1,720,966 research outputs found
Recombinant human renalase, a putative flavoenzyme involved in hypertension and cardiovascular diseases : expression and purification
No full textEXPRESSION, PURIFICATION AND CHARACTERIZATION OF TWO RECOMBINANT FORMS OF HUMAN RENALASE, A NEW PROTEIN INVOLVED IN BLOOD PRESSURE AND CARDIOVASCULAR FUNCTION REGULATION
No full textCardiovascular complications represent one of the leading causes of morbidity and mortality in Chronic Kidney Disease (CKD). Even mild to moderate renal injuries, besides total functionality loss, are proven to correlate with increased cardiovascular risk. The hypothesis that the kidney may fill additional unknown assignments for its endocrine function, which may be very relevant for the development of cardiovascular diseases, prompted some authors (Xu et al., 2005) to sift the human genome to find novel proteins secreted by the kidney, finally leading to the discovery of renalase.
The cDNA library of the Mammalian Gene Collection Project was screened for proteins with secretory features and low similarity with proteins of known function. A single gene, encoding a new human protein was found, named renalase, after its main secretion site. Based on the detection of renalase in plasma, on its putative monoamine oxidase activity on catecholamines in vitro, on the identification of a FAD-binding motif and a low degree of similarity with monoamine oxidases (MAOs), on the observation of in vivo effects on blood pressure and hemodynamics, and on the absence on plasma renalase in patients with severe renal failure, it has been proposed that renalase is a FAD-dependent enzyme secreted by the kidney, which metabolizes plasma catecholamines, resulting in the decrease of blood pressure and heart rate. For this reason, renalase is also considered as a new member of the MAO family of enzymes, and it has been also called MAO-C.
The hypothesis of the fundamental role of renalase in the regulation of circulating catecholamine levels and its involvement in the control of blood pressure and cardiac function and, consequently, in the pathogenesis of cardiovascular diseases was supported by population genetic analysis and by in vivo studies (Zhao et al., 2007; Ghosh et al., 2006a; Ghosh et al., 2006b; Desir et al., 2008; Li et al., 2008; Desir et al., 2007). However, the data about the catalytic activity of renalase on catecholamines reported by Xu et al. (2005) were severely criticized (Boomsma and Tipton, 2007). Above that, the lack of a thorough biochemical characterization of renalase prompted us to start the study of this protein.
Here we report the development and optimization of two different protocols for the expression of recombinant human renalase1 in E. coli, the predominant human isoform, and its purification in a holoprotein form (Pandini, Ciriello et al., 2010), and provide a detailed biochemical and structural characterization of this protein. The cDNA encoding renalase1 was purchased from imaGenes GmbH (Berlin, Germany), carried by an expression plasmid which drive the production of a fusion protein with an N-terminal His-tag in frame with the coding sequence of renalase1. Starting from this plasmid, we generated a second construct for the production of an N-tagged SUMO-renalase fusion protein, which is easily cleaved by a specific SUMO protease (Senp2 protease), resulting in a protein which carries only an additional serine residues at its N-terminus, followed by the native sequence of human renalase1 (Ser-renalase).
Both recombinant forms were purified to homogeneity in amounts sufficient for their biochemical and structural characterization, and were found to be highly stable and monomeric. Considering the proposed MAO-like nature of renalase, its biochemical characterization has initially focused on the possibility that renalase could actually be ascribed to the MAO family of enzymes. We demonstrated for the first time that renalase contains non-covalently bound FAD, while the flavin cofactor in MAOs is bound in a covalent manner. Moreover, other biochemical differences emerged between renalase and monoamine oxidases, in terms of activity of the protein and reactivity of the flavin cofactor, suggesting that it is improper to classify it as a MAO-like enzyme. On the contrary, further experiments showed that renalase might rather be an NADPH-dependent flavoenzyme, with an enzymatic activity possibly similar to that of bacterial NAD(P)H-dependent flavin monooxygenases, such as p-hydroxybenzoate hydroxylase
Renalase : a new human flavoprotein possibly involved in the regulation of cardiac function and blood pressure
No full textRenalase, a putative monoamine oxidase, has been recently identified by the group of G.V. Desir (1). It is considered to be a new renal hormone which is involved in the regulation of cardiac function and blood pressure by way of its assumed monoamino oxidase activity. The renalase protein contains a signal peptide for secretion (1-17 aa) overlapping a FAD binding region (3-42 aa) and an amino oxidase region (75-335 aa). The similarity to MAO A and B is only 13% and it is restricted to the first 80-100 aa. We have expressed the full-length human cDNA in Escherichia coli and purified the soluble protein. Here we report on its biochemical properties. We have demonstrated for the first time that indeed this renalase is a flavoprotein and shown that contains non-covalently bound FAD at variance with the MAO enzymes. Furthermore, our protein is devoid of catecholamine oxidase activity, thus casting doubts on the real substrate of this enzyme. 1. Xu J, Li G, Wang P, Velazquez H, Yao X, Li Y, Wu Y, Peixoto A, Crowley S, Desir GV., J Clin Invest (2005), 115(5):1275-80
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
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