Lateral diffusion in an archipelago. Single-particle diffusion.

Several laboratories have measured lateral diffusion of single particles on the cell surface, and these measurements may reveal an otherwise inaccessible level of submicroscopic organization of cell membranes. Pitfalls in the interpretation of these experiments are analyzed. Random walks in unobstructed systems show structure that could be interpreted as free diffusion, obstructed diffusion, directed motion, or trapping in finite domains. To interpret observed trajectories correctly, one must consider not only the trajectories themselves but also the probabilities of occurrence of various trajectories. Measures of the asymmetry of obstructed and unobstructed random walks are calculated, and probabilities are evaluated for random trajectories that resemble either directed motion or diffusion in a bounded region.     

Structure of branched poly-alpha-amino acids in dimethylformamide. II. Viscosity, sedimentation, and diffusion

The intrinsic viscosity, sedimentation and diffusion of a series of branched, multichain poly-α-amino acids having a poly(L -lysine) backbone and poly(γ-benzyl L -glutamate) and poly (β-benzyl L -aspartate) side chains was studied at room temperature in dimethylformamide. The molecules were found to be extremely compact structures in which the molecular backbone is either lying along the major axis in a slightly twisted configuration (the longer the side chain the smaller the twist) or is coiled up in the form of a disk with backbone and side chains coplanar. Heat treatment (to 70°C.) introduces only small changes in the hydrodynamic parameters showing that the heat-labile aggregates detected by light scattering are reversibly broken up during the hydrodynamic measurements. The above structural information concerns the initial metastable conformation of the molecules which is irreversibly destroyed by heat treatment.     

Reaction mechanism of chalcone isomerase. pH dependence, diffusion control, and product binding differences.

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)-flavanones. The activity of CHI is essential for the biosynthesis of flavanone precursors of floral pigments and phenylpropanoid plant defense compounds. We have examined the spontaneous and CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, 2',4'-dihydroxychalcone, and 4,2'-dihydroxychalcone into the corresponding flavanones. The pH dependence of flavanone formation indicates that both the non-enzymatic and enzymatic reactions first require the bulk phase ionization of the substrate 2'-hydroxyl group and subsequently on the reactivity of the newly formed 2'-oxyanion during C-ring formation. Solvent viscosity experiments demonstrate that at pH 7.5 the CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, and 2',4'-dihydroxychalcone are approximately 90% diffusion-controlled, whereas cyclization of 4,2'-dihydroxychalcone is limited by a chemical step that likely reflects the higher pK(a) of the 2'-hydroxyl group. At pH 6.0, the reactions with 4,2',4',6'-tetrahydroxychalcone and 4,2',4'-trihydroxychalcone are approximately 50% diffusion-limited, whereas the reactions of both dihydroxychalcones are limited by chemical steps. Comparisons of the 2.1-2.3 A resolution crystal structures of CHI complexed with the products 7,4'-dihydroxyflavanone, 7-hydroxyflavanone, and 4'-hydroxyflavanone show that the 7-hydroxyflavanones all share a common binding mode, whereas 4'-hydroxyflavanone binds in an altered orientation at the active site. Our functional and structural studies support the proposal that CHI accelerates the stereochemically defined intramolecular cyclization of chalcones into biologically active (2S)-flavanones by selectively binding an ionized chalcone in a conformation conducive to ring closure in a diffusion-controlled reaction.     

Quantitative, radial diffusion slide assay for staphylocoagulase.

A simple, quantitative radial diffusion assay for staphylocoagulase in culture fluids, using microscope slides coated with a thin layer of agar containing plasma and fibrinogen, was developed. No prior purification of the enzyme was needed, and only small quantities, 7 microliter, were required for each test. This method is particularly suitable for objectively comparing the relative amounts of coagulase produced by different cultures of Staphylococcus aureus.     

Lateral diffusion in an archipelago. Distance dependence of the diffusion coefficient.

An understanding of the distance dependence of the lateral diffusion coefficient is useful in comparing the results of diffusion measurements made over different length scales, and in analyzing the kinetics of mobile redox carriers in organelles. A distance-dependent, concentration-dependent diffusion coefficient is defined, and it is evaluated by Monte Carlo calculations of a random walk by mobile point tracers in the presence of immobile obstacles on a triangular lattice, representing the diffusion of a lipid or a small protein in the presence of immobile membrane proteins. This work confirms and extends the milling crowd model of Eisinger, J., J. Flores, and W. P. Petersen (1986. Biophys J. 49:987-1001). Similar calculations for diffusion of mobile particles interacting by a hard-core repulsion yield the distance dependence of the self-diffusion coefficient. An expression for the range of short-range diffusion is obtained, and the distance scales for various diffusion measurements are summarized.     

Leaf diffusion resistance, illuminance, and transpiration

Stepwise increases in fluorescent illuminance, imposed as a single variable in a controlled environment, induced progressive stomatal opening in 8 plant species, as evidenced by a consistent decrease in leaf diffusion resistance (R_L), ranging from 15 to 70 sec cm⁻¹ in darkness to about 1 sec cm⁻¹ at approximately 40 kilolux. The minimum R_L values were the same for the upper and the lower epidermis, provided that stomatal density was adequate. Saturation illuminance was not achieved in any species; extrapolation indicates that 50 kilolux would bring about full stomatal opening (R_L ≤ 0.1 sec cm⁻¹).

In 4 species, reasonable agreement was obtained in a controlled environment between transpiration as measured by weight loss and that calculated from determination of (a) the difference in water vapor density from leaf to air, (b) the boundary layer resistance, and (c) the leaf diffusion resistance. This result confirms the physical validity of the resistance measurement procedure.

     

Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: application to photobleaching measurements of multicomponent and anomalous diffusion.

Fluorescence recovery after photobleaching (FRAP) is widely used to measure fluorophore diffusion in artificial solutions and cellular compartments. Two new strategies to analyze FRAP data were investigated theoretically and applied to complex systems with anomalous diffusion or multiple diffusing species: 1) continuous distributions of diffusion coefficients, alpha(D), and 2) time-dependent diffusion coefficients, D(t). A regression procedure utilizing the maximum entropy method was developed to resolve alpha(D) from fluorescence recovery curves, F(t). The recovery of multi-component alpha(D) from simulated F(t) with random noise was demonstrated and limitations of the method were defined. Single narrow Gaussian alpha(D) were recovered for FRAP measurements of thin films of fluorescein and size-fractionated FITC-dextrans and Ficolls, and multi-component alpha(D) were recovered for defined fluorophore mixtures. Single Gaussian alpha(D) were also recovered for solute diffusion in viscous media containing high dextran concentrations. To identify anomalous diffusion from FRAP data, a theory was developed to compute F(t) and alpha(D) for anomalous diffusion models defined by arbitrary nonlinear mean-squared displacement <x2> versus time relations. Several characteristic alpha(D) profiles for anomalous diffusion were found, including broad alpha(D) for subdiffusion, and alpha(D) with negative amplitudes for superdiffusion. A method to deduce apparent D(t) from F(t) was also developed and shown to provide useful complementary information to alpha(D). alpha(D) and D(t) were determined from photobleaching measurements of systems with apparent anomalous subdiffusion (nonuniform solution layer) and superdiffusion (moving fluid layer). The results establish a practical strategy to characterize complex diffusive phenomena from photobleaching recovery measurements.     

MR diffusion tensor spectroscopy and imaging.

This paper describes a new NMR imaging modality--MR diffusion tensor imaging. It consists of estimating an effective diffusion tensor, Deff, within a voxel, and then displaying useful quantities derived from it. We show how the phenomenon of anisotropic diffusion of water (or metabolites) in anisotropic tissues, measured noninvasively by these NMR methods, is exploited to determine fiber tract orientation and mean particle displacements. Once Deff is estimated from a series of NMR pulsed-gradient, spin-echo experiments, a tissue's three orthotropic axes can be determined. They coincide with the eigenvectors of Deff, while the effective diffusivities along these orthotropic directions are the eigenvalues of Deff. Diffusion ellipsoids, constructed in each voxel from Deff, depict both these orthotropic axes and the mean diffusion distances in these directions. Moreover, the three scalar invariants of Deff, which are independent of the tissue's orientation in the laboratory frame of reference, reveal useful information about molecular mobility reflective of local microstructure and anatomy. Inherently tensors (like Deff) describing transport processes in anisotropic media contain new information within a macroscopic voxel that scalars (such as the apparent diffusivity, proton density, T1, and T2) do not.     

Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2.

We studied intracellular binding and possible compartmentalization of the fluorescent Ca2+ indicators, indo-1 and fura-2, in single mammalian cardiac ventricular cells that had been loaded with indo-1 and fura-2 by exposure to the acetoxymethylester form of the indicators (indo-1/AM and fura-2/AM). Techniques similar to those used in experiments on fluorescence recovery after photobleaching (FRAP) were used. It was assumed that reversible binding in myoplasm would be evident as slowed recovery of fluorescence after photobleaching, and that irreversible binding of the indicators to immobile myoplasmic sites (or "compartmentalization" in organelles) would be evident as incomplete recovery. Through the use of a mask, one half of a cell was exposed to high-intensity ultraviolet (UV) light to bleach the indo-1 or fura-2 in only that part of the cell. Upon removal of the mask and termination of the high-intensity UV illumination, fluorescence recovered in the bleached half of the cell, indicating diffusion of indo-1 and fura-2. Mathematical modeling of the diffusional redistribution of the indicators indicated that in these cells the apparent diffusion coefficient for indo-1 is 1.57 x 10(-7) cm2 s-1 (SD 0.48 x 10(-7) cm2 s-1; n = 5 cells, 21 degrees C), and for fura-2 is 3.19 x 10(-7) cm2 s-1 (SD 1.85 x 10(-7) cm2 s-1; n = 6 cells, 21 degrees C). These values are approximately 6 and 3, respectively, times smaller than those expected for free diffusion in the myoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of Ru-Arene complexes of Me-Duphos. X-ray, PGSE NMR diffusion and catalytic studies

Cationic [RuCl(arene)(Me-Duphos)]Cl complexes, arene=η⁶-benzene and η⁶-p-cymene, Me-Duphos=1,2-bis-((2R,5R)-2,5-dimethylphospholano) benzene) have been prepared and studied by X-ray crystallography and NMR spectroscopy. PGSE NMR diffusion studies have been used to recognize (a) ion pairing as a function of solvent and (b) larger molecular volumes. Several arene-Ru-complexes have been shown to be useful catalyst precursors in the hydrolysis of terminal aryl alkynes to afford acetophenones.     

Concentration dependence of protein diffusion.

The concentration dependence of protein self-diffusion constants is described by a free volume diffusion theory which accounts for the effects of local protein concentration fluctuations.