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In vitro evolution of a dimeric variant of human pancreatic ribonuclease
No full textSite-directed mutagenesis of human pancreatic RNase (HP-RNase) was used as a model system for investigating the genetic events underlying the evolutionary origins of protein oligomers. HP-RNase is a monomeric enzyme with no natural tendency to oligomerize (K(d) for any dimers in solution of > 280 mM). Nevertheless, deletion of five amino acid residues in the loop linking the N-terminal helix of HP-RNase to the rest of the protein was found to drive polypeptide chains to fold into dimers. These dimers could not be dissociated by heating at 70 °C, and small amounts of monomer were detected only in highly diluted samples. Measurement of dimer and monomer concentrations under equilibrium conditions yielded a K(d) of 1.5 μM. This implies that the deletion increases the protein propensity to dimerize at least 5.2 orders of magnitude. Moreover, the HP-RNase dimers were found to be over 4.6 orders of magnitude more stable than the dimers of bovine pancreatic RNase A obtained by lyophilization from acetic acid (K(d) > 73 mM). Cross- linking experiments with divinyl sulfone indicated that the HP-RNase dimers are stabilized by the exchange between subunits of their N-terminal helices. This generates composite active sites, i.e., each contributed by two subunit chains, that retain full enzymatic activity. Overall, these results show that a deletion of few residues in a key region of a monomeric protein can be the primary event irreversibly leading to oligomerization of the protein through the swap of a secondary structure element between protomers
ENGINEERING THE REFOLDING PATHWAY AND THE QUATERNARY STRUCTURE OF SEMINAL RIBONUCLEASE BY NEWLY INTRODUCED DISULFIDE BRIDGES.
No full textSeminal RNase (BS-RNase), a ribonuclease from bovine seminal vesicles, is a homodimeric enzyme with a strong cytotoxic activity selective for tumor cells. It displays the unusual structural feature of existing in solution as an equilibrium mixture of two quaternary isoforms. The major one is characterized by the swap between subunits of their N-terminal ends, whereas the minor isoform shows no swap. The tendency of the two isolated isoforms to interconvert into each other has so far made it difficult to attribute the functional properties of BS-RNase to either isoform. Herein, molecular modeling and site-directed mutagenesis were used to engineer the refolding pathway of BS-RNase and obtain a stable variant of its non-swapping isoform. The protein was engineered with two extra disulfide bridges linking the N-terminal helix of each subunit to the main body of the same subunit. Purified as an active enzyme, the BS-RNase variant was found to be very resistant to thermal denaturation. Its functional characterization revealed that the lack of swapping has a negative effect on the cytotoxic activity of BS-RNase
Engineering the refolding pathway and the quaternary structure of seminal ribonuclease by newly introduced disulfide bridges
No full textSeminal RNase (BS-RNase), a ribonuclease from bovine seminal vesicles, is a homodimeric enzyme with a strong cytotoxic activity selective for tumor cells. It displays the unusual structural feature of existing in solution as an equilibrium mixture of two quaternary isoforms. The major one is characterized by the swap between subunits of their N-terminal ends, whereas the minor isoform shows no swap. The tendency of the two isolated isoforms to interconvert into each other has so far made it difficult to attribute the functional properties of BS-RNase to either isoform. Herein, molecular modeling and site-directed mutagenesis were used to engineer the refolding pathway of BS-RNase and obtain a stable variant of its non-swapping isoform. The protein was engineered with two extra disulfide bridges linking the N-terminal helix of each subunit to the main body of the same subunit. Purified as an active enzyme, the BS-RNase variant was found to be very resistant to thermal denaturation. Its functional characterization revealed that the lack of swapping has a negative effect on the cytotoxic activity of BS-RNase
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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