Transient electric birefringence study of highly dilute agarose solutions

In order to characterize the first step of agarose gelation, highly dilute solutions (2·10^?3 to 0.5 g/L) have been studied by means of the transient electric birefringence technique. The field-free decay curves of the birefringence are well described by a stretched-exponential B(t) ≈ exp(?t/τ)^β; the value of the exponent β is close to 0.5 whatever the agarose concentration. The suspended particles observed by electron microscopy present a rod-like shape with a constant diameter (～50 Å), without any branching; they are polydisperse with a distribution of lengths approximately exponential. The mean length of these fibers, deduced from their mean rotational diffusion coefficient, is proportional to the 0.37 power of the agarose concentration in the solution. Furthermore, these particles possess a strong permanent electrical dipole confirming the side-to-side arrangement of helices into bundles; this dipole is roughly proportional to the particle length, indicating a self-similarity in the unidirectional growth of the agarose fibers, even when approaching the gelling concentration.     

Electric and hydrodynamic properties of polypeptides in solution. II. Conformation of poly(L-glutamic acid) in various organic solvents

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol, dimethyl sulfoxide, dimethylformamide, N-methylacetamide, trifluoroacetic acid, dioxane–water mixtures (3:1 and 4:1 by volume), and dioxane–formamide mixture (1:1 by volume) has been measured by the use of the rectangular pulse technique at 30 °C. The intrinsic viscosity has also been measured at the same temperature. The magnitude of the specific Kerr constant and the intrinsic viscosity suggests that PLGA is helical and has a large dipole moment in methanol, dimethyl sulfoxide, dimelhylformamide, N-methylacetamide, and dioxane–water mixtures. In this case we have obtained the length distribution curve and the mean length of PLGA molecules from the decay of the electric birefringence, by applying the method recently developed for helical polypeptides. Furthermore, we have proposed and applied a method of obtaining the mean dipole moment and the optical anisotropy factor from the field strength dependence of the electric birefringence for polydisperse systems on the basis of the knowledge on the length distribution. The results show that PLGA may have a different helical conformation in dimethyl sulfoxide. The specific Kerr constant of PLGA in trifluoroacetic acid is very small, which suggests that PLGA is a random coil in this solvent.     

Theory of boundary spreading in velocity ultracentrifugation of polydisperse solutes

Under the assumptions that the rectangular approximation to the sector-shaped cell is valid and that both the sedimentation coefficient s and the diffusion coefficient D are independent of concentration, asymptotic solutions to the boundary spreading equation for velocity ultracentrifugation of polydisperse solutes have been derived for three cases: case A, D = constant for all s; case B, sD = constant; case C, \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {sD} = {\rm constant} $\end{document}. Case A is the situation treated by all of the previous authors but supposed to be unrealistic for ordinary macromolecular solutes. Cases B and C may be associated with synthetic polymers under theta conditions and globular proteins in aqueous media, respectively. The solutions obtained have been used to explore the theoretical background of the empirical Gralén method for evaluating the distribution of s from sedimentation boundary curves, with special interest in the behavior of a plot for Sversus 1/t. Here S is the value of s for a fixed value of the apparent integral distribution of s and t is the time of centrifugation. It was found that when the distribution of s is Gaussian-like and fairly narrow, this plot becomes linear over a more extended range of t in the order case B > case C > case A.     

Transient state isoelectric focusing: theory

Mathematical theory is developed to describe the transient state of isoelectric focusing (pH-gradient electrophoresis) in a linear pH gradient under highly idealized conditions. This theory makes it possible to predict the concentration profile (distribution) for the protein or other amphoteric species of interest as a function of time, when the sample is applied in a zone of infinitesimal thickness at one end of the column, or in a uniform distribution throughout the column. Further, the position of the centroid, and the second moment around the mean, σ², (square of the standard deviation of peak width) are described as a function of time, irrespective of the initial distribution of the protein in the column. Three arbitrary stages of the “focusing” experiment are considered: (1) Focusing, wherein the sample is applied to a preformed pH gradient; (2) Defocusing, which occurs when the electrical field is abolished after an arbitrary time (usually after the concentration profile has begun to approach its steady state) and diffusion is allowed to occur. (3) Refocusing, which occurs after the electrical field is reapplied. Although stages 1 and 3 are conceptually identical aside from the difference in initial conditions, they may differ in several important respects in practice, both with regard to technical problems of measurement, and with regard to the closeness of conditions to the stated assumptions.This theory should make it possible to predict the time necessary to achieve any desired degree of focusing, i.e., approach to the steady-state distribution. Further, this theory and the techniques of analytical scanning isoelectric focusing provide the basis for measurement of the apparent diffusion coefficient (D), the derivative of velocity with respect to position, and if the field strength is known, the slope of the mobility-pH curve at the isoelectric point, {$\text{dM}\text{d(}\text{pH}\text{)}$}.     

Electric and hydrodynamic properties of polypeptides in solution. IV. A new conformational change of poly(L-glutamic acid) in dimethylsulfoxide–methanol mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in dimethylsulfoxide (DMSO)–methanol mixtures has been measured by use of the rectangular pulse technique. The length distribution curve, the mean molecular length, and the mean apparent permanent dipole moment of PLGA in solution have been obtained from the decaycurve and field strength dependence of the steady-state birefringence according to the method developed for analyzing the electric birefringence of a polydisperse system. The length distribution curve exhibits one or two peaks. The length corresponding to a high peak and the mean length of PLGA undergo an abrupt change in the vicinity of 50 to 60 vol % DMSO at 30°C. Moreover, a sharp change of the Moffitt b₀ parameter with the solvent composition is observed. These results provide evidence for the existence of a solvent-induced transition from a helical conformation (presumably α-helix) to another helical conformation with shorter length per amino acid residue. Further, the temperature dependence of the length distribution of PLGA in 50 vol % DMSO suggests the existence of a temperature-induced helix ? helix transition.     

Dependence of laser light scattering of DNA on NaCl concentration

We show that the persistence length a of DNA, derived from total intensity laser light scattering of linear Col E₁ DNA and corrected for excluded-volume effects, varies from about 68 nm in 0.005M NaCl to about 40 nm in 0.2M NaCl, leveling off to a constant value (about 27 nm) at high NaCl (1–4M) concentration. These observations do not agree with current views on the effect of electrostatic charge and ionic conditions on DNA dimensions. The apparent diffusion constant D_app, determined from laser light scattering autocorrelation as a function of scattering vector q, at NaCl concentrations 0.005–4M, correctly yields the translational diffusion coefficient D_t at low values of q and scales with molecular dimensions rather than segment length at high values of q; thus, D_app/D_t yields a universal curve when plotted against q²R, where R_g is the radius of the gyration. The sedimentation coefficients s at 0.1 and 0.2M NaCl concentration closely agree with the well-tested empirical relations, and a combination of s, D_t, and the appropriate density increments yield correct molar masses over the whole salt concentration range. Approximate constancy of D_tR_g indicates limited draining in translational flow. We present some observations and thoughts on the regimes in which a dependence of the correlation decay times on q³ rather than q² applies. We conclude that quasielastic laser light scattering discloses little information about dynamics of internal motion of DNA chains.     

Rare earth element uptake by the marine diatom

Abstract

Rare earth distribution coefficients, DT = (moles cm^-3, cells)/(moles cm^-3, solution), obtained using seawater (S = 36.4, t = 25°C, pH ～ 8.2, pCO₂ ～ 345 μatm) and the marine diatom, Skeletonema costatum, exhibited a strong tendency toward the order Ce > Gd > Yb. Observations of rapid initial uptake, with subsequent gradual uptake over time, are suggestive of initial adsorption onto cell surfaces followed by slow transport to interior cell sites. The average volume concentration factors (DT) obtained in our study are: DTCe = (3.33 ± 0.9) × 10⁵; DTGd = (2.41 ± 0.7) × 10⁵; DTYb = (1.64 ± 0.3) × 10⁵. Distribution coefficient results, expressed as a competition between solution and solid-state complexation terms, indicate that rare earth element complexation, both in solution and on surfaces, strongly increases with atomic number. Relatively small differences in rare earth element distribution coefficients (DT) with atomic number are the result of small differences between large solution and solid-state complexation terms.     

Electrical and hydrodynamical properties of polypeptides in solution. I. Poly(L-glutamic acid) in methanol-water mixtures

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol–water mixtures has been measured by the use of the rectangular pulse technique at 25°C. The permanent dipole moment, the anisotropy of electrical polarizability, and the optical anisotropy factor of PLGA in solution were obtained from the dependence of the steady-state birefringence on the electric field strength. Further, the mean length of PLGA in solution was calculated by a parameter method developed for analyzing the decay curve of electric birefringence. The permanent dipole moment per unit length obtained from these studies was 2.9₆, 2.4₈, 2.3₀, 2.6₆ D/Å in pure methanol, 10, 30, and 50 vol-% water, respectively. The increase of water content caused the decrease of the mean length and broadened the length distribution of PLGA. These results are discussed in relation to the viscosity and the electrical conductivity of PLGA solutions.     

On the use of non-linear regression with the logistic equation for changes with time of percentage root length colonized by arbuscular mycorrhizal fungi

For the regression of sigmoid-shaped responses with time t of colonization C of roots by arbuscular mycorrhizal fungi, C=C _p/1+[e ^−k(t−ti) ] is the most useful form of the logistic equation. At the time of inflection t _i the slope is maximal and directly proportional to the product of the colonization plateau C _p and the abruptness k of the curve. Coefficient k has a high value when the curve rises abruptly following and preceding long shallow phases. The logistic equation has a curve that is symmetrical about t _i such that C=C _p/2 at inflection. Although the logistic equation can generate a good fit to many data sets for changes in colonization with time, there are cases that are not sigmoid and the logistic equation does not apply. For sigmoid curves, the lag in the development of colonization is directly related to both t _i and k but not to the plateau and not to the value of the maximum slope. Higher values of k or t _i reflect longer lag. When considered alone, t _i and k do not fully summarize the lag in colonization, and so a numerical method to combine them is presented here which allows lag to be compared between curves. In this method, the lag is evaluated by calculating the time during early colonization when the slope equals half of the value of the maximum slope. In summary, use of the logistic equation for regression of sigmoid curves of colonization with time allows numerical comparison between curves of the lag, the period of steep ascent, and the plateau. The logistic equation does not model directly the fundamental processes at work in the development of the mycorrhizae. Instead, it can be used as described here to gain insight into the colonization process by comparing the dynamics of that colonization for different species under various conditions. Accepted: 20 October 2000     

Dependence of the internal relaxation time for DNA on salt type as inferred by quasielastic light scattering

Quasielastic light scattering methods were used to study calf thymus DNA in solutions of LiCl, NaCl, NH₄Ac, and NH₄Cl. Plots of the reciprocal relaxation time (1/τ) vs sin²(θ/2), where θ is the scattering angle, exhibit two linear regions, in accordance with theories for semiflexible polymers based on the t → 0 approximation. In these theories the slope of the linear region at low angles is associated with the translational diffusion coefficient (D_t), whereas the slope of the linear region at high angles is associated with the segmental diffusion coefficient (D_s = kT/?_s). The midpoint of the “transition” between these two linear regions is associated with the mean displacement between segments (b). Data presented here indicate that the Rouse-Zimm parameters b and ?_s are significantly different for DNA in 0.4M NH₄Cl relative to the other salts at comparable ionic strengths. It is suggested that this difference reflects local solvent structure and that both b and D_s are sensitive to the local water structure.     

Molecular characterization of the HMW glutenin genes Dt x1.5?+?Dt y10 from Aegilops tauschii and their PCR-mediated recombinants

The high molecular weight (HMW) glutenin subunits, D^tx1.5 + D^ty10, are special types of storage proteins found in Aegilops tauschii that are never found in common wheat (Triticum aestivum). This study reports the characterization of the complete open reading frames (ORFs) of the HMW glutenin genes, D^tx1.5 and D^ty10, using a restrict-enzyme based method named the restricted deletion method (RDM). The D^tx1.5 and D^ty10 were found to have an identical structure compared with the other published HMW glutenin genes. Comparison of the deduced protein sequences also indicated that the D^ty10 in Ae. tauschii differed from its counterpart Dy10 in common wheat, by having insertions and deletions in the central repetitive domain. This result confirms the two subunits with same mobility in SDS-PAGE are different types of HMW glutenin subunits. In addition, four PCR-mediated recombinants of the D^tx1.5 and D^ty10 genes were amplified using a PCR program with shorter extension time. The recombinants had a similar structure to their corresponding natural genes, but a significantly different central repetitive domain. Western blot analysis exhibited a normal expression of the recombinants in E. coli. In addition to its usefulness for studying structure and function of the HMW glutenin subunits, the PCR-mediated recombination may provide an efficient method to generate novel HMW glutenin genes for wheat breeding.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

QELS-SEF study on poly(lysine) in the extraordinary phase: Effect of electric field strength on the apparent diffusion coefficient

The technique of quasielastic light scattering (QELS) in the presence of a sinusoidal electric field (QELS-SEF) was used to examine the diffusion of poly(lysine) in the extraordinary phase, which is defined by the anomalously small value in the apparent diffusion coefficient (D_app, obtained by QELS methods) as the added salt concentration is lowered below 10 mM. Spectra were obtained for high-molecular-weight poly(lysine) (800,000) in 0.5 mM KCl, using a driving frequency of 90 Hz. It was observed that the linewidth, hence apparent diffusion coefficient (D_SF), increased with electric field strength (E) over the range 6 < E < 30 V/cm. As might be anticipated, the asymptotic limit, D_SF(E = 0), was almost numerically equivalent to D_app at this KCl concentration. As E approached the upper limit value of 31 V/cm, the value of D_SF approached the high salt (500 mM KCl) value of D_app. These observations are discussed in terms of small-ion dispersion effects.     

A novel computational framework for D(t) from Fluorescence Recovery after Photobleaching data reveals various anomalous diffusion types in live cell membranes

Diffusion of proteins and lipids in lipid membranes plays a pivotal role in almost all aspects of cellular biology, including motility, exo?/endocytosis and signal transduction. For this reason, gaining a detailed understanding of membrane structure and function has long been a major area of cell biology research. To better elucidate this structure‐function relationship, various tools have been developed for diffusion measurements, including Fluorescence Recovery After Photobleaching (FRAP). Because of the complexity of cellular microenvironments, biological diffusion is often correlated over time and described by a time‐dependent diffusion coefficient, D(t) , although the underlying mechanisms are not fully understood. Since D(t) provides important information regarding cellular structures, such as the existence of subresolution barriers to diffusion, many efforts have been made to quantify D(t) by FRAP assuming a single power law, D(t) = Γt ^{α ? 1} where Γ and α are transport coefficient and anomalous exponent. However, straightforward approaches to quantify a general form of D(t) are lacking. In this study, we develop a novel mathematical and computational framework to compute the mean square displacement of diffusing molecules and diffusion coefficient D(t) from each individual time point of confocal FRAP data without the single power law assumption. Additionally, we developed an auxiliary equation for D(t) which can readily distinguish normal diffusion or single power law anomalous diffusion from other types of anomalous diffusion directly from FRAP data. Importantly, by applying this approach to FRAP data from a variety of membrane markers, we demonstrate the single power law anomalous diffusion assumption is not sufficient to describe various types of D(t) of membrane proteins. Lastly, we discuss how our new approaches can be applied to other fluorescence microscopy tools such as Fluorescence Correlation Spectroscopy (FCS) and Single Particle Tracking (SPT).     

Current-voltage relationships and voltage sensitivity of the Cl− pump inHalicystis parvula

Summary The current-voltage (I–V) relationships for internally perfused and nonperfused cells ofHalicystis parvula were determined. In both types of cells theI–V curve shows a conspicuous region of negative slope, beginning at vacuole potentials around –30 mV and continuing to values of +20 to +40mV. The negative slope in perfused cells is abolished by the metabolic inhibitors, darkness and low temperature. In order to determine the origin of this negative slope, we measured the voltage sensitivity of the unidirectional fluxes of Cl^–, Na⁺ and K⁺ in perfused cells. The results show that the Cl^– influx, which is mediated primarily by a Cl^– pump, increases as the vacuole potential is clamped at increasingly morenegative values up to –50 mV, while the other fluxes measured changed in the directions predicted by the change in electrical driving force. The voltage sensitivity of the Cl^– pump quantitatively accounts for the negative slope of theI–V curve. Also, we observed a large transient outward current of 10–20-sec duration following an abrupt depolarization by voltage clamping. This transient current was reduced or abolished by low temperature, which suggests that it may be due to the voltage-sensitive Cl^– pump. Finally, we found an inverse relationship between the transprotoplasm resistance (R _m ) and thePD under standard conditions, which suggests that the activity of the electrogenic Cl^– pump lowerR _m , i.e., it is a conductive pump.     

Bionanocomposites based on pea starch and cellulose nanowhiskers hydrolyzed from pea hull fibre: Effect of hydrolysis time

By hydrolyzing pea hull fibres (PHF) for different times (t) with sulfuric acid, a series of pea hull fibre-derived nanowhisker (PHFNW-t) dispersions was prepared. The PHFNW-t dispersions were then blended with pea starch (PS) to fabricate bionanocomposite films (PS/PHFNW-t). The effect of t on the structure and properties of the PHFNW-t nanowhiskers and those of the corresponding PS/PHFNW-t nanocomposite films were investigated. The length (L), diameter (D) and L/D values of the PHFNW-t ranged from 240 to 400 nm, 7 to 12 nm, and 32.22 to 36.00, respectively. The PS/PHFNW-t nanocomposite films exhibited higher ultraviolet absorption, transparency, tensile strength, elongation at break, and water-resistance than both the neat PS film and the PS/PHF film (as a control with a t of 0 h). The PS/PHFNW-8 film showed the highest transparency, tensile strength, and elongation at break among the PS/PHFNW-t films, due to the high L/D of PHFNW-8. These results revealed that the length of hydrolysis time had a great effect on the structure (including L, D and L/D) of the PHFNW-t nanowhiskers, as well as on the structure and performance of the resulting PS/PHFNW-t films. For preparation of PHFNW-t nanowhiskers and corresponding PS/PHFNW-t films, the most suitable hydrolysis time, in this work, is 8 h.     

Molecular Dynamics Studies of Diffusion in Model Cylindrical Pores at Very Low Densities

Abstract

Molecular dynamics simulation has been used to study diffusion of methane at ambient temperature in cylindrical pores at very low densities. The cylinders were modelled as a continuum solid which interacts with the methane in the radial direction only. At the lowest densities, the VACF method does not yield reliable values of the self diffusion coefficient, D_s , but a suitable choice of time step and run length enables values of D_s to be found from MSD plots that are below the classical Knudsen diffusion coefficients. When density is increased, D_s passes through a maximum although the adsorption isotherm remains inside the Henry law region. Maxima are found for two cylinder radii and for two adsorbent field strengths. The existence of a maximum is attributed to transient intermolecular interactions. Analysis of a molecular trajectory demonstrates that long diffusion paths can be triggered by the rare event of an intermolecular encounter which forces a molecule into the repulsive part of the wall potential. At sufficiently high density, subsequent collisions quench the tendency towards long paths, and D_s decreases again. The issue of simulation artefact as a source of these observations is discussed.     

137Cs mobility in soils and its long-term effect on the external radiation exposure

To predict the external gamma-dose rate of Chernobyl-derived ¹³⁷Cs for a period of about 100 years after its deposition, the vertical distribution of radiocesium in several meadow soils in the Chernobyl area and in Germany was determined, and the corresponding residence half-times of this radionuclide in the various soil layers were evaluated using a compartment model. The resulting residence half-times were subsequently used to calculate the vertical distribution of ¹³⁷Cs in the soil as a function of time and finally to predict the external gamma-dose rates in air for these sites at various times. A regression analysis of the data obtained showed that the time dependence of the relative gamma-dose rate in air D(t) at the Chernobyl sites can be described by an exponential equation D(t) = a + b ⋅ exp(–t/c), where t is the time after deposition. For the ten German sites the best fit was obtained using the two-exponential equation D(t) = a ⋅ exp(–t/b) + c ⋅ exp(–t/d). The gamma-dose rate of ¹³⁷Cs at the Chernobyl sites decreases significantly more slowly with time than at the German sites. This means that after e.g. 30 years the mean relative gamma-dose rate at the German sites will have decreased from 100% (corresponding to an infinite plane source on a smooth surface) to 9% (95% confidence interval 8%–10%), while at the sites in the Chernobyl area it will have decreased only to 21% (20%–23%). This difference is the result of the longer residence half-times of ¹³⁷Cs in the soils at the Chernobyl sites. All results are compared with estimates from earlier studies. Received: 16 October 1996 / Accepted in revised form: 28 November 1996     

Brownian Dynamics Simulations of Colloidal Liquids: Hydrodynamics and Stress Relaxation

Abstract

We describe the statistical mechanics background and additional algorithmic features of a recently proposed simple mean-field Brownian Dynamics algorithm formulated to include many-body hydrodynamics, using a local density approximation for the friction coefficient. We show that the equations of motion satisfy the incompressibility of phase space. We make further developments to the model, computing the hydrodynamic effects on the shear stress relaxation function. We show that stress relaxation takes place over two well-defined regimes, in both cases with and without mean field hydrodynamics, MFH. At short times ta ²/D ₀ < 10^?3, where a is the radius of the colloidal particle and D ₀ is the self-diffusion coefficient at infinite dilution, decay of the stress autocorrelation function, C_s(t) is essentially independent of volume fraction and does not fit to a simple analytic form. At longer times than ta ²/D ₀ < 10^?2 the decay has the fractional exponential form ～exp(-t ^β) with β ? 1. The transition between these two regimes coincides with a rapid fall in the time-dependent diffusion coefficient from the so-called short-time to long-time values. We do not find any evidence for power law decay in the C_s(t) as predicted by recent mode-coupling based analytical expansions.     

Transient state isoelectric focusing. Experimental determination of apparent diffusion coefficients in polyacrylamide gels

A photoelectric scanning assembly utilizing uv absorption optics and an on-line digital data acquisition and processing system has been used to follow kinetically zone spreading during the defocusing stage (absence of electric field) of transient state isoelectric focusing (TRANSIF) in polyacrylamide gels. Measurement of the variance (σ²) of a diffusing zone as a function of time yields a linear relationship, the slope of which corresponds to the apparent diffusion coefficient (D) of the protein. A linear relationship is also obtained when the logarithm of the apparent diffusion coefficients (logD) are plotted vs acrylamide concentration (T). This relationship can be used to extrapolate D to zero gel concentration. The apparent diffusion coefficient measured in this way is significantly larger than the true diffusion coefficient. The slope of the plot logD vs T, designated C_R, is expected to be a measure of molecular size related to the retardation coefficient in polyacrylamide gel electrophoresis.