1,720,955 research outputs found
Analysis of Chemokine Receptor Trafficking by Site-Specific Biotinylation.
No full textChemokine receptors undergo internalization and desensitization in response to ligand activation. Internalized receptors are either preferentially directed towards recycling pathways (e.g. CCR5) or sorted for proteasomal degradation (e.g. CXCR4). Here we describe a method for the analysis of receptor internalization and recycling based on specific Bir A-mediated biotinylation of an acceptor peptide coupled to the receptor, which allows a more detailed analysis of receptor trafficking compared to classical antibody-based detection methods. Studies on constitutive internalization of the chemokine receptors CXCR4 (12.1% ± 0.99% receptor internalization/h) and CCR5 (13.7% ± 0.68%/h) reveals modulation of these processes by inverse (TAK779; 10.9% ± 0.95%/h) or partial agonists (Met-CCL5; 15.6% ± 0.5%/h). These results suggest an actively driven internalization process. We also demonstrate the advantages of specific biotinylation compared to classical antibody detection during agonist-induced receptor internalization, which may be used for immunofluorescence analysis as well. Site-specific biotinylation may be applicable to studies on trafficking of transmembrane proteins, in general
Functional consequences of chemically-induced β-arrestin binding to chemokine receptors CXCR4 and CCR5 in the absence of ligand stimulation
No full textChemokine receptors CXCR4 and CCR5: Cell surface expression, signaling and modulation by β-arrestin 2
Get PDFG protein-coupled receptors which mediate a large variety of different cellular effects have always been a field of significant interest for pharmaceutical research. Since it was shown that the chemokine receptors CXCR4 and CCR5 act as essential co-receptors for HIV-1 entry the interest in understanding the regulation of cell surface expression of these specific GPCR increased even more. The receptor expression at the cell surface is regulated by different mechanisms such as agonist induced receptor endocytosis and subsequent receptor recycling, whereas boths effects are more or less distinct for different receptors. During recent years it was also shown that constitutive receptor endocytosis plays a prominent role in the circulation of receptors. Previous methods for the analysis of receptor cycling which used receptor-specific antibodies were not sensitive enough and lacked the potential to monitor constitutive receptor endocytosis in quantitative terms.
In this work an antibody-independent method based on specific biotinylation of an acceptor peptide (AP) by the enzyme biotin ligase A was established. Exemplified by the quantification of the ligand induced internalization and recycling of CXCR4 and CCR5 the robustness and high sensivity of the assay was demonstrated. Furthermore, the assay was not influenced by receptors which were freshly translocated to the cell surface without prior ligand binding. As an additional feature the assay provided the possibility for a detailed quantification of constitutive receptor endocytosis. In order to detect all receptors within a cell regardless of whether they had previously been expressed at the cell surface AP-specific monoclonal antibodies were generated which can be used for double immunofluorescence microscopy. These antibodies allow discriminations of biotinylated and non biotinylated receptors or detection of transmembrane proteins lacking high specific antibodies.
β-Arrestin 2 is a multivalent adaptor protein involved in receptor signaling as well as endocytosis which binds to various intracellular proteins. Recent reports challenged the classical concept of GPCR signaling via heterotrimeric G proteins and postulated a higher relevance of receptor homodimerization or binding of β-arrestins to the receptor. To circumvent G protein activation after ligand binding a chemical-induced dimerization system consisting of three components was used. Either a FKBP12 (DmrA) or FRB (DmrC) domain was fused to the C-terminus of CXCR4/CCR5 and β arrestin 2. Treatment of Rec DmrA/βArr DmrC cell lines with AP21967 led to dose- and time dependent recruitment of β arrestin 2 to the receptor in the absence of ligand stimulation. AP21967-induced translocation of β arrestin 2 to the receptor significantly decreased ligand-induced G protein mediated calcium release. In cell lines without βArr-DmrC expression no alterations were obtained. AP21967-binding also provoked a ligand-independent internalization of CXCR4/CCR5 which was on a comparable level as ligand-induced internalization. Interestingly, the AP21967 induced recruitment of β-arrestin 2 to the receptor was sufficient to mimic the specific, ligand-induced intracellular receptor distribution of either CXCR4 or CCR5. Whereas AP21967 treatment led to a β arrestin 2 receptor desensitization and internalization it was not sufficient to mediate receptor signaling via the MAP kinases ERK 1/2. AP20187-induced receptor homodimerization had no detectable effect on either receptor desensitization or the phsophorylation level of ERK 1/2. However AP20187 pretreament led to an enhanced ligand-induced internalization in Rec-DmrA cell lines. In summary, the results obtained within this work contribute to a more detailed understanding of β arrestin-mediated functions during chemokine receptor trafficking and demonstrated the applicability of a highly sensitive, biotin-based detection system for the analysis of trafficking of transmembrane proteins
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
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