261 research outputs found
Detection and quantification of protein carbonyl-quenching activities by high-resolution mass spectrometry
No full textReactive carbonyl species (RCS) are highly electrophilic compounds generated in the organism upon oxidative stress. These molecules are implicated in the pathogenesis and progression of different oxidative-based disorders, such as diabetes, fibrosis and Alzheimer’s disease. Detoxification of RCS by nucleophilic compounds able to form unreactive adducts (carbonyl quenching agents) represents a promising strategy to reduce RCS concentration and to prevent their spontaneous and detrimental reaction with nucleophilic moieties of DNA, lipids and proteins1.
We analyzed and compared the quenching ability of known carbonyl quenching agents including aminoguanidine, hydralazine, pyridoxamine and carnosine. Their ability to prevent protein carbonylation was evaluated by testing different RCS such as 4-hydroxy-trans-2-nonenal, methylglyoxal, glyoxal and malondialdehyde. The quenching ability was quantified by using an innovative approach based on high-resolution mass spectrometry and on ubiquitin, as model protein2.
An approach based on mass spectrometry was applied to identify and characterize the reaction products between RCS and the nucleophilic residues of ubiquitin. Increasing amounts of carbonyl quenchers prevented the formation of protein adducts, as determined by calculating the UC50 values, that is the concentration required to inhibit ubiquitin carbonylation by 50%. Quantitative analyses showed different carbonyl quenching activities: carnosine efficiently quenched the 4-hydroxy-trans-2-nonenal, while aminoguanidine was more active on methylglyoxal and glyoxal. Hydralazine efficiently quenched all reactive carbonyl species, while pyridoxamine was particularly active on malondialdehyde. Selectivity was evaluated by testing the reactivity of the tested compounds towards pyridoxal (an endogenous aldehyde); the results indicated that only carnosine and pyridoxamine are highly selective.
The reaction products between RCS and the different carbonyl quenchers were fully characterized by high-resolution mass spectrometry, leading to the elucidation of the quenching reaction mechanisms.
We then tested the quenching ability of complex mixtures, such as natural extracts. This revealed the ability of green coffee bean extract and procyanidins from Vitis vinifera to prevent protein carbonylation, thus demonstrating that the proposed analytical strategy can be used to test the ability of pure compounds as well as of natural extracts as carbonyl quenching agents.
References
(1) Aldini, G.; Vistoli, G.; Stefek, M.; Chondrogianni, N.; Grune, T.; Sereikaite, J.; Sadowska-Bartosz, I.; Bartosz, G. Molecular Strategies to Prevent, Inhibit and Degrade Advanced Glycoxidation and Advanced Lipoxidation End Products. Free Radic. Res. 2013.
(2) Colzani, M.; Criscuolo, A.; De Maddis, D.; Garzon, D.; Yeum, K.-J.; Vistoli, G.; Carini, M.; Aldini, G. A Novel High Resolution MS Approach for the Screening of 4-Hydroxy-Trans-2-Nonenal Sequestering Agents. J. Pharm. Biomed. Anal. 2014, 91, 108–118
SPORT E INCLUSIONE: L’ESPERIENZA DEL RUGBY MIXED ABILITY NELLA PROSPETTIVA DELL’EMBODIED COGNITION
No full textlo scopo del presente contributo è quello di evidenziare le prospettive di studio e d ricerca in un campo di confine tra sport, pedagogia e neuroscienze, volte a potenziare i processi inclusivi. L’approccio dell’Embodied Cognition, in particolare, favorisce la messa in relazione delle “Subjective dimensions” (corporee ed emotivo-relazionali) con la dimensione inclusiva. Il setting dello sport Mixed Ability risulta coerente e favorevole alla realizzazione dei processi di apprendimento e partecipazione secondo il modello dell’Embodied Cognition. Viene pertanto presentato un progetto di ricerca per la comprensione e l’applicazione delle attività del Rugby Mixed Ability, secondo la prospettiva pedagogica inclusiv
Almost everywhere convergence of inverse fourier transforms
No full textWe show that if log(2 - Delta)f epsilon L-2(R-d), then the inverse Fourier transform of f converges almost everywhere. Here the partial integrals in the Fourier inversion formula come from dilates of a closed bounded neighbourhood of the origin which is star shaped with respect to 0. Our proof is based on a simple application of the Rademacher-Menshov Theorem. In the special case of spherical partial integrals, the theorem was proved by Carbery and Soria. We obtain some partial results when root(log(2 - Delta))f epsilon L-2(R-d) and log log(4 - Delta)f epsilon L-2(R-d). We also consider sequential convergence for general elements of L-2(R-d)
Tangential convergence of temperatures and harmonic functions in Besov and in Triebel-Lizorkin spaces
No full text'L'INTERPRETAZIONE GIUDIZIALE DEI TRATTATI INTERNAZIONALI'
No full textThe thesis is devoted to treaty interpretation by international judges. The subject has already been studied in depth by international law’s scholars: this work is an attempt of analysis from the philosophy of law’s perspective. In particular, the thesis focuses on three main topics: the first chapter is devoted to the concept of “international treaty”, the second chapter is devoted to the question if interpretation is an art or a science and the third chapter concerns the possibility to interpret a treaty using domestic rules of interpretation. These topics allow the author to highlight three different issues:
1) the relationship between international law system and national law systems;
2) the problems related to interpretation of multilingual texts;
3) the problems related to multicultural comunication.
One of the main topic discussed in this work, the more relevant for philosophy of law, concerns the role of article 33 of Vienna Convention on multilingual texts: differently from what international law scholars think, this article, according to the author, is the most important rule of treaty interpretation.
The reason is that the interpreter is obliged to manage/face with languages wich are different from his own native language: in this sense, from the interpretation of a treaty a lot of problems due to linguistic and cultural differences could rise
Mass spectrometric strategies for studying albumin covalent modifications induced by xenobiotics and endogenous electrophilic cytotoxic compounds
No full textProtein covalent modification is involved in the toxic mechanisms of electrophilic xenobiotics as well as of endogenous cytotoxic compounds such as reactive carbonyl species generated by oxidative stress. Protein covalent modification is irreversible and unrepairable and besides inducing a loss of protein conformation and of functional integrity, it can make the adducted proteins immunogenic and pro-inflammatory. Identification and characterization of covalent protein adducts represent an important aspect of pharmaceutical analysis, not only to predict idiosyncratic reactions but also to elucidate cytotoxic mechanism at cellular levels and to find novel biomarkers of electrophilic/oxidative stress.
Human serum albumin (HSA) is considered the main blood protein target of circulating electrophilic compounds and this is due not only to its relevant plasma concentration (≈ 0.6 mM) but also because the presence of several accessible nucleophilic sites such as Cys34, Lys199 and 525. For this reason, HSA protein adducts are searched by using different analytical techniques and among these, mass spectrometry has attained a central role because of the wealth of structural and molecular information that can be obtained. MS approach not only reveals the identity of the adducted protein but also clarifies the stoichiometry of reaction, the aminoacid site undergoing biotransformation, the reaction products and hence the mechanism of reaction. Such an approach has been applied in our laboratory to fully elucidate the HSA binding by amoxicillin (AX) and by 4-hydroxy-trans-2-nonenal (HNE) which is an endogenous electrophilic RCS generated by lipid-peroxidation.
The binding of HSA with amoxicillin was investigated since it is considered a key process for the allergic response of the drug. HSA binding by AX was firstly studied by a top-down MS approach and then the identification of the adducted HSA sites was carried out by a novel bottom-up approach based on a precursor ion approach. At the lowest AX concentration, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.
HNE is one of most abundant and toxic lipid-peroxidation derived compound, which reacts with HSA. In a previous study we fully identified the protein adduct in terms of stoichiometry, reaction kinetic and sites of adduction [1]. We now set-up a LC-ESI-MS/MS approach in order to quantitate the protein adduct. The method consists to digest the protein and quantitate the adducted peptides by using a triple quadrupole MS analyzer working in multiple reaction monitoring mode. The isotope dilution technique was used for quantitative analysis and using deuterium labelled HNE adducted peptides. The method was firstly validated and it is now applied to measure HNE-adducted albumin in the sera of patients as a novel biomarker of oxidative stress.
References
1. Aldini G et al.Albumin is the main nucleophilic target of human plasma: a protective role against pro-atherogenic electrophilic reactive carbonyl species? Chem Res Toxicol. 2008,21,824-35
Hepatitis C viraemia and antibody to core epitopes in anti-HCV ELISA negative blood donors
No full textAn inclusive numerical framework to assess the role of feedstock features on the quality of cold spray deposits
No full textCold spray technology provides unique capabilities for coating, repair, and additive manufacturing. However experimental trial and error to optimize deposit quality indexes, that rely on powder characteristics, can be costly and tedious. In this research, multiple finite element modelling approaches were compared regarding their capability and shortcomings to analyze the role of major powder features on deposit properties. Lagrangian and CEL methods were selected as the most robust approaches for analyzing the single/multi-particle impact condition, respectively. An intricate algorithm was introduced to accurately evaluate the effect of parameters including particle size, shape, oxidation extent, and impact angle on the deposition indicators such as critical velocity, particle flattening, and deposit porosity. The results exhibited a good agreement with the reported experimental data, confirming the capacity of the proposed numerical framework to tune the deposit properties as a function of a wide range of feedstock characteristics. It was concluded that for successful deposition, the effect of various powder properties and their respective effect and contribution towards plastic deformation should be taken into account. The results indicated that for a given particle velocity, smaller particle, more irregular morphology, thicker oxide layer, and smaller impact angle will result in limited particle deformation and a lower possibility of successful adhesion
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