Latest PUBLICATIONS

  • The microRNA 15a/16-1 cluster down-regulates protein repair isoaspartyl methyltransferase in hepatoma cells: implications for apoptosis regulation.
    Publication Date: 23/12/2011, on The Journal of biological chemistry
    by Sambri I, Capasso R, Pucci P, Perna AF, Ingrosso D
    DOI: 10.1074/jbc.M111.290437

    Asparaginyl deamidation, a spontaneous protein post-biosynthetic modification, determines isoaspartyl formation and structure-function impairment. The isoaspartyl protein carboxyl-O-methyltransferase (PCMT1; EC 2.1.1.77) catalyzes the repair of the isopeptide bonds at isoaspartyl sites, preventing deamidation-related functional impairment. Protein deamidation affects key apoptosis mediators, such as BclxL, thus increasing susceptibility to apoptosis, whereas PCMT1 activity may effectively counteract such alterations. The aim of this work was to establish the role of RNAi as a potential mechanism for regulating PCMT1 expression and its possible implications in apoptosis. We investigated the regulatory properties of the microRNA 15a/16-1 cluster on PCMT1 expression on HepG2 cells. MicroRNA 15a or microRNA 16-1 transfection, as well as their relevant antagonists, showed that PCMT1 is effectively regulated by this microRNA cluster. The direct interaction of these two microRNAs with the seed sequence at the 3' UTR of PCMT1 transcripts was demonstrated by the luciferase assay system. The role of PCMT1 down-regulation in conditioning the susceptibility to apoptosis was investigated using various specific siRNA or shRNA approaches, to prevent non-PCMT1-specific pleiotropic effects to take place. We found that PCMT1 silencing is associated with an increase of the BclxL isoform reported to be inactivated by deamidation, thus making cells more susceptible to apoptosis induced by cisplatinum. We conclude that PCMT1 is effectively regulated by the microRNA 15a/16-1 cluster and is involved in apoptosis by preserving the structural stability and biological function of BclxL from deamidation. Control of PCMT1 expression by microRNA 15a/16-1 may thus represent a late checkpoint in apoptosis regulation.

  • Early identification of left ventricular dysfunction induced by trastuzumab.
    Publication Date: 13/12/2011, on Journal of the American College of Cardiology
    by Tocchetti CG, Coppola C, Arra C, Maurea N
    DOI: 10.1016/j.jacc.2011.08.056

  • Design, synthesis and characterisation of guanosine-based amphiphiles.
    Publication Date: 02/12/2011, on Chemistry (Weinheim an der Bergstrasse, Germany)
    by Simeone L, Milano D, De Napoli L, Irace C, Di Pascale A, Boccalon M, Tecilla P, Montesarchio D
    DOI: 10.1002/chem.201101827

    A small library of sugar-modified guanosine derivatives has been prepared, starting from a common intermediate, fully protected on the nucleobase. Insertion of myristoyl chains and of diverse hydrophilic groups, such as an oligoethylene glycol, an amino acid or a disaccharide chain, connected through in vivo reversible ester linkages, or of a charged functional group provided different examples of amphiphilic guanosine analogues, named G1-G7 herein. All of the sugar-modified derivatives were positive in the potassium picrate test, showing an ability to form G-tetrads. CD spectra demonstrated that, as dilute solutions in CHCl(3), distinctive G-quadruplex systems may be formed, with spatial organisations dependent upon the structural modifications. Two compounds, G1 and G2, proved to be good low-molecular-weight organogelators in polar organic solvents, such as methanol, ethanol and acetonitrile. Ion transportation experiments through phospholipid bilayers were carried out to evaluate their ability to mediate H(+) transportation, with G5 showing the highest activity within the investigated series. Moreover, G3 and G5 exhibited a significant cytotoxic profile against human MCF-7 cancer cells in in vitro bioassays.

  • Determination of dimethyl fumarate in desiccant and antimould sachets by reversed-phase liquid chromatography.
    Publication Date: 01/12/2011, on Biomedical chromatography : BMC
    by Gennari O, Montesano D, Salzano A, Albrizio S, Grumetto L
    DOI: 10.1002/bmc.1602

    A method for the determination of dimethyl fumarate (DMF) in desiccant and antimould sachets, employed for protecting consumer products from humidity and mould, has been developed. The method is based on a solid-liquid extraction followed by HPLC-UV analysis. The method was validated with respect to recovery, linearity, limits of detection and quantitation and precision. The recovery was 98%. The correlation coefficient value (r) was equal to 0.94. Both intra- and inter-day precisions were studied at several concentration levels, being satisfactory in all cases (RSD < 5). Limits of detection and quantification values were in the low microgram per gram level, thus allowing the determination of DMF at concentrations below the limit established (0.1 mg/kg) by the recent EU Directive (Decision 2009/251/EC). The proposed procedure was applied for the determination of the target compound in 41 desiccant and antimould samples. DMF was detected in 39.0% of samples and its content in many samples exceeded the legal limits. The results of our analysis highlight the high risk of exposure to this powerful allergic sensitizer for consumers.

  • N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells.
    Publication Date: 01/12/2011, on Molecular biology of the cell
    by Imjeti NS, Lebreton S, Paladino S, de la Fuente E, Gonzalez A, Zurzolo C
    DOI: 10.1091/mbc.E11-04-0320

    Sorting of glycosylphosphatidyl-inositol--anchored proteins (GPI-APs) in polarized epithelial cells is not fully understood. Oligomerization in the Golgi complex has emerged as the crucial event driving apical segregation of GPI-APs in two different kind of epithelial cells, Madin-Darby canine kidney (MDCK) and Fisher rat thyroid (FRT) cells, but whether the mechanism is conserved is unknown. In MDCK cells cholesterol promotes GPI-AP oligomerization, as well as apical sorting of GPI-APs. Here we show that FRT cells lack this cholesterol-driven oligomerization as apical sorting mechanism. In these cells both apical and basolateral GPI-APs display restricted diffusion in the Golgi likely due to a cholesterol-enriched membrane environment. It is striking that N-glycosylation is the critical event for oligomerization and apical sorting of GPI-APs in FRT cells but not in MDCK cells. Our data indicate that at least two mechanisms exist to determine oligomerization in the Golgi leading to apical sorting of GPI-APs. One depends on cholesterol, and the other depends on N-glycosylation and is insensitive to cholesterol addition or depletion.

  • Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease.
    Publication Date: 01/12/2011, on Biochimica et biophysica acta
    by D'Errico G, Ercole C, Lista M, Pizzo E, Falanga A, Galdiero S, Spadaccini R, Picone D
    DOI: 10.1016/j.bbamem.2011.08.009

    Binding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential.

  • Early subclinical cochlear dysfunction in myotonic dystrophy type 1.
    Publication Date: 01/12/2011, on European journal of neurology
    by Pisani V, Tirabasso A, Mazzone S, Terracciano C, Botta A, Novelli G, Bernardi G, Massa R, Di Girolamo S
    DOI: 10.1111/j.1468-1331.2011.03470.x

    Myotonic dystrophy type 1 (DM1) is an autosomal-dominant inherited disorder clinically characterized by variable systemic manifestations. Among clinical features of the disease, 'precocious presbyacusis' has been previously reported. The underlying mechanism of this auditory impairment remains still poorly understood. Hearing is an active process located in the cochlea, where the outer hair cells (OHCs) play an important role in sound perception through a 'contractile' like movement resembling skeletal muscle fibers dynamics. OHCs status has not yet been investigated in DM1 patients. OHCs integrity can be assessed by measuring transient-evoked otoacoustic emissions (TEOAE), a non-invasive, repeatable, and objective quantitative tool.

  • Comprehensive mutation analysis (20 families) of the choroideremia gene reveals a missense variant that prevents the binding of REP1 with Rab geranylgeranyl transferase.
    Publication Date: 01/12/2011, on Human mutation
    by Esposito G, De Falco F, Tinto N, Testa F, Vitagliano L, Tandurella IC, Iannone L, Rossi S, Rinaldi E, Simonelli F, Zagari A, Salvatore F
    DOI: 10.1002/humu.21591

    Choroideremia (CHM), an X-linked degeneration of the retinal pigmented epithelium (RPE), photoreceptors, and choroid, ultimately leads to blindness. It is caused by loss-of-function of the CHM gene product, the Rab escort protein 1 (REP1) that is involved, together with its homologue REP2, in prenylation of Rab GTPases, key regulators of intracellular vesicular traffic. Here, we report the molecular characterization of 20 unrelated Italian families affected by CHM. We identified 19 different mutations, nine of which are new. In most cases, we analyzed the effect of the mutations at the mRNA level. Furthermore, we demonstrated, by in vitro trancription/translation assays, that the mutated mRNAs produced truncated proteins in all cases but one. In fact, we also identified a novel REP1 missense variant (c.1520A>G; p.H507R) associated to CHM. Thus far, only two other CHM-associated missense mutations have been identified, one of which was a splicing alteration. We investigated the impact of the p.H507R amino acid change on REP1 structure and function, thus providing the first experimental demonstration that correlates a missense mutation in CHM with a functional impairment of REP1. Overall, our results indicate that the REP1-Rab geranyl-geranyl transferase interaction and consequently REP1-mediated Rab prenylation is essential for RPE and photoreceptor function.

  • Nonlinear Heart Rate Variability features for real-life stress detection. Case study: students under stress due to university examination.
    Publication Date: 07/11/2011, on Biomedical engineering online
    by Melillo P, Bracale M, Pecchia L
    DOI: 10.1186/1475-925X-10-96

    This study investigates the variations of Heart Rate Variability (HRV) due to a real-life stressor and proposes a classifier based on nonlinear features of HRV for automatic stress detection.

  • Nucleolipid nanovectors as molecular carriers for potential applications in drug delivery.
    Publication Date: 01/11/2011, on Molecular bioSystems
    by Simeone L, Mangiapia G, Irace C, Di Pascale A, Colonna A, Ortona O, De Napoli L, Montesarchio D, Paduano L
    DOI: 10.1039/c1mb05143a

    Novel thymidine- or uridine-based nucleolipids, containing one hydrophilic oligo(ethylene glycol) chain and one or two oleic acid residues (called ToThy, HoThy and DoHu), have been synthesized with the aim to develop bio-compatible nanocarriers for drug delivery and/or produce pro-drugs. Microstructural characterization of their aggregates has been determined in pure water and in pseudo-physiological conditions through DLS and SANS experiments. In all cases stable vesicles, with mean hydrodynamic radii ranging between 120 nm and 250 nm have been revealed. Biological validation of the nucleolipidic nanocarriers was ensured by evaluation of their toxicological profiles, performed by administration of the nanoaggregates to a panel of different cell lines. ToThy exhibited a weak cytotoxicity and, at high concentration, some ability to interfere with cell viability and/or proliferation. In contrast, DoHu and HoThy exhibited no toxicological relevance, behaving similarly to POPC-based liposomes, widely used for systemic drug delivery. Taken together, these results show nucleolipid-based nanocarriers as finely tunable, multi-functional self-assembling materials of interest for the in vivo transport of biomolecules or drugs.

  • The protective effect of tianeptine on Gp120-induced apoptosis in astroglial cells: role of GS and NOS, and NF-κB suppression.
    Publication Date: 01/11/2011, on British journal of pharmacology
    by Janda E, Visalli V, Colica C, Aprigliano S, Musolino V, Vadalà N, Muscoli C, Sacco I, Iannone M, Rotiroti D, Spedding M, Mollace V
    DOI: 10.1111/j.1476-5381.2010.01172.x

    Tianeptine is an antidepressant affecting the glutamatergic system. In spite of its proven clinical efficacy, molecular effects of tianeptine are not entirely clear. Tianeptine modulates cytokine expression in the CNS and protects the hippocampus from chronic stress effects. HIV infection is associated with inflammation and neuronal loss, causing HIV-associated dementia (HAD). The human immunodeficiency virus type-1 glycoprotein gp120 has been proposed as a likely aetiological agent of HAD. In this study, we determined whether tianeptine protects astroglial cells from the neurodegenerative effects of gp120.

  • Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands.
    Publication Date: 01/11/2011, on Bioorganic & medicinal chemistry
    by Peduto A, Pagano B, Petronzi C, Massa A, Esposito V, Virgilio A, Paduano F, Trapasso F, Fiorito F, Florio S, Giancola C, Galeone A, Filosa R
    DOI: 10.1016/j.bmc.2011.08.062

    A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.

  • Variably protease-sensitive prionopathy: a novel disease of the prion protein.
    Publication Date: 01/11/2011, on Journal of molecular neuroscience : MN
    by Gambetti P, Puoti G, Zou WQ
    DOI: 10.1007/s12031-011-9543-1

    Variably protease-sensitive prionopathy (VPSPr) is a novel disease involving the prion protein (PrP) that has clinical similarities with non-Alzheimer's dementias especially frontotemporal dementia, diffuse Lewis body disease, and normal pressure hydrocephalus. VPSPr can be distinguished from sporadic Creutzfeldt-Jakob disease (sCJD) especially for the characteristics of the abnormal PrP. Furthermore, although VPSPr like sCJD affects patients with the three PrP genotypes as determined by the common methionine/valine polymorphism, the allelic prevalence is very different in the two diseases. These findings suggest that VPSPr is basically different from classical prion diseases such as sCJD being perhaps more akin to other neurodegenerative dementias.

  • Evaluation of the antiproliferative activity of diterpene isonitriles from the sponge Pseudoaxinella flava in apoptosis-sensitive and apoptosis-resistant cancer cell lines.
    Publication Date: 28/10/2011, on Journal of natural products
    by Lamoral-Theys D, Fattorusso E, Mangoni A, Perinu C, Kiss R, Costantino V
    DOI: 10.1021/np2005055

    One new (1) and three known (2-4) isonitrile diterpenes, isolated from the Caribbean sponge Pseudoaxinella flava, were assayed in human cancer cell lines in vitro using an MTT colorimetric assay and quantitative videomicroscopy. Compounds 1-4 displayed activity for human PC3 prostate apoptosis-sensitive cancer cell lines. Compounds 3 and 4 demonstrated similar growth inhibitory effects for three apoptosis-sensitive and three apoptosis-resistant cancer cell lines. Quantitative videomicroscopy analysis revealed that compounds 1 and 2 exerted their activity through cytotoxic effects, and compounds 3 and 4 through cytostatic effects. These results identify marine diterpene isonitriles as potential lead compounds for anticancer drug discovery.

  • Chelation of lysosomal iron protects dopaminergic SH-SY5Y neuroblastoma cells from hydrogen peroxide toxicity by precluding autophagy and Akt dephosphorylation.
    Publication Date: 01/10/2011, on Toxicological sciences : an official journal of the Society of Toxicology
    by Castino R, Fiorentino I, Cagnin M, Giovia A, Isidoro C
    DOI: 10.1093/toxsci/kfr179

    In human neuroblastoma SH-SY5Y cells, hydrogen peroxide (H(2)O(2), 200μM) rapidly (< 5 min) induced autophagy, as shown by processing and vacuolar relocation of light chain 3(LC3). Accumulation of autophagosome peaked at 30 min of H(2)O(2) exposure. The continuous presence of H(2)O(2) eventually (at > 60 min) caused autophagy-dependent annexin V-positive cell death. However, the cells exposed to H(2)O(2) for 30 min and then cultivated in fresh medium could recover and grow, despite ongoing autophagy. H(2)O(2) rapidly (5 min) triggered the formation of dichlorofluorescein-sensitive HO(·)-free radicals within mitochondria, whereas the mitochondria-associated oxidoradicals revealed by MitoSox (O(2)(·-)) became apparent after 30 min of exposure to H(2)O(2). 3-Methyladenine inhibited autophagy and cell death, but not the generation of HO(·). Genetic silencing of beclin-1 prevented bax- and annexin V-positive cell death induced by H(2)O(2), confirming the involvement of canonical autophagy in peroxide toxicity. The lysosomotropic iron chelator deferoxamine (DFO) prevented the mitochondrial generation of both HO(.) and O(2)(·-) and suppressed the induction of autophagy and of cell death by H(2)O(2). Upon exposure to H(2)O(2), Akt was intensely phosphorylated in the first 30 min, concurrently with mammalian target of rapamycin inactivation and autophagy, and it was dephosphorylated at 2 h, when > 50% of the cells were dead. DFO did not impede Akt phosphorylation, which therefore was independent of reactive oxygen species (ROS) generation but inhibited Akt dephosphorylation. In conclusion, exogenous H(2)O(2) triggers two parallel independent pathways, one leading to autophagy and autophagy-dependent apoptosis, the other to transient Akt phosphorylation, and both are inhibited by DFO. The present work establishes HO(·) as the autophagy-inducing ROS and highlights the need for free lysosomal iron for its production within mitochondria in response to hydrogen peroxide.