1,721,027 research outputs found
Studies on Active site Mutants of Human Non-Pancreatic secreted Phospholipase A2
No full textPhospholipase A2 (PLA2) is an enzyme that catalyses the hydrolysis of phospholipids at the sn-2 fatty-acyl bond, liberating free fatty acids and lysophospholipids. As many as eleven different forms of the enzyme have been characterised, including both secreted and cytosolic, calcium dependent and independent varieties. These PLA2S are involved in a range of physiological processes including phospholipid digestion, signal transduction and host defence, and have been isolated from a diverse range of organisms and cells.This thesis is concerned with the human non-pancreatic secreted form of PLA2 (hnpsPLA2), which is secreted by a variety of cells including platelets, macrophages and smooth muscle cells. HnpsPLA2 is a 14 kDa, calcium-dependent extracellular enzyme believed to be an acute phase protein, with an important antimicrobial role. It is the binding of this enzyme to the phospholipid interface that is crucial in the physiological regulation of the enzyme. In order to study interfacial binding in the absence of catalysis, structurally intact but inactive mutants were required. The production of the mutants H48Q, H48N and H48A appeared to be the obvious strategy based on previous work on other secreted phospholipases A2 from bovine pancreas and bee venom. However, the mutant H48Q unexpectedly retained significant (~4%) catalytic activity that was contrary to all previous work in the field.In this thesis it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions. The crystallisation of the H48Q mutant to a resolution of 1.5 Å allowed a comparison with the corresponding wild-type enzyme (N1A) that was also crystallised. This comparison revealed that all the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins and the two catalytic water molecules were clearly resolved in the H48Q mutant. A 'two-water mechanism' in which the second water molecule at the active site hydrogen bonds to the glutamine could explain the residual activity of the H48Q. A second active site mutant (H48N) showed minimal activity and was used to study the interfacial binding properties of the enzyme. The results highlighted the use of this mutant in interfacial binding assays and confirmed the high affinity of this enzyme for anionic interfaces. The third active site mutant, H48A, was completely inactive. However, this mutant could not be expressed in significant amounts and was not further investigated.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Get PDFWe 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
Get PDFWe 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
The crystal structure of the H48Q active site mutant of human group IIA secreted phospholipase A(2) at 1.5 angstrom resolution provides an insight into the catalytic mechanism
No full textThe human group IIA secreted PLA2 is a 14 kDa calcium-dependent extracellular enzyme that has been characterized as an acute phase protein with important antimicrobial activity and has been implicated in signal transduction. The selective binding of this enzyme to the phospholipid substrate interface plays a crucial role in its physiological function. To study interfacial binding in the absence of catalysis, one strategy is to produce structurally intact but catalytically inactive mutants. The active site mutants H48Q, H48N, and H48A had been prepared for the secreted PLA2s from bovine pancreas and bee venom and retained minimal catalytic activity while the H48Q mutant showed the maximum structural integrity. Preparation of the mutant H48Q of the human group IIA enzyme unexpectedly produced an enzyme that retained significant (2-4%) catalytic activity that was contrary to expectations in view of the accepted catalytic mechanism. In this paper it is established that the high residual activity of the H48Q mutant is genuine, not due to contamination, and can be seen under a variety of assay conditions including assays in the presence of Co2+ and Ni2+ in place of Ca2+. The crystallization of the H48Q mutant, yielding diffraction data to a resolution of 1.5 Å, allowed a comparison with the corresponding recombinant wild-type enzyme (N1A) that was also crystallized. This comparison revealed that all of the important features of the catalytic machinery were in place and the two structures were virtually superimposable. In particular, the catalytic calcium ion occupied an identical position in the active site of the two proteins, and the catalytic water molecule (w6) was clearly resolved in the H48Q mutant. We propose that a variation of the calcium-coordinated oxyanion ("two water") mechanism involving hydrogen bonding rather than the anticipated full proton transfer to the histidine will best explain the ability of an active site glutamine to allow significant catalytic activity
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