Immunoglobulins Against Tyrosine-Nitrated Epitopes in Coronary Artery Disease

Background—Several lines of evidence support a pathophysiological role of immunity in atherosclerosis. Tyrosine-nitrated proteins, a footprint of oxygen- and nitrogen-derived oxidants generated by cells of the immune system, are enriched in atheromatous lesions and in circulation of patients with coronary artery disease (CAD). However, the consequences of possible immune reactions triggered by the presence of nitrated proteins in subjects with clinically documented atherosclerosis have not been explored. Methods and Results—Specific immunoglobulins that recognize 3-nitrotyrosine epitopes were identified in human lesions, as well as in circulation of patients with CAD. The levels of circulating immunoglobulins against 3-nitrotyrosine epitopes were quantified in patients with CAD (n=374) and subjects without CAD (non-CAD controls, n=313). A 10-fold increase in the mean level of circulating immunoglobulins against protein-bound 3-nitrotyrosine was documented in patients with CAD (3.75±1.8 μg antibody Eq/mL plasma versus 0.36±0.8 μg antibody Eq/mL plasma), and was strongly associated with angiographic evidence of significant CAD. Conclusions—The results of this cross-sectional study suggest that posttranslational modification of proteins via nitration within atherosclerotic plaque-laden arteries and in circulation serve as neo-epitopes for the elaboration of immunoglobulins, thereby providing an association between oxidant production and the activation of the immune system in CAD

Lysosomal iron: its role in the molecular mechanisms of DNA damage and apoptosis under condition of oxidative stress

The results of the present study show that redox active iron (iron that is able to react with Η₂Ο₂) is not homogenously distributed inside cells. In addition, it was shown that the initial site of Η₂Ο₂ reaction with redox-active iron is the lysosomal compartment resulting in lysosomal damage. This damage leads to the destabilization of lysosomal membrane with ensuing release of redox active iron to the cytosol. Delocalized lysosomal iron, in the presence of Η₂Ο₂, mediates the induction of DNA damage and apoptosis. It was also observed that over-expression of the mutated and constitutively active form of IRP1 (IRP1c437s) results in increased levels of redox-active iron and renders cells sensitive to Η₂Ο₂, whereas protection of the lysosomes eliminated the sensitivity. Moreover, Hsp70 was found to modulate the stability of lysosomal membrane and in this way most probably is involved in the regulation of intracellular redox-active iron levels and therefore affects the sensitivity of nuclear DNA towards Η₂Ο₂.Στην παρούσα μελέτη δείχθηκε ότι ο οξειδοαναγωγικά ενεργός σίδηρος, ο σίδηρος δηλαδή που είναι ικανός να αντιδρά με Η₂Ο₂, δεν είναι ομοιογενώς κατανεμημένος στο κύτταρο. Δείχθηκε ότι μετά από έκθεση των κυττάρων σε Η₂Ο₂, πρωταρχικό σημείο δράσης του μορίου αυτού είναι τα λυσοσωμάτια όπου αντιδρώντας με τον οξειδοαναγωγικά ενεργό σίδηρο που αυτά περιέχουν επάγει βλάβη στην μεμβράνη των οργανιδίων αυτών με επακόλουθη απελευθέρωση ιόντων σιδήρου στο κυτταρόπλασμα. Τα ιόντα αυτά στη συνέχεια και παρουσία Η₂Ο₂ διαμεσολαβούν την πρόκληση βλαβών στο DNA και την απόπτωση. Βρέθηκε επίσης ότι κύτταρα που υπερέκφράζαν την μεταλλαγμένη και συνεχώς ενεργοποιημένη πρωτεΐνη ρύθμισης του σιδήρου 1 (IRP1c437s) παρουσίαζαν αυξημένα επίπεδα οξειδοαναγωγικά ενεργού σιδήρου και μεγαλύτερη ευαισθησία στο Η₂Ο₂, η οποία ανεστάλη μετά από προστασία των λυσοσωματίων τους. Επιπλέον, η κυτταροπροστατετευτική δράση της πρωτεΐνης θερμικού στρες 70 (Hsp 70) έναντι του Η₂Ο₂ φαίνεται να οφείλεται στην συμβολή της πρωτεΐνης αυτής στην διατήρηση της σταθερότητας των λυσοσωματίων, η οποία πιθανά είναι απαραίτητη για τη ρύθμιση των επιπέδων του οξειδοαναγωγικά ενεργού σιδήρου

Strategies and tools to explore protein S-nitrosylation

BACKGROUND: A biochemical pathway by which nitric oxide accomplishes functional diversity is the specific modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, interactions and location. However, comprehensive studies exploring protein signaling pathways or interrelated protein clusters that are regulated by S-nitrosylation have not been performed on a global scale. SCOPE OF REVIEW: To provide insights to these important biological questions, sensitive, validated and quantitative proteomic approaches are required. This review summarizes current approaches for the global identification of S-nitrosylated proteins. MAJOR CONCLUSIONS: The application of novel methods for identifying S-nitrosylated proteins, especially when combined with mass-spectrometry based proteomics to provide site-specific identification of the modified cysteine residues, promises to deliver critical clues for the regulatory role of this dynamic posttranslational modification in cellular processes. GENERAL SIGNIFICANCE: Though several studies have established S-nitrosylation as a regulator of protein function in individual proteins, the biological chemistry and the structural elements that govern the specificity of this modification in vivo are vastly unknown. Additionally, a gap in knowledge exists concerning the potential global regulatory role(s) this modification may play in cellular physiology. By further studying S-nitrosylation at a global scale, a greater appreciation of nitric oxide and protein S-nitrosylation in cellular function can be achieved

Strategies for Correcting Very Long Chain Acyl-CoA Dehydrogenase Deficiency

Nuestra conductora, Ceida Rosell, resuelve las consultas de redes sociales, referente a temas de protección al consumidor en pasajes de aerolíneas, servicios bancarios en casos de emergencia, cumplimiento de términos

Does the calcein-AM method assay the total cellular ‘labile iron pool’ or only a fraction of it?

The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular ‘labile iron pool’ (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at <pH 5. In the present study we show that the calcein-AM method does not capture lysosomal low-mass iron and, therefore, that the method seriously underestimates total cellular labile iron