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Featured researches published by Claudio Seppi.


Mechanisms of Ageing and Development | 1991

Evidence for membrane protein oxidation during in vivo aging of human erythrocytes.

Claudio Seppi; Maria A. Castellana; Giampaolo Minetti; Giampiero Piccinini; Cesare Balduini; Augusta Brovelli

Oxidative lesions to membrane proteins were studied in human erythrocytes of different age and were evaluated on ghost membrane preparations by assaying thiol and methionine sulphoxide groups, and in situ on intact cells, after treating erythrocytes with the fluorochrome N-(7-dimethyl-amino-4-methyl-coumarinyl) maleimide (DACM). DACM reacts with thiol groups and the amount of this reagent bound by membrane proteins was quantified after SDS-PAGE separation. Results obtained show that during aging of normal cells the oxidative state of membrane proteins increases: this was better shown by the assay of methionine sulphoxide residues rather than by the thiol titration, when studies were carried out on ghost membranes. After separation of individual membrane proteins by SDS-PAGE, decreased accessibility of DACM to thiol groups of band 3 and of the main proteins of the membrane skeleton was evident in senescent erythrocytes. These results show that during aging, band 3 and membrane skeleton proteins undergo conformational changes and/or oxidation. Similar results were obtained when thiol distribution was studied in membrane proteins separated by SDS-PAGE in both reducing and non-reducing conditions.


Advances in Experimental Medicine and Biology | 1991

Conformational changes and oxidation of membrane proteins in senescent human erythrocytes.

Augusta Brovelli; Maria A. Castellana; Giampaolo Minetti; Giampiero Piccinini; Claudio Seppi; Maria R. De Renzis; Cesare Balduini

Human red cells spend 120 days in the circulation and are then removed in an age-dependent manner (1). Since cell destruction is age-dependent, studies about red cell senescence focused on the mechanisms by which the aging of the cell leads to its destruction. The presence of autoantibodies on the surface of senescent cells produced the development of the autoimmune hypothesis for senescent cell removal from the circulation (2–4), and raised questions about the presence of senescence markers on the cell surface that permit such recognition and the mechanisms of their development during red cell life span. Studies on surface changes taking place during red cell senescence have been carried out mainly on density-separated red cells (5). A reduction in membrane surface area in the dense cell population is evident as a decrease in membrane cholesterol and phospholipid content (6,7) and in acetylcholinesterase activity and sialic acid content (8). Cell deformability decreases (9–12) and at the level of the membrane slight modifications of the covalent structure of some components have been described, produced by processes like oxidation (13–15), proteolysis (16, 17), glycation (18), methylation and transamidation (19), phosphorylation (20), and modifications of phospholipid asimmetry (21) and of topology and topography of proteins have been reported or hypothesized (22–25). Most of these modifications are effective in promoting autoantibody binding and/or phagocytosis in vitro, thus supporting a possible role of these mechanisms in determining recognition and removal of senescent cells. Investigations carried out with in vivo (26,27) and in vitro models (28,29) for red cell senescence and studies with mutant erythrocytes showed that oxidation plays a relevant role in determining surface properties of senescent cells and of many pathological cells with a decreased life span (30–32). Since the oxidative state of membrane proteins in human red cells of different age has not been investigated in detail in the past, we tried to quantitate the oxidative lesion the membrane proteins undergo during red cell life-span, in an attempt to understand what kind of membrane processes expressed in senescent red cells can be related to oxidation.


PLOS ONE | 2015

γ-PGA Hydrolases of Phage Origin in Bacillus subtilis and Other Microbial Genomes.

Stefania Mamberti; Paola Prati; Paolo Cremaschi; Claudio Seppi; Carlo F. Morelli; Alessandro Galizzi; Massimo Fabbi; Cinzia Calvio

Poly-γ-glutamate (γ-PGA) is an industrially interesting polymer secreted mainly by members of the class Bacilli which forms a shield able to protect bacteria from phagocytosis and phages. Few enzymes are known to degrade γ-PGA; among them is a phage-encoded γ-PGA hydrolase, PghP. The supposed role of PghP in phages is to ensure access to the surface of bacterial cells by dismantling the γ-PGA barrier. We identified four unannotated B. subtilis genes through similarity of their encoded products to PghP; in fact these genes reside in prophage elements of B. subtilis genome. The recombinant products of two of them demonstrate efficient polymer degradation, confirming that sequence similarity reflects functional homology. Genes encoding similar γ-PGA hydrolases were identified in phages specific for the order Bacillales and in numerous microbial genomes, not only belonging to that order. The distribution of the γ-PGA biosynthesis operon was also investigated with a bioinformatics approach; it was found that the list of organisms endowed with γ-PGA biosynthetic functions is larger than expected and includes several pathogenic species. Moreover in non-Bacillales bacteria the predicted γ-PGA hydrolase genes are preferentially found in species that do not have the genetic asset for polymer production. Our findings suggest that γ-PGA hydrolase genes might have spread across microbial genomes via horizontal exchanges rather than via phage infection. We hypothesize that, in natural habitats rich in γ-PGA supplied by producer organisms, the availability of hydrolases that release glutamate oligomers from γ-PGA might be a beneficial trait under positive selection.


International Journal of Biochemistry | 1984

Modification of membrane protein organization during in vitro aging of human erythrocytes

Augusta Brovelli; Claudio Seppi; Carlo L. Balduini

In in vitro aged human erythrocytes, the presence of protein clusters can be found on the membrane; these clusters are made up of peptides held together by disulfide bridges, since they can be nearly completely dissociated by dithiothreitol treatment. SDS-polyacrylamide gel electrophoresis after dithiothreitol dissociation indicates that the aggregates are made of peptide fragments with a molecular weight ranging from 20 to approximately 110 kdalton; none of these fragments correspond to an intact protein component of the membrane. Their formation results from oxidation and proteolysis of membrane, and perhaps cytoplasmic proteins.


Advances in Experimental Medicine and Biology | 1992

Membrane Properties of Senescent and Carrier Human Erythrocytes

Maria A. Castellana; Maria R. De Renzis; Giampiero Piccinini; Giampaolo Minetti; Claudio Seppi; Cesare Balduini; Augusta Brovelli

Studies about human red cell senescence have shown that the viability and post-transfusion survival of red cells is related to the structure of their plasma membrane.1 In an attempt to analyze the survival potential in the circulation of red cells manipulated for loading with drugs or biosubstances, we addressed our investigation to the identification of new parameters useful to describe membrane characteristics of red cells with a decreased life expectancy. In this study we have analyzed membrane properties of young, middle-aged, and senescent red cells, and compared them with those of red cells manipulated for loading, in order to discern the membrane structural lesions leading to a decreased survival potential. Removal from the circulation of senescent red cells seems to be triggered by the binding of autologous antibodies2 recognizing band 3 (B3) protein3–5, α-galactosyl groups, probably belonging to glycolipids6, and other epitopes not yet defined.5 Although the role played by autoantibodies in the removal of senescent cells has not been completely elucidated, their presence on the surface of senescent erythrocytes focused on plasma membrane studies about cell aging and raised questions about the mechanisms leading to the expression on the cell surface of the senescence antigens. Among the processes modifying the structure of membrane components and described to occur during red cell senescence, oxidation seems to play an important role.7–11 Therefore we have analyzed the oxidative state of membrane proteins in young, middle-aged and senescent normal red cells and tried to relate it with the functional activity of B3 protein12, considering that the involvement of B3 in the expression of the senescence antigen has been recognized by different authors.3–5,11 The same investigation was carried out on red cells submitted to hypotonic dialysis and resealed. The aim of this investigation was to identify steps of cell loading processes producing cell suffering and decrease of the survival potential, in order to prevent or minimize the cellular damage with appropriate protocols.


TH Open | 2017

Release of Prometastatic Platelet-Derived Microparticles Induced by Breast Cancer Cells: A Novel Positive Feedback Mechanism for Metastasis

Marta Zarà; Gianni F. Guidetti; Daniela Boselli; Chiara Villa; Ilaria Canobbio; Claudio Seppi; Caterina Visconte; Jessica Canino; Mauro Torti

Circulating platelets and platelet-derived microparticles are regulators of cancer metastasis. In this study, we show that breast cancer cells induce platelet aggregation and lead to the release of platelet-derived microparticles. Although able to cause comparable aggregation, the highly aggressive MDA-MB-231 cells were more potent than the poorly aggressive MCF7 cells in inducing platelet-derived microparticles release, which was comparable to that promoted by thrombin. MDA-MB-231 cells were able to bind and internalize both MCF7- and MDA-MB-231-induced platelet-derived microparticles with comparable efficiency. By contrast, MCF7 cells did not interact with either type of platelet-derived microparticles. Upon internalization, only platelet-derived microparticles released by platelet stimulation with MDA-MB-231 cells, but not those released upon stimulation with MCF7 cells, caused activation of MDA-MB-231 cells and promoted the phosphorylation of selected signaling proteins, including p38MAPK and myosin light chain. Accordingly, MDA-MB-231-induced, but not MCF7-induced, platelet-derived microparticles dose-dependently stimulated migration and invasion of targeted MDA-MB-231 cells. These results identify a novel paracrine positive feedback mechanism initiated by aggressive breast cancer cell types to potentiate their invasive phenotype through the release of platelet-derived microparticles.


Cellular Signalling | 2018

Amyloid precursor protein is required for in vitro platelet adhesion to amyloid peptides and potentiation of thrombus formation

Caterina Visconte; Jessica Canino; Gianni F. Guidetti; Marta Zarà; Claudio Seppi; Aisha Alsheikh Abubaker; Giordano Pula; Mauro Torti; Ilaria Canobbio

Amyloid precursor protein (APP) is the precursor of amyloid β (Aβ) peptides, whose accumulation in the brain is associated with Alzheimers disease. APP is also expressed on the platelet surface and Aβ peptides are platelet agonists. The physiological role of APP is largely unknown. In neurons, APP acts as an adhesive receptor, facilitating integrin-mediated cell adhesion, while in platelets it regulates coagulation and venous thrombosis. In this work, we analyzed platelets from APP KO mice to investigate whether membrane APP supports platelet adhesion to physiological and pathological substrates. We found that APP-null platelets adhered and spread normally on collagen, von Willebrand Factor or fibrinogen. However, adhesion on immobilized Aβ peptides Aβ1-40, Aβ1-42 and Aβ25-35 was completely abolished in platelets lacking APP. By contrast, platelet activation and aggregation induced by Aβ peptides occurred normally in the absence of APP. Adhesion of APP-transfected HEK293 to Aβ peptides was significantly higher than that of control cells expressing low levels of APP. Co-coating of Aβ1-42 and Aβ25-35 with collagen strongly potentiated platelet adhesion when whole blood from wild type mice was perfused at arterial shear rate, but had no effects with blood from APP KO mice. These results demonstrate that APP selectively mediates platelet adhesion to Aβ under static condition but not platelet aggregation, and is responsible for Aβ-promoted potentiation of thrombus formation under flow. Therefore, APP may facilitate an early step in thrombus formation when Aβ peptides accumulate in cerebral vessel walls or atherosclerotic plaques.


Archive | 1992

Membrane Processes in Human Red Cell Aging

Augusta Brovelli; Maria A. Castellana; Giampaolo Minetti; Giampiero Piccinini; Claudio Seppi; M. R. DeRenzis; Cesare Balduini

During maturation, mammalian red cells lose organelles and subcellular structures; due to the absence of protein synthesis, circulating erythrocytes represent a good model for the study of how protein molecules and plasma membranes age. Human red cells spend 120 days in the circulation and are then removed in an age-dependent manner [1]. The presence of autoantibodies on the surface of senescent cells [2, 3] led to the development of the autoimmune hypothesis for senescent cell removal from the circulation and focus on plasma membrane studies about red cell aging. Since this presentation deals with membrane processes occurring during red cells senescence, the most important structural properties of plasma membrane in human red cells will be briefly summarized (Fig. 1). The most relevant ones concerning the integral domain of the membrane are the asymmetry in the distribution of phospholipids in the two halves of the bilayer and the topology and topography of integral proteins; in the peripheral domain, the supramolecular structure of the membrane skeleton and of its junctions with the integral domain are critical for the preservation of cell shape and deformability. It is interesting that the phospholipid distribution in the two halves of the bilayer, in particular phosphatidylserine, is entirely confined to the cytoplasmic side [5]; its exposure on the external surface increases cell adhesion to endothelium and macrophages [6].


Biochemical Journal | 1996

Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes.

Giampaolo Minetti; Giampiero Piccinini; Cesare Balduini; Claudio Seppi; Augusta Brovelli


Journal of Proteome Research | 2005

Proteomic Analysis of Erythrocyte Membranes by Soft Immobiline Gels Combined with Differential Protein Extraction

Maurizio Bruschi; Claudio Seppi; Simona Arena; Luca Musante; Laura Santucci; Cesare Balduini; Andrea Scaloni; Marina Lanciotti; Pier Giorgio Righetti; Giovanni Candiano

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