2022, Articolo in rivista, ENG
Percivale G., Angelini C., Falugi C., Picco C. and Prestipino G.
In this work, the presence of calcium-dependent calcium channels and their receptors (RyR) has been investigated in Paracentrotus lividus eggs and early embryos, from unfertilized egg to four-blastomere stages. Electrophysiological recordings of RyR single-channel current fluctuations showed that RyRs are functional during the first developmental events with a maximum at zygote stage, c. 40 min after fertilization, corresponding to the first cleavage. The nature of vertebrate-like RyRs active at this stage was established by specific activation/blockade experiments.
2022, Articolo in rivista, ENG
Alloisio Susanna, Clericuzio Marco, Nobile Mario, Salis Annalisa, Damonte Gianluca, Canali Claudia, Fortuna-Perez Ana Paula, Cornara Laura and Burlando Bruno
Zornia latifoliais a plant suspected to possess psychoactive properties and marketed as a marijuana substitute under the name `maconha brava'. In this study, the effects of fractions obtained from a 2-propanol extract of aerial portions of the plant were determined by multielectrode array (MEA) analyses on cultured networks of rat cortical neurons. Lipophilic (ZL_lipo, mainly containing flavonoid aglycones), and hydrophilic (ZL_hydro, mainly containing flavonoid glycosides) fractions were initially obtained from the raw extract. ZL_lipo significantly inhibited mean firing rate (MFR) and mean bursting rate (MBR) of MEA recordings, while ZL_hydro induced no inhibition. Column chromatography separation of ZL_lipo yielded five fractions (ZL1-ZL5), among which ZL1 induced the strongest MFR and MBR inhibition. NMR and HPLC-MS analyses of ZL1 revealed the prevalence of the common flavonoids genistein (1) and apigenin (2) (in about a 1:1 ratio), and the presence of the rare flavone syzalterin (6,8-dimethylapigenin) (3) as a minor compound. Exposures of MEA to apigenin and genistein standards did not induce the MFR and MBR inhibition observed with ZL1, whereas exposure to syzalterin standard or to a 1:9 mixture syzalterin-genistein induced effects similar to ZL1. These inhibitory effects were comparable to that observed with high-THC hashish, possibly accounting for the plant psychoactive properties. Data indicate thatZ. latifolia, currently marketed as a free herbal product, should be subjected to measures of control. In addition, syzalterin showed distinctive pharmacological properties, opening the way to its possible exploitation as a neuroactive drug.
2022, Articolo in rivista, ENG
Nappi Mario, Barrese Vincenzo, Carotenuto Lidia, Lesca Gaetan, Labalme Audrey, Ville Dorothee, Smol Thomas, Rama Melanie, Dieux-Coeslier Anne, Rivier-Ringenbach Clotilde, Soldovieri Maria Virginia, Ambrosino Paolo, Mosca Ilaria, Pusch Michael, Miceli Francesco and Taglialatela Maurizio
Developmental and epileptic encephalopathies (DEEs) are neurodevelopmental diseases characterized by refractory epilepsy, distinct electroencephalographic and neuroradiological features, and various degrees of developmental delay. Mutations in KCNQ2, KCNQ3, and, more rarely, KCNQ5 genes encoding voltage-gated potassium channel subunits variably contributing to excitability control of specific neuronal populations at distinct developmental stages have been associated to DEEs. In the present work, the clinical features of two DEE patients carrying de novo KCNQ5 variants affecting the same residue in the pore region of the Kv7.5 subunit (G347S/A) are described. The in vitro functional properties of channels incorporating these variants were investigated with electrophysiological and biochemical techniques to highlight pathophysiological disease mechanisms. Currents carried by Kv7.5 G347 S/A channels displayed: 1) large (>10 times) increases in maximal current density, 2) the occurrence of a voltage-independent component, 3) slower deactivation kinetics, and 4) hyperpolarization shift in activation. All these functional features are consistent with a gain-of-function (GoF) pathogenetic mechanism. Similar functional changes were also observed when the same variants were introduced at the corresponding position in Kv7.2 subunits. Nonstationary noise analysis revealed that GoF effects observed for both Kv7.2 and Kv7.5 variants were mainly attributable to an increase in single-channel open probability, without changes in membrane abundance or single-channel conductance. The mutation-induced increase in channel opening probability was insensitive to manipulation of membrane levels of the critical Kv7 channel regulator PIP2. These results reveal a pathophysiological mechanism for KCNQ5-related DEEs, which might be exploited to implement personalized treatments.
2021, Articolo in rivista, ENG
Bosi M.; Seravalli L.; Mazzolini P.; Mezzadri F.; Fornari R.
In this paper, we focus on the growth of ?- and ?/?-Ga2O3 thin films via metal-organic vapor phase epitaxy on c-plane sapphire using water and trimethyl-gallium at temperatures between 610 and 650 °C. Using these precursors, the monoclinic ?-phase is usually obtained only at temperatures higher than 700 °C. We show here, for the first time, that both ?- and ?-Ga2O3 can also be obtained by tuning the growth rate of the film, that is, by controlling the supersaturation of the vapor phase. The experimental findings are discussed in the framework of classical nucleation theory and Ostwald's step rule, showing the interplay of thermodynamic (related to different chemical potentials for the metastable ?/? phase and the stable ? phase) and kinetic effects (mainly related to different surface energy barriers for nuclei of different crystallographic phases/ planes). The experimental conditions that permit the nucleation and growth of the desired Ga2O3 polymorph are identified and thoroughly explained, giving to this work a fundamental as well as a technological relevance.
2020, Articolo in rivista, ENG
Ridone Pietro; Pandzic Elvis; Vassalli Massimo; Cox Charles D.; Macmillan Alexander; Gottlieb Philip A.; Martinac Boris
The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-beta-cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP's response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure-response curve, increased channel latency in response to mechanical stimuli, and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM superresolution imaging revealed that, at the nanoscale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of polyunsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane composition and lateral organization of membrane cholesterol domains, which coordinate the activity of clustered PIEZO1 channels.
2020, Articolo in rivista, ENG
Gao Yimeng; Vasic, Radovan; Song Yuanbin; Teng Rhea; Liu Chengyang; Gbyli Rana; Biancon Giulia; Nelakanti Raman; Lobben Kirsten; Kudo Eriko; Liu Wei; Ardasheva Anastasia; Fu Xiaoying; Wang Xiaman; Joshi Poorval; Lee Veronica; Dura Burak; Viero Gabriella; Iwasaki Akiko; Fan Rong; Xiao Andrew; Flavell Richard A.; Li Hua-Bing; Tebaldi Toma and Halene Stephanie
N-6-methyladenosine (m(6)A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m(6)A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m(6)A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m(6)A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mays or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m(6)A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.
2016, Articolo in rivista, ENG
Fresia C, Vigliarolo T, Guida L, Booz V, Bruzzone S, Sturla L, Di Bona M, Pesce M, Usai C, De Flora A, Zocchi E
Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the a subunit of a G(i) protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation.
2013, Articolo in rivista, ENG
Lombardo, Emanuele; Sabellico, Cristian; Hajek, Jan; Stankova, Veronika; Filipsky, Tomas; Balducci, Valentina; De Vito, Paolo; Leone, Stefano; Bavavea, Eugenia I.; Silvestri, Ilaria Proietti; Righi, Giuliana; Luly, Paolo; Saso, Luciano; Bovicelli, Paolo; Pedersen, Jens Z.; Incerpi, Sandra
Natural polyphenol compounds are often good antioxidants, but they also cause damage to cells through more or less specific interactions with proteins. To distinguish antioxidant activity from cytotoxic effects we have tested four structurally related hydroxyflavones (baicalein, mosloflavone, negletein, and 5,6-dihydroxyflavone) at very low and physiologically relevant levels, using two different cell lines, L-6 myoblasts and THP-1 monocytes. Measurements using intracellular fluorescent probes and electron paramagnetic resonance spectroscopy in combination with cytotoxicity assays showed strong antioxidant activities for baicalein and 5,6-dihydroxyflavone at picomolar concentrations, while 10 nM partially protected monocytes against the strong oxidative stress induced by 200 mu M cumene hydroperoxide. Wide range dose-dependence curves were introduced to characterize and distinguish the mechanism and targets of different flavone antioxidants, and identify cytotoxic effects which only became detectable at micromolar concentrations. Analysis of these dose-dependence curves made it possible to exclude a protein-mediated antioxidant response, as well as a mechanism based on the simple stoichiometric scavenging of radicals. The results demonstrate that these flavones do not act on the same radicals as the flavonol quercetin. Considering the normal concentrations of all the endogenous antioxidants in cells, the addition of picomolar or nanomolar levels of these flavones should not be expected to produce any detectable increase in the total cellular antioxidant capacity. The significant intracellular antioxidant activity observed with 1 pM baicalein means that it must be scavenging radicals that for some reason are not eliminated by the endogenous antioxidants. The strong antioxidant effects found suggest these flavones, as well as quercetin and similar polyphenolic antioxidants, at physiologically relevant concentrations act as redox mediators to enable endogenous antioxidants to reach and scavenge different pools of otherwise inaccessible radicals.
2010, Articolo in rivista, ENG
Papi, Massimiliano and Maulucci, Giuseppe and De Spirito, Marco and Missori, Mauro and Arcovito, Giuseppe and Lancellotti, Stefano and Di Stasio, Enrico and De Cristofaro, Raimondo and Arcovito, Alessandro
Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.
2010, Articolo in rivista, ENG
Canettieri G; Di Marcotullio L; Greco A; Coni S; Antonucci L; Infante P; Pietrosanti L; De Smaele E; Ferretti E; Miele E; Pelloni M; De Simone G; Pedone EM; Gallinari P; Giorgi A; Steinkühler C; Vitagliano L; Pedone C; Schinin ME; Screpanti I; Gulino A.
Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling
2009, Articolo in rivista
Eleonora Cominelli; Chiara Tonelli
MYB proteins are transcription factors present in all eukaryotes, sharing a common DNA-binding domain that consists of one to three imperfect helix-helix-turn-helix repeats of about 50 amino acids, called R1, R2, and R3 respectively 1. In animals and yeast these proteins represent a small gene family 1. Animal R1R2R3-MYB proteins have been described for their role in cell cycle regulation mainly at the G1/S, but also at the G2/M transition, as firstly demonstrated in Drosophila 2.
2007, Articolo in rivista, ENG
Nipoti, Roberta
With the aim to set a starting point for future investigations on the relevance of the heating ramp on the annealing of ion implanted SiC, a review study is presented here. This study focuses on the heating rate of different annealing setups and presents results that highlight the relevance of the heating ramp on the morphological, structural and electrical properties of ion implanted < 0001 > 4H- and 6H-SiC. The post-implantation annealing results of hot and room temperature implanted SiC are so different that their presentation is kept distinct.
2007, Articolo in rivista
Kranjc, A; Anselmi, C; Carloni, P; Blaney, FE
Human big conductance Ca2+- and voltage-gated K+ channels (hBK) are putative drug targets for cardiovascular, respiratory and urological diseases. Here we have used molecular simulation and bioinformatics approaches to construct models of two domains important for Ca2+ binding and channel gating, namely the regulator of conductance for K+ (RCK1) domain and the so-called calcium bowl (CB). As templates for RCK1 were used the corresponding domains from a K+ channel from E. coli and the K+ channel from Methanobacterium thermoautothropicum (MthK). CB was modeled upon the structure of the human thrombospondin-1 C-terminal fragment and allowing the domain to relax in a simulated aqueous environment for 10-ns molecular dynamics simulations. The relevance of these models for interpreting the available molecular biology data is then discussed. (C) 2007 Elsevier B.V. All rights reserved.
2007, Contributo in atti di convegno, ENG
Iucolano F, Giannazzo F, Roccaforte F, Puglisi V, Grimaldi MG, Raineri V
The effects of thermal annealing either on the electrical activation of implanted species or device isolation were investigated. Silicon implantation was used for n-type doping, Magnesium for p-type doping and/or devices edge termination, while Nitrogen for devices isolation. The ions species were implanted on n-type GaN films (2E16 cm-3) at energies between 30 and 180 keV and fluences in the range 0.1 - 5E14 cm-2. After implantation, the samples were annealed in N2 at high temperatures (about 1000 C) and different ramp rates (5 - 100 C/min). Scanning Capacitance Microscopy (SCM) was used to estimate the electrical activation and/or determine the doping concentration profile in the implanted region. For n-type Si-implantation, annealing temperatures of 1200 C were necessary to achieve a significant electrical activation of the implanted specie. An active fraction of 63% was achieved combining a conventional furnace annealing at 1200 C with a rapid annealing at 1100 C. On the other hand, in the case of Mg-implantation, SCM analyses showed a compensation of the n-type dopant after rapid annealing at 1150 C, and the formation of a p-type region upon rapid annealing at 1200 C.
2006, Articolo in rivista
Gomila, G; Casuso, I; Errachid, A; Ruiz, O; Pajot, E; Minic, J; Gorojankina, T; Persuy, MA; Aioun, J; Salesse, R; Bausells, J; Villanueva, G; Rius, G; Hou, Y; Jaffrezic, N; Pennetta, C; Alfinito, E; Akimov, V; Reggiani, L; Ferrari, G; Fumagalli, L; Sampie
The animal olfactory system represents the gold standard of olfactory biosensors with its capability to identify and discriminate thousands of odorant compounds. In order to mimic the performances of natural olfactory sensors it is necessary to develop methods and techniques for the production, immobilization and electrical characterization of olfactory receptors. We review in this paper some of the advances we obtained in these fields. (c) 2006 Elsevier B.V. All rights reserved.
2006, Articolo in rivista
Macri, MA; Garreffa, G; Giove, F; Moraschi, M; Giulietti, G; Modugno, N; Colonnese, C; Maraviglia, B
Parkinson's disease is a neurological disorder associated with disfunction of dopaminergic pathways of the basal ganglia. In this study, we report the effects of decreasing plasma concentrations of the dopamine-agonist apomorphine on the size and extents of activity clusters observed with functional magnetic resonance imaging during a simple motor task. Eight patients at advanced disease stage and six healthy volunteers were studied during four consecutive sessions. We observed consistent activations in the primary sensorimotor area of the contralateral side and in the supplementary motor area of both patients and controls during the first session. During subsequent sessions, while the drug concentration gradually decreased in patients, they showed a fragmentation of the activity areas, with an overall decrease of involved volume and a decline of activity in the supplementary motor area. The appearing of activity in the ipsilateral motor area matched a partial recovery of supplementary motor area activation. During the last session, when patients showed severe dyskinesia, a widespread region of positive and negative correlations between signal and task was observed. We conclude that the lack of subcortical circuitry is partially reversible by apomorphine and that when the drug effects are reduced, there is a possible mechanism recruitment of alternate subcortical pathways. (c) 2006 Elsevier Inc. All rights reserved.
2005, Articolo in rivista, ENG
Bergamini, A and Rao, SP and Saddow, SE and Nipoti, R
Al+ implanted p(+)/n 4H-SiC diodes were realized via planar technology. The p(+)/n junctions were obtained by hot implantation at 400&DEG; C, followed by a post implantation annealing at 1600&DEG; C in Silane ambient. 136 diodes and other test structures were measured: the current voltage curves and the resistivity of the implanted layer were investigated at room temperature. The majority of the measured diodes had a turn on voltage of about 1.75 V, a forward characteristic with exponential trend and ideality factor equal to 1.2, and a very. low spread in the distribution of the reverse leakage current values at -100V. The average reverse leakage current value is (9.7 &PLUSMN; 0.4) x 10(-9) A/cm(2). The breakdown voltage of these diodes approached the theoretical value for the use epitaxial 4H-SiC layer, i.e. 0.75 - 1.0 kV. All these positive results are penalized by the high resistivity value of the implanted Al+ layer, which amounts to 11 &OHM;&BULL; cm that is one order of magnitude higher than the desired value.
1996, Articolo in rivista, ENG
Calabro, V; Strazzullo, M; LaMantia, G; Fedele, M; Paulin, C; Fusco, A; Lania, L
The p16(INK4) tumor-suppressor gene (also known as CDKN2, CDK41 and MTS1) encodes a negative regulator of the cell cycle. This gene, located in 9p21, is mutated or homozygously deleted in a high percentage of tumor cell lines and specific types of primary tumors. We have examined the status of the p16(INK4) gene in 31 thyroid tumors and 7 thyroid cell liner. No DNA abnormalities were found in primary tumors. Conversely, p16(INK4) gene structural alterations, deletions and point mutations were found in 4 thyroid cell lines. The expression of the 2 different p16(INK4) mRNAs, the p16 alpha and p16 beta transcripts, was determined by RNA-PCR experiments. All the primary thyroid tumors expressed the beta transcript, while the p16 alpha was barely detectable. The thyroid cell lines always expressed the p16 beta transcript, while the a transcript was absent or, whenever present, coded for a mutated form of the p16(INK4) gene product. Taken together, our results suggest that loss of p16(INK4) function is not directly involved in the process of thyroid-tumor development, but it probably gives cells in tissue culture a selective growth advantage.
1993, Articolo in rivista, ENG
Franco Palla, Celestino Bonurat, Letizia Anello, Caterina Casanot, Mirella Ciaccio and Giovanni Spinelli
To shed some light on the mechanisms involved in the coordinate regulation of the early histone gene set during sea urchin development, we tested the hypothesis that the upstream sequence element USE1, previously identified in the early H2A modulator, could also participate in the transcription of the early histone H3 gene. We found by DNase I protection analysis and by competition in electrophoretic mobility- shift experiments that two sequence elements of the H3 promoter closely resembled the USE1-H2A sequence in their binding activity for nuclear factors from 64-cell stage embryos. These modulator binding factor 1 (MBF-1)-related factors seem to recognize the ACAGA motif that is conserved between the USEl-like sequences of both H2A and H3 promoters. In fact, excess oligonucleotide containing a mutated USE1-H2A element in which the ACAGA sequence was mutated to AGTCA failed to compete with the USE1 sites of both H2A and H3 genes for interaction with MBF-1. Finally, in vivo transcriptional analysis in both Xenopus and sea urchin showed that an excess of USE1-H2A element efficiently competed for the activity of the H3 promoter. From these results we condude that MBF-1 is a transcription factor conserved between sea urchin and frog and that MBF-1 or related transcription factors are involved in the coordinate expression of both H2A and H3 early histone genes.