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Role and function of the Ca2+-dependent protease calpain-1 associated to membrane lipid rafts
No full textThe Ca2+-dependent protease calpain-1 has been found constitutively present in lipid raft/caveolin-1 membrane microdomains isolated from endothelial bEnd5 cells. In this membrane localization calpain-1 is associated with some of its substrates, such as endothelial Nitric Oxide Synthase (eNOS) and the chaperone Heat Shock Protein 90 (HSP90), whereas the calpain-2 and the inhibitor calpastatin have not been detected.
Alteration in intracellular Ca2+ homeostasis, induced by either treatment with Ca2+-ionophore A23187 or prolonged cell exposure to high glucose, leads to a significant decrease in the level of eNOS associated to lipid rafts, followed by a recruitment of HSP90 at this site. The decrease of eNOS is due not only to its Ca2+-dependent release from the caveolin-1 microdomains, but also to its digestion mediated by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2). However, the modifications observed in lipid rafts protein composition make bEnd5 cells more resistant against cell death caused by Ca2+ overload. In this context, calpain-1 seems to play a protective role against NO overproduction.
Alterations in eNOS, calpain-1 and HSP90 levels have been also detected in aorta of Zucker Diabetic Rats (ZDR). The digestion of HSP90 indicates an aberrant activation of calpain, due to a chronic alteration in Ca2+ homeostasis occurring in these animals, and thereby the transition from a physiological to a pathological cell condition. These changes cannot be detected in brain cortex, suggesting a tissue-specificity of the alteration in Ca2+ homeostasis related to diabetes.
A similar protein organization of lipid rafts has been also observed in neuroblastoma SK-N-BE cells, where calpain-1 is associated with NMDAR, HSP90 and neuronal NOS (nNOS), as well as in brain cortex of normotensive rats. Preliminary results have demonstrated that the increase in [Ca2+]i, that characterizes the nervous tissue of hypertensive rats, leads to a protein reorganization of the lipid rafts.
The results presented in this thesis provide new information on a selective localization and role of calpain-1. In endothelial cells the protease and its associated substrates seem to have a crucial role in responding to fluctuations of [Ca2+]i. Further investigations will be necessary to analyse the role of the protease associated to lipid rafts in nervous tissue both in physiological and pathological conditions
Calpain-1 resident in lipid raft/caveolin-1 membrane microdomains plays a protective role in endothelial cells.
No full textWe are here reporting that calpain-1 is a constitutive component of a distinct lipid raft/caveolin-1 microdomain isolated from bEnd5 cells in association with endothelial nitric oxide synthase (eNOS) and heat shock protein 90 (HSP90). Perturbations in intracellular calcium concentration by Ca2+-ionophore A23187 or prolonged cell exposure to high glucose induce a significant decrease in the level of eNOS accompanied by a recruitment of additional HSP90 molecules at this site. In these conditions the cells are more resistant to cell death by Ca2+ overload. The decrease of eNOS has been due not only to its Ca2+-mediated release from the caveolin-1 aggregates but also to its digestion by calpain-1. The specific involvement of calpain-1 in digestion of eNOS is supported by the preventive effect of a synthetic calpain inhibitor (CI-2) and by the absence of calpain-2 and calpastatin in the caveolin-1 microdomain. These results suggest that the protein adjustments observed in lipid raft/caveolin-1 microdomains could be visualized as a process required to protect the cells against NO overproduction and aberrant calpain activation. Alterations in eNOS, calpain-1 and HSP90 levels have been observed in aorta of Zucker Diabetic Rats (ZDR). The loss of HSP90 occurring in these animals indicates an aberrant activation of calpain and thereby the transition from a physiological to a pathological cell condition
Role of the regulatory domain of calpastatin in the autoproteolytic activation of calpain
No full textCalpastatin (CST), the natural inhibitor of calpain (CALP), is a protein composed by four repetitive inhibitory domains and a regulatory domain without inhibitory properties. Depending on the CST form, the regulatory region can be constituted by L- or XL-L domains which exist in different splicing forms. We have previously observed that all the CSTs, containing the XL-L and L-domains, lose inhibitory efficiency at concentrations expected to promote complete CALP inhibition. It is known that the CST inhibitory domain binds to CALP catalytic site in the presence of Ca2+, and that the L-domain binds at the N-terminus of CALP in a region close to the same site. Hence, we explored the possible role of the L-domain during the activation of human erythrocyte calpain. Activation of CALP proceeds following conformational changes reorganizing the catalytic triad, through the conversion of the 80kD to 75kD which requires lower Ca2+ for activity.
Our experiments indicate that CALP conversion is reduced in the presence of cast600 (containing the L-domain and one inhibitory unit), and is delayed in the presence of cast300 (single inhibitory unit, without L-domain). Moreover, while addition of free L-domain to cast300 reduces CALP autoproteolysis, its addition to cast600 has no effect. We can postulate that both CSTs prevent the access of the substrate to CALP active site, but cast600 acts also on the autolytic process, inhibiting the formation of the 75kD form. Hence, the CALP/CST interaction is controlled by the L-domain whose presence can address the formation of different enzyme-inhibitor complexes. Since the L-domain binds to CALP also when the protease is in the inactive conformation, we explored if it affects CALP activation at low [Ca2+]. We analyzed whether the 75kD CALP can activate native 80kD CALP in 5 μM Ca2+, a concentration too low for activating erythrocyte calpain. We observed that, the active 75kD CALP does not catalyze the intermolecular conversion of the native CALP to the 75kD form, but it degrades the protease to inactive fragments. When we performed the same experiments with increasing amounts of free L-domain, we observed that CALP degradation mediated by the 75kD CALP is nearly abolished. This was evidenced both from the limited appearance of calpain fragments and, most importantly, by the activity of the 80kD CALP assayed in the presence of substrate.
Thus, at low [Ca2+] the L-domain bound to native CALP protects the protease from the degradation mediated by activated CALP and leaves the active site available for substrate digestion. Since the recombinant L-domain (RNCAST110) used in our experiments is a truncated mRNA for CST, detected in rat brain, we are now looking for a similar mRNA in human nervous tumors where we have already observed different ratios of native/activated calpain. These data can be the starting point for exploring new possibilities, not based on CALP inhibition, to correct aberrant CALP activity
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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