Bidimensional Tandem Mass Spectrometry for Selective Identification of Nitration Sites in Proteins.

Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures

The phenoxyl group-modulated interplay of cation-π and σ-type interactions in the alkali metal series.

An extensive exploration of the interaction PESs of phenol and catechol complexes with alkali metal cations reveals a striking effect of –OH substitution on the balance between cation-π and σ-type noncovalent interactions

Fattori prognostici nelle pazienti con carcinoma ovarico in stadio avanzato sottoposte a chirurgia citoriduttiva primaria e chemioterapia a base di platino e taxolo

Il carcinoma ovarico è la più comune causa di morte per neoplasia ginecologica nel mondo occidentale: circa 125 000 donne all’anno muoiono per questa patologia. Più del 50% delle pazienti raggiunge una remissione clinica completa dopo la prima linea di trattamenti, costituiti da chirurgia citoriduttiva primaria, seguita da terapia medica a base di carboplatino e taxolo. Purtroppo la maggior parte delle pazienti va incontro a recidiva e morte nell’arco di 5 anni. L’elevata frequenza di recidiva indica la necessità di individuare degli indicatori prognostici da prendere in considerazione per il successivo follow-up delle pazienti in risposta completa. Lo scopo di questa tesi è stato quello di valutare la correlazione tra le variabili clinico- patologiche e la sopravvivenza libera da progressione e la sopravvivenza globale in 247 pazienti con carcinoma ovarico in stadio avanzato, sottoposte, presso il nostro istituto, a chirurgia citoriduttiva primaria, seguita da sei cicli di chemioterapia a base di platino e taxolo. All’analisi univariata, la sopravvivenza libera da malattia e la sopravvivenza globale erano correlate significativamente al performance status (rispettivamente p = 0.04 e p = 0.00002), alla malattia residua (p = 0.00002 e p = 0.001), alla presenza di ascite (p = 0.00001 e p = 0.0003) e ai livelli di CA 125 post-chemioterapia, utilizzando come cut-off 10.8 U/ml (corrispondente al 50° percentile dei livelli dell’antigene al termine della terapia) (p = 0.0001 e p = 0.01). La sopravvivenza libera da progressione era correlata, inoltre, ai valori di CA 125 post-chemioterapia, usando come cut-off 7.1 U/ml (p = 0.02), 18.5 U/ml (p < 0.000001) (rispettivamente corrispondenti al 25° e 75° percentile dei valori dell’antigene al termine della terapia) e 35 U/ml (p = 0.0001), mentre la sopravvivenza globale allo stadio secondo FIGO (p = 0.02). L’analisi multivariata ha confermato come variabili prognostiche indipendenti per la sopravvivenza libera da progressione: la malattia residua, l’ascite e i livelli sierici di CA 125 post-chemioterapia, utilizzando come cut-off 7.1 U/ml, 10.8 U/ml, 18.5 U/ml e 35 U/ml. Il performance status, la malattia residua e i livelli sierici di CA 125 post- chemioterapia, usando come cut-off 10.8, sono risultati, invece, variabili prognostiche indipendenti per la sopravvivenza globale. Alla luce di questi risultati, appare evidente che le pazienti con malattia residua macroscopica assente dopo chirurgia citoriduttiva primaria, hanno una sopravvivenza libera da progressione e una sopravvivenza globale significativamente migliori rispetto a quelle con malattia residua persistente, anche se di dimensioni inferiori al centimetro. Ciò indica che, per offrire alle pazienti le migliori chance terapeutiche, è necessario un team chirurgico dedicato con expertise specifico, che preveda la sistematica collaborazione del ginecologo oncologo e del chirurgo addominale dedicato. È interessante notare che anche il valore del CA 125 al termine della chemioterapia è una variabile prognostica indipendente sia per la sopravvivenza libera da malattia, sia per la sopravvivenza globale. Generalmente, nelle pazienti con carcinoma ovarico in stadio avanzato, si considera un predittore biochimico di buona prognosi il raggiungimento di valori sierici di CA 125 < 35 U/ml al termine della chemioterapia. Invece, solo al raggiungimento di valori sierici dell’antigene più bassi (per esempio 10,8 U/ml, che corrisponde al 50° percentile dei livelli del CA 125 post-chemioterpia) è associato un outcome clinico significativamente migliore

N-Methyl-d-aspartate Receptor Stimulation Activates Tyrosinase and Promotes Melanin Synthesis in the Ink Gland of the Cuttlefish Sepia officinalis through the Nitric Oxide/cGMP Signal Transduction Pathway: A NOVEL POSSIBLE ROLE FOR GLUTAMATE AS PHYSIOLOGIC ACTIVATOR OF MELANOGENESIS *

Abstract The tyrosinase-catalyzed conversion of l-tyrosine to melanin represents the most distinctive biochemical pathway in the ink gland of the cuttlefishSepia officinalis; however, the molecular mechanisms underlying its activation have remained so far largely uncharted. In this paper we demonstrate for the first time thatl-glutamate can stimulate tyrosinase activity and promote melanin synthesis in Sepia ink gland via theN-methyl-d-aspartate (NMDA) receptor/NO/cGMP signal transduction pathway. Incubation of intact ink glands with either l-glutamate or NMDA resulted in an up to 18-fold increase of tyrosinase activity and a more than 6-fold elevation of cGMP levels. Comparable stimulation of tyrosinase was induced by an NO donor and by 8-bromo-cGMP. An NMDA receptor antagonist, NO synthase (NOS) inhibitors, and a guanylate cyclase blocker suppressed NMDA-induced effects. Immunohistochemical evidence indicated that enhanced cGMP production was localized largely in the mature part of the ink gland. Increased de novo synthesis of melanin was demonstrated in NMDA- and NO-stimulated ink glands by a combined microanalytical approach based on spectrophotometric determination of pigment levels and high performance liquid chromatography quantitation of pyrrole-2,3,5-tricarboxylic acid, a specific melanin marker, in melanosome-containing fractions. These results fill a longstanding gap in the understanding of the complex biochemical mechanisms underlying activation of melanogenesis in the mature ink gland cells of S. officinalis and disclose a novel physiologic role of the excitatory neurotransmitter glutamate mediated by the NMDA receptor/NO/cGMP signaling pathway

Unimolecular Variant of the Fluorescence Turn-On Oxidative Coupling of Catecholamines with Resorcinols

Reported herein is a unimolecular variant of the fluorescence turn-on oxidative coupling of catecholamines with resorcinols ("FluoResCat") based on the easily accessible conjugate 4-(2-((2,4-dihydroxybenzyl)amino)ethyl)benzene-1,2-diol (1). The process involves an alkali-activatable sequence of autoxidation and intramolecular cyclization steps with loss of carbon, leading to a fluorescent methanobenzofuroazocinone product identical to that obtained from the oxidative coupling of dopamine with resorcinol. A mechanistic route for this unexpected reaction, mimicking the synthesis of the natural fluorophore matlaline, would involve highly constrained polycyclic spiro intermediates (liquid chromatography–mass spectrometry analysis of intermediates, model reactions, and density functional theory calculations). Emission turn-on from 1 in response to oxygen, superoxide-generating systems, or gaseous ammonia/volatile amines may be of interest for sensing applications, for example, in smart packaging

Anomalous evolution of broadband optical absorption reveals dynamic solid state reorganization during eumelanin build-up in thin films

The origin of eumelanin optical properties remains a formidable conundrum preventing a detailed understanding of the complex photo-protective role of these widespread natural pigments and the rational design of innovative bioinspired materials for optoelectronic applications. Here we report the unusual kinetic and thickness-dependent evolution of the optical properties of black eumelanin polymers generated by spontaneous aerial polymerization of 5,6-dihydroxyindole (DHI) thin films (0.1-1 μm), consistent with peculiar solid state reorganization mechanisms governing broadband absorption. The complete reversal of eumelanin UV-visible transmittance spectrum curvature on passing from 0.2 to 0.5 μm thick films, the marked increase in visible extinction coefficients with increasing film thickness and the higher UV extinction coefficients in slowly vs. rapidly generated polymers concur to support distinct dynamic regimes of solid-state molecular reorganization at the nanoscale level and to do affect the development of broadband visible absorption. Solid state control of molecular reorganization disclosed herein may delineate new rational strategies for tuning optical properties in eumelanin thin films for optoelectronic applications

Identification of black sturgeon caviar pigment as eumelanin

Reported herein is the purification of the pigment of black sturgeon caviar and its unambiguous identification as a typical eumelanin by means of chemical degradation coupled with electron paramagnetic resonance (EPR) evidence. HPLC and LC-MS analysis of oxidative degradation mixtures revealed the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA), a specific marker of eumelanin pigments, in yields compatible with a 6.5% w/w pigment content. EPR spectral features and parameters were in close agreement with those reported for a typical natural eumelanin such as Sepia melanin from squid ink. The identification for the first time of eumelanin in a fish roe is expected to provide a novel molecular basis for the valorization of black caviar and production wastes thereof in food chemistry and diet

Current experience in testing mitochondrial nutrients in disorders featuring oxidative stress and mitochondrial dysfunction: rational design of chemoprevention trials

An extensive number of pathologies are associated with mitochondrial dysfunction (MDF) and oxidative stress (OS). Thus, mitochondrial cofactors termed “mitochondrial nutrients” (MN), such as α-lipoic acid (ALA), Coenzyme Q10 (CoQ10), and l-carnitine (CARN) (or its derivatives) have been tested in a number of clinical trials, and this review is focused on the use of MN-based clinical trials. The papers reporting on MN-based clinical trials were retrieved in MedLine up to July 2014, and evaluated for the following endpoints: (a) treated diseases; (b) dosages, number of enrolled patients and duration of treatment; (c) trial success for each MN or MN combinations as reported by authors. The reports satisfying the above endpoints included total numbers of trials and frequencies of randomized, controlled studies, i.e., 81 trials testing ALA, 107 reports testing CoQ10, and 74 reports testing CARN, while only 7 reports were retrieved testing double MN associations, while no report was found testing a triple MN combination. A total of 28 reports tested MN associations with “classical” antioxidants, such as antioxidant nutrients or drugs. Combinations of MN showed better outcomes than individual MN, suggesting forthcoming clinical studies. The criteria in study design and monitoring MN-based clinical trials are discussed

Antioxidant activities of hydroxylated naphthalenes: the role of aryloxyl radicals

Herein is delineated a first systematic framework for the definition of structure-antioxidant property relationships in the dihydroxynaphthalene (DHN) series. The results obtained by a combined experimental and theoretical approach revealed that 1,8-DHN is the best performing antioxidant platform, with its unique hydrogen-bonded peri-hydroxylation pattern contributing to a fast H atom transfer process. Moreover, the comparative analysis of the antioxidant properties of DHNs carried out by performing DPPH and FRAP assays and laser flash photolysis experiments, revealed the higher antioxidant power associated with an α-substitution pattern (i. e. in 1,8- and 1,6-DHN) with respect to DHNs exhibiting a β-substitution pattern (i. e. in 2,6- and 2,7-DHN). DFT calculations and isolation and characterization of the main oligomer intermediates formed during the oxidative polymerization of DHNs supported this evidence by providing unprecedented insight into the generation and fate of the intermediate naphthoxyl radicals, which emerged as the main factor governing the antioxidant activity of DHNs