Biotinylated proteins as molecular weight standards on Western blots

Protein molecular weight standards were biotinylated by reaction with biotinyl-N-hydroxysuccinimide ester. The biotinylated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The resolved protein bands were detected by formation of a streptavidin-biotin/horseradish peroxidase complex and reaction with 4-chloro-1-naphthol and hydrogen peroxide. The biotinylated proteins are easy to prepare and are useful as molecular weight standards with most procedures employing immunodetection of proteins following transfer to nitrocellulose paper.     

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.     

Immune serum to protein molecular weight standards for calibrating Western blots

A method for calibrating proteins by Western blot analysis is described. Rabbit immune serum, specific for seven protein molecular weight standards, and goat anti-rabbit horseradish peroxidase conjugate are used to visualize standards after electrotransfer onto nitrocellulose. This method can be used as a reagent and procedure control.     

Plesiomorphic character states cause systematic errors in molecular phylogenetic analyses: a simulation study

Analysis of sequence data using time‐reversible substitution models and maximum likelihood (ML) algorithms is currently the most popular method to infer phylogenies, despite the fact that results often contradict each other. Searching for sources of error we focus on a hitherto neglected feature of these methods: character polarity is usually thought to be irrelevant in ML analyses. Mechanisms that lead to wrong tree topologies were analysed at the level of split‐supporting site patterns. In simulations, plesiomorphic site patterns can be identified by comparison with known root sequences. These patterns cause some surprising effects: Using data sets generated with simulations of sequence evolution along a variety of topologies and inferring trees using the same (correct) model, we show for cases of branch‐length heterogeneity that (i) as already known, ML analyses can fail to recover the correct tree even when the correct substitution model is used, but also that (ii) plesiomorphic character states cause substantial mistakes and therefore character polarity is relevant, and (iii) accumulating chance similarities on long branches are far less misleading than plesiomorphic states accumulating on shorter branches. The artefacts occur when branch lengths are heterogeneous. The systematic errors disappear for the most part when the sites with symplesiomorphies supporting false clades are deleted from the data set. We conclude that many of the phylogenies published during the past decades may be false due to the neglected effects of symplesiomorphies.     

Heparin can liberate high molecular weight DNA from secondary necrotic cells

The borderline between necrosis and apoptosis is indistinct, but that between types of cell death is important because necrosis may lead to local inflammation, whereas apoptosis usually does not. In certain autoimmune disorders, inhibition of cell death is crucial, since macromolecules released from the dead cells may accelerate the autoimmune processes. We have used various cell death inhibitors to block cell death induced by 4HPR [N‐(4‐hydroxyphenil)‐retinamide] the BL41 and U937 cell lines. VD‐FMK, a general caspase inhibitor, inhibited DNA fragmentation induced by 4HPR, but not PI (propidium iodide) uptake and necrosis. Interestingly heparin, a serine‐protease inhibitor, lowered the PI fluorescence of the dead cell population and increased the sub‐G1 population as measured by flow cytometry. Regarding these changes, we found that heparin failed to increase DNA fragmentation, but merely liberated high molecular mass DNA fragments from dead cells. The exact mechanism is unclear, but heparin during secondary necrosis might enter the cells, bind RNPs (ribonucleoproteins), and pull them out with the attached DNA, where they would be sensitive to enzymatic degradation. Thus, the results suggest that heparin treatment helps in the clearance of cell debris and decreases the immunogenity of secondary necrotic cells.     

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.     

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.     

hSMUG1 can functionally compensate for Ung1 in the yeast Saccharomyces cerevisiae

There are at least four distinct families of enzymes that recognize and remove uracil from DNA. Family-3 (SMUG1) enzymes have recently been identified and have a preference for uracil in single-stranded DNA when assayed in vitro. Here we investigate the in vivo function of SMUG1 using the yeast Saccharomyces cerevisiae as a model system. These organisms lack a SMUG1 homologue and use a single enzyme, Ung1 to carry out uracil-repair. When a wild-type strain is treated with antifolate agents to induce uracil misincorporation into DNA, S-phase arrest and cellular toxicity occurs. The arrest is characteristic of checkpoint activation due to single-strand breaks caused by continuous uracil removal and self-defeating DNA repair. When uracil-DNA glycosylase is deleted (deltaung1), cells continue through S-phase and arrest at G(2)/M, presumably due to the effects of stable uracil misincorporation in DNA. Pulsed field gel electrophoresis (PFGE) demonstrates that cells are able to complete DNA replication with uracil-substituted DNA and do not experience the extensive strand breakage attributed to uracil-DNA glycosylase-mediated repair. As a result, these cells experience early protection from antifolate-induced cytotoxicity. When either UNG1 or SMUG1 functions are reintroduced back into the null strain and then subjected to antifolate treatment, the cells revert back to the wild-type phenotype as shown by a restored sensitivity to drug and S-phase arrest. The arrest is accompanied by the accumulation of replication intermediates as determined by PFGE. Collectively, these data indicate that SMUG1 can act as a functional homolog of the family-1 uracil-DNA glycosylase enzymes.     

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.     

Detecting systematic errors in multi-clinic observational data.

In multi-clinic studies it is hard to maintain a uniformly high quality of measurement and coding. Systematic errors almost always occur, in spite of the best of intentions and the most rigid protocols. It is the statistician's responsibility to plan for the detection of these errors, as well as to try to avoid them and not be misled by them. The practice of examining the univariate and multivariate sample frequency distributions of the variables under study, with an eye open for anything that looks puzzling, can be very helpful in detecting and trying to correct systematic errors that would bias the analysis. Examples are given from a 21-clinic study on pregnancy and child development.     

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.     

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.