on Journal of agricultural and food chemistry
by Servillo L, Giovane A, Casale R, D'Onofrio N, Ferrari G, Cautela D, Balestrieri ML, Castaldo D
Citrus genus is characterized by a specific presence of indole metabolites deriving from the N-methylation of tryptamine and its hydroxylated form, 5-hydroxytryptamine (serotonin), which are likely involved in plant defense mechanisms. In this study, we identified for the first time the occurrence in Citrus plants of serotonin 5-O-β-glucoside and all its N-methylated derivatives, that is, N-methylserotonin 5-O-β-glucoside, N,N-dimethylserotonin (bufotenine) 5-O-β-glucoside, and N,N,N-trimethylserotonin (bufotenidine) 5-O-β-glucoside. The identification of the glucosylated compounds was based on mass spectrometric studies, hydrolysis by glucosidase, and in some cases, comparison to authentic compounds. Beside leaves, the distribution of the glucosylated forms and their aglycones in some Citrus species was evaluated in flavedo, albedo, juice, and seeds. The simultaneous presence of serotonin and its N-methylated derivatives, together with the corresponding glucosylated forms, is consistent with the occurrence of a metabolic pathway, specific for Citrus, aimed at potentiating the defensive response to biotic stress through the optimization of the production and use of the most toxic of such metabolites.
by Balestrieri ML, Rizzo MR, Barbieri M, Paolisso P, D'Onofrio N, Giovane A, Siniscalchi M, Minicucci F, Sardu C, D'Andrea D, Mauro C, Ferraraccio F, Servillo L, Chirico F, Caiazzo P, Paolisso G, Marfella R
The role of sirtuin 6 (SIRT6) in atherosclerotic progression of diabetic patients is unknown. We evaluated SIRT6 expression and the effect of incretin-based therapies in carotid plaques of asymptomatic diabetic and nondiabetic patients. Plaques were obtained from 52 type 2 diabetic and 30 nondiabetic patients undergoing carotid endarterectomy. Twenty-two diabetic patients were treated with drugs that work on the incretin system, GLP-1 receptor agonists, and dipeptidyl peptidase-4 inhibitors for 26 ± 8 months before undergoing the endarterectomy. Compared with nondiabetic plaques, diabetic plaques had more inflammation and oxidative stress, along with a lesser SIRT6 expression and collagen content. Compared with non-GLP-1 therapy-treated plaques, GLP-1 therapy-treated plaques presented greater SIRT6 expression and collagen content, and less inflammation and oxidative stress, indicating a more stable plaque phenotype. These results were supported by in vitro observations on endothelial progenitor cells (EPCs) and endothelial cells (ECs). Indeed, both EPCs and ECs treated with high glucose (25 mmol/L) in the presence of GLP-1 (100 nmol/L liraglutide) presented a greater SIRT6 and lower nuclear factor-κB expression compared with cells treated only with high glucose. These findings establish the involvement of SIRT6 in the inflammatory pathways of diabetic atherosclerotic lesions and suggest its possible positive modulation by incretin, the effect of which is associated with morphological and compositional characteristics of a potential stable plaque phenotype.
on Free radical biology & medicine
by Servillo L, Castaldo D, Casale R, D'Onofrio N, Giovane A, Cautela D, Balestrieri ML
Ergothioneine (ESH), an aromatic thiol occurring in the human diet and which accumulates in particular cells, is believed to act as an antioxidant. However, its redox mechanism remains unclear and it does not seem to provide any advantage compared to other antioxidants, such as alkylthiols, which are better reducing agents and generally present in cells at higher levels. Here, we investigated by ESI-MS the products of ESH oxidation produced by neutrophils during oxidative burst and, to further elucidate ESH redox behavior, we also analyzed the oxidation products of the reaction of ESH with hypochlorite in cell-free solutions. Indeed, neutrophils are the main source of hypochlorite in humans. Furthermore, we also tested other biologically relevant oxidants, such as peroxynitrite and hydrogen peroxide. Our results indicate that treatment of human neutrophils with phorbol 12-myristate 13-acetate in the presence of ESH leads to a remarkable production of the sulfonated form (ESO3H), a compound never described before, and hercynine (EH), the desulfurated form of ESH. Similar results were obtained when ESH was subjected to cell-free oxidation in the presence of hypochlorite, as well as hydrogen peroxide or peroxynitrite. Furthermore, when the disulfide of ESH was reacted with those oxidants, we found that it was also oxidized, with production of EH and ESO3H, whose amount was dependent on the oxidant strength. These data reveal a unique ESH redox behavior, entirely different from that of alkylthiols, and suggest a mechanism, so far overlooked, through which ESH performs its antioxidant action in cells.
on Journal of cellular biochemistry
by Sirangelo I, Giovane A, Maritato R, D'Onofrio N, Iannuzzi C, Giordano A, Irace G, Balestrieri ML
W7FW14F apomyoglobin (W7FW14F ApoMb) amyloid aggregates induce cytotoxicity in SH-SY5Y human neuroblastoma cells through a mechanism not fully elucidated. Amyloid neurotoxicity process involves calcium dyshomeostasis and reactive oxygen species (ROS) production. Another key mediator of the amyloid neurotoxicity is Platelet-Activating Factor (PAF), an inflammatory phospholipid implicated in neurodegenerative diseases. Here, with the aim at evaluating the possible involvement of PAF signaling in the W7FW14F ApoMb-induced cytotoxicity, we show that the presence of CV3899, a PAF receptor (PAF-R) antagonist, prevented the detrimental effect of W7FW14F ApoMb aggregates on SH-SY5Y cell viability. Noticeably, we found that the activation of PAF signaling, following treatment with W7FW14F ApoMb, involves a decreased expression of the PAF acetylhydroase II (PAF-AH II). Interestingly, the reduced PAF-AH II expression was associated with a decreased acetylhydrolase (AH) activity and to an increased sphingosine-transacetylase activity (TA(S)) with production of N-acetylsphingosine (C2-ceramide), a well known mediator of neuronal caspase-dependent apoptosis. These findings suggest that an altered PAF catabolism takes part to the molecular events leading to W7FW14F ApoMb amyloid aggregates-induced cell death.
by Porru M, Zappavigna S, Salzano G, Luce A, Stoppacciaro A, Balestrieri ML, Artuso S, Lusa S, De Rosa G, Leonetti C, Caraglia M
Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB.
on Biochimica et biophysica acta
by D'Onofrio N, Caraglia M, Grimaldi A, Marfella R, Servillo L, Paolisso G, Balestrieri ML
The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.
on Journal of agricultural and food chemistry
by Servillo L, Giovane A, D'Onofrio N, Casale R, Cautela D, Ferrari G, Balestrieri ML, Castaldo D
The distribution of tyramine and its methylated derivatives, N-methyltyramine and N,N-dimethyltyramine, was investigated in tissue parts (leaves and fruits) of several plants of Citrus genus by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In the course of our study we discovered the occurrence of N,N,N-trimethyltyramine in all citrus plants examined. This quaternary ammonium compound, known to act in animals as a neurotoxin, was recognized and characterized by mass spectrometric analysis. The substance, never described before in the Citrus genus, is also known as candicine or maltoxin. Results indicate that N,N,N-trimethyltyramine is consistently expressed in leaves of clementine, bitter orange, and lemon. Conversely, low levels were found in the leaves of orange, mandarin, chinotto (Citrus myrtifolia), bergamot, citron, and pomelo. In the edible part of the fruits, N,N,N-trimethyltyramine was found at trace levels.
on Food chemistry
by Sessa M, Balestrieri ML, Ferrari G, Servillo L, Castaldo D, D'Onofrio N, Donsì F, Tsao R
Different O/W nanoemulsion-based delivery systems were developed in order to optimize the bioavailability of encapsulated resveratrol for potential oral administration. Blank formulations without resveratrol had no negative effect on cell viability or the cytoskeleton structure of Caco-2 cells (XTT viability assay and confocal microscopy). All nanoemulsions were then evaluated based on permeability tests on Caco-2 cells. As a result, the most efficient formulations were lecithin-based nanoemulsions which were able to transport resveratrol through cell monolayers in characteristically shorter times (1-6h) than those required for their metabolization (3-12h), allowing for better preservation of the integrity of the emulsion droplets, thus better protecting the resveratrol molecule. Fluorescence spectroscopy studies confirmed that resveratrol was encapsulated in the inner core of the nanoemulsions, which provides protection against chemical degradation. Furthermore, the developed systems also demonstrated the capability of nanoemulsions in sustained release of resveratrol from dialysis bags compared to the unencapsulated compound.
on PloS one
by Strazzullo M, Gasparrini B, Neglia G, Balestrieri ML, Francioso R, Rossetti C, Nassa G, De Filippo MR, Weisz A, Di Francesco S, Vecchio D, D'Esposito M, D'Occhio MJ, Zicarelli L, Campanile G
The transcriptome profiles were compared for buffalo embryos with normal growth and embryos with retarded growth on Day 25 after mating. Embryos with retarded growth on Day 25 after mating have a reduced likelihood of undergoing attachment to the uterine endometrium and establishing a pregnancy. Italian Mediterranean buffaloes were mated by AI and on Day 25 underwent trans-rectal ultrasonography to ascertain embryo development. Embryos with an embryonic width (EW)>2.7 mm were classed as normal embryos and embryos with an EW<2.7 mm were classed as retarded embryos. Three buffaloes with embryos of the largest EW (3.7, 3.7 and 3.9 mm) and three buffaloes with embryos of the smallest EW (1.5, 1.6 and 1.9 mm) were slaughtered on Day 27 to recover embryos for transcriptome analysis using a bovine custom designed oligo array. A total of 1,047 transcripts were differentially expressed between embryos with normal growth and embryos with retarded growth. Retarded embryos showed 773/1,047 (74%) transcripts that were down-regulated and 274/1,047 (26%) transcripts that were up-regulated relative to normal embryos; in silico analyses focused on 680/1,047 (65%) of the differentially expressed transcripts. The most altered transcripts observed in retarded embryos were associated with membrane structure and function and with metabolic and homeostasis maintenance functions. Other notable functions altered in retarded embryos were developmental processes and in particular nervous system differentiation and function. Specific biochemical pathways such as the complement cascade and coagulation were also altered in retarded embryos. It was concluded from the findings that buffalo embryos with retarded growth on Day 25 after mating show altered gene expression compared with normal embryos, and some de-regulated functions are associated with attachment to the uterine endometrium.
on PloS one
by Servillo L, Giovane A, Cautela D, Castaldo D, Balestrieri ML
N(ε)-trimethyllysine (TML) is a non-protein amino acid which takes part in the biosynthesis of carnitine. In mammals, the breakdown of endogenous proteins containing TML residues is recognized as starting point for the carnitine biosynthesis. Here, we document that one of the main sources of TML could be the vegetables which represent an important part of daily alimentation for most mammals. A HPLC-ESI-MS/MS method, which we previously developed for the analysis of N(G)-methylarginines, was utilized to quantitate TML in numerous vegetables. We report that TML, believed to be rather rare in plants as free amino acid, is, instead, ubiquitous in them and at not negligible levels. The occurrence of TML has been also confirmed in some vegetables by a HPLC method with fluorescence detection. Our results establish that TML can be introduced as free amino acid in conspicuous amounts from vegetables. The current opinion is that mammals utilize the breakdown of their endogenous proteins containing TML residues as starting point for carnitine biosynthesis. However, our finding raises the question of whether a tortuous and energy expensive route as the one of TML formation from the breakdown of endogenous proteins is really preferred when the substance is so easily available in vegetable foods. On the basis of this result, it must be taken into account that in mammals TML might be mainly introduced by diet. However, when the alimentary intake becomes insufficient, as during starvation, it might be supplied by endogenous protein breakdown.