1,721,041 research outputs found
Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains
No full textMICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 μM) and increases kcat (10-25 s-1) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼10-fold higher than that of the other forms (3-5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues
Characterization of a new bioadhesive material from fish parasites by a proteomic approach
No full textMonogenoids are fish parasites which are able to quickly and reversibly attach to their host through the secretion of a proteic glue. Unlike most adhesive secretions found in the animal world which stick to abiotic substrates, monogenoidean adhesive secretion works on living tissues. Moreover, the adhesion of the parasite to its host takes place in an aqueous environment, in the presence of strong water currents. All these features make it a very promising material for exploitation in the surgical field. Another unique and very interesting feature of monogenoidean adhesion is that it can be easily reverted through the secretion of a still uncharacterized “detaching” protein (S3), likely a protease. The adhesive secretion is produced by glands located besides the pharynx or more specifically, in the antero-lateral region of the animal. Demonstration of the proteinaceous nature of the glue came in 2002 (Hamwood TE et al., Folia parasitologica; 49(1):39-49) who showed that the material is SDS insoluble without any trace of glycosylation. No further characterization of the adhesive proteins has been performed since this initial report. Besides very promising potential clinical applications, the study of these proteins represents something completely new also from the biochemical point of view. Understanding the structure of these adhesive proteins, the way they combine to yield the insoluble glue working in an aqueous environment and the mechanism of interaction with a biological substrate, like an epithelium, represents a major challenge which will widen our knowledge about protein structure/function relationship. The aim of this work is the characterization of the bio-adhesive material by means of a proteomic approach. The first step of the project was to set up the conditions to obtain and solubilise the secreted material before separation by electrophoresis and characterization by tandem mass spectrometry.
OBTAINING THE SECRETED ADHESIVE MATERIAL
The secreted material was obtained by electrostimulation of the parasites in a 50% PBS solution using 40 volts electric field and 2 Hz frequency. The secretion of 30 parasites was collected in a test tube and the SDS soluble components were discharged. The remaining material was solubilised in buffer A (7M Urea, 2M Tiourea, 4% Chaps, 40mM TrisHCl). Upon sonication and centrifugation the supernatant was collected and the protein content was evaluated measuring the absorbance at 280 nm before electrophoresis.
PROTEIN SEPARATION BY 2D ELECTROPHORESIS
The proteins soluble in buffer A were separated by 2-D electrophoresis upon reduction and derivatization by iodoacetamide. Isoelectric focusing (IEF) was performed in 7 cm immobilised pH gradient (IPG) strips with non linear pH ranges of 3-10. The second dimension was performed on 12% SDS-polyacrylamide gels using a Mini Protean cell. Running proceeded at 20 mA/gel. After running, the 2-D gels were stained with a mass-compatible silver.
TANDEM MASS SPECTROMETRY AND DE NOVO SEQUENCING
For mass analysis, each spot was excised, destained with 50% acetonitrile in ammonium bicarbonate 0.1 M (40 min at 25 °C), dried in a Speed Vac, soaked with ammonium bicarbonate 0.1 M and digested overnight with trypsin sequencing grade (Roche, Monza, Italy) at 37 °C. The in gel tryptic digest was extracted with 50% acetonitrile in 0.1% trifluoroacetic acid. Digested aliquots were removed and subjected to a desalting/concentration step on a μZipTipC18 (Millipore) using 40% CH3CN in 0.1% TFA as eluent. LC-ESI-MS/MS analysis was performed on a Dionex UltiMate 3000 HPLC System with a Hypersil Gold column (150 mm, internal diameter of 180 μm) filled with 3μm Reprosil-Pur C18-AQ resin. The gradient consisted of 5-15 % acetonitrile in 0.1% formic acid for 10 min, 15-40% acetonitrile in 0.1% formic acid for 52 min and 40-95 % acetonitrile in 0.1% formic for 68 min at a flow rate of 1.2 μl/min . The eluate was electrosprayed into an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) through a Proxeon nanoelectrospray ion source for de novo sequencing since the genome of these parasites is still unknown. Tandem mass spectrometry analysis was performed with each full scan (400-1700 m/z) followed by 5 data-dependent MS/MS scan at 35% normalized collision energy. The full scans were acquired with 2-microscan averaging at resolution 30000, AGC target 500000, maximum inection time 500 ms. All MS/MS data were analyzed with the Sequest program and the Peak Studio 5.1 program
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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