1,721,119 research outputs found
Aggresome formation by anti-Ras intracellular scFv fragments. The fate of the antigen-antibody complex
Full text linkDiverting the antigen from its normal intracellular location to other compartments in an antibody-mediated way represents a mode of action for intracellular antibodies [Cardinale, A., Lener, M., Messina, S., Cattaneo, A. & Biocca, S. (1998) FEBS Lett., 439, 197-202; Lener, M., Horn, I.R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A. & Biocca, S. (2000) Eur J Biochem. 267, 1196-205]. In the case of p21Ras, the sequestration of the antigen in aggregated structures in the cytoplasm of transfected cells leads to the inhibition of its biological function. We have further investigated the intracellular fate of the antigen-antibody complex by analyzing the effect of proteasome inhibitors on the formation and the intracellular localization of the aggregates. Overexpression of anti-Ras scFv fragments or inhibition of proteasomes activity leads to the formation of large perinuclear aggresomes formed of ubiquitinated-scFv fragments in which p21Ras is sequestered and degraded in an antibody-mediated way. Disruption of microtubules by nocodazole completely abrogates the accumulation of scFv fragments in a single aggresome and induces the dispersion of these structures in the periphery of the cell. Cotransfection of the GFP-scFv with a myc-tagged ubiquitin and colocalization with specific anti-proteasome antibodies indicate the recruitment of exogenous ubiquitin and proteasomes to the newly formed aggresomes. Taken together these results suggest that the intracellular antigen-antibody complex is naturally addressed to the ubiquitin-proteasome pathway and that the mechanism of ubiquitination does not inhibit the antibody binding properties and the capacity to block the antigen function
Evidence for proteasome dysfunction in cytotoxicity mediated by anti-Ras intracellular antibodies
Full text linkAnti-Ras intracellular antibodies inhibit cell proliferation in vivo by sequestering the antigen and diverting it from its physiological location [Lener, M., Horn, I. R., Cardinale, A., Messina, S., Nielsen, U.B., Rybak, S.M., Hoogenboom, H.R., Cattaneo, A., Biocca, S. (2000) Eur. J. Biochem.267, 1196-1205]. Here we demonstrate that strongly aggregating single-chain antibody fragments (scFv), binding to Ras, induce apoptosis, and this effect is strictly related to the antibody-mediated aggregation of p21Ras. Proteasomes are quickly recruited to the newly formed aggregates, and their activity is strongly inhibited. This leads to the formation of aggresome-like structures, which become evident in the vast majority of apoptotic cells. A combination of anti-Ras scFv fragments with a nontoxic concentration of the proteasome inhibitor, lactacystin, markedly increases proteasome dysfunction and apoptosis. The dominant-negative H-ras (N17-H-ras), which is mostly soluble and does not induce aggresome formation or inhibit proteasome activity, only affects cell viability slightly. Together, these observations suggest a mechanism linking antibody-mediated Ras aggregation, impairment of the ubiquitin-proteasome system, and cytotoxicity
R–Al–Si systems (R: Pr, Nd): Experimental investigation of phase equilibria in the Al-rich corner
No full textThe effect of the two light rare earths Pr and Nd addition to the Al–Si system, in the Al-rich corner, has been experimentally studied. The constitution of the alloys and the liquidus surface projection have been determined by means of differential thermal analysis, scanning electron microscopy, electron microprobe analysis and X-ray powder diffraction. In the two systems investigated have been identified, respectively, six regions of primary crystallization [αPr3Al11, PrAl(1−x)Si(1+x), PrAl2Si2, βPrAlxSi2−x, (Al) and (Si)] for R = Pr and four [NdAl(1−x)Si(1+x), NdAl2Si2, (Al) and (Si)] for R = Nd
Can intrabodies targeted to the secretory compartment interact with a cytosolic protein? A comment on the article by Sawahata et al. "Cytoplasmic expression and specific binding of the VH/VL single domain intrabodies in transfected NIH3T3 cells",
No full textCombating protein misfolding and aggregation by intracellular antibodies
No full textConformational or misfolding diseases are a large class of devastating human disorders associated with protein misfolding and aggregation. Most conformational diseases are caused by a combination of genetic and environmental factors, suggesting that spontaneous events can destabilize the protein involved in the pathology or impair the clearance mechanisms of misfolded aggregates. Aging is one of the risk factors associated to these events, and the clinical relevance of conformational disorders is growing dramatically, as they begin to reach epidemic proportions due to increases in mean lifespan. Currently, there are no effective strategies to slow or prevent these diseases. Intrabodies are promising therapeutic agents for the treatment of misfolding diseases, because of their virtually infinite ability to specifically recognize the different conformations of a protein, including pathological isoforms, and because they can be tarqeted to the potential sites of aggregation (both intra- and extracellular sites). These molecules can work as neutralizing agents bgainst amyloidogenic proteins by preventing their aggregation, and/or as molecular shunters of intracellular traffic by rerouting the protein from its potential aggregation site. The fast-developing field of recombinant antibody technology provides intrabodies with enhanced binding specificity and stability, together with lower immunogenicity, for use in a clinical setting. This review provides an update on the applications of intrabodies in misfolding diseases, with particular emphasis on an evaluation of their multipie and feasible modes of action. © 2008 Bentham Science Publishers Ltd
The potential of intracellular antibodies for therapeutic targeting of protein-misfolding diseases
No full textMisfolding diseases are a wide group of devastating disorders characterized by the accumulation of pathological protein aggregates. Although these disorders still lack an effective treatment, new antibody-based strategies are emerging and entering clinical trials. The intrabody approach is a gene-based technology developed to neutralize or modify the function of intracellular and extracellular target antigens. Because intrabodies can potentially target all the different isoforms of a misfolding-prone protein, including pathological conformations, they are emerging as therapeutic molecules for the treatment of misfolding diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases. This review will provide a description of the intrabody approach, an update of preclinical studies on misfolding diseases and an outlook on the intrabody delivery issue for therapeutic purposes. © 2008 Elsevier Ltd. All rights reserved
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
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