Alkaline Water and Longevity: A Murine Study

The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline water provides higher longevity in terms of \u201cdeceleration aging factor\u201d as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline water of long-lived mouse models

Citrinin mycotoxin recognition and removal by naked magnetic nanoparticles

Citrinin is a nephrotoxic mycotoxin which can be synthesized by Monascus mold during the fermentation process in foods. Monascus, generally described as red mold, is a red-pigmented filamentous fungus attracting a great interest for the production of natural dyes and cholesterol-lowering statins. We individuated a specie of Monascus producing high amount of natural dyes. However, this high pigmentation was correlated with the production of citrinin. Peculiar magnetic nanoparticles, synthesized in-house and called \u201cSurface Active Maghemite Nanoparticles\u201d (SAMNs), are proposed as an efficient and reliable mean for citrinin removal from Monascus treated foods. The nanomaterial efficiency for citrinin binding was proved on Monascus suspensions, and SAMN@citrinin complex was characterized by M\u4e7ssbauer spectroscopy and magnetization measurements, showing that SAMNs resulted structurally and magnetically well conserved after citrinin binding. SAMNs are excellent and stable magnetic nano-carrier for toxin removal, which can be applied in food industry

Ammino Ossidasi umana sensibile alla semicarbazide: dalla caratterizzazione cinetica agli studi cellulari in adipociti.

Human Semicarbazide-Sensitive Amine Oxidase (SSAO/VAP-1) is a transmembrane glycoprotein, which catalyzes the oxidative deammination of primary amines to aldehydes, with the release of the ammonium ion and hydrogen peroxide. The physiological role of SSAO/VAP-1 is not still clear: in adipocytes, SSAO/VAP-1 activity has an “insuline” like effect, while in vascular and sinusoidal hepatic endothelium, SSAO/VAP-1 is involved in the leucocyte extravasation at sites of inflammation. On these basis, the development of new specific inhibitors for SSAO/VAP-1 is an important target for the development of new anti-inflammatory drugs, while the development of new specific substrates can help to clarify the physio-pathological functions of this enzyme. To obtain information about the characteristics of a molecule to be recognized by human SSAO active site, a kinetic characterization of SSAO/VAP-1 from adipocytes was performed. Primary amines, with different size, sterical hindrance and charge distribution were used as “probe-substrates”. 1,12 diaminododecane and cis-4-chloro-butenylamine resulted the substrates with the higher catalytic efficiency among those tested. From these measurements, it was possible to estimate the value of the dielectric constant of the active site of SSAO/VAP-1, that was found to be in the range 10-20. From the effect of pH on Vmax/KM of SSAO/VAP-1 for some substrates, it was possible to highlight that a residue in its protonated form impedes the catalysis. The preliminary analysis of the 3D structures of VAP-1 shows that this residue could be Lys 393. From the effect of ionic strength and of temperature on the enzyme activity it appeared that the hydrophobic effect has an important role in the active site-substrate recognition step. Various classes of compounds (phosphonium and ammonium salts, pyridine and aniline derivatives and N-oxides) as SSAO/VAP-1 inhibitors were also tested . Although they are good inhibitors of the bovine serum enzyme and of 82% of sequence homology between the two enzymes, they resulted poor inhibitors of the human SSAO/VAP-1 (with Ki=3-4mM). From these studies, it emerges that a potential good substrate (or inhibitor) for the human SSAO/VAP-1 should be a molecule characterized by a large apolar moiety and by the presence of a positive charge at a distance higher than 10-12 ? from the reactive amino group. To obviate the use of radioactive isotopes, a discontinuos fluorimetric method for the detection of SSAO/VAP-1 activity in human plasma was set up. The method was validated and applied to the measurement of soluble SSAO/VAP-1 activity in human plasma from healthy controls and from patients with various diseases. A significant increase of soluble SSAO/VAP-1 activity was found in patients with “moderate-severe” Alzheimer disease and in patients with coronary stent implantation. In these patients in addition to SSAO/VAP-1 augment, an increase of circulating extracellular superoxide dismutase activity was also found, suggesting a condition of “oxidative stress”, probably due to the presence of the stent. In the patients with hepatic cirrhosis (induced by alcohol abuse) it was possible to highlight an increase of soluble SSAO/VAP-1 activity in the patients with “mild-moderate” hepatic cirrhosis (according to Child score), while in the patients with “severe” hepatic cirrhosis this increase of activity is lower. These results suggest that high levels of soluble SSAO/VAP-1 are the consequence of an intense inflammatory response, while the relative decrease of the SSAO/VAP-1 level in plasma in the severe state of cirrhosis, could be due to a fall down of the immune defences in these patients. To investigate a possible role of SSAO/VAP-1 activity in adipocytes, a preliminary study was carried out incubating the cells with insuline, methylamine, catalase and malvidine 3-glucoside. From this study, it appears that some proteins are differently expressed in the presence of these factors. In particular, the presence of insuline, methylamine and malvidine 3-glucoside appears to modulate the expression of SSAO/VAP-1 and of MAO, suggesting that both the enzymes are involved in a common signaling pathway, mediated by the insuline. In future the identification of the expressed proteins by Mass-Spectrometry, associated with Immunoblotting, using specific antibodies may contribute to clarify the connection between SSAO/VAP-1 and insuline pathway and will contribute to obtain new information about the role of this enzyme.L’ammino ossidasi umana sensibile alla semicarbazide (SSAO/VAP-1) è una glicoproteina transmembrana, che catalizza la deamminazione ossidativa di ammine primarie ad aldeidi, con rilascio dello ione ammonio e perossido di idrogeno. Il suo ruolo fisiologico non è ancora chiaro: in adipociti, l’attività SSAO/VAP-1 ha un effetto che mima quello dell’insulina, mentre nell’endotelio vascolare e sinusoidale epatico, SSAO/VAP-1 è coinvolta nell’extravasazione dei leucociti ai siti di infiammazione. Su queste basi, lo sviluppo di nuovi specifici inibitori per SSAO/VAP-1 è un target importante per lo sviluppo di nuovi farmaci antinfiammatori, mentre lo sviluppo di nuovi specifici substrati può contribuire a chiarire le funzioni fisio-patologiche di questo enzima. Per ottenere informazioni sulle caratteristiche che una molecola dovrebbe possedere per essere riconosciuta dal sito attivo dell’enzima umano SSAO, è stata eseguita una caratterizzazione cinetica di SSAO/VAP-1 da adipociti, utilizzando ammine primarie, caratterizzate da differente lunghezza, ingombro sterico e distribuzione di carica. L’1,12 diamminododecano e la cis-4-cloro-butenilammina sono risultati i substrati con la più alta efficienza catalitica fra quelli provati. E’ stato possibile stimare il valore di costante dielettrica del sito attivo di SSAO/VAP-1, che è compreso in un intervallo di circa 10-20. Dall’effetto del pH sul rapporto Vmax/KM della SSAO/VAP-1 per alcuni substrati, è stato possibile evidenziare che un residuo nella sua forma protonata inibisce la catalisi. Da un’analisi preliminare delle strutture 3D di VAP-1, questo residuo potrebbe essere la lisina 393. Dall’effetto della forza ionica e della temperatura sull’attività enzimatica è stato possibile dedurre che l’effetto idrofobico sembra avere un ruolo importante nella fase di riconoscimento substrato - sito attivo della SSAO/VAP-1. Sono state anche provate varie classi di composti (sali di fosfonio ed ammonio, piridina, anilina e i suoi derivati e gli N-ossidi), che sebbene siano buoni inibitori per l’enzima da siero bovino, si sono rivelati inibitori poco efficienti per l’enzima umano SSAO/VAP-1 (valori minimi di Ki=3-4 mM), anche se l’omologia di sequenza fra i due enzimi è elevata (82%). Dall’insieme di questi studi, è emerso che un buon substrato o inibitore per l’enzima umano SSAO/VAP-1 dovrebbe essere una molecola caratterizzata da una larga porzione apolare e dalla presenza di una carica positiva ad una distanza maggiore di 10-12 ? dal gruppo amminico reattivo. Per ovviare all’impiego di isotopi radioattivi, è stato messo a punto un metodo fluorimetrico discontinuo per la misura dell’attività di SSAO/VAP-1 nel plasma umano. Il metodo è stato validato e applicato alla misura dell’attività della forma solubile di SSAO/VAP-1 nel plasma umano di individui controllo e in pazienti affetti da diverse patologie. Un aumento significativo dei livelli di attività SSAO/VAP-1 plasmatica è stato trovato in pazienti con Alzheimer “moderato-grave” ed in pazienti con impianto di stent coronarico. In questi pazienti oltre a SSAO/VAP-1, è stato trovato inoltre un aumento dei livelli di attività della superossido dismutasi circolante nel plasma, suggerendo una condizione di “stress ossidativo”, probabilmente dovuta alla presenza dello stent. Nel caso dei pazienti con cirrosi epatica esotossica (indotta da abuso di alcool), è stato messo in luce un aumento dell’attività della forma solubile si SSAO/VAP-1 nei pazienti con cirrosi epatica “lieve-moderata” (classificazione Child), mentre nei pazienti con cirrosi epatica “grave”, questo aumento si riduce. I risultati ottenuti suggeriscono che elevati livelli della forma solubile di SSAO/VAP-1, sono la conseguenza di un’intensa risposta infiammatoria, mentre la relativa diminuzione del livello di SSAO/VAP-1 nello stato più grave della patologia, potrebbe essere dovuto ad un crollo delle difese immunitarie in questi pazienti. Per cercare di chiarire un possibile ruolo dell’attività di SSAO/VAP-1 negli adipociti, è stato condotto uno studio preliminare trattando le cellule con fattori quali insulina, metilammina, catalasi e malvidina 3-glucoside. L’analisi SDS-PAGE, evidenzia che alcune proteine sono espresse in maniera differente nell’incubazione degli adipociti con questi fattori. In particolare, la presenza di questi composti modula l’espressione di SSAO/VAP-1 e delle MAO, suggerendo che entrambi gli enzimi sono coinvolti in una comune via di segnale, mediata dall’insulina. In futuro l’identificazione delle proteine espresse tramite Spettrometria di Massa, associata ad analisi tramite Immunoblotting con anticorpi specifici, potrà contribuire a chiarire il collegamento tra SSAO/VAP-1 e la via di segnale mediata dall’insulina e a dare nuove informazioni sul ruolo di questo enzima

New Perspectives on Biomedical Applications of Iron Oxide Nanoparticles

Iron oxide nanomaterials are considered promising tools for improved therapeutic efficacy and diagnostic applications in biomedicine. Accordingly, engineered iron oxide nanomaterials are increasingly proposed in biomedicine, and the interdisciplinary researches involving physics, chemistry, biology (nanotechnology) and medicine have led to exciting developments in the last decades. The progresses of the development of magnetic nanoparticles with tailored physico-chemical and surface properties produced a variety of clinically relevant applications, spanning from magnetic resonance imaging (MRI), drug delivery, magnetic hyperthermia, to in vitro diagnostics. Notwithstanding the well-known conventional synthetic procedures and their wide use, recent advances in the synthetic methods open the door to new generations of naked iron oxide nanoparticles possessing peculiar surface chemistries, suitable for other competitive biomedical applications. New abilities to rationally manipulate iron oxides and their physical, chemical, and biological properties, allow the emersion of additional possibilities for designing novel nanomaterials for theranostic applications

A structure-activity study to identify novel and efficient substrates of the human semicarbazide-sensitive amine oxidase/VAP-1 enzyme

Kinetic studies were performed with various alkanamines as "substrate probes" of the properties of the active site of the human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1). We found that the enzyme-substrate recognition step is mainly controlled by apolar interactions and that a "good" substrate has a molecular structure containing a long aliphatic chain and a second positive charge at a distance greater than 12 A from the reactive amino group. In this context, we identified a novel substrate for the human SSAO/VAP-1, 1,12-diaminododecane (DIADO), which is characterised by the highest catalytic efficiency reported to date in comparison to the prototypic substrate benzylamine. Computational docking studies revealed the structural basis of this behaviour, highlighting the key role played by Lys393 in hindering substrate docking. Maximum SSAO/VAP-1 activity is reached at relatively low concentrations of DIADO (10-30 microM), and, in these conditions, it has good selectivity: it is a good substrate of SSAO/VAP-1 but not of human adipocyte monoamine oxidases or pig kidney diamine oxidase. From these findings, it appears that DIADO can be used as a new substrate for human SSAO/VAP-1 to elicit glucose transport into adipocytes, and may consequently have potential pharmacological applications in the design of anti-diabetic agents

Protein corona for proteome fingerprinting

Proteome modifications in a biological system can potentially mirror the occurrence of pathologies, even if the individuation of the proteome fingerprint correlated to a specific disease represents a very complex task. When a nanomaterial is introduced in a biological fluid, protein compete for the formation of a protein corona on the nanoparticle surface, thus, depending on the specific proteome, different patterns of proteins will form the final protein corona shell, depending on their affinity for the nanoparticle surface. Novel surface active maghemite nanoparticles (SAMNs) display a remarkable selectivity toward protein corona formation, thus they are able to concentrate proteins and peptides presenting high affinity for their surface even if present in very low amount. Among 3000 proteins present in fetal calf serum, SAMNs lead to the formation of a selfassembled corona shell with 22 selected proteins, representing minor plasma proteins, conversely bovine serum albumin (BSA), representing 80% of the total serum proteins, shows negligible absorption. Moreover, SAMNs were introduced in milk samples from healthy cows and from animals affected by mastitis, and the selectively bound protein corona shell was easily analyzed and quantified by gel electrophoresis and characterized by mass spectrometry. Upon incubation in mastitic milk, SAMNs were able to selectively bind \u3b1s2-casein fragments containing the FALPQYLK sequence, which were not present in healthy samples. The present report proposes protein competition for SAMN protein corona formation as a mean for mirroring proteome modifications, thus the selected protein shell on nanoparticles can result in a fingerprint of a specific pathology

Removal of Cr(VI) in contaminated water of Stoppani S.p.a. site (Liguria, Italy) by surface active maghemite nanoparticles

The importance of the interface between geosphere and biosphere represents one of the most fascinating frontiers of mineral science.Heavy metal release, transport and dispersion into the biosphere have a direct impact on the environment and on human health.New technologies for water and soils remediation represent a main task, and magnetic nanoparticles emerge as one of the most promising mean in this field.Environmental applications and risk assessment of manufactured nanoparticles greatly depend on the understanding of their interactions with water and soils.Novel superparamagnetic nanoparticles (Surface Active Maghemite Nanoparticles, SAMNs) constituted of stoichiometric maghemite, characterized by specific surface chemical behavior without any coating or superficial modification, stable in water for months as colloidal suspensions, were used as adsorbent of chromium(VI) in water.SAMNs can be superficially modified by simple incubation in presence of K2Cr2O7, forming a hybrid nanomaterial, SAMN@Cr(VI), stable without any release of Cr(VI) in solution.The aim of this work is to test the efficiency of SAMNs for Cr(VI) removal from Stoppani Spa site, an extreme environmental polluted Cr(VI) site.Stoppani industry located in the Cogoleto and Arenzano Area (Genova, Italy), transformed Cr(III) from chromite mineral (FeCr2O4) (Piccardo et al., 1989) to Cr(VI).It ceased the activity at the beginning of 2003, and since 2001, the Stoppani S.p.A. has been included, with DM n.468, into the national program of environmental remediation and restoration.In the 1918-1982 period, it discharged up to 1 million tons of post-treatment mud on the neighbouring beaches, groundwater resulted heavily polluted by Cr(VI). The water samples object of this work are representative of 3 pumping wells distinguished on the basis on the Cr(VI)-content in: sample W1(527< Cr(VI) < 11700 g/lt); W6 (83800 < Cr(VI) < 146000 g/lt); W9 (10500 < Cr(VI) < 232000 g/lt).Water temperature, electrical conductivity, alkalinity by acidimetric titration, pH, and Eh were determined during sampling. In the laboratory, waters have been analyzed for: Mg, and Ca by AAS, Na and K by AES Cl, SO42-, and NO3- by ion-chromatography, Si, Fe, minor and trace elements by ICP-OES.The application of SAMNs (100 mg mL-1) of three water samples removed 75-80% of Cr(VI), while, if the same treatment was accomplished at pH 3.0, Cr(VI) removal was about 95% with respect the initial concentration.Temperature, in the 4-25 \ub0C range, did not influence Cr(VI) removal by SAMNs.A second SAMN treatment on the same water samples increased Cr(VI) removal efficiency up to 98 %, leading to a final Cr(VI) concentration below the limits stated by Italian law.SAMNs represent efficient candidates for Cr(VI) removal from aqueous polluted environments