1,721,092 research outputs found
Functional reconstitution and characterization of AqpZ, the E. coli water channel protein
No full textUnderstanding the selectivity of aquaporin water channels will require
structural and functional studies of wild-type and modi®ed proteins;
however, expression systems have not previously yielded aquaporins in
the necessary milligram quantities. Here we report expression of a histidine-
tagged form of Escherichia coli aquaporin-Z (AqpZ) in its homologous
expression system. 10-His-AqpZ is solubilized and puri®ed to near
homogeneity in a single step with a ®nal yield of 2.5 mg/l of culture.
The histidine tag is removed by trypsin, yielding the native protein with
the addition of three N-terminal residues, as con®rmed by microsequencing.
Sucrose gradient sedimentation analysis showed that the native,
solubilized AqpZ protein is a trypsin-resistant tetramer. Unlike other
known aquaporins, AqpZ tetramers are not readily dissociated by 1%
SDS at neutral pH. Hydrophilic reducing agents have a limited effect on
the stability of the tetramer in 1% SDS, whereas incubations for more
than 24 hours, pH values below 5.6, or exposure to the hydrophobic
reducing agent ethanedithiol cause dissociation into monomers. Cys20,
but not Cys9, is necessary for the stability of the AqpZ tetramer in SDS.
Upon reconstitution into proteoliposomes, AqpZ displays very high
osmotic water permeability (pf 5 10 10ÿ14 cm3 sÿ1 subunitÿ1) and low
Arrhenius activation energy (Ea
3.7 kcal/mol), similar to mammalian
aquaporin-1 (AQP1). No permeation by glycerol, urea or sorbitol was
detected. Expression of native and modi®ed AqpZ in milligram quantities
has permitted biophysical characterization of this remarkably stable aquaporin
tetramer, which is being utilized for high-resolution structural studies
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
Purification and Functional Characterization of Aquaporin-8
No full textBackground information. Aquaporins (AQPs) are a family of channels permeable to water and some small solutes. In mammals, 13 members (AQPO-AQP12) have been found. AQP8 is widely distributed in many tissues and organs. Previous studies in frog oocytes suggested that AQP8 was permeable to water, urea and ammonium, but no direct characterization had yet been reported.Results. We expressed recombinant rAQP8, hAQP8 and mAQP8 (rat, human and mouse AQP8 respectively) in yeast, purified the proteins to homogeneity and reconstituted them into proteoliposomes. Although showing high sequence similarity, AQP8 proteins from the three species had to be purified with different detergents prior to reconstitution. In stopped-flow studies, all three AQP8 proteoliposomes showed water permeability, which was inhibited by mercuric chloride and rescued by 2-mercaptoethanol. rAQP8 and hAQP8 proteoliposomes did not transport glycerol or urea but were permeable to formamide, which was also inhibited by mercuric chloride. In the oocyte transport assay, hAQP8-injected oocytes showed significantly higher [C-14]methylammonium uptake than water-injected oocytes.Conclusions. In the present study, we successfully purified rAQP8, hAQP8 and mAQP8 proteins and characterized their biochemical and biophysical properties. All three AQP8 proteins transport water. rAQP8 and hAQP8 are not permeable to urea or glycerol. Moreover, hAQP8 is permeable to ammonium analogues (formamide and methylammonium). Our results suggest that AQP8 may transport ammonium in vivo and physiologically contribute to the acid-base equilibrium
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
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