Following autophagy step by step

Autophagy is an evolutionarily conserved lysosomal degradation route for soluble components of the cytosol and organelles. There is great interest in identifying compounds that modulate autophagy because they may have applications in the treatment of major diseases including cancer and neurodegenerative disease. Hundeshagen and colleagues describe this month in BMC Biology a screening assay based on flow cytometry that makes it possible to track distinct steps in the autophagic process and thereby identify novel modulators of autophagy.     

Making tubes: step by step

Branched hollow tubes form the architectural basis of many mammalian organs. The growth factor HGF/SF and its receptor, the Met receptor tyrosine kinase, stimulate epithelial cells to undergo tubulogenesis in vitro. In this issue of Developmental Cell, O'Brien et al. (2004) look at temporal regulation and the role of two HGF/SF effectors, the ERK 1/2 MAP kinases and matrix metalloproteases, in this process.     

A step by step guide to phylogeny reconstruction

The aim of this paper is to enable those who have never reconstructed a phylogeny to do so from scratch. The paper does not attempt to be a comprehensive theoretical guide, but describes one rigorous way of obtaining phylogenetic trees. Those who follow the methods outlined should be able to understand the basic ideas behind the steps taken, the meaning of the phylogenetic trees obtained and the scope of questions that can be answered with phylogenetic methods. The protocols have been successfully tested by volunteers with no phylogenetic experience.     

MitraClip step by step; how to simplify the procedure

The MitraClip system is a device for percutaneous edge-to-edge reconstruction of the mitral valve in patients with severe mitral regurgitation who are deemed at high risk for surgery. Studies have underlined the therapeutic benefit of the MitraClip system for patients at extreme and high risk for mitral valve surgery, suffering from either degenerative or functional mitral regurgitation. The MitraClip procedure shows low peri-procedural complication rates, and a significant reduction in mitral regurgitation, as well as an improvement in functional capacity and most importantly quality of life. It hereby widens the spectrum of mitral valve repair for the Heart Team. The current review underscores the efficacy of the procedure and describes the technique to simplify the procedure.     

一步法RT-PCR和两步法RT-PCR对蜜蜂病毒诊断研究的比较

【目的】探讨适合蜜蜂病毒病诊断的RT-PCR技术。【方法】从感染蜜蜂以色列急性麻痹病毒、残翅病毒、囊状幼虫病毒、急性麻痹病毒、黑蜂王台病毒以及慢性麻痹病毒的阳性样品中,分别提取这6种病毒的RNA。然后,同时应用一步法RT-PCR和两步法RT-PCR的反应体系对6种病毒扩增,并对两种方法的灵敏性进行比较和分析。【结果】上述两种方法分别得到158(IAPV)、269(DWV)、342(SBV)、460(ABPV)、536(BQCV)和774 bp(CBPV)的扩增片段,测序结果证实扩增片段符合预期。两步法RT-PCR可检测到更低浓度的病毒颗粒。【结论】结果表明,该2种方法均可快速、有效地诊断蜜蜂病毒。但两步法RT-PCR灵敏性更高。     

Submit your interaction data the IMEx way: a step by step guide to trouble-free deposition

The ever-increasing generation of, and corresponding interest in, molecular interaction data has lead to the establishment of a number of high-quality molecular interaction databases which manually curate interaction data extracted from the literature. In order to effectively share the curation load, and ensure that data is stored in and accessible from multiple sources, these databases have united to form the IMEx consortium. All of the IMEx databases also accept direct deposition of interaction data from authors prior to publication, thus both assisting the scientist in preparing the dataset for publication and ensuring that its subsequent representation in the public domain databases is fully accurate. This article walks the potential submitter through the various routes by which data may be deposited with the databases and describes the tools which have been developed to assist in this process.     

One step purification of chymosin by mixed mode chromatography

Mixed mode Sepharose and Perloza bead cellulose matrices were prepared using various chemistries. These matrices contained hydrophobic (aliphatic and/or aromatic) and ionic (carboxylate or alkylamine) groups. Hydrophobic amine ligands were attached to epichlorohydrin activated Sepharose (mixed mode amine matrices). Hexylamine, aminophenylpropanediol and phenylethylamine were the preferred ligands, on the basis of cost and performance. Other mixed mode matrices were produced by incomplete attachment (0-80%) of the same amine ligands to carboxylate matrices. The best results were obtained using unmodified or partially ligand-modified aminocaproic acid Sepharose and Perloza. High ligand densities were used, resulting in high capacity. Furthermore, chymosin was adsorbed at high and low ionic strengths, which reduced sample preparation requirements. Chymosin, essentially homogeneous by electrophoresis, was recovered by a small pH change. The methods described were simple, efficient, inexpensive and provided very good resolution of chymosin from a crude recombinant source. The carboxylate matrices had the best combination of capacity and regeneration properties. The performance of Sepharose and Perloza carboxylate matrices was similar, but higher capacities were found for the latter. Because it is cheaper and can be used at higher flow rates, Perloza should be better suited to large scale application. High capacity chymosin adsorption was found with carboxymethyl ion exchange matrices, but low ionic strength was essential for adsorption and the purity was inferior to that of the mixed mode matrices. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 45-55, 1997.     

The proton transfer step catalyzed by yeast pyruvate kinase

The nature of the proton donor to the C-3 of the enolate of pyruvate, the intermediate in the reaction catalyzed by yeast pyruvate kinase, was investigated by site-directed mutagenesis and physical and kinetic analyses. Thr-298 is correctly located to function as the proton donor. T298S and T298A were constructed and purified. Both mutants are catalytically active with a decrease in k(cat) and k(cat)/K(m)(,PEP). Mn(2+)-activated T298S and T298A do not exhibit homotropic kinetic cooperativity with phosphoenolpyruvate (PEP) in the absence of fructose 1,6-bisphosphate, although PEP binding to enzyme-Mn(2+) is cooperative. The pH dependence of k(cat) for T298A indicates the loss of pK(a)(,2) = 6.4-6.9. Thr-298 affects the ionization (pK(a) approximately 6.5) responsible for modulation of k(cat). Fluorescence studies show altered dissociation constants of ligands to each enzyme complex upon Thr-298 mutations. The rates of the phosphoryl transfer and proton transfer steps in the pyruvate kinase-catalyzed reaction are altered; pyruvate enolization is affected to a greater extent. Proton inventory studies demonstrate solvent isotope effects on k(cat) and k(cat)/K(m)(,PEP). Fractionation factors are metal-dependent and significantly <1. The data suggest that a water molecule in a water channel is the direct proton donor to enolpyruvate and that Thr-298 affects a late step in catalysis.     

Taking it step by step: mechanistic insights from structural studies of ubiquitin/ubiquitin-like protein modification pathways

Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins regulate a diverse array of cellular pathways through post-translational attachment to protein substrates. Ub/Ubl-mediated signaling is initiated through E1, E2, and E3-mediated conjugation, transduced by proteins that recognize Ub/Ubl-modified substrates, and terminated by proteases which remove the Ub/Ubl from the substrate. Recent structural studies have elucidated mechanisms pertinent to Ub/Ubl conjugation, recognition, and deconjugation, highlighting essential steps during Ub/Ubl modification that illustrate common and divergent mechanistic themes within this important process.     

Lithium increases actin polymerization rates by enhancing the nucleation step

Lithium affects the polymerization mechanism of some cytoskeletal proteins in vitro, so its biological activity could also reflect lithium influence on assembly processes. Our data demonstrate that lithium nucleates actin polymerization and, in parallel, is less effective in the elongation step. Furthermore, falling-ball and fluorimetric tests suggested that lithium-induced actin polymers at steady-state are shorter than K(+)-polymerized actin filamentous structures. The lithium-induced actin assembly seems to follow the "reversible polymerization model" and the critical concentration of Li(+)-assembled actin at steady-state is markedly lower than that of sister actin samples polymerized by potassium chloride. Finally, the stabilization of actin nuclei induced by lithium ions could be related to their effect of lowering the dissociation rate constant.     

Single-strand DNA translation initiation step analyzed by Isothermal Titration Calorimetry

Is single-strand DNA translatable? Since the 60s, the question still remains whether or not DNA could be directly translated into protein. Some discrepancies in the results were reported about functional translation of single-strand DNA but all results converged on a similar behavior of RNA and ssDNA in the initiation step. Isothermal Titration Calorimetry method was used to determine thermodynamic constants of interaction between single-strand DNA and S30 extract of Escherichia coli. Our results showed that the binding was not affected by the nature of the template tested and the dissociation constants were in the same range when ssDNA (K_d = 3.62 ± 2.1 × 10⁻⁸ M) or the RNA corresponding sequence (K_d = 2.7 ± 0.82 × 10⁻⁸ M) bearing SD/ATG sequences were used. The binding specificity was confirmed by antibiotic interferences which block the initiation complex formation. These results suggest that the limiting step in translation of ssDNA is the elongation process.