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

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

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

Effect of halloysite nanotubes filler on polydopamine properties

Hypothesis: Polydopamine (PDA) is widely used as hydrophilic coating for several applications. However, most of the methods studied to improve or manipulate PDA properties are multistep and time-consuming, and there is a need for versatile strategies aimed at controlling and modifying the properties of PDA. Experiments: PDA-halloysite nanocomposites were produced under different oxidation conditions in alkaline and acidic media and were characterized by UV–visible and attenuated total refraction- Fourier Transform Infrared spectroscopies, thermogravimetric analysis, porosimetry, scanning electron microscopy, X-ray diffraction and contact angle measurements against the reference PDA polymer. Findings: Inclusion of the inorganic halloysite nanofiller in the PDA component was found to affect the thermal properties of the nanocomposite as well as its structure, depending on the experimental conditions. The ability of the nanocomposites to adsorb organic dyes as possible membrane coatings for environmental remediation was also investigated by different models, suggesting promising applications as adsorbents for the treatment of wastewaters

Antioxidant activity of film forming systems based on melanins from 5,6-dihydroxyindole derivatives

The development of innovative dip-coating technologies for surface functionalization has been a very active issue over the past decade following the discovery of the adhesion properties of polydopamine, a eumelanin-like material. New opportunities have derived from the discovery that hexamethylenediamine (HMDA) markedly enhances film deposition from a variety of catechol, including the key eumelanin precursor 5,6-dihydroxyindole (DHI). The remarkable antioxidant properties of synthetic eumelanins from the other main melanogenic precursor 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and its methyl ester (MeDHICA) have recently been described. In this work the film forming properties of MeDHICA melanin generated in the presence of HMDA or other diamines/monoamines and the antioxidant activity of the resulting films were investigated. Further to a systematic investigation, the most promising results were obtained running the aerobic oxidative polymerization of MeDHICA in aqueous buffers at pH 9.0 at 1 mM in the presence HMDA at 1:1.5 molar ratio. Under these conditions a dark yellow pigment is formed over 24 h exhibiting good film forming properties on different supports. HPLC analysis of the film solubilized in organic solvents indicated a mixture of oligomers of MeDHICA up to hexamers. Further polymerization of the film was obtained by exposure to ammonia vapors. The films showed high antioxidant activities in the 2,2-diphenyl-1-picrylhydrazyl and Ferric Reducing Antioxidant Power assays. Biocompatibility of MeDHICA/HMDA films was assessed on HaCat cells

Eumelanin broadband absorption develops from aggregation-modulated chromophore interactions under structural and redox control

Eumelanins, the chief photoprotective pigments in man and mammals, owe their black color to an unusual broadband absorption spectrum whose origin is still a conundrum. Excitonic effects from the interplay of geometric order and disorder in 5,6-dihydroxyindole (DHI)-based oligomeric/polymeric structures play a central role, however the contributions of structural (scaffold-controlled) and redox ( €-electron-controlled) disorder have remained uncharted. Herein, we report an integrated experimental-theoretical entry to eumelanin chromophore dynamics based on poly(vinyl alcohol)-controlled polymerization of a large set of 5,6-dihydroxyindoles and related dimers. The results a) uncover the impact of the structural scaffold on eumelanin optical properties, disproving the widespread assumption of a universal monotonic chromophore; b) delineate eumelanin chromophore buildup as a three-step dynamic process involving the rapid generation of oxidized oligomers, termed melanochromes (phase I), followed by a slow oxidant-independent band broadening (phase II) leading eventually to scattering (phase III); c) point to a slow reorganization-stabilization of melanochromes via intermolecular redox interactions as the main determinant of visible broadband absorption