration jumps were also recorded in the presence of the enzyme α-chymotrypsin. The effect of acylphosphatase (AcP), a cytosolic enzyme that may affect the functioning of Na +,K +-ATPase by hydrolyzing its acylphosphorylated intermediate, was investigated by performing ATP concentration jumps both in the presence and in the absence of AcP. In the presence of Na + but not of K +, the addition of AcP causes the charge translocated as a consequence of ATP concentration jumps to decrease by about 50% over the pH range from 6 to 7, and to increase by about 20% at pH 8. Conversely, no appreciable effect of pH upon the translocated charge is observed in the absence of AcP. The above behavior suggests that protons are involved in the AcP-catalyzed dephosphorylation of the acylphosphorylated intermediate of Na +,K +-ATPase.

Numerical analysis of the effects of temperature and concentration jumps on transient evaporation of moderately large (0.01 ≲ Kn ≲ 0.3) droplets in non-isothermal multicomponent gaseous mixtures

In this study transient evaporation of a single moderately large (0.01 ≲ Kn ≲ 0.3) droplet in non-isothermal multicomponent gaseous mixture taking into account the effects of temperature and concentration jumps is investigated numerically. System of nonstationary nonlinear energy and mass conservation equations is solved using anelastic approximation. The performed analysis is pertinent to slow droplet evaporation when Mach number is much less then unity (M≪1). Transport coefficients are calculated as functions of temperature and concentrations of the gaseous species. The dependence of the droplet surface temperature on time is taken into account. Comparison of numerical results obtained using models with different types of boundary conditions at the gas-liquid interface is performed. It is found that the effect of temperature and concentration jumps on the evaporation of moderately large droplet during warming-up period is significant when the temperature and vapor concentration at the droplet surface is relatively low. It is shown that in case of large concentrations at the droplet surface boundary conditions for concentration jump obtained from kinetic theory and those obtained from the Fick's law yield the same results. It is shown also that taking into account the kinetic effects results in a significant deviation from the D2-law.

The Problem on Nonstationary Growth and Evaporation of Drops

A general theory is suggested of nonstationary processes of evaporation and condensation growth of a quiescent spherical drop, when the boundary conditions in the case of simultaneous solution of nonstationary equations of diffusion and heat conduction include jumps of concentration and temperature on the Knudsen layer. Integral Laplace transforms are used to derive a general expression for the concentration distribution, as well as equations for the temperature fields on the drop surface and in the surrounding medium. Their dependence on the composite coefficients of concentration and temperature jumps is observed. The asymptotic behavior of general equations of the fields of concentration and temperature, as well as of the distributions of the rate of temperature variation on the drop surface, is investigated. A general (for all times) expression for the rate of variation of the drop radius is derived and investigated using asymptotic expansions, and the limits of validity of some results of previous theories are established.

The Ca/Ca exchange mode of the Na/Ca exchanger investigated by photolytic Ca2+ concentration jumps.

The Ca/Ca exchange mode of the Na/Ca exchanger investigated by photolytic Ca2+ concentration jumps.

Equilibrium and kinetic folding of hen egg-white lysozyme under acidic conditions.

The equilibrium and kinetic folding of hen egg-white lysozyme was studied by means of circular dichroism spectra in the far- and near-ultraviolet (UV) regions at 25 degrees C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl-induced unfolding experiment. However, in the GdnCl-induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time-dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped-flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far-UV CD change during the dead time (<10 ms) of the stopped-flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single-exponential function, and the rate constants obtained in the far- and near-UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three-state mechanism, U<-->I<-->N, in which the burst-phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate-determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed.

Slow phases of GABAA receptor desensitization: structural determinants and possible relevance for synaptic function

GABA(A) receptor fast desensitization is thought to shape the time course of individual IPSCs. Although GABA(A) receptors also exhibit slower phases of desensitization, the possible role of slow desensitization in modifying synaptic function is poorly understood. In transiently transfected human embryonic kidney (HEK293T) cells, rat alpha1beta3delta and alpha1beta3gamma2L GABA(A) receptors showed distinct desensitization patterns during long (28 s) concentration jumps using a saturating (1 mM) GABA concentration. alpha1beta3gamma2L receptors desensitized extensively (approximately 90%), with four phases (tau(1) approximately 20 ms, tau(2) approximately 400 ms, tau(3) approximately 2 s, tau(4) approximately 10 s), while alpha1beta3delta receptors desensitized slowly and less extensively (approximately 35 %), with one or two slow phases with time constants similar to tau(3) and tau(4) of alpha1beta3gamma2L receptors. To determine the structural basis of subunit-specific desensitization, delta-gamma2L chimera subunits were expressed with alpha1 and beta3 subunits. Replacing the entire N-terminus of the gamma2L subunit with delta subunit sequence did not alter the number of phases or the extent of desensitization. Although extension of delta subunit sequence into transmembrane domain 1 (TM1) abolished the fast and intermediate components of desensitization, the two slow phases still accounted for substantial current loss (approximately 65 %). However, when delta subunit sequence was extended through TM2, the extent of desensitization was significantly decreased and indistinguishable from that of alpha1beta3delta receptors. The importance of TM2 sequence was confirmed by introducing gamma2 subunit TM2 residues into the delta subunit, which significantly increased the extent of desensitization, without introducing either the fast or intermediate desensitization phases. However, introducing delta subunit TM2 sequence into the gamma2L subunit had minimal effect on the rates or extent of desensitization. The results suggest that distinct delta subunit structures are responsible for its unique desensitization properties: lack of fast and intermediate desensitization and small contribution of the slow phases of desensitization. Finally, to investigate the possible role of slow desensitization in synaptic function, we used a pulse train protocol. We observed inhibition of peak current amplitude that depended on the frequency and duration of GABA pulses for receptors exhibiting extensive desensitization, whether fast phases were present or not. The minimally desensitizing alpha1beta3delta receptor exhibited negligible inhibition during pulse trains. Because receptors that desensitized without the fast and intermediate phases showed pulse train inhibition, we concluded that receptors can accumulate in slowly equilibrating desensitized states during repetitive receptor activation. These results may indicate a previously unrecognized role for the slow phases of desensitization for synaptic function under conditions of repeated GABA(A) receptor activation.