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Glycoglycerolipid analogues inhibit PKC translocation to the plasma membrane and downstream signaling pathways in PMA-treated fibroblasts and human glioblastoma cells, U87MG
No full textGlycoglycerolipid analogues, derived from 2-O-β-d-galactosylglycerol, have been synthesized on the base of the structure of natural glycoglycerolipids showing anti-tumor and anti-inflammatory efficacy. These compounds have been previously demonstrated to inhibit phorbol 12-myristate-13-acetate (PMA) induced tumor promotion in mouse skin, but their mechanism of action has never been elucidated. In this work, we studied the effects of glycoglycerolipid analogues on PKC activation induced by PMA and its downstream target molecules, in human fibroblasts. Our results proved that: a) the tested compounds were able to block PKC translocation to the plasma membrane, promoted by PMA, in a dose-dependent manner (IC(50): 0.48 μM for the most active compound 2); b) the efficacy of these compounds was strongly connected to their acyl chain linked to galactose; in particular, the addition of hexanoyl and branched chains enhanced PKC inhibition, the presence of a cyclohexane ring and an excessive length of the acyl chain, or its lack, exerted a negative effect; c) the inhibition of PKC translocation blocked enzyme activation and downstream signaling pathways, MAPK and FAK, involved in proliferation and adhesion/migration control. In addition, the branched glycoglycerolipid (compound 2) was able to inhibit PKC translocation and activation in naturally highly PKC activating glioblastoma cells, U87MG. As consequence, U87MG cell proliferation and, especially, migration potential resulted to be markedly reduced (-30% and -84%, respectively). Thus, these results reveal the role of a PKC-dependent mechanism in glycoglycerolipid analogues mediated protective effects and highlight their possible employment in the field of prevention/treatment of cancer
Crystal Structure of the Human Cytosolic Sialidase Neu2 : evidence for the dynamic nature of substrate recognition
No full textGangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade -propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr334, conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu111 and the catalytic aspartate Asp46 are disordered in the apo form but upon binding of DANA become ordered to adopt two short -helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses
Extracellular sphingosine-1-phosphate acts as a survival factor for human glioblastoma stem cells
No full textSialidase Neu4L overexpression up-regulates Wnt/beta catenin signaling in SK-N-BE neiroblastoma cells, promoting the proliferation on undifferentiated neuroblasts
No full textNeuroblastoma (NB) is a frequently lethal tumor of childhood which originates from embryonic neural crest cells. The malignant and aggressive phenotype of NB is strictly related to the de-regulation of pivotal pathways governing the proliferation/differentiation status of neural crest precursor cells, such as MYCN, Delta/Notch, Wnt/β-catenin (CTNNB1) signaling. In this article, we demonstrated that sialidase Neu4L influences the differentiation/proliferation behavior of NB SK-N-BE cells, determining an hyper-activation of the Wnt/ß-catenin signaling pathway. NEU4L over-expression in SK-N-BE cells induced a significant increase of active, non-phosphorylated β-catenin content, of β-catenin/TCF transcriptional activity, and of β-catenin gene target expression, MYCN, MYC, CCND2 (cyclin D2). In turn, these molecular features deeply modified the behavior of NEU4L SK-N-BE over-expressing cells, promoting: a) an enhanced proliferation rate, mainly due to a faster transition from G1 to S cell cycle phase; b) a more undifferentiated cell phenotype similar to stem-like NB cells, possibly mediated by an increase of the expression of the pluripotency genes, MYC, NANOG, OCT-4, CD133, NES (nestin); c) the failure of NB cell differentiation after serum withdrawal. The molecular link between Neu4L and the Wnt/β-catenin signaling most likely seemed to rely on the capability of the enzyme to modify the sialylation level of cell glycoproteins. In conclusion, NB cells may deregulate Wnt/β-catenin signaling and other related oncogenic genes altering NEU4L expression and activity. These findings could provide a new potential candidate for therapeutic treatment
Glycoglycerolipid analogues effect on PKC translocation and downstream signaling pathways in PMA-treated human fibroblasts and U87MG glioblastoma cells
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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
Down regulation of membrane-bound Neu3 constitutes a new potential marker for childhood acute lymphoblastic leukemia and induces apoptosis suppression of neoplastic cells
No full textMembrane-linked sialidase Neu3 is a key enzyme for the extralysosomal catabolism of gangliosides. In this respect, it regulates pivotal cell surface events, including trans-membrane signaling, and plays an essential role in carcinogenesis. In this report, we demonstrated that acute lymphoblastic leukemia (ALL), lymphoblasts (primary cells from patients and cell lines) are characterized by a marked down-regulation of Neu3 in terms of both gene expression (-30 to 40%) and enzymatic activity toward ganglioside GD1a (-25.6 to 30.6%), when compared with cells from healthy controls. Induced overexpression of Neu3 in the ALL-cell line, MOLT-4, led to a significant increase of ceramide (+66%) and to a parallel decrease of lactosylceramide (-55%). These events strongly guided lymphoblasts to apoptosis, as we assessed by the decrease in Bcl2/Bax ratio, the accumulation of Neu3 transfected cells in the sub G0-G1 phase of the cell cycle, the enhanced annexin-V positivity, the higher cleavage of procaspase-3. Therefore, the reduced expression of Neu3 in ALL could help lymphoblasts to survive, maintaining the cellular content of ceramide below a critical level. Interestingly, we found that Neu3 activity varied in relation to disease progression, increasing in clinical remission after chemotherapy, and decreasing again in patients that relapsed. In addition, a negative correlation was observed between Neu3 expression and the percentage of the ALL marker 9-OAcGD3 positive cells. Consequently, Neu3 could represent a new potent biomarker in childhood ALL, to assess the efficacy of therapeutic protocols and to rapidly identify an eventual relapse
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
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