Light-scattering Method Research Articles

Electrostatic effects of smooth muscle calponin on actin assembly.

The contribution of electrostatic interactions to the effects of chicken gizzard calponin on the kinetics of actin polymerization and the bundling of F-actin were characterized by a combination of fluorescence, light-scattering, co-sedimentation, and electron-microscopic methods. Stoichiometric amounts of calponin accelerate actin polymerization in low-ionic-strength solutions, but this effect is diminished at [KCI] = 150 mM. At low ionic strengths, micromolar concentrations of calponin induce the formation of large bundles of actin filaments, and lower concentrations of calponin quench the fluorescence of pyrene-labeled F-actin. The latter effect is related to binding of calponin to F-actin rather than to bundling of the filaments. The concentration of calponin required to bundle a fixed concentration of actin filaments increases with increasing ionic strength, as the average diameter of the bundles decreases. Millimolar concentrations of ATP, GTP or ITP are equally efficient at dispersing actin bundles to single filaments or smaller aggregates, even though a significant fraction of calponin remains bound to F-actin. Our findings show that the binding of calponin to actin is determined at least in part by electrostatic interactions, and that the polycationic nature of calponin is primarily responsible for the formation of F-actin bundles via its ability to reduce the electrostatic repulsion between the negatively charged actin filaments.

Pulsed Electric Linear Dichroism of Poly(dA)·Poly(dT)·Poly(dT) and Poly(rA)·Poly(rU)·Poly(rU) in Solutions

Abstract The dependence of reduced dichroism (Δε/ε) of sonicated rodlike title triplexes with the counterion Mg2+, (dA)n·(dT)n·(dT′)n and (rA)n·(rU)n·(rU′)n, on the wavelength (190—300 nm) and on the applied-field strength (3.8—23.1 kV cm−1) was studied in aqueous ionic solutions at 7 °C. The sonicated samples were fractionated and well-characterized by the gel-permeation chromatography/low-angle laser light-scattering method. The reduced dichroism saturated at infinitely high electric fields ((Δε/ε)s) was estimated to be −1.22 for (dA)n·(dT)n·(dT′)n and −1.09 for (rA)n·(rU)n·(rU′)n, by fitting the observed Δε/ε values at 260 nm to a theoretical orientation function. The electric linear dichroism (ELD) spectra, i.e., the dependence of (Δε/ε)s on the wavelength, of (dA)n·(dT)n·(dT′)n and (rA)n·(rU)n·(rU′)n were determined. The measured ELD spectra were simulated by assigning the appropriate roll (θR) and tilt (θT) angles to the constituent bases. The most probable θR and θT values, determined for each triplex, indicate that the structures of triple helices both belong to an “A-form” family. The constituent bases in base-triple do not lie on a common plane. From an analysis of transient decay signals, the translation per base-triple (h) was estimated for each triplex. The structures of the present high molecular-weight triple-stranded helices in aqueous solution were determined for the first time and the plausible structural models were constructed from the experimentally evaluated sets of θR and θT.

Phosphorylation of Serine 256 Is Required for cAMP-dependent Regulatory Exocytosis of the Aquaporin-2 Water Channel

The aquaporin-2 (AQP2) vasopressin water channel is translocated to the apical membrane upon vasopressin stimulation. Phosphorylation of serine 256 of AQP2 by cAMP-dependent protein kinase has been shown, but its relation to vasopressin-regulated translocation has not been elucidated. To address this question, wild type (WT) AQP2 and a mutant with alanine in place of serine 256 of AQP2 (S256A) were expressed in LLC-PK1 cells by electroporation. Measurements by a stopped-flow light-scattering method revealed that the osmotic water permeability (Pf) of LLC-PK1 cells transfected with WT was 69.6 +/- 6.5 microm/s (24.8 +/- 2.2 microm/s for mock-transfected), and stimulation by 500 microM 8-(4-chlorophenylthio)-cAMP increased the Pf by 85 +/- 12%. When S256A AQP2 was transfected, the cAMP-dependent increase in the Pf was only 8 +/- 5%. After cAMP stimulation, the increase in surface expression of AQP2 determined by surface biotin labeling was 4 +/- 10%, significantly less than that for WT (88 +/- 5%). In addition, an in vivo [32P]orthophosphate labeling assay demonstrated significant phosphorylation of WT AQP2 and only minimal phosphorylation of S256A AQP2 in LLC-PK1 cells. Our results indicated that serine 256 of AQP2 is necessary for regulatory exocytosis and that cAMP-responsive redistribution of AQP2 may be regulated by phosphorylation of AQP2.

Demonstration of a Metabolically Active Glucose-6-phosphate Pool in the Lumen of Liver Microsomal Vesicles

Glucose-6-phosphate transport was investigated in rat or human liver microsomal vesicles using rapid filtration and light-scattering methods. Upon addition of glucose-6-phosphate, rat liver microsomes accumulated the radioactive tracer, reaching a steady-state level of uptake. In this phase, the majority of the accumulated tracer was glucose, but a significant intraluminal glucose-6-phosphate pool could also be observed. The extent of the intravesicular glucose pool was proportional with glucose-6-phosphatase activity. The relative size of the intravesicular glucose-6-phosphate pool (irrespective of the concentration of the extravesicular concentration of added glucose-6-phosphate) expressed as the apparent intravesicular space of the hexose phosphate was inversely dependent on glucose-6-phosphatase activity. The increase of hydrolysis by elevating the extravesicular glucose-6-phosphate concentration or temperature resulted in lower apparent intravesicular glucose-6-phosphate spaces and, thus, in a higher transmembrane gradient of glucose-6-phosphate concentrations. In contrast, inhibition of glucose-6-phosphate hydrolysis by vanadate, inactivation of glucose-6-phosphatase by acidic pH, or genetically determined low or absent glucose-6-phosphatase activity in human hepatic microsomes of patients suffering from glycogen storage disease type 1a led to relatively high intravesicular glucose-6-phosphate levels. Glucose-6-phosphate transport investigated by light-scattering technique resulted in similar traces in control and vanadate-treated rat microsomes as well as in microsomes from human patients with glycogen storage disease type 1a. It is concluded that liver microsomes take up glucose-6-phosphate, constituting a pool directly accessible to intraluminal glucose-6-phosphatase activity. In addition, normal glucose-6-phosphate uptake can take place in the absence of the glucose-6-phosphatase enzyme protein, confirming the existence of separate transport proteins.

Role of Complement in Rats Injected with Liposome-Encapsulated Hemoglobin

Previous studies have documented that liposome-encapsulated hemoglobin (LEH) can cause a rapid and transient thrombocytopenia following intravenous injection into small animals. The present study evaluated the role of complement during the LEH-induced thrombocytopenia in rats. We have compared changes in platelet levels in the blood, platelet organ distribution, and total hemolytic complement levels following intravenous administration of LEH in control and complement-depleted rats. Changes in platelet organ distribution at various times after LEH administration were monitored by labeling autologous platelets with indium-111 (111In)-oxine and imaging the111In-platelets with a gamma camera after reinjection. Platelet counts were determined by light-scattering methods and by following111In radioactivity at various times after LEH administration. Platelet levels did not significantly change for the complement-depleted rats during the 60 min following an injection of LEH, whereas thrombocytopenia (40% decrease) was noted within 4 min post-LEH-injection for control rats with a gradual return to baseline circulating platelet levels within 60 min. This drop in circulating platelets was correlated with a rapid redistribution of111In-platelets from the circulation to the lungs and liver, whereas complement-depleted rats showed no transient movement of the111In-platelets from the circulation. Baseline complement levels of 21.6 ± 2.2 CH50/ml for control rats and 0.2 ± 0.1 CH50/ml for complement-depleted rats did not significantly change during the 60 min following LEH administration. This study suggests that complement must be present during LEH-induced transient thrombocytopenia, as complement-depleted rats underwent no thrombocytopenia, and that the transient LEH-induced thrombocytopenia may be associated with complement activation.

Liposome Behavior in Capillary Electrophoresis

The behavior of liposomes in capillary electrophoresis is studied for the purpose of developing a potential method for characterizing liposomes prepared for use in industrial and analytical applications. This study characterizes the electrophoretic behavior of liposomes under various conditions to provide information about electrophoretic mobility and liposome-capillary surface interactions. The results of this method are compared with the results obtained using traditional laser light-scattering methods to obtain size information about liposome preparations. Additionally, reactions of liposomes and the surfactant n-octyl-β-d-glucopyranoside are performed off-line in bulk solution experiments and on-line in the capillary. Automated delivery of lysis agents by multiple electrokinetic injections is demonstrated as a general method for inducing on-capillary reactions between liposomes and other reagents. Furthermore, some preliminary evidence on the use of liposomes as a hydrophobic partitioning medium for analytical separations is presented.

Absolute real-time measurement of particle size distribution with the flying light-scattering indicatrix method

The flying light-scattering indicatrix (FLSI, angular dependency of the intensity of light scattered by a moving individual particle) method, based on a scanning flow cytometer (SFC) that permits measurement of individual particle characteristics from light-scattering data, has been used for the determination of size distribution of the following particles: polystyrene latex, milk fat, and spores (Penicillium levitum, Aspergillus pseudoglaucus). The optical system of the SPC and empirical equations provided absolute sizing at the rate of 50 particles/s. Size distributions obtained with the FLSI method and a best-fit procedure using Mie scattering theory have been compared.

Kinetics of volume variation of Corynebacterium glutamicum following saline osmotic upshifts

The variation over time of the volume of Corynebacterium glutamicum following osmotic upshocks produced by the addition of increasing NaCl quantities was measured by a light-scattering method. During the first seconds the cell volume rapidly decreased. The extent of volume reduction varied between 30 % at 1.5 osmol/kg and 65 % at 3.3 osmo/kg. In a second phase the cell volume increased again over a time period lasting 10 minutes or more depending on the magnitude of the saline upshock.

Development of Large Platelet Aggregates from Small Aggregates as Determined by Laser-light Scattering: Effects of Aggregant Concentration and Antiplatelet Medication

Particle-counting methods that employ light scattering (LS) quantify changes in the number of platelet aggregates of different sizes after the application of an aggregating stimulus. Using the LS method, we studied the effects of aggregant concentration, aspirin administration, and ticlopidine administration on aggregate formation and compared the results with those obtained using the conventional optical density (OD) method. Subjects were 47 controls, 31 patients treated with aspirin (330 mg/day), and 37 patients treated with ticlopidine (200 mg/day). Platelet aggregation after stimulation by 0.5, 1.0 or 5.0 muM ADP, or 0.5, 1.0 or 2.0 micrograms/ml collagen was determined using both methods. Using the LS method, small (9-25 micrograms), medium (25-50 micrograms), and large (50-70 micrograms) aggregates were counted. In patients untreated with antiplatelet medication, greater concentrations of ADP or collagen generated larger aggregates. Generation of small and medium-sized aggregates showed a significant positive correlation with OD levels after stimulation with 0.5 or 1.0 muM ADP, or 0.5 or 1.0 micrograms/ml collagen. In patients treated with aspirin, the development of small aggregates into large aggregates was inhibited. Thus, the number of small aggregates increased. Inhibition induced by aspirin was more effective against aggregation after stimulation with collagen than with ADP. In patients treated with ticlopidine, small and medium-sized aggregate formation was inhibited after stimulation with low concentrations of ADP or collagen, but was promoted after stimulation with high aggregant concentrations. The capability of the LS method to quantify different sizes of aggregates after stimulation with low concentration agonists may facilitate investigation of the aggregation process, and of how this process is affected by antiplatelet agents.

FTIR Spectroscopy of UF6 Clustering in a Supersonic Laval Nozzle

The clustering of UF6 seeded in Ar was observed in a continuous supersonic Laval nozzle flow. The onset conditions for UF6 clustering were investigated by measuring the FTIR spectra of UF6 monomer and clusters in the nozzle. The onset conditions for the clustering, temperature, density of UF6 (or partial pressure), and cooling rate, were determined as {130 K, 0.009 mol m-3 (10 Pa), 0.6 × 106 K s-1} and {155 K, 0.06 mol m-3 (80 Pa), 1.5 × 106 K s-1}. The onset temperature determined here was higher by 40−50 K than that determined by a light-scattering method. The frequency shift of the main peak of the UF6 clusters from the monomer peak was about −17 cm-1, which was smaller than the shift of the crystalline UF6 by about 11 cm-1. The increase in temperature caused by the heat of condensation and the change of the spectra of UF6 clusters with the growth after the onset were also observed. It was shown that the clustering rate due to the collision between the monomer and cluster is much higher than that due t...

Effect of Temperature on the Interaction between the Nonionic Surfactant C12E5 and Poly(ethylene oxide) Investigated by Dynamic Light Scattering and Fluorescence Methods

The interaction between the nonionic surfactant C12E5 and a high molar mass (M = 5.94 × 105) poly(ethylene oxide) (PEO) in aqueous solution has been examined as a function of temperature by dynamic light scattering and fluorescence methods over a broad concentration range. Clusters of small surfactant micelles form within the PEO coil, leading to its extension. The hydrodynamic radius of the complex increases strongly with temperature as well as with the concentrations of surfactant and polymer. At high concentrations of the surfactant, the coil/micellar cluster complex coexists with free C12E5 micelles in the solution. Fluorescence quenching measurements show a moderate micellar growth from 155 to 203 monomers in PEO-free solutions of C12E5 over a wide concentration range (0.02−2.5%) at 8 °C. Below 0.25% C12E5, the average aggregation number (N) of the micelles is smaller in the presence of PEO than in its absence. However, N increases with increasing surfactant concentration up to a plateau value of abo...

Interaction of the Nonionic Surfactant C12E8 with High Molar Mass Poly(ethylene oxide) Studied by Dynamic Light Scattering and Fluorescence Quenching Methods

Dynamic light scattering has been used to investigate ternary aqueous solutions of n-dodecyl octaoxyethylene glycol monoether (C12E8) with high molar mass poly(ethylene oxide) (PEO). The measurements were made at 20 °C, always below the cloud point temperature (Tc) of the mixed solutions. The relaxation time distributions are bimodal at higher PEO and surfactant concentrations, owing to the presence of free surfactant micelles, which coexist with the slower component, representing the polymer coil/micellar cluster complex. As the surfactant concentration is increased, the apparent hydrodynamic radius (RH) of the coil becomes progressively larger. It is suggested that the complex structure consists of clusters of micelles sited within the polymer coil, as previously concluded for the PEO−C12E5−water system. However, C12E8 interacts less strongly than C12E5 with PEO; at low concentrations of surfactant the complex does not contribute significantly to the total scattered intensity. The perturbation of the PE...

Inspection Of Recombination Active Defects For Sige And Solar Cells

AbstractMapping Low Angle Light Scattering method (MLALS) is proposed to study defect structure in materials used for solar cell production. Several types of defects are observed in Czochralski Si1−xGex (0.022<x<0.047) single crystals. Recombination activity of these defects is investigated. The possibility of contactless visualisation of grain boundary recombination in polysilicon is also demonstrated.

An Immiscibility Loop in Isotactic Polypropylene/Partially Hydrogenated Oligo(styrene-co-indene) Blend

We found an immiscibility loop in isotactic polypropylene/partially hydrogenated oligo(styrene-co-indene) blend by the cloud point method; the blend had a lower critical solution temperature-type phase boundary at low temperatures and an upper critical solution temperature-type boundary at high temperatures. The existence of the immiscibility loop was justified by the kinetic analysis of the liquid−liquid phase separation, that is, the apparent diffusion coefficient of spinodal decomposition, obtained by the time-resolved light-scattering method, increased and then decreased with increasing temperature inside the immiscibility loop, suggesting the presence of two spinodal temperatures. The existence of the immiscibility loop was also supported by the kinetics of the crystallization around the lower spinodal temperature and the observation of spherulite formation inside the immiscibility loop.

Study of 1,4-butanediol dimethacrylate by optical Kerr effect and light scattering methods

We report the results of a temperature study of the optical Kerr effect in 1,4-butanediol dimethacrylate revealing a phase-transition-like change in the temperature range from 295 to 302 K. Using the Brillouin light-scattering method, we studied the temperature behavior of the sound velocity and the elasto-optic constant of 1,4-butanediol dimethacrylate. The preceding temperature dependences display also characteristic discontinuity at about 298 K. To verify these results, a study of the Raman light scattering in 1,4-butanediol dimethacrylate was performed in the same temperature range. It was found that the temperature dependence of the relative intensities of the bands corresponding to the C = C, C = 0, and = C-H bonds reveals a discontinuity at about 294 to 297 K, which coincides with the observed temperature discontinuity of the optical Kerr effect and the Brillouin light scattering. The obtained results are discussed in terms of changes in molecular interactions and the arrangement in the liquid compound investigated.

Direct estimation of dynamic characteristics and interaction potential of latex particles interacting with a glass surface by evanescent wave light-scattering microscope method

The dynamic and static characteristics of a polystyrene latex particle in dispersion interacting with a glass surface were studied by the evanescent wave light-scattering microscope (EVLSM) technique originally proposed by Prieve et al. for static studies. The dynamic behavior of the thermal vibration of the particle in a potential well created by electrostatic interaction between the particle and glass and gravity was clearly and quantitatively estimated, in addition to the estimation of the potential profile itself. The potential minimum became shallower with increasing added salt concentration. It was also clearly observed that the vibrational motion of the particle in the well became large in amplitude and the probability of the occurrence of the large vibration became large with increasing salt concentration. Such information on the dynamics is essential for the correct under-standing of the interaction potential. The EVLSM method is shown to be a very powerful technique for the estimation of not only the potential profile but also dynamic characteristics.

Laser Ablation/Inductively Coupled Plasma Mass Spectrometry with Aerosol Density Normalization

In order to correct the variation of aerosol density in laser ablation/inductively coupled plasma mass spectrometry, a laboratory-made laser light-scattering cell was inserted into the aerosol transport tube between the laser ablation system and the torch. The temporal variation of signals from the laser light-scattering cell correlated well with that of the ion signals from the mass spectrometer. The precision of ion intensity measurement corrected with the laser light-scattering method was improved to the same level as that attained with the conventional internal standard method even in the single pulse mode.

Determination of Serum Urea by Isotope Dilution Method Using Discharge-Assisted Thermospray Liquid Chromatography/ Mass Spectrometry

In order to correct the variation of aerosol density in laser ablation/inductively coupled plasma mass spectrometry, a laboratory-made laser light-scattering cell was inserted into the aerosol transport tube between the laser ablation system and the torch. The temporal variation of signals from the laser light-scattering cell correlated well with that of the ion signals from the mass spectrometer. The precision of ion intensity measurement corrected with the laser light-scattering method was improved to the same level as that attained with the conventional internal standard method even in the single pulse mode.

Electro-Optic Effects in Colloidal Crystals of Spherical Silica Particles

The electro-optic behavior of isotropic colloidal crystals of spherical silica particles is investigated by the electric light-scattering method. The electro-optic responses observed at low frequencies (hertz range) are consistent with particle concentration fluctuations induced by charge transport and convection. A resonance frequency is detected, at which the decay of the electro-optic response is viscoelastic. The increase of field intensity introduces electrohydrodynamic effects similar to those observed in the liquid crystals.

Light scattering study on the micellar systems solubilizing a fatty acid

The effect of oleic acid on the aggregational behavior of the α-sulfonated fatty acid methyl ester sodium salt (α-SFMe) and sodium linear C12/C14-alkylbenzene-sulfonate (LAS) has been studied using a light-scattering method. The solubilizing capacity of α-SFMe was larger than that of LAS, and depended both on the number of micelles in the surfactant solution and the solubilized amount per micelle.