Simulation of saturation transfer electron paramagnetic resonance spectra for rotational motion with restricted angular amplitude.

We have simulated both conventional (V1) and saturation transfer (V'2) electron paramagnetic resonance spectra for the case of Brownian rotational diffusion restricted in angular amplitude. Numerical solutions of the diffusion-coupled Bloch equations were obtained for an axially symmetric 14N nitroxide spin label with its principal axis rotating within a Gaussian angular distribution of full width delta theta at half maximum. Spectra were first calculated for a macroscopically oriented system with cylindrical symmetry (e.g., a bundle of muscle fibers or a stack of membrane bilayers), with the Gaussian angular distribution centered at theta 0 with respect to the magnetic field. These spectra were then summed over theta 0 to obtain the spectrum of a randomly oriented sample (e.g., a dispersion of myofibrils or membrane vesicles). The angular amplitude delta theta was varied from 0 degrees, corresponding to isotropic motion (order parameter = 0). For each value of delta theta, the rotational correlation time, tau r, was varied from 10(-7) to 10(-2) s, spanning the range from maximal to minimal saturation transfer. We provide plots that illustrate the dependence of spectral parameters on delta theta and tau r. For an oriented system, the effects of changing delta theta and tau r are easily distinguishable, and both parameters can be determined unambiguously by comparing simulated and experimental spectra. For a macroscopically disordered system, the simulated spectra are still quite sensitive to delta theta, but a decrease in tau r produces changes similar to those from an increase in delta theta. If delta theta can be determined independently, then the results of the present study can be used to determine tau r from experimental spectra. Similarly, if tau r is known, then delta theta can be determined.     

Phagocytosis of Dictyostelium discoideum studied by the particle-tracking method

Single phagocytic events of cellular slime mold Dictyostelium discoideum were studied by the method of particle tracking. A 2-microm polystyrene bead, which had been covalently coated with folate, was attached to the advancing edge of a Dictyostelium ameba with the aid of an optical trap. The bead was transported backward on the cell surface. Forty-five percent of the transported beads were internalized. The bead motion was analyzed by determining every 33 ms the x-y coordinate of the centroid of the phase-contrast image of the bead. The x(t) and y(t) traces were smoothed over 1 s and the difference between the smoothed (x(t) and y(t)) and the original traces, delta(x) identical with x(t) - x(t) and delta(y) identical with y(t) - y(t), were calculated, which represented relatively rapid components of the bead motion. The plot of delta(2) = (delta(x)(2) + delta(y)(2)) against time could be divided into three phases on the basis of the variance of delta(2). Comparison of the plot with the video sequence indicated that the first phase corresponded to the transport, the second phase to the internalization, and the third phase to the postinternalization process (intracellular movement). Cytochalasin A at 5 microM completely inhibited phagocytosis without affecting the binding of bead to the cell surface, indicating the importance of actin cytoskeleton in all the phases. At 1 microM cytochalasin A the variance of the postinternalization process decreased, and the duration of the transport phase increased. At 0.25 microM cytochalasin A the duration of the internalization phase exhibited a significant increase, but other parameters did not change appreciably. The complex and differential effects of cytochalasin A on the parameters characterizing the three phases in the phagocytic process indicate that various aspects of actin dynamics are involved in the individual process of phagocytosis.     

A possible mechanism of stochastic resonance in the light of an extra-classical receptive field model of retinal ganglion cells

Traditionally the intensity discontinuities in an image are detected as zero-crossings of the second derivative with the help of a Laplacian of Gaussian (LOG) operator that models the receptive field of retinal Ganglion cells. Such zero-crossings supposedly form a raw primal sketch edge map of the external world in the primary visual cortex of the brain. Based on a new operator which is a linear combination of the LOG and a Dirac-delta function that models the extra-classical receptive field of the ganglion cells, we find that zero-crossing points thus generated, store in presence of noise, apart from the edge information, the shading information of the image in the form of density variation of these points. We have also shown that an optimal image contrast produces best mapping of the shading information to such zero-crossing density variation for a given amount of noise contamination. Furthermore, we have observed that an optimal amount of noise contamination reproduces the minimum optimal contrast and hence gives rise to the best representation of the original image. We show that this phenomenon is similar in nature to that of stochastic resonance phenomenon observed in psychophysical experiments.     

Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity

DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.     

Bound ligand motion in crystalline carboxypeptidase A.

Deuterium NMR spectra for the phenyl ring deuterons have been obtained for D-phenylalanine, L-phenylalanine, phenylacetic acid, and phenyl propionic acid in randomly oriented crystals of carboxypeptidase A as a function of water content. The spectra are analyzed using a two-site jump model for phenyl ring pi-flips when the ligand is bound to the protein, and the model includes the possibility that the ligand may exchange with isotropic or unbound environments within the crystal. Although the binding pocket may impose local dynamical constraints, a complete pi-flip motion is consistent with the spectra of all ligands at all water contents. The rate constants for the pi-flip at 298 K are found to be 7.5 x 10(5) S-1, 1.9 x 10(6) S-1, 4.0 x 10(6) S-1, and 4.0 x 10(6) S-1 for L-phenylalanine, D-phenylalanine, phenyl propionic acid, and phenylacetic acid, respectively, at water activity of 0.98. The pi-flip rate for the ligand bound to the enzyme increases with water content. Assuming that the activation barrier may be written, delta G+2 = delta G+2o + baw, where aw is the water activity, and the value of b is -1.9 kcal/mol for phenylacetic acid and phenyl propionic acid, -1.3 kcal/mol for L-phenylalanine, and -2.1 kcal/mol for D-phenylalanine. Phenylacetic acid crystals were studied as an example of a phenyl ring motion that is highly constrained by a known and symmetrical packing environment. The deuterium spectra are complex and are not consistent with pi-flip motions, but they are consistent with a superposition of ring jump motions of 24 degrees, 34 degrees, and 72 degrees, with probabilities in the ratio of 1:1:2. Because of the limited space for motion imposed by the tight packing in the crystal, these motions must be highly cooperative and probably locally coherent; however, the spectra by themselves do not prove this intuitively reasonable hypothesis.     

Light-induced axial and radial shrinkage effects and changes of the refractive index in isolated bovine rod outer segments and disc vesicles

Flash-induced transients in the near-infrared scattering of bovine rod outer segments and isolated discs are investigated. Their common characteristic is the saturation at a rhodopsin bleaching of ca. 10%, which was previously described for the so-called signalP. The theory is based on the Rayleigh-Gans-approximation and on a cylindrical particle shape. This treatment is shown to be applicable in the measured angular range (in general30), in spite of the polydisperse shape of the real particles. Using the angular dependence of the relative intensity change (difference scattering curve), changes of the polarizability (refractive index) and of the particle shape can be distinguished. Model difference scattering curves are calculated for the dimensions of the rod outer segments. Static scattering measurements are used for an estimation of the average particle shape: the isolated disc samples appear to contain flat discs as well as an admixture of rod-like structures (ca. 1% of the total scattering mass); in rod outer segment preparations, a contribution of non-rodlike scattering is found which is strongly dependent on the treatment of the sample. The flash induced transients were measured using randomly oriented particles (discs and rod outer segments) and axially oriented rod outer segments. The angular dependence of the amplitude yields its difference scattering curve. On suspensions of isolated discs, which were re-loaded with the proteins extracted at low ionic strength, one single signal is observed (termedP _D, first order,=0.6–1.2 s). Using randomly oriented rod outer segments, a signal with complex millisecond kinetics (termed signalP) and a slow signal (termedP _S, first order,=5–25 s) can be distinguished kinetically. In the axially oriented rod outer segments, theP-signal splits into a fast axial (10 ms) and a slower radial component (50–100 ms). The slow signalP _S observed in ROS and the signalP _D in discs have one common physical interpretation as local changes of the polarizability, directly observed in light-scattering as a change of the refractive index. The fast signalP in ROS, however, has no detectable local component but represents a pure shrinkage effect. On the axially oriented system, this shrinkage turns out to be axial and radial with different kinetics. Only rough estimations for the relative shrinkage effects and refractive index changes can be given. One obtains for 1% rhodopsin bleaching:n/n10^–4,L/L10^–2,R/R5×10^–4. Assuming a fluid plane for the disc membrane, the planar shrinkage induced by one bleached rhodopsin is estimated from the radial shrinkage as ca. 300 å². This high value is discussed in relation to the binding of rhodopsin to the GTP-binding protein which is involved in comparable effects described by Kühn et al. (1981). According to our data, a chemical binding process in milliseconds is only indicated in the isolated disc; in the closed disc stack of the rod outer segment, only weak (fast) local interactions are consistent with the difference scattering data. A turn or lift of the GTPase would better satisfy this condition and explain the above high value for the individual shrinkage effect.Abbreviations ROS rod outer segments - RGA Rayleigh-Gans-approximation     

Novel nucleotide-binding sites in ATP-sensitive potassium channels formed at gating interfaces

The coupling of cell metabolism to membrane electrical activity is a vital process that regulates insulin secretion, cardiac and neuronal excitability and the responses of cells to ischemia. ATP-sensitive potassium channels (K(ATP); Kir6.x) are a major part of this metabolic-electrical coupling system and translate metabolic signals such as the ATP:ADP ratio to changes in the open or closed state (gate) of the channel. The localization of the nucleotide-binding site (NBS) on Kir6.x channels and how nucleotide binding gates these K(ATP) channels remain unclear. Here, we use fluorescent nucleotide binding to purified Kir6.x proteins to define the peptide segments forming the NBS on Kir6.x channels and show that unique N- and C-terminal interactions from adjacent subunits are required for high-affinity nucleotide binding. The short N- and C-terminal segments comprising the novel intermolecular NBS are next to helices that likely move with channel opening/closing, suggesting a lock-and-key model for ligand gating.     

Streptozocin-induced neuropathy is associated with altered expression of voltage-gated calcium channel subunit mRNAs in rat dorsal root ganglion neurones.

Voltage-gated calcium channels (VGCCs) within sensory neurones are believed to perform an important role in neuropathic pain. In the present study we examine the changes in VGCC mRNA which occur following streptozocin- (STZ) induced diabetic neuropathy using in situ hybridization. STZ caused a significant increase in alpha(2)delta(1), alpha(2)delta(2), and alpha(2)delta(3) mRNA levels in all neuronal cell types. Similarly, mRNA levels of alpha(1F), alpha(1I), and alpha(1S) were increased in all cell types studied whilst alpha(1A) and alpha(1G) mRNAs were specifically upregulated in medium and large diameter neurones. In conclusion, we demonstrate that the induction of diabetic neuropathy is associated with dramatic changes in the expression of VGCCs.     

Dominantly inherited expression of BID, an invariant undiversified T cell receptor delta chain

In BALB/c lung and lymph node gamma delta T cells, a large fraction of the expressed V delta 5 genes consist of an invariant sequence, BID (for BALB/c invariant delta). BID results from a direct joining of the V delta 5, D delta 2, and J delta 1 segments, which conserve their complete germline coding sequences. In C57BL/6 (H-2b) mice, where identical and functional segments are present in the germline, BID is absent. It appears that BID+ gamma delta T cells are positively selected by factors encoded outside of the classical MHC region, as indicated by their dominance in F1(C57BL/6 x BALB/c) and in BALB.B (H-2b) mice. Additional observations, including the expression of BID in BALB/c nu/nu but not in C57BL/6 nu/nu mice, suggest that the expansion of BID+ T cells essentially occurs extrathymically.     

Hydrophobicity regained.

A widespread practice is to use free energies of transfer between organic solvents and water (delta G0transfer to define hydrophobicity scales for the amino acid side chains. A comparison of four delta G0transfer scales reveals that the values for hydrogen-bonding side chains are highly dependent on the non-aqueous environment. This property of polar side chains violates the assumptions underlying the paradigm of equating delta G0transfer with hydrophobicity or even with a generic solvation energy that is directly relevant to protein stability and ligand binding energetics. This simple regaining of the original concept of hydrophobicity reveals a flaw in approaches that use delta G0transfer values to derive generic estimates of the energetics of the burial of polar groups, and allows the introduction of a "pure" hydrophobicity scale for the amino acid residues.     

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.     

Segmentation of microscopic images of small intestinal glands with directional 2-D filters

We present an image segmentation algorithm for small intestinal glands consisting of goblet cells that are evenly distributed and arranged in parallel at the base. Making use of the properties of the chain distribution of the goblet cells, directional 2-dimensional (2-D) linear filters with different orientations were designed to enhance the rims of the intestinal glands. Segmentations are based on the combined responses of the multiple zero-phase directional 2-D linear filters. For comparisons, outputs of combined directional filters are shown along with those of the comparable nondirectional Gaussian filters. Segmentation results of small intestinal glands of both normal and cancer cases are provided.     

Antibody/antigen affinity behavior in liquid environment with electrical impedance analysis of quartz crystal microbalances

Electrical impedance analysis has been used to study anti-human immunoglobulin G (anti-h IgG) adsorption and the subsequent human immunoglobulin G (hIgG) or rabbit immunoglobulin G (rIgG) affinity reaction in aqueous liquids on a polystyrene (PS)-modified quartz crystal microbalance (QCM) surface. Time-dependent adsorption data of both the frequency shift and the electrical equivalent parameters (motional resistance, shunt capacitance, quality factor, etc) are monitored. It was found that the motional resistance, R, increases while the resonance frequency, f, decreases during both the anti-h IgG immobilization and the subsequent affinity process. Decreasing f primarily arises from the increased mass loading. Increasing R indicates more power dissipation (increased losses) in the system. The change in motional resistance, delta R, in the affinity reaction is considerably larger than that in anti-h IgG immobilization adsorption process, although the resonant frequency shifts, delta f, are very close in these two processes. Specifically, for a saturated solution, the ratio of delta R/delta f is 9.45 x 10 (-3) Omega/Hz for anti-h IgG adsorption and 28.1 x 10 (-3) omega/Hz for anti-h IgG/hIgG binding respectively, indicating the increased power dissipation with the increasing binding molecules. The shunt capacitance changes little in the hIgG binding process ( approximately 0.01 pF).     

Functional architecture of the inner pore of a voltage-gated Ca2+ channel

The inner pore of voltage-gated Ca2+ channels (VGCCs) is functionally important, but little is known about the architecture of this region. In K+ channels, this part of the pore is formed by the S6/M2 transmembrane segments from four symmetrically arranged subunits. The Ca2+ channel pore, however, is formed by four asymmetric domains of the same (alpha1) subunit. Here we investigated the architecture of the inner pore of P/Q-type Ca2+ channels using the substituted-cysteine accessibility method. Many positions in the S6 segments of all four repeats of the alpha1 subunit (Ca(v)2.1) were modified by internal methanethiosulfonate ethyltrimethylammonium (MTSET). However, the pattern of modification does not fit any known sequence alignment with K+ channels. In IIS6, five consecutive positions showed clear modification, suggesting a likely aqueous crevice and a loose packing between S6 and S5 segments, a notion further supported by the observation that some S5 positions were also accessible to internal MTSET. These results indicate that the inner pore of VGCCs is indeed formed by the S6 segments but is different from that of K+ channels. Interestingly some residues in IIIS6 and IVS6 whose mutations in L-type Ca2+ channels affect the binding of dihydropyridines and phenylalkylamines and are thought to face the pore appeared not to react with internal MTSET. Probing with qBBr, a rigid thiol-reactive agent with a dimension of 12 angstroms x 10 angstroms x 6 angstroms suggests that the inner pore can open to >10 angstroms. This work provides an impetus for future studies on ion permeation, gating, and drug binding of VGCCs.     

TOP-IDP-scale: a new amino acid scale measuring propensity for intrinsic disorder

Intrinsically disordered proteins carry out various biological functions while lacking ordered secondary and/or tertiary structure. In order to find general intrinsic properties of amino acid residues that are responsible for the absence of ordered structure in intrinsically disordered proteins we surveyed 517 amino acid scales. Each of these scales was taken as an independent attribute for the subsequent analysis. For a given attribute value X, which is averaged over a consecutive string of amino acids, and for a given data set having both ordered and disordered segments, the conditional probabilities P(s(o) | x) and P(s(d) | x) for order and disorder, respectively, can be determined for all possible values of X. Plots of the conditional probabilities P(s(o) | x) and P(s(o) | x) versus X give a pair of curves. The area between these two curves divided by the total area of the graph gives the area ratio value (ARV), which is proportional to the degree of separation of the two probability curves and, therefore, provides a measure of the given attribute's power to discriminate between order and disorder. As ARV falls between zero and one, larger ARV corresponds to the better discrimination between order and disorder. Starting from the scale with the highest ARV, we applied a simulated annealing procedure to search for alternative scale values and have managed to increase the ARV by more than 10%. The ranking of the amino acids in this new TOP-IDP scale is as follows (from order promoting to disorder promoting): W, F, Y, I, M, L, V, N, C, T, A, G, R, D, H, Q, K, S, E, P. A web-based server has been created to apply the TOP-IDP scale to predict intrinsically disordered proteins (http://www.disprot.org/dev/disindex.php).     

Modeling the rod outer segment birefringence change correlated with metarhodopsin II formation.

A rapid birefringence loss associated with metarhodopsin II formation, delta (delta n) MII, is produced when frog rod outer segments are exposed to a bleaching light flash. To analyze the nature of the underlying structure change, measurements of delta (delta n) MII were made in rod outer segments perfused with glycerol solutions to increase the refractive index of the cytoplasmic and intradisk spaces. Comparisons of experimental results with computed changes in the form birefringence component using two- and three-dielectric outer segment models for several putative structure changes were made. It is concluded that delta (delta n) MII can be due to either a change in the intrinsic birefringence component caused by the reorientation of anisotropic molecules, or to a change in the form birefringence component caused by small changes in the cytoplasmic and/or intradisk volumes.     

Binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal) to human leukocyte elastase: a thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH 8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTI--Ka = 6.3 x 10(4) M-1, delta G degree = -26.9 kJ/mol, delta H degree = +11.7 kJ/mol, and delta S degree = +1.3 x 10(2) entropy units; porcine PSTI--Ka = 7.0 x 10(3) M-1, delta G degree = -21.5 kJ/mol, delta H degree = +13.0 kJ/mol, and delta S degree = +1.2 x 10(2) entropy units (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature independent over the range (between 5.0 degrees C and 45.0 degrees C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from congruent to 7.0, in the free enzyme, to congruent to 5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae

DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.