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Fluorescent Assay to Demonstrate the Interaction of Thioredoxin Reductase and Protein Disulfide Isomerase.
No full textFluorescent Assay to Demonstrate the Interaction of Thioredoxin Reductase and Protein Disulfide Isomerase Tomazzolli R.1, Guella G.2, Bellisola G.3, Colombatti M.3 and Menestrina G.1 1: ITC-CNR IBF Unit at Trento, Via Sommarive 18, 38050 Povo (TN), Italy 2: Lab Bioorg Chem, Dep Physics, Uni Trento, Via Sommarive 14, 38050 Povo, Italy 3: Dep Pathology, Sect of Immunol, Uni Verona, L.go Scuro 10, 37134 Verona, Italy In eukaryotic cells, the ubiquitous thioredoxin reductase-thioredoxin system (TrxR-Trx) and the glutathione reductase-glutathione system (GSHR-GSSG) catalyse substrate disulfide reduction using NADPH as a source of reducing equivalents. TrxR reduces the redox protein Trx as well as other endogenous and exogenous compounds, while the glutathione system plays a key role in protecting cellular macromolecules from damage caused by reactive oxygen species. Due to a second redox-active site, mammalian TrxR has a broader substrate specificity than glutathione reductase and Escherichia coli TrxR [1]. Thioredoxin forms a superfamily with several other proteins sharing little sequence similarity but possessing a common active site [2]. Among these, protein disulfide isomerase (PDI) is colocalized, in cells, with TrxR. In vitro there are now some evidences that PDI can be a substrate of TrxR [3, 4]. To further demonstrate this interaction we have produced a fluorogenic substrate suitable to test TrxR-dependent PDI activity. The substrate is constituted by two identical cys-containing tri-peptides linked at the N-terminus to a molecule of fluorescein-5-isothiocyanate (FITC) and held together by a disulfide bridge between the cysteines. The preparation follows a few steps: the tri-peptide is labelled with FITC; it is separated from FITC excess by two reverse phase liquid chromatography elutions and analysed by electrospray ionization mass spectrometry (ESI-MS); oxidation occurs spontaneously or is induced by DMSO. The final substrate, named pep-FITC, is characterized by NMR and ESI-MS. The reduction of pep-FITC was measured at 25°C in a Fluoromax spectrofluorimeter and was indicated by an increase of the FITC fluorescence at 520nm with excitation at 494nm. All the fluorescence measurements were taken in NaP 0.1M, EDTA 2mM pH 7.5. We demonstrated that the TrxR (both mammalian and bacterial)-PDI system successfully reduced pep-FITC while the single components didn’t. GSHR didn’t reduce pep-FITC, in spite of its aminoacidic sequence similarity to GSSG but was active on a fluorogenic GSSG, labelled at N-termini with FITC, thus demonstrating the narrow substrate specificity of GSHR. Further proofs of the interaction between PDI and TrxR we obtained by fluorescence resonant energy transfer (FRET). In this case we labelled the enzymes with Alexa Fluor 546 and Alexa Fluor 488 respectively, two dyes forming a donor/acceptor pair with a Forster distance of 58 Å [5]. A FRET was observed under suitable conditions confirming tight interaction. (1) Mustacich D, Powis G. 2000. Biochem. J., 346: 1-8. (2). Hirota K, Nakamura H, Masutani H, Yodoi J. 2002. Ann. NY Acad. Sci., 957:189-99. (3). Lundstrom J, Holmgren A. 1990. J. Biol. Chem., 265: 9114-9120. (4). Bellisola G, Fracasso G, Ippoliti R, Menestrina G, Rosen A, Solda S, Udali S, Tomazzolli R, Tridente G, Colombatti M. 2004. Biochem. Pharmacol., 67: 1721-1731. (5). Epe B, Steinhauser KG, Woolley P. 1983. Proc. Natl. Acad. Sci. USA 80 (May), 2579-2583
Ionic factors regulating the interaction of Gardnerella vaginalis hemolysin with red blood cells
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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
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