Short algorithm to compensate for sampling-volume errors in diffusion-cell studies

This communication describes a short algorithm enabling arbitrarilylarge but equal samples to be taken from the two chambers ofa diaphragm diffusion cell without introducing any systematicerror. It creates a transformed time-scale, dependent on samplingvolume, which linearizes the output, fits this to a straightline by simple linear regression, and computes the diffusioncoefficient from the slope. The advantages of sampling equalfractions from both chambers are discussed in terms of the greaterprecision this allows. The procedure may have uses in otherfields.     

Protein molecular weight computation from sedimentation velocity data

In ultracentrifugation, the concentration gradient of mono-disperse samples obtained by sedimentation velocity experiments is described by Gehatia's equation which holds several parameters including the sedimentation and diffusion constants. Once these two constants are known, the molecular weight follows from the Svedberg equation. A least squares method has been developed to derive the transport constants from the refractive index gradient curves. The method employs a mathematical model based on Gehatia's theory. A main feature of the model is the application of two sets of intermediate parameters via which the transport coefficients are much casier calculated than along a direct way. Furthermore some difficult to observe quantities cancel out. The square residues are minimised numerically. The potential errors introduced by this numerical minimalisation are shown to be unimportant compared to the unavoidable experimental errors.     

N-cofilin can compensate for the loss of ADF in excitatory synapses

Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content.     

Diffusion coefficient and molecular weight of type 5 adenovirus by photon-correlation spectroscopy

The technique of photon-correlation spectroscopy (intensity fluctuation spectroscopy) is applied to light scattered from type 5 adenovirus undergoing Brownian motion in solution and the translation diffusion coefficient (D₂₀,W) measured to be 0.367 ± 0.003 Fick units. Using Svedberg's equation with previously determined parameters, a molecular weight of 165 · 10⁶ ± 5 · 10⁶ is obtained.     

Detecting and overcoming systematic errors in genome-scale phylogenies

Genome-scale data sets result in an enhanced resolution of the phylogenetic inference by reducing stochastic errors. However, there is also an increase of systematic errors due to model violations, which can lead to erroneous phylogenies. Here, we explore the impact of systematic errors on the resolution of the eukaryotic phylogeny using a data set of 143 nuclear-encoded proteins from 37 species. The initial observation was that, despite the impressive amount of data, some branches had no significant statistical support. To demonstrate that this lack of resolution is due to a mutual annihilation of phylogenetic and nonphylogenetic signals, we created a series of data sets with slightly different taxon sampling. As expected, these data sets yielded strongly supported but mutually exclusive trees, thus confirming the presence of conflicting phylogenetic and nonphylogenetic signals in the original data set. To decide on the correct tree, we applied several methods expected to reduce the impact of some kinds of systematic error. Briefly, we show that (i) removing fast-evolving positions, (ii) recoding amino acids into functional categories, and (iii) using a site-heterogeneous mixture model (CAT) are three effective means of increasing the ratio of phylogenetic to nonphylogenetic signal. Finally, our results allow us to formulate guidelines for detecting and overcoming phylogenetic artefacts in genome-scale phylogenetic analyses.     

Statistical analysis of systematic errors in high-throughput screening

High-throughput screening (HTS) is an efficient technology for drug discovery. It allows for screening of more than 100,000 compounds a day per screen and requires effective procedures for quality control. The authors have developed a method for evaluating a background surface of an HTS assay; it can be used to correct raw HTS data. This correction is necessary to take into account systematic errors that may affect the procedure of hit selection. The described method allows one to analyze experimental HTS data and determine trends and local fluctuations of the corresponding background surfaces. For an assay with a large number of plates, the deviations of the background surface from a plane are caused by systematic errors. Their influence can be minimized by the subtraction of the systematic background from the raw data. Two experimental HTS assays from the ChemBank database are examined in this article. The systematic error present in these data was estimated and removed from them. It enabled the authors to correct the hit selection procedure for both assays.     

Novel hydrophobic standards for membrane protein molecular weight determinations via sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally employed technique that separates proteins on the basis of molecular weight (MW). However, membrane proteins are known to size anomalously on SDS-PAGE calibrated with conventional standards, an issue that complicates interpretation of protein identity, purity, degradation, and/or stoichiometry. Here we describe the preparation of novel polyleucine hydrophobic standards for SDS-PAGE that reduce the average deviation of the apparent MW from the formula MW of natural membrane proteins to 7% versus 20% with commercially available standards. Our results suggest that gel calibration with hydrophobic standards may facilitate the interpretation of membrane protein SDS-PAGE experiments.     

Intracellular Ca can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells

The aim of the present study is to determine the role of intracellular Ca²⁺ in VEGF signaling. We demonstrate that reduction in Ca²⁺ by chelating compound BAPTA-AM or by IP₃-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca²⁺. These results suggest an essential role for Ca²⁺ in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation.     

Plants impaired in state transitions can to a large degree compensate for their defect

Arabidopsis thaliana plants lacking the PSI-H or PSI-L subunit of photosystem I have been shown to be severely affected in their ability to perform state transitions, but no visual phenotype was observed when these plants were grown under different light quantities and qualities. However, the chloroplasts in the PSI-H- and PSI-L-less plants contained fewer and more extended grana stacks. The plants lacking PSI-H or PSI-L were characterised with respect to their photosynthetic performance. Wild-type plants adjusted the non-photochemical fluorescence quenching to maintain constant levels of PSII quantum yield and reduction of the plastoquinone pool. In contrast, the plants deficient in state transitions had a more reduced plastoquinone pool and consequently, a less efficient PSII-photochemistry under growth-light conditions and in state 2. The maximal photosynthetic capacity and the quantum efficiency of oxygen evolution were diminished by 8-14% in the PSI-H-less plants. Under growth-light conditions, the stroma was similarly reduced in the PSI-H-less plants and the rate of cyclic electron transport was unchanged. Pigment analysis showed that the xanthophyll cycle was not upregulated in order to compensate for the lack of state transitions. In general, the plants lacking PSI-H and PSI-L showed a decreased ability to optimise photosynthesis according to the light conditions.     

Further consideration of systematic errors present in protein-ligand interactions

Relatively minor systematic errors present during measurements of protein-ligand interaction can lead to large inaccuracies in the calculated values of the equilibrium dissociation constant and the total concentration of the binding protein. These errors, which include binding of the ligand to low affinity material and underestimation of bound ligand, cause the calculation of the concentration of free ligand at equilibrium to be overestimated. We report herein a model of ligandprotein binding which incorporates these errors into the mathematical formulation of the equilibrium binding equation. The effect of these errors on the Scatchard plot is presented.     

Pulling geometry-induced errors in single molecule force spectroscopy measurements

In AFM-based single molecule force spectroscopy, it is tacitly assumed that the pulling direction coincides with the end-to-end vector of the molecule fragment being stretched. By systematically varying the position of the attachment point on the substrate relative to the AFM tip, we investigate empirically and theoretically the effect of the pulling geometry on force-extension characteristics of double-stranded DNA. We find that increasing the pulling angle can significantly lower the force of the characteristic overstretching transition and increase the width of the plateau feature beyond the canonical 70%. These effects, when neglected, can adversely affect the interpretation of measured force-extension relationships. We quantitatively evaluate force and extension errors originating from this "pulling angle effect" and stress the need to correct the pulling geometry when stretching rigid molecules with an AFM.     

The effect of anisotropic systematic errors in estimating helical angles

A common question in movement studies is how the results should be interpreted with respect to systematic and random errors. In this study, simulations are made in order to see how a rigid body's orientation in space (i.e. helical angle between two orientations) is affected by (1) a systematic error added to a single marker (2) a combination of this systematic error and Gaussian white noise. The orientation was estimated after adding a systematic error to one marker within the rigid body. This procedure was repeated with Gaussian noise added to each marker. In conclusion, results show that the systematic error's effect on estimated orientation depends on number of markers in the rigid body and also on which direction the systematic error is added. The systematic error has no effect if the error is added along the radial axis (i.e. the line connecting centre of mass and the affected marker).     

Diffusion-ordered NMR spectroscopy: a versatile tool for the molecular weight determination of uncharged polysaccharides

Diffusion-ordered NMR spectroscopy (DOSY) experiments have been carried out on dilute aqueous solutions of uncharged saccharide systems and, in particular, on six well characterized pullulan fractions of different molecular weights. The values of diffusion coefficients and hydrodynamic radii determined for the pullulan fractions are in good agreement with the results obtained with other methodologies such as light scattering. Fitting the diffusion coefficients data as a function of the molecular weight allows for the determination of a calibration curve that can be applied to a wide range of mono-, oligo-, and polysaccharides. Therefore, DOSY is proposed as a versatile tool for achieving a simple estimation of the molecular weight of uncharged polysaccharides. Mixtures of homopolymers of different molecular weight can be nicely separated. An advantage of the method is that the same sample used for the NMR characterization can be used for the molecular weight determination without any further manipulation. Other water soluble polymers, such as poly(ethylene oxide) and poly(vinylpyrrolidone), can be roughly characterized using the same calibration curve.     

A mutation in integrase can compensate for mutations in the simian immunodeficiency virus att site.

Sequences at the left terminus of U3 in the left long terminal repeat (LTR) and at the right terminus of U5 in the right LTR are important for integration of retroviral DNA. In the infectious pathogenic molecular clone of simian immunodeficiency virus strain mac239 (SIVmac239), 10 of the 12 terminal base pairs form an imperfect inverted repeat structure (5' TGGAAGGGATTT 3' [nucleotides 1 to 12] and 3' ACGATCCCTAAA 5' [nucleotides 10279 to 10268]). Nineteen different mutant forms of SIVmac239 proviral DNA with changes at one or more of the positions in each of the 12-terminal-base-pair regions were constructed. Viral replication was severely or completely compromised with nine of these mutants. Revertants appeared 40 to 50 days after transfection in two independent experiments with mutant 7, which contained changes of AGG to TAC at positions 5 to 7 in U3 and TCC to GAA at positions 10275 to 10273 in U5. Virus produced at these times from mutant 7 transfection replicated upon reinfection with only a slight delay when compared to the wild type. Sequence analysis of the LTR and integrase regions from infected cultures revealed two predominant changes: G to A at position 10275 in U5 and Glu to Lys at position 136 in integrase. Derivatives of clone 7 in which these changes were introduced individually and together were constructed by site-specific mutagenesis. Each change individually restored replication capacity only partially. However, the combination of both mutations restored replicative capacity to that of the original revertants. These results indicate that changes in integrase can compensate for mutations in the terminal nucleotides of the SIV LTR. The results further indicate that resistance to integrase inhibitors may include both integrase and LTR mutations.