GRK2 svolge un ruolo cruciale nella regolazione dei meccanismi di recupero mitocondriale attivati dall'esposizione a radiazioni ionizzanti.

Abstract Una radiazione nell'area del mediastino puó determinare alterazioni cardiache nei pazienti sottoposti a radioterapia. Ad oggi, per questi pazienti, non si conosce ancora la dosa di radiazione necessaria minima per il cuore, in quanto una radiazione puo determinare alterazioni nella contrattilità, ed in particolare determinare danno ai singoli cardiomiciti e alle sue componenti subcellulari. Una radiazione per definizione, determia un amento della produzione di radicali liberi dell’ossigeno ROS in pochi minuti dall'evento acuto , in maniera diretta se interagisce direttamente sul DNA, che in maniera indiretta interagendo con le proteine cellulari. Una quota di questi radicali sono fisiologicamente prodotti dai mitocondri per generare ATP, che in particolare nel cardiomiocita svolgono un ruolo cardine per fornire energia sufficiente alla forza contrattile . I ROS nella cellula fungono un ruolo di secondi messageri, ed in particolare essi determinano la regolazione di moltre proteine , una di queste e GRK2. GRK2 È una proteina ubiquitaria che appare essere sensibile alle risposte dello stress cellulare attraverso l’interazione con diverse chaperonine , come HSP90. Tuttavia il ruolo di GRK2 non è ancora chiaro in risposta allo stress. Per valutare il ruolo di GRK2 sulle risposte allo stress da X –Ray nel cuore abbiamo esposto a singoli radiazioni i topi C57BL / J6 ed abbiamo eseguito una valutazione ecocardiografia transtoracica a 3 e 24 ore dopo la radiazione per valutare la funzione cardiaca. L'ecocardiografia ha mostrato che a 3 ore dopo la radiazione la camera ventricolare sinistra era dilatata e la funzione di eiezione (EF%) e le frazioni accorciamento (FS%) erano ridotte in risposta acuta allo stress rispetto a sham. A 24 ore dalla radiazione la funzionalita cardiaca era recuperata. L’ Analisi di Wester Blot sui cuori dopo l'irradiazione ha mostrato una diversa localizzazione subcellulare di GRK2 a 3 e 24 ore. Inoltre la localizzazione subcellulare di GRK2 è stata valutata anche su cellule e successivamente abbiamo valutato la morfologia mitocondriale tramite TEM e la funzionalità mitocondriale. A 3 ore da irradiazione abbiamo osservato che GRK2 è localizzata principalmente nelle membrane ed è ridotta nei mitocondri, e con una consequente riduzione della massa mitocondriale e quindi un amento di ROS, mentre le immagini di miscroscopia mostrano alterazioni mitocondriale con mitocondri vacuolizzati. Ad 8 ore dalla radiazione, abbiamo osservato che GRK2 si accumula nel mitocondriale ed il fenotipo mitocondriale e la funzione viene recuperato. Per capire se la localizzazione di GRK2 è essenziale per la risposta mitocondriale dopo irradiazione, abbiamo regolato i livelli di GRK2 tramite (Si-RNA) ed overespressione (pc-DNA-GRK2). 3 dopo dalla radiazione in cellule con overespressione di GRK2, non abbiamo valutato modifiche nella morfologia mitocondriale e nella funzione mitocondriale. Al contrario il silenziamento di GRK2 mostra una riduzione della massa mitocondrialea 3 ed 8 ore , così come la produzione di ROS mitocondriali che risultato essere aumentati a a 3 e 8 ore dopo l'esposizione. Questi dati suggeriscono che la localizzazione subcellulare di GRK2 è regolata in modo dipendente dal tempo in risposta allo stress radiazioni, ed è associata con la modifica della funzione mitocondriale e nella regolazione del “quality control” mitocondriale

Mitochondria clumping vs. mitochondria fusion in CMT2A diseases

Phenotypic variations in Charcot-Marie-Tooth disease type 2A (CMT2A) result from the many mutations in the mitochondrial fusion protein, mitofusin 2 (MFN2). While the GTPase domain mutations of MFN2 lack the ability to hydrolyze GTP and complete mitochondrial fusion, the mechanism of dysfunction in HR1 domain mutations has yet to be explored. Usin

Reciprocal regulation of mitofusin 2-mediated mitophagy and mitochondrial fusion by different PINK1 phosphorylation events

Mitochondrial repair is essential to metabolic homeostasis. Outer mitochondrial membrane mitofusin (MFN) proteins orchestrate mitochondrial fusion that opposes mitochondrial degeneration caused by senescence. Depending upon physiological context, MFN2 can either mediate mitochondrial fusion or recruit cytosolic Parkin to initiate mitophagic elimination. Because it is not clear how these events are counter-regulated we engineered and expressed MFN2 mutants that mimic phosphorylated or non-phosphorylatable MFN2 at its PINK1 phosphorylation sites: T111, S378, and S442. By interrogating mitochondrial fusion, polarization status, and Parkin binding/mitophagy as a function of inferred MFN2 phosphorylation, we discovered that individual MFN2 phosphorylation events act as a biological bar-code , directing mitochondrial fate based on phosphorylation site state. Experiments i

Assessment of ambient air quality in the port of Naples

Two experimental monitoring campaigns were carried out in 2012 to investigate the air quality in the port of Naples, the most important in southern Italy for traffic of passengers and one of the most important for goods. Therefore, it represents an important air pollution source located close to the city of Naples. The concentrations of sulfur dioxide (SO2), nitrogen dioxide (NO2), and BTEX (benzene, toluene, ethylbenzene, and xylenes) in the air were measured at 15 points inside the Naples port area through the use of passive samplers. In addition, a mobile laboratory was positioned in a fixed point inside the port area to measure continuous concentration of pollutants together with particulate matter, ambient parameters, and wind direction and intensity. The pollution levels monitored were compared with those observed in the urban area of Naples and in other Mediterranean ports. Even though the observation time was limited, measured concentrations were also compared with limit values established by European legislation. All the measured pollutants were below the limits with the exception of nitrogen dioxide: its average concentration during the exposition time exceeded the yearly limit value. A spatial analysis of data, according to the measured wind direction and intensity, provided information about the effects that ship emissions have on ambient air quality in the port area. The main evidence indicates that ship emissions influence sulfur dioxide concentration more than any other pollutants analyze

Piperine derivatives enhance fusion and axonal transport of mitochondria by activating mitofusins

Piperine (1-piperoylpiperidine) is the major pungent component of black pepper (Piper nigrum) and exhibits a spectrum of pharmacological activities. The molecular bases for many of piperine’s biological effects are incompletely defined. We noted that the chemical structure of piperine generally conforms to a pharmacophore model for small bioactive molecules that activate mitofusin (MFN)-mediated mitochondrial fusion. Piperine, but not its isomer chavicine, stimulated mitochondrial fusion in MFN-deficient cells with EC50 of ~8 nM. We synthesized piperine analogs having structural features predicted to optimize mitofusin activation and defined structure-activity relationships (SAR) in live-cell mitochondrial elongation assays. When optimal spacing was maintained between amide and aromatic groups the derivatives were potent mitofusin activators. Compared to the prototype phenylhexanamide mitofusin activator, 2, novel molecules containing the piperidine structure of piperine exhibited markedly enhanced passive membrane permeability with no loss of fusogenic potency. Lead compounds 5 and 8 enhanced mitochondrial motility in cultured murine Charcot-Marie-Tooth disease type 2A (CMT2A) neurons, but only 8 improved mitochondrial transport in sciatic nerve axons of CMT2A mice. Piperine analogs represent a new chemical class of mitofusin activators with potential pharmaceutical advantages

ST 1535: a preferential A2A adenosine receptor antagonist.

Antagonism of the A2A adenosine function has proved beneficial in the treatment of Parkinson's disease, in that it increases L-dopa therapeutical effects without concomitant worsening of its side-effects. In this paper we describe a preferential A2A adenosine antagonist, ST 1535, with long-lasting pharmacodynamic effects. It competitively antagonizes the effects of the A2A adenosine agonist NECA on cAMP in cells cloned with the human A2A adenosine receptor (IC50=353+/-30 nM), and the effects of the A1 adenosine agonist CHA on cAMP in cells cloned with the human A1 adenosine receptor (IC50=510+/-38 nM). ST 1535, at oral doses of 5 and 10 mg/kg, antagonizes catalepsy induced by intracerebroventricular administration of the A2A adenosine agonist CGS 21680 (10 microg/5 microl) in mice. At oral doses ranging between 5 and 20 mg/kg, ST 1535 induces hypermotility and antagonizes haloperidol-induced catalepsy in mice up to 7 h. Oral ST 1535, at 1.25 and 2.5 mg/kg, potentiates L-dopa effects in reducing haloperidol-induced catalepsy. ST 1535 represents a potential new compound, with long-lasting activity, for the treatment of Parkinson's disease

Synthesis and Biological Evaluation of Metabolites of 2-n-Butyl-9-methyl-8-[1,2,3]triazol-2-yl-9H-purin-6-ylamine (ST1535), A Potent Antagonist of the A2AAdenosine Receptor for the Treatment of Parkinson’s Disease

none9The synthesis and preliminary in vitro evaluation of five metabolites of the A2A antagonist ST1535 (1) are reported. The metabolites, originating in vivo from enzymatic oxidation of the 2-butyl group of the parent compound, were synthesized from 6-chloro-2-iodo-9-methyl-9H-purine (2) by selective C–C bond formation via halogen/magnesium exchange reaction and/or palladium-catalyzed reactions. The metabolites behaved in vitro as antagonist ligands of cloned human A2A receptor with affinities (Ki 7.5–53 nM) comparable to that of compound 1 (Ki 10.7 nM), thus showing that the long duration of action of 1 could be in part due to its metabolites. General behavior after oral administration in mice was also analyzed.openGiovanni Piersanti;Francesca Bartoccini;Simone Lucarini;Walter Cabri;Maria Antonietta Stasi;Teresa Riccioni;Franco Borsini;Giorgio Tarzia;Patrizia MinettiPiersanti, Giovanni; Bartoccini, Francesca; Lucarini, Simone; Walter, Cabri; Maria Antonietta, Stasi; Teresa, Riccioni; Franco, Borsini; Tarzia, Giorgio; Patrizia, Minett

In vitro fermentation patterns and methane production of sainfoin (Onobrychis viciifolia Scop.) hay with different condensed tannin contents.

Sainfoin (Onobrychis viciifolia Scop.) is a perennial legume recently reappraised for some positive charac- teristics leading to highly satisfactory animal perfor- mance. Sainfoin’s characteristics may be partly explained by the presence of moderate levels of condensed tannins (CTs) able to protect dietary protein from microbial degradation in the rumen. Decreased CH4 emissions have been reported for ruminants consuming CT-containing forage. The aim of this study was to evaluate the effects of CT content on the in vitro fermentation characteristics and kinetics and methane production of four samples of O. viciifolia cut at different phenological stages. Sainfoin hays and one sample of alfalfa hay were incubated at 39C in anaerobiosis using the in vitro gas production technique. The chemical composition, tannin content and fermentation charac- teristics and kinetics of sainfoin samples were signifi- cantly affected by phenological stage. After 48 h, the CH4 production in sainfoin hays showed a tendency to increase with the advancement of phenological stage [from 38Æ6 to 49 Æ8 mL g )1 of degraded organic matter (OM)]. The best period to cut sainfoin for hay making is between early and late flowering, when the forage combines high OM digestibility, low CH4 production and more efficient microbial fermentation

Restoring mitofusin balance prevents axonal degeneration in a Charcot-Marie-Tooth type 2A model

Mitofusin-2 (MFN2) is a mitochondrial outer-membrane protein that plays a pivotal role in mitochondrial dynamics in most tissues, yet mutations in MFN2, which cause Charcot-Marie-Tooth disease type 2A (CMT2A), primarily affect the nervous system. We generated a transgenic mouse model of CMT2A that developed severe early onset vision loss and neurological deficits, axonal degeneration without cell body loss, and cytoplasmic and axonal accumulations of fragmented mitochondria. While mitochondrial aggregates were labeled for mitophagy, mutant MFN2 did not inhibit Parkin-mediated degradation, but instead had a dominant negative effect on mitochondrial fusion only when MFN1 was at low levels, as occurs in neurons. Finally, using a transgenic approach, we found that augmenting the level of MFN1 in the nervous system in vivo rescued all phenotypes in mutant MFN2R94Q-expressing mice. These data demonstrate that the MFN1/MFN2 ratio is a key determinant of tissue specificity in CMT2A and indicate that augmentation of MFN1 in the nervous system is a viable therapeutic strategy for the disease

Liver transplantation for hepatocellular carcinoma: further considerations on selection criteria

The selection criteria in liver transplantation for HCC are a matter of debate. We reviewed our series, comparing two periods: before and after 1996, when we started to apply the Milan criteria. The study population was composed of patients with a preoperative diagnosis of HCC, confirmed by the pathological report and with a survival of > 1 year. Preoperative staging as revealed by radiological imagining was distinguished from postoperative data, including the variable of tumor volume. After 1996 tumor recurrences significantly decreased (6 out of 15 cases, 40% vs. 3 out of 48, 6.3%, P < .005) and 5-year patient survival improved (42% vs. 83%, P < .005). Not meeting the Milan criteria was significantly related to higher recurrence rate (37.5% vs. 12.7%, P < .05) and to lower 5-year patient survival (38% vs. 78%, P < .005%) in the preoperative analysis, but not in the postoperative one. The alfa-fetoprotein level of more than 30 ng/dL and the preoperative tumor volume of more than 28 cm3 predicted HCC recurrences in the univariate and mutivariate analysis (P < .005 and P < .05, respectively). The ROC curve showed a linear correlation between preoperative tumor volume and HCC recurrence. Milan criteria significantly reduced tumor recurrences after liver transplantation, improving long-term survival. In conclusion, the efficacy of tumor selection criteria must be analyzed with the use of preoperative data, to avoid bias of the postoperative evaluation. Tumor volume and alfa-fetoprotein level may improve the selection of patients. Copyright © 2004 by the American Association for the Study of Liver Diseases