1,720,963 research outputs found
Purification and Characterization of Native and Recombinant Dipeptidyl Aminopeptidase 1 of Plasmodium falciparum
No full textPlasmodium falciparum dipeptidyl aminopeptidase 1 (DPAP1) contributes to the degradation of hemoglobin by releasing dipeptides from globin oligopeptides in the food vacuole. The lack of success at DPAP1 gene disruption suggests that this exopeptidase is important for efficient growth during the erythrocytic asexual stage. DPAP1 is therefore an attractive target for the development of anti-malarial drugs that block the catabolism of hemoglobin. To guide the design of selective, potent DPAP1 inhibitors, it is necessary to characterize the substrate specificity of this enzyme along with its human homolog cathepsin C. Although native purification of DPAP1 is possible, the amount of purified enzyme obtained is insufficient for extensive biochemical characterization. To overcome this obstacle, a strategy was developed for the recombinant expression of soluble DPAP1 in the bacterium Escherichia coli and for its activation in vitro. The production of active recombinant DPAP1 presents three challenges: 1) expression of the protein in soluble form, 2) generation of the native N-terminus, and 3) cleavage of the pro-domain. Soluble expression of DPAP1 was achieved by fusing it to the C-terminus of maltose-binding protein (MBP). A linker sequence encoding a tobacco etch virus protease (TEVp) cleavage site was introduced between MBP and DPAP1 such that TEVp cleavage would generate the presumed native N-terminus of DPAP1. Incubation of the MBP-DPAP1 fusion with TEVp resulted in the release of free DPAP1which hydrolyzed the fluorogenic substrate proyly-arginyl-7-amido-4 methyl coumarin (Pro-Arg-AMC). Various proteases were tested for the ability to excise the pro-region. Treatment with both trypsin and papain removed the pro-region and increased DPAP1 activity two to three fold. When assayed with Pro-Arg-AMC, trypsin-treated DPAP1 had kinetic properties similar to native enzyme whereas papain-treated DPAP1 deviated from Michaelis-Menten kinetics. Using a combinational dipeptidyl substrate library, the substrate specificities of native and recombinant (trypsin-activated) DPAP1, as well as of human cathepsin C were profiled. We find that both DPAP1 and human cathepsin C accept a wide spectrum of amino acid side chains at the substrate P1 and P2 positions. Interestingly, several P2 residues show high selectivity for DPAP1 or cathepsin C. The collected data point to the feasibility of designing inhibitors that are specific for DPAP1 over cathepsin C.Master of Science in Life Science
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
Generation of active immunotoxins containing recombinant restrictocin
No full textRestrictocin, a toxin produced by the fungusAspergillus restrictus,is a potent inhibitor of eukaryotic protein synthesis. Recombinant restrictocin was made inEscherichia coliand purified to homogeneity in large amounts. The recombinant protein was found to be poorly immunogenic in mice with low toxicity, when injected intraperitoneally. Two immunotoxins were constructed by coupling the recombinant restrictocin to an antibody to the human transferrin receptor, using a cleavable and a stable linkage. The immunotoxins so generated showed specific cytotoxic activity toward receptor bearing cells in tissue culture. Immunotoxin with a cleavable linkage, however, was more active than that containing a stable linkage. Restrictocin appears to be a promising candidate to be developed as a chimeric toxin for targeted therapy
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
Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action
Get PDFRestrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)-restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5' end of that encoding restrictocin. The other chimaeric toxin, restrictocin-anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3' end of the DNA encoding restrictocin. These gene fusions were expressed in Escherichia coli, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free in vitro translation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin-anti-TFR(scFv) was more active than anti-TFR(scFv)-restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus
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