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PubMed2004

Transition state and encounter complex for fast association of cytochrome c2 with bacterial reaction center.

Electrostatic interactions strongly enhance the electron transfer reaction between cytochrome (Cyt) c(2) and reaction center (RC) from photosynthetic bacteria, yielding a second-order rate constant, k(2) approximately 10(9) s(-1).M(-1), close to the diffusion limit. The proposed mechanism involves an encounter complex (EC) stabilized by electrostatic interactions, followed by a transition state (TS), leading to the bound complex active in electron transfer. The effect of electrostatic interactions was previously studied by Tetreault et al. [Tetreault, M., Cusanovich, M., Meyer, T., Axelrod, H. & Okamura, M. Y. (2002) Biochemistry 41, 5807-5815] by measuring k(2) for RC and Cyt molecules with modified charged residues at the binding interface. The present work is a computational analysis of this kinetic study to determine the ensemble of configurations of the TS and EC. Changes in the TS energies due to different mutations were compared with differences in the calculated electrostatic energies for a wide range of Cyt/RC configurations. The TS ensemble, obtained from structures having the highest correlation coefficients in the comparison with experimental data, has the Cyt displaced by approximately 10 A from its position in x-ray crystal structure, close to the average position of the EC ensemble, with strong electrostatic interactions between Cyt on the M subunit side of the RC surface. The heme of the Cyt is oriented toward Tyr L162 on the RC, the tunneling contact in the bound final state on the RC. The similarity between the structures of the EC, TS, and bound state can account for the rapid rate of association responsible for fast diffusion-controlled electron transfer.

Cytochromes c2↗
PubMed2002

Microphone array measurement system for analysis of directional and spatial variations of sound fields.

A measurement system has been developed that is capable of analyzing the directional and spatial variations in a reverberant sound field. A spherical, 32-element array of microphones is used to generate a narrow beam that is steered in 60 directions. Using an omnidirectional loudspeaker as excitation, the sound pressure arriving from each steering direction is measured as a function of time, in the form of pressure impulse responses. By subsequent analysis of these responses, the variation of arriving energy with direction is studied. The directional diffusion and directivity index of the arriving sound can be computed, as can the energy decay rate in each direction. An analysis of the 32 microphone responses themselves allows computation of the point-to-point variation of reverberation time and of sound pressure level, as well as the spatial cross-correlation coefficient, over the extent of the array. The system has been validated in simple sound fields in an anechoic chamber and in a reverberation chamber. The system characterizes these sound fields as expected, both quantitatively from the measures and qualitatively from plots of the arriving energy versus direction. It is anticipated that the system will be of value in evaluating the directional distribution of arriving energy and the degree and diffuseness of sound fields in rooms.

Amplifiers, Electronic↗
PubMed1999

Depolarization-induced mitochondrial Ca accumulation in sympathetic neurons: spatial and temporal characteristics.

Several lines of evidence suggest that neuronal mitochondria accumulate calcium when the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) is elevated to levels approaching approximately 500 nM, but the spatial, temporal, and quantitative characteristics of net mitochondrial Ca uptake during stimulus-evoked [Ca(2+)](i) elevations are not well understood. Here, we report direct measurements of depolarization-induced changes in intramitochondrial total Ca concentration ([Ca](mito)) obtained by x-ray microanalysis of rapidly frozen neurons from frog sympathetic ganglia. Unstimulated control cells exhibited undetectably low [Ca](mito), but high K(+) depolarization (50 mM, 45 sec), which elevates [Ca(2+)](i) to approximately 600 nM, increased [Ca](mito) to 13.0 +/- 1.5 mmol/kg dry weight; this increase was abolished by carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). The elevation of [Ca](mito) was a function of both depolarization strength and duration. After repolarization, [Ca](mito) recovered to prestimulation levels with a time course that paralleled the decline in [Ca(2+)](i). Depolarization-induced increases in [Ca](mito) were spatially heterogeneous. At the level of single mitochondria, [Ca](mito) elevations depended on proximity to the plasma membrane, consistent with predictions of a diffusion model that considers radial [Ca(2+)](i) gradients that exist early during depolarization. Within individual mitochondria, Ca was concentrated in small, discrete sites, possibly reflecting a high-capacity intramitochondrial Ca storage mechanism. These findings demonstrate that in situ Ca accumulation by mitochondria, now directly identified as the structural correlate of the "FCCP-sensitive store, " is robust, reversible, graded with stimulus strength and duration, and dependent on spatial location.

Animals↗
PubMed1986

Direct measurement of longitudinal endolymph flow rate in the guinea pig cochlea.

The rate of longitudinal endolymph flow in the guinea pig cochlea has been measured with a novel tracer technique. The tracer we utilized was the tetramethylammonium (TMA) ion, the movement of which was monitored by ion-sensitive microelectrodes. Extremely small amounts of tracer were required as the electrodes could readily detect TMA concentrations in endolymph as low as 10 microM. TMA was introduced into scala media in the form of a small bolus, varying from 2-20 nl in volume. To examine whether longitudinal flow affects the dispersion of TMA in endolymph, we compared the characteristics of TMA spread to turn I following injection into turn II, with those of TMA spread to turn II following injection into turn I. The comparison of these data with an analytical model combining the processes of diffusion and volume flow demonstrates that the spread of tracer is dominated by passive diffusion processes with very little contribution from longitudinal endolymph flow. The rate of longitudinal endolymph flow between turn I and turn II was estimated to be less than 0.01 mm/min directed towards the basal turn. This value is considerably lower than recently published estimates using other techniques.

Cochlea↗
PubMed1994

Water diffusion and acute stroke.

The occlusion of the middle cerebral artery was used as an experimental acute stroke model in 30 cats. The diffusion of water was followed by diffusion-sensitized MRI between 1 and 15 h after induction of stroke. It is demonstrated that images representing the trace of the diffusion tensor provide a much more accurate delineation of affected area than images representing the diffusion in one direction only. The reason is that the strong contrast caused by the anisotropy and orientation of myelin fibers is completely removed in the trace of the diffusion tensor. The trace images show a small contrast between white and gray matter. The diffusion coefficient of white matter is decreased in acute stroke to approximately the same extent as gray matter. It is further shown that the average lifetime of water in extra and intracellular space is shorter than 20 ms both for healthy and ischemic tissue indicating that myelin fibers are permeable to water. The anisotropy contrast did not change before or after induction of stroke, nor after sacrifice. Together, these observations are consistent with the view that the changes in water diffusion during acute stroke are directly related to cytotoxic oedema, i.e., to the change in relative volume of intra- and extracellular spaces. Changes in membrane permeability do not appear to contribute significantly to the changes in diffusion.

Acute Disease↗
PubMed2005

Transport coefficients and orientational distributions of rodlike particles with magnetic moment normal to the particle axis under circumstances of a simple shear flow.

We have investigated the influences of the magnetic field strength, shear rate, and random forces on transport coefficients such as viscosity and diffusion coefficient, and also on the orientational distributions of rodlike particles of a dilute colloidal dispersion. This dispersion is composed of ferromagnetic spheroidal particles with a magnetic moment normal to the particle axis. In the present analysis, these spheroidal particles are assumed to conduct the rotational Brownian motion in a simple shear flow as well as an external magnetic field. The basic equation of the orientational distribution function has been derived from the balance of the torques and solved numerically. The results obtained here are summarized as follows. For a very strong magnetic field, the rodlike particle is significantly restricted in the field direction, so that the particle points to a direction normal to the flow direction (and also to the magnetic field direction). However, the present particle does not exhibit a strong directional characteristic, which is one of the typical properties for the previous particle with a magnetic moment parallel to the particle axis. That is, the particle can rotate around the axis of the magnetic moment, although the magnetic moment nearly points to the field direction. The viscosity significantly increases with the field strength, as in the previous particle model. The particle of a larger aspect ratio leads to the larger increase in the viscosity, since such elongated particles induce larger resistance in a flow field. The diffusion coefficient under circumstances of an applied magnetic field is in reasonable agreement between theoretical and experimental results.

Journal Article↗
PubMed2006

Diffuse source apportionment of the Po river eutrophying load to the Adriatic sea: assessment of Lombardy contribution to Po river nutrient load apportionment by means of an integrated modelling approach.

The source apportionment of the annual nutrient load carried by the Po river to the Adriatic sea has been studied. An integrated modelling approach was applied to the Lombardy plain area, which covers about 34% of the Po river watershed area and accounts for about 50% of the point sources' loads carried by the river. To extract all the information available from direct instream measurements, two different modelling tools were alternatively used. The source apportionment was investigated considering both dry and wet weather scenarios. In order to quantify the apportionment in dry-weather conditions, the Lombardy portion of the Po river basin was modelled by using the US-EPA QUAL2E model. Such a simulation allowed to assess a significant contribution (about 50% of the total dry-weather load) of a not rain-driven diffuse pollution component (i.e. groundwater, springs, lake emissaries). Moreover, to estimate the rain-driven surface runoff contribution to the instream total load, the Lombardy plain area was also modelled by means of the US-DA SWAT model. SWAT results indicate a runoff contribution to the Po river instream total load of about 10000 t N yr(-1) and 1300 t P yr(-1) (i.e. approximately the 10-20% of the total annual Lombardy nutrient load). At the event scale (i.e. the single rainstorm event) the runoff contribution may rise up to 30-80% of the total instream load. Finally, the total annual nitrogen load at the Po basin closure was estimated for the period 1985-2001. Out of a total annual load of 140000 t N yr(-1), Lombardy accounts for 43% (point plus diffuse sources). The rain-driven diffuse sources constitute the 20% of the overall total load, the point sources account for 40%, whereas the remaining 40% is mainly constituted by "dry-weather diffuse sources" (i.e. groundwater, springs, lake emissaries).

Calibration↗
PubMed1993

Molecular dynamics simulation of NO recombination to myoglobin mutants.

Molecular dynamics simulations on two coupled electronic surfaces are employed to investigate the geminate recombination of nitric oxide to mutants of sperm whale myoglobin. A model for the ground and the excited states is constructed based on experimental data. The crossing between the surfaces is treated using the Landau-Zener formula. The reaction probability and the recombination curves are calculated directly by histogramming the results of an ensemble of trajectories. The experimental trend is reproduced in which the picosecond recombination rate of different mutants increases in the order Phe29 > Leu29 > Val29 > Ala29. Furthermore, in accord with the experiment on significantly longer time scales an opposite trend is obtained, in which the recombination rate for Ala29 is larger than for Phe29. These results are explained by constrained diffusion of the ligand in the heme pocket. The average and the transient volume of the heme pocket is modified by the 29 mutants.

Amino Acid Sequence↗
PubMed1996

Motion discrimination of single targets: comparison of preliminary findings in normal subjects and patients with glaucoma.

BACKGROUND: Luminance, global motion and flicker sensitivities are affected in patients with primary open-angle glaucoma. Although no theoretical model has been put forward to explain the observed reduction in sensitivity in this patient group, these findings have often been attributed to diffuse and selective loss of large optic nerve fibres. METHODS: Movement processing was investigated using an optical projection system that generates smooth, continuous motion with control of speed, displacement and motion direction. Motion-displacement and direction-discrimination thresholds were measured in eight normal subjects and in three patients with diagnosed glaucoma. At each speed tested, targets were presented for a range of displacements and thresholds were extracted after probit analysis. The measurements were carried out both foveally and at 19 degrees in the periphery and provided the data necessary to develop and optimise a model of motion perception based on multiple time delays for the correlation of signals that map progressively more distant parts of the visual field. RESULTS: Our preliminary findings show that direction discrimination can be at chance level even for large displacements when motion is detected 80% of the time. Model simulations show that specific changes in the spatial sampling interval and the speed of transmission of the motion signals involved can cause the observed reduction in motion sensitivity and direction discrimination in patients with glaucoma. CONCLUSIONS: A model for motion detection and direction discrimination of single targets has been proposed to account for the measured functional relationship between motion displacement thresholds and target speed in normal subjects. Tested patients with glaucoma show reduced motion sensitivity and poor discrimination of motion direction. The type of degraded performance observed experimentally in glaucoma patients is also predicted by the model. Such predictions require specific changes in model parameters that may be indicative of changes in the retina caused by the disease.

Adult↗
PubMed1998

Ultrafast DNA analysis by capillary electrophoresis/laser-induced fluorescence detection.

The limits of ultrafast DNA analysis by CE were determined by investigating the influence of the effective capillary length and the electric field strength on the analysis time for a given peak resolution (10 bp). In accordance with theory, the use of a fast ramp power supply for narrow plug electrokinetic injection was found to be essential to minimize the extra column effects on peak dispersion. Two major column dispersion factors, longitudinal diffusion and thermal dispersion, were determined experimentally, as well as the influence of the electric field strength on the electrophoretic mobilities and diffusion coefficients of DNA. It was found that higher field strengths can be applied with lower thermal dispersion than predicted by classical CE models. This was attributed to the faster mass transport in the radial direction due to field-induced DNA orientation. Short capillaries (approximately 3-7 cm effective length) and moderate to high electric field strengths (approximately 600-800 V/cm) were used to perform a series of fast DNA separations. The dsDNA fragment standards phiX174/HaeIII and pBR322/HaeIII were separated within 30 s. The possibility for fast mutation detection was demonstrated using constant denaturant capillary electrophoresis (CDCE) for the analysis of a single base mutation in mitochondrial DNA in 72 s. The potential for fast DNA sequencing was illustrated by separating 300 ssDNA fragments within 180 s.

DNA↗
PubMed1997

Spatial and density effects in evolutionary game theory.

Two models are considered for the study of game dynamics in a spatial domain. Both models are continuous in space and time and give rise to reaction-diffusion equations. The spatial domain is homogeneous but the mobility of the individuals is allowed to depend upon the strategy. The models are analysed for spatial patterns (via a Turing instability) and also for the direction of the travelling wave that replaces one strategy by another. It is shown that the qualitative behaviour of the two models is quite different. When considering the existence of spatial patterns and deciding whether increased mobility is helpful or not, it is shown that the answers depend crucially upon the model equations. Since both models (in the absence of spatial variation) are quite standard, it is clear that considerable care has to be exercised in the formulation of spatial models and in their interpretation.

Animals↗
PubMed1988

Longitudinal mixing in dog lungs during high-frequency forced flow oscillation.

Longitudinal mixing in the conducting airways of eight intubated anesthetized beagles (10.8 +/- 0.9 kg) was studied at functional residual capacity in the presence of forced sinusoidal flow oscillations and in the absence of fresh air bias flow. The ranges of oscillation conditions were: frequencies, f, from 3 to 18 Hz and minute volumes, Vosc, from 50 to 150 ml/sec, corresponding to tidal volumes, Vosc/f, from 0.3 to 4.5 ml/kg body mass. Oscillations were imposed during a breath holding interval incorporated into a modified single-breath nitrogen (N2) washout maneuver. The expired N2 fraction curves were analyzed with a Fickian diffusion model by adjusting the value of a global mixing parameter, (DA2), to achieve an optimal fit of the model to the data. The mixing parameter was an increasing function of minute volume and a decreasing function of frequency, which is well represented by the equation: (DA2) = 2.72 Vosc 1.74 f-1.57 By comparison to available theory and previous measurements in physical systems, this formula implies that Taylor-type dispersion is the dominant mixing mechanism in the conducting airways. Also, the diffusion model predicted, and the data verified, the existence of a mouth-ward 'diffusion flow' during breath holding. This effect, caused by the non-uniform nature of the summed airway cross-section, is directly correlated with the value of (DA2).

Animals↗
PubMed2006

Smoothing and cluster thresholding for cortical surface-based group analysis of fMRI data.

Cortical surface-based analysis of fMRI data has proven to be a useful method with several advantages over 3-dimensional volumetric analyses. Many of the statistical methods used in 3D analyses can be adapted for use with surface-based analyses. Operating within the framework of the FreeSurfer software package, we have implemented a surface-based version of the cluster size exclusion method used for multiple comparisons correction. Furthermore, we have a developed a new method for generating regions of interest on the cortical surface using a sliding threshold of cluster exclusion followed by cluster growth. Cluster size limits for multiple probability thresholds were estimated using random field theory and validated with Monte Carlo simulation. A prerequisite of RFT or cluster size simulation is an estimate of the smoothness of the data. In order to estimate the intrinsic smoothness of group analysis statistics, independent of true activations, we conducted a group analysis of simulated noise data sets. Because smoothing on a cortical surface mesh is typically implemented using an iterative method, rather than directly applying a Gaussian blurring kernel, it is also necessary to determine the width of the equivalent Gaussian blurring kernel as a function of smoothing steps. Iterative smoothing has previously been modeled as continuous heat diffusion, providing a theoretical basis for predicting the equivalent kernel width, but the predictions of the model were not empirically tested. We generated an empirical heat diffusion kernel width function by performing surface-based smoothing simulations and found a large disparity between the expected and actual kernel widths.

Cerebral Cortex↗
PubMed1993

Collagen packing and mineralization. An x-ray scattering investigation of turkey leg tendon.

Several recent results are suggesting that the collagen packing in mineralized tissues is much less regular than in the case of other nonmineralizing collagen, e.g., rat tail tendon. To clarify this question we have investigated the molecular arrangement in mineralized and unmineralized turkey leg tendon as a model for the collagen of mineralized tissues. Using a combination of diffuse x-ray scattering and computer simulation, it could be shown quantitatively that, although the collagen fibril structure is periodic in the axial direction, it is similar to a two-dimensional fluid in the lateral plane. This has important consequences for the understanding of the mineralization process, which is also discussed.

Animals↗
PubMed2004

Iterative absolute electroanalytical approach to characterization of bulk redox conducting systems.

A novel electroanalytical approach is proposed here, and it is demonstrated with the direct and simultaneous determination of two unknowns: the concentration of redox sites and the apparent diffusion coefficient for charge propagation in a single crystal of dodecatungstophosphoric acid. This Keggin-type polyoxometalate serves as a model bulk redox conducting inorganic material for solid-state voltammetry. The system has been investigated using an ultramicrodisk working electrode in the absence of external liquid supporting electrolyte. The analytical method requires numerical solution of the combination of two equations in which the first one describes current (or charge) in a well-defined (either spherical or linear) diffusional regime and the second general equation describes chronoamperometric (or normal pulse voltammetric current) under mixed (linear-spherical) conditions. The iterative approach is based on successive approximations through calculation and minimizing the least-squares error function. The method is fairly universal, and in principle, it can be extended to the investigation of other bulk systems including sol-gel processed materials, redox melts, and solutions on condition that they are electroactive and well behaved, they contain redox centers at sufficiently high level, and a number of electrons for the redox reaction considered is known.

Journal Article↗
PubMed1998

Characterization of the regional intestinal kinetics of drug efflux in rat and human intestine and in Caco-2 cells.

PURPOSE: The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro. METHODS: Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 microns to 1000 microns. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C4 (100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3 degrees C to 37 degrees C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis. RESULTS: Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Ileal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 microns. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Each of the intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number. CONCLUSIONS: Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Km's were in the micro-molar range, and involved an energy dependent mechanism(s).

Adenosine Triphosphate↗
PubMed1995

Improved therapeutic window for treatment of histotoxic hypoxia with a free radical spin trap.

The therapeutic time window for N-methyl-D-aspartate (NMDA) antagonists, non-NMDA antagonists, and glutamate release inhibitors in focal models of ischemia appears to be about 1-2 h. In contrast, a free radical spin trap was found to have an improved therapeutic window. We compared the therapeutic time windows of the NMDA antagonist dizolcilpine maleate (MK-801), the glutamate release inhibitor lamotrigine, and the free radical spin trap n-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) against striatal lesions produced by the mitochondrial toxin malonate, which produces histotoxic hypoxia. Lamotrigine exerted neuroprotective effects when administered at 1 h before malonate injections. MK-801 protected at 1 h before and 1 h after malonate injections, whereas S-PBN showed efficacy when administered up to 6 h after malonate injections. Striatal injections of malonate produced a rapid increase in lactate production and early changes in diffusion-weighted imaging as assessed by magnetic resonance imaging. Therefore, the time course to evolve a lesion in our model of histotoxic hypoxia is comparable with that of other models of focal ischemia. These findings provide direct evidence that a free radical spin trap has an improved therapeutic window compared to an NMDA antagonist and a glutamate release inhibitor. This could be a therapeutic advantage in the treatment of clinical stroke patients.

Animals↗
PubMed2004

Transport coefficients of aqueous dodecyltrimethylammonium bromide solutions: comparison between experiments, analytical calculations and numerical simulations.

We study dynamical properties of ionic species in aqueous solutions of dodecyltrimethylammonium bromide, for several concentrations below and above the critical micellar concentration (cmc). New experimental determinations of the electrical conductivity are given which are compared to results obtained from an analytical transport theory; transport coefficients of ions in these solutions above the cmc are also computed from Brownian dynamics simulations. Analytical calculations as well as the simulation treat the solution within the framework of the continuous solvent model. Above the cmc, three ionic species are considered: the monomer surfactant, the micelle and the counterion. The analytical transport theory describes the structural properties of the electrolyte solution within the mean spherical approximation and assumes that the dominant forces which determine the deviations of transport processes from the ideal behavior (i.e., without any interactions between ions) are hydrodynamic interactions and electrostatic relaxation forces. In the simulations, both direct interactions and hydrodynamic interactions between solutes are taken into account. The interaction potential is modeled by pairwise repulsive 1/r(12) interactions and Coulomb interactions. The input parameters of the simulation (radii and self-diffusion coefficients of ions at infinite dilution) are partially obtained from the analytical transport theory which fits the experimental determinations of the electrical conductivity. Both the electrical conductivity of the solution and the self-diffusion coefficients of each species computed from Brownian dynamics are compared to available experimental data. In every case, the influence of hydrodynamic interactions (HIs) on the transport coefficients is investigated. It is shown that HIs are crucial to obtain agreement with experiments. In particular, the self-diffusion coefficient of the micelle, which is the largest and most charged species in the present system, is enhanced when HIs are included whereas the diffusion coefficients of the monomer and the counterion are roughly not influenced by HIs.

Journal Article↗