1,720,977 research outputs found
Protein kinase C mediates caspase 3 activation: A role for erythrocyte morphology changes
No full textWe have previously showed that morphological alterations in Red Blood Cells (RBCs) are correlated to an impaired eNOS enzymatic activity and a concomitant reduced NO derived metabolites formation. Here we extend our previous observations, reporting that RBC morphology is regulated by a series of specific cell signaling events linked to Protein Kinase C (PKC)-mediated activation of caspase 3. Pretreatment of RBCs with the PKC inhibitor chelerythrine, prior to the addition of phorbol-12-myristate-13-acetate (PMA), an activator of PKC, blocks the appearance of the morphology alterations and the sustained decrease in nitrates and nitrites levels induced by PMA. Inhibition of PKC also completely inhibits PMA mediated caspase-3 activation. On the other hand, caspase 3 inhibition, lessens the PMA induced-effects on the appearance of RBC morphology alterations, although it enhances PMA-mediated effects on nitric oxide (NO) derived metabolites levels. These data demonstrate that PKC-mediated activation of caspase 3 is an integral and essential part of signaling pathway in RBCs, that may be a regulatory factor of RBC mechanical properties, through regulation of NO metabolism
Understanding the binding of daunorubicin and doxorubicin to NADPH-dependent cytosolic reductases by computational methods
No full textThe anthracycline anticancer agents daunorubicin (DAUN) and doxorubicin (DOX) are reduced by different NADPH-dependent cytosolic reductases into their corresponding alcohol metabolites daunorubicinol (DAUNol) and doxorubicinol (DOXol), which have been implicated in the development of chronic cardiomyopathy. To better understand the individual importance of each enzyme in the reduction and to provide deeper insight into the binding at atomic level we performed molecular docking and dynamics simulations of DAUN and DOX into the active sites of human carbonyl reductase 1 (CBR1) and human aldehyde reductase (AKR1A1). Such simulations evidenced a different behavior between the reductases with respect to DAUN and DOX suggesting major contribution of CBR1 in the reduction. The results are in agreement with available experimental data and for each enzyme and anthracycline pair provided the identification of key residues involved in the interactions. The structural models that we have derived could serve as a useful tool for structure-guided drug design studies
NO Metabolites Levels in Human Red Blood Cells are Affected by Palytoxin, an Inhibitor of Na(+)/K(+)-ATPase Pump.
Get PDFPalytoxin (PTX), a marine toxin, represents an increasing hazard for human health. Despite its high toxicity for biological systems, the mechanisms triggered by PTX, are not well understood. The high affinity of PTX for erythrocyte Na(+)/K(+)-ATPase pump is largely known, and it indicates PTX as a sensitive tool to characterize the signal transducer role for Na(+)/K(+)-ATPase pump. Previously, it has been reported that in red blood cells (RBC), probably via a signal transduction generated by the formation of a PTX-Na(+)/K(+)-ATPase complex, PTX alters band 3 functions and glucose metabolism. The present study addresses the question of which other signaling pathways are regulated by Na(+)/K(+)-ATPase in RBC. Here it has been evidenced that PTX following its interaction with Na(+)/K(+)-ATPase pump, alters RBC morphology and this event is correlated to decreases by 30% in nitrites and nitrates levels, known as markers of plasma membrane eNOS activity. Orthovanadate (OV), an antagonist of PTX binding to Na(+)/K(+)-ATPase pump, was able to reverse the effects elicited by PTX. Finally, current investigation firstly suggests that Na(+)/K(+)-ATPase pump, following its interaction with PTX, triggers a signal transduction involved in NO metabolism regulatio
Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation
No full textThe aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine-haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO2 thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme-protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silica docking and molecular dynamics studies and by what we may call the ``caspase silence{''; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing powe
Identification and modelling of a GT-A fold in the α-dystroglycan glycosylating enzyme LARGE1
Get PDFLARGE xylosyl- and glucuronyltransferase 1 (LARGE1)is an enzyme responsible for the final steps of the post-translational modifications of dystroglycan (DG), a membrane receptor that links the cytoskeleton with the extracellular matrix in skeletal muscle and in a variety of other tissues. LARGE1 acts by adding the repeating disaccharide unit [-3Xyl-α1,3GlcAβ1-] to the extracellular portion of the DG complex (α-DG); defects in the LARGE1 gene result in an aberrant glycosylation of α-DG and consequent impairment of its binding to laminin, eventually affecting the connection between the cell and the extracellular environment. In skeletal muscle, this leads to degeneration of the muscular tissue and muscular dystrophy. So far, a few missense mutations have been identified within the LARGE1 protein and linked to congenital muscular dystrophy and, since no structural information is available on this enzyme, our understanding of the molecular mechanisms underlying these pathologies is still very limited. Here, we generated a 3D model structure of the two catalytic domains of LARGE1, combining different molecular modelling approaches. Furthermore, by using molecular dynamics simulations we analyzed the effect on the structure and stability of the first catalytic domain of the pathological missense mutation S331F that gives rise to a severe form of muscle-eye-brain disease
Identification and antitumor activity of a novel inhibitor of the NIMA-related kinase NEK6
Get PDFThe NIMA (never in mitosis, gene A)-related kinase-6 (NEK6), which is implicated in cell cycle control and plays significant roles in tumorigenesis, is an attractive target for the development of novel anti-cancer drugs. Here we describe the discovery of a potent ATP site-directed inhibitor of NEK6 identified by virtual screening, adopting both structure- and ligand-based techniques. Using a homology-built model of NEK6 as well as the pharmacophoric features of known NEK6 inhibitors we identified novel binding scaffolds. Twenty-five compounds from the top ranking hits were subjected to in vitro kinase assays. The best compound, i.e. compound 8 ((5Z)-2-hydroxy-4-methyl-6-oxo-5-[(5-phenylfuran-2-yl)methylidene]-5,6-dihydropyridine-3-carbonitrile), was able to inhibit NEK6 with low micromolar IC50 value, also displaying antiproliferative activity against a panel of human cancer cell lines. Our results suggest that the identified inhibitor can be used as lead candidate for the development of novel anti-cancer agents, thus opening the possibility of new therapeutic strategies
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
A second Ig-like domain identified in dystroglycan by molecular modelling and dynamics
No full textDystroglycan (DG) is a cell surface receptor which is composed of two subunits that interact noncovalently, namely alpha- and beta-DG. In skeletal muscle, DG is the central component of the dystrophin-glycoprotein complex (DGC) that anchors the actin cytoskeleton to the extracellular matrix. To date only the three-dimensional structure of the N-terminal region of alpha-DG has been solved by X-ray crystallography. To expand such a structural analysis, a theoretical molecular model of the murine alpha-DG C-terminal region was built based on folding recognition/threading techniques. Although there is no a significant (<30\%) sequence homology with the N-terminal region of alpha-DG, protein fold recognition methods found a significant resemblance to the alpha-DG N-terminal crystallographic structure. Our in silico structural prediction identified two subdomains in this region. Amino acid residues similar to 500-600 of alpha-DG were predicted to adopt an immunoglobulin-like (Ig-like) beta-sandwich fold. Such modeled domain includes the beta-DG binding epitope of alpha-DG and, confirming our previous experimental results, suggests that the linear epitope (residues 550-565) assumes a beta-strand conformation. The remaining segment of the alpha-DG C-terminal region (residues 601-653) is organized in a coil-helix-coil motif. A 20-ns molecular dynamics simulation in explicit water solvent provided support to the predicted Ig-like model structure. The identification of a second Ig-like domain in DG represents another important step towards a full structural and functional description of the alpha/beta DG interface. Preliminary characterization of a novel recombinant peptide (505-600) encompassing this second lg-like domain demonstrates that it is soluble and stable, further corroborating our in silico analysi
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
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