Latest PUBLICATIONS

  • hnRNP H1 and intronic G runs in the splicing control of the human rpL3 gene.
    Publication Date: 01/05/2010, on Biochimica et biophysica acta
    by Russo A, Siciliano G, Catillo M, Giangrande C, Amoresano A, Pucci P, Pietropaolo C, Russo G
    DOI: 10.1016/j.bbagrm.2010.01.008

    By generating mRNA containing a premature termination codon (PTC), alternative splicing (AS) can quantitatively regulate the expression of genes that are degraded by nonsense-mediated mRNA decay (NMD). We previously demonstrated that AS-induced retention of part of intron 3 of rpL3 pre-mRNA produces an mRNA isoform that contains a PTC and is targeted for decay by NMD. We also demonstrated that overexpression of rpL3 downregulates canonical splicing and upregulates the alternative splicing of its pre-mRNA. We are currently investigating the molecular mechanism underlying rpL3 autoregulation. Here we report that the heterogeneous nuclear ribonucleoprotein (hnRNP) H1 is a transacting factor able to interact in vitro and in vivo with rpL3 and with intron 3 of the rpL3 gene. We investigated the role played by hnRNP H1 in the regulation of splicing of rpL3 pre-mRNA by manipulating its expression level. Depletion of hnRNP H1 reduced the level of the PTC-containing mRNA isoform, whereas its overexpression favored the selection of the cryptic 3' splice site of intron 3. We also identified and characterized the cis-acting regulatory elements involved in hnRNP H1-mediated regulation of splicing. RNA electromobility shift assay demonstrated that hnRNP H1 specifically recognizes and binds directly to the intron 3 region that contains seven copies of G-rich elements. Site-directed mutagenesis analysis and in vivo studies showed that the G3 and G6 elements are required for hnRNP H1-mediated regulation of rpL3 pre-mRNA splicing. We propose a working model in which rpL3 recruits hnRNP H1 and, through cooperation with other splicing factors, promotes selection of the alternative splice site.

  • Differential role of cathepsins B and L in autophagy-associated cell death induced by arsenic trioxide in U87 human glioblastoma cells.
    Publication Date: 01/05/2010, on Biological chemistry
    by Pucer A, Castino R, Mirković B, Falnoga I, Slejkovec Z, Isidoro C, Lah TT
    DOI: 10.1515/BC.2010.050

    Arsenic trioxide (arsenite) was the first chemotherapeutic drug to be described and is now being rediscovered in cancer treatment, including glioblastoma multiforme. Arsenite toxicity triggers autophagy in cancer cells, although final stages of the process involve executive caspases, suggesting an interplay between autophagic and apoptotic pathways that awaits to be explained at a molecular level. We evaluated the contribution of the lysosomal cathepsins (Cat) L and B, which are upregulated in glioblastomas, in the mechanism of arsenite toxicity in human glioblastoma cells. Arsenite treatment induced autophagosome formation and permeabilization of mitochondria, followed by caspase 3/7-mediated apoptosis. The autophagy inhibitor 3-methyladenine protected from arsenite toxicity, whereas bafilomycin A1 did not. Furthermore, arsenite significantly decreased CatB levels and selectively inhibited its cellular and recombinant protein activity, while not affecting CatL. However, downregulation of CatL greatly enhanced apoptosis by arsenite. Our results show that arsenite toxicity involves a complex interplay between autophagy and apoptosis in human glioblastoma cells and is associated with inhibition of CatB, and that this toxicity is highly exacerbated by simultaneous CatL inhibition. The latter points to a synergy that could be used in clinical treatment to lower the therapeutic dose, thus avoiding the toxic side effects of arsenite in glioblastoma management.

  • The co-chaperone BAG3 interacts with the cytosolic chaperonin CCT: new hints for actin folding.
    Publication Date: 01/05/2010, on The international journal of biochemistry & cell biology
    by Fontanella B, Birolo L, Infusini G, Cirulli C, Marzullo L, Pucci P, Turco MC, Tosco A
    DOI: 10.1016/j.biocel.2009.12.008

    It has been recently hypothesized that BAG3 protein, a co-chaperone of Hsp70/Hsc70, is involved in the regulation of several cell processes, such as apoptosis, autophagy and cell motility. Following the identification of Hsc70/Hsp70, further BAG3 molecular partners such as PLC-gamma and HspB8 were likewise identified, thus contributing to the characterization of the mechanisms and the biological roles carried out by this versatile protein. By using a His-tagged BAG3 protein as bait, we fished out and identified the cytosolic chaperonin CCT, a new unreported BAG3 partner. The interaction between BAG3 and CCT was confirmed and characterized by co-immunoprecipitation experiments and surface plasmon resonance techniques. Furthermore, our analyses showed a slower CCT association and a faster dissociation with a truncated form of BAG3 containing the BAG domain, thus indicating that other protein regions are essential for a high-affinity interaction. ATP or ADP does not seem to significantly influence the chaperonin binding to BAG3 protein. On the other hand, our experiments showed that BAG3 silencing by small interfering RNA slowed down cell migration and influence the availability of correctly folded monomeric actin, analyzed by DNAse I binding assays and latrunculin A depolymerization studies. To our knowledge, this is the first report showing a biologically relevant interaction between the chaperonin CCT and BAG3 protein, thus suggesting interesting involvement in the folding processes regulated by CCT.

  • Partial silencing of methyl cytosine protein binding 2 (MECP2) in mesenchymal stem cells induces senescence with an increase in damaged DNA.
    Publication Date: 01/05/2010, on FASEB journal : official publication of the Federation of American Societies for Experimental Biology
    by Squillaro T, Alessio N, Cipollaro M, Renieri A, Giordano A, Galderisi U
    DOI: 10.1096/fj.09-143057

    DNA methylation is an epigenetic modification that occurs almost exclusively on CpG dinucleotides. MECP2 is a member of a family of proteins that preferentially bind to methylated CpGs. We analyzed the contribution of MECP2 to the physiology of mesenchymal stem cells (MSCs). Partial silencing of MECP2 in human MSCs induced a significant reduction of S-phase cells, along with an increase in G(1) cells. These changes were accompanied by a reduction of apoptosis, the triggering of senescence, a decrease in telomerase activity, and the down-regulation of genes involved in maintaining stem cell properties. Senescence appeared to rely on impairment of DNA damage repair and seemed to occur through RB- and P53-related pathways. The effects of MECP2 silencing could be related to the modification of the DNA methylation status. Our results indicate that the silencing of MECP2 induces an increase in methylated cytosines in the genome. Nevertheless, MECP2 partial silencing did not change the methylation of promoters, whose expression is affected by MECP2 down-regulation.

  • Radial extracorporeal shock-wave therapy in rotator cuff calcific tendinosis.
    Publication Date: 01/05/2010, on Clinical cases in mineral and bone metabolism : the official journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases
    by Mangone G, Veliaj A, Postiglione M, Viliani T, Pasquetti P
    DOI:

    The objective of the study is to evaluate the effectiveness of Radial Extracorporeal Shock-wave Therapy (RESWT) compared with High Power LASER Therapy (HPLT) for the treatment of patients with Rotator Cuff Calcific Tendinosis (RCCT). RCCT is widely diffused, it is painful and invalidating. It is an important public health problem with social and economic implications. The most common therapeutic approach is a physiotherapic one. Both HPLT and RESWT give positive results. There is a debate on which is to be preferred. Therefore there is need to obtain scientific evidence to support either case. An observational study was carried out in the period between October 2008 and September 2009 in our outpatient clinic with 62 patients, divided into 3 groups: group A 36 patients treated only with RESWT, group B 26 patients treated only with HPLT and group C 16 patients with only short term improvement with HPLT retreated with RESWT. Patients were evaluated with Constant-Murley scale before and after treatment (immediately, 1 month and 3 months) for mean constant score, pain and range of movement. Data were examined statistically with SPSS. Criteria for inclusion and exclusion were defined. Patients treated with HPLT have shown good clinical results but have returned to original syndrome 1 month after treatment. RESWT has given improvement after treatment extended in time (3 months) in terms of pain and recover of functionality with a limited number of applications. The evidence collected indicates that RESWT is the method of choice.

  • Identification of DeltaNp63alpha protein interactions by mass spectrometry.
    Publication Date: 05/04/2010, on Journal of proteome research
    by Amoresano A, Di Costanzo A, Leo G, Di Cunto F, La Mantia G, Guerrini L, Calabrò V
    DOI: 10.1021/pr9011156

    p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (DeltaN) a transactivation domain. DeltaNp63alpha is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of DeltaNp63alpha, we set out to identify DeltaNp63alpha interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential DeltaNp63alpha binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of DeltaNp63alpha with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that DeltaNp63alpha exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of DeltaNp63alpha in mRNA metabolism and transport.

  • DNA damage and repair in a model of rat vascular injury.
    Publication Date: 01/04/2010, on Clinical science (London, England : 1979)
    by Forte A, Finicelli M, Grossi M, Vicchio M, Alessio N, Santé P, De Feo M, Cotrufo M, Berrino L, Rossi F, Galderisi U, Cipollaro M
    DOI: 10.1042/CS20090416

    Restenosis rate following vascular interventions still limits their long-term success. Oxidative stress plays a relevant role in this pathophysiological phenomenon, but less attention has been devoted to its effects on DNA damage and to the subsequent mechanisms of repair. We analysed in a model of arteriotomy-induced stenosis in rat carotids the time-dependent expression of DNA damage markers and of DNA repair genes, together with the assessment of proliferation and apoptosis indexes. The expression of the oxidative DNA damage marker 7,8-dihydro-8-oxo-2'-deoxyguanosine was increased at 3 and 7 days after arteriotomy, with immunostaining distributed in the injured vascular wall and in perivascular tissue. The expression of the DNA damage marker phospho-H2A.X was less relevant but increasing from 4 hrs to 7 days after arteriotomy, with immunostaining prevalently present in the adventitia and, to a lesser extent, in medial smooth muscle cells at the injury site. RT-PCR indicated a decrease of 8 out of 12 genes of the DNA repair machinery we selected from 4 hrs to 7 days after arteriotomy with the exception of increased Muyth and Slk genes (p<0.05). Western Blot revealed a decrease of p53 and catalase at 3 days after arteriotomy (p<0.05). A maximal 7% of BrdU-positive cells in endothelium and media occurred at 7 days after arteriotomy, while the apoptotic index peaked at 3 days after injury (p<0.05). Our results highlight a persistent DNA damage presumably related to a temporary decreased expression of the DNA repair machinery and of the antioxidant enzyme catalase, playing a role in stenosis progression.

  • Therapeutic angiogenesis in diabetic apolipoprotein E-deficient mice using bone marrow cells, functional hemangioblasts and metabolic intervention.
    Publication Date: 01/04/2010, on Atherosclerosis
    by Balestrieri ML, Lu SJ, de Nigris F, Giovane A, Williams-Ignarro S, D'Armiento FP, Feng Q, Fiorito C, Testa G, Pastore L, Cacciatore F, Mancini FP, Servillo L, De Rosa G, Pagliarulo C, Rienzo M, Minucci PB, Farzati B, Salvatore F, Rengo F, Ignarro LJ, Giordano A, Baker A, Lanza R, Napoli C
    DOI: 10.1016/j.atherosclerosis.2009.10.022

    Peripheral arterial disease (PAD) is a major health problem especially when associated to concomitant diabetes and hypercholesterolemia. Hyperglycemia with an overwhelming generation of oxygen radicals and formation of glycation end-products exacerbates oxidation-sensitive mechanisms activated by tissue ischemia. Administration of autologous bone marrow cells (BMC) is an increasing notable intervention to induce therapeutic angiogenesis, ameliorated by metabolic intervention (MT). Recently, hemangioblasts (HS) with functional properties were isolated.

  • Agonist monoclonal antibodies against HGF receptor protect cardiac muscle cells from apoptosis.
    Publication Date: 01/04/2010, on American journal of physiology. Heart and circulatory physiology
    by Pietronave S, Forte G, Locarno D, Merlin S, Zamperone A, Nicotra G, Isidoro C, Nardo PD, Prat M
    DOI: 10.1152/ajpheart.01323.2008

    Hepatocyte growth factor (HGF), a pleiotropic cytokine with mitogenic, motogenic, morphogenic, and antiapoptotic effects in various cell types, is a cardioprotective growth factor that can counteract the loss of cardiomyocytes usually observed in cardiac diseases. HGF is a quite unstable molecule in its biologically active heterodimeric form. Since all HGF-induced biological responses are mediated by its high-affinity tyrosine kinase receptor (Met/HGF-R) encoded by the Met gene, we asked whether a monoclonal antibody (MAb) that displays receptor full agonist activity could protect cardiac muscle cell lines from hydrogen peroxide-induced apoptosis. We report that the MAb efficiently inhibited hydrogen peroxide-induced cell shrinkage, DNA fragmentation, annexin V positivity, mitochondrial translocation of bax, and caspase activation. The MAb was thus able to counteract apoptosis evaluated by both morphological and biochemical criteria. The agonist activity of the MAb was mediated by Met/HGF-R, since a Met/HGF-R-specific short hairpin RNA (shRNA) inhibited both activation of transduction pathways and motility triggered by MAb DO-24. The protective antiapoptotic effect of MAb DO-24 was dependent on activation of the ras-MAPK Erk1/2 and phosphatidylinositol 3-kinase (PI3-kinase)-Akt transduction pathways, since it was abrogated by treatments with their specific pharmacological inhibitors, PD-98059 and wortmannin. Moreover, the MAb induced a motogenic, but not mitogenic, response in these cells, mimicking in all aspects the natural ligand HGF but displaying a significant higher stability than HGF in culture. This MAb may thus be a valuable substitute for HGF, being more easily available in a biologically active, highly stable, and purified form.

  • Atorvastatin combined to interferon to verify the efficacy (ACTIVE) in relapsing-remitting active multiple sclerosis patients: a longitudinal controlled trial of combination therapy.
    Publication Date: 01/04/2010, on Multiple sclerosis (Houndmills, Basingstoke, England)
    by Lanzillo R, Orefice G, Quarantelli M, Rinaldi C, Prinster A, Ventrella G, Spitaleri D, Lus G, Vacca G, Carotenuto B, Salvatore E, Brunetti A, Tedeschi G, Brescia Morra V
    DOI: 10.1177/1352458509358909

    A large body of evidence suggests that, besides their cholesterol-lowering effect, statins exert anti-inflammatory action. Consequently, statins may have therapeutic potential in immune-mediated disorders such as multiple sclerosis. Our objectives were to determine safety, tolerability and efficacy of low-dose atorvastatin plus high-dose interferon beta-1a in multiple sclerosis patients responding poorly to interferon beta-1a alone. Relapsing-remitting multiple sclerosis patients, aged 18-50 years, with contrast-enhanced lesions or relapses while on therapy with interferon beta-1a 44 microg (three times weekly) for 12 months, were randomized to combination therapy (interferon + atorvastatin 20 mg per day; group A) or interferon alone (group B) for 24 months. Patients underwent blood analysis and clinical assessment with the Expanded Disability Status Scale every 3 months, and brain gadolinium-enhanced magnetic resonance imaging at screening, and 12 and 24 months thereafter. Primary outcome measure was contrast-enhanced lesion number. Secondary outcome measures were number of relapses, EDSS variation and safety laboratory data. Forty-five patients were randomized to group A (n = 21) or B (n = 24). At 24 months, group A had significantly fewer contrast-enhanced lesions versus baseline (p = 0.007) and significantly fewer relapses versus the two pre-randomization years (p < 0.001). At survival analysis, the risk for a 1-point EDSS increase was slightly higher in group B than in group A (p = 0.053). Low-dose atorvastatin may be beneficial, as add-on therapy, in poor responders to high-dose interferon beta-1a alone.

  • Non-Alcoholic Wernicke Encephalopathy: MR Imaging and Review of the Literature.
    Publication Date: 01/04/2010, on The neuroradiology journal
    by Saturnino PP, Conforti R, Amoroso V, D'Agostino V, Ferrara M, Cirillo S
    DOI: 10.1177/197140091002300202

    We describe a patient with non-alcoholic Wernicke's encephalopathy caused by long-term parenteral nutrition. The diagnosis is based on clinical and magnetic resonance findings. We also reviewed the literature review in typical and atypical findings at MR examination.

  • A preamplification approach to GMO detection in processed foods.
    Publication Date: 01/03/2010, on Analytical and bioanalytical chemistry
    by Del Gaudio S, Cirillo A, Di Bernardo G, Galderisi U, Cipollaro M
    DOI: 10.1007/s00216-009-3199-5

    DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.

  • Neuropsychologic assessment and cognitive rehabilitation in a patient with locked-in syndrome and left neglect.
    Publication Date: 01/03/2010, on Archives of physical medicine and rehabilitation
    by Trojano L, Moretta P, Estraneo A, Santoro L
    DOI: 10.1016/j.apmr.2009.10.033

    We describe a patient affected by severe incomplete locked-in syndrome (LIS) and left neglect caused by a combination of vascular lesions. Our patient's neglect prevented the use of augmentative communication devices based on a computerized eye-tracker system. For this reason, we adapted a visual scanning training for neglect rehabilitation. At the end of the rehabilitative training, the patient had regained full exploration of the monitor and could use the eye-tracker system for communicative purposes. This case report shows that specific rehabilitative approaches can be devised in severely disabled LIS patients with additional brain lesions and specific cognitive defects.

  • Structural characterization of the transmembrane proximal region of the hepatitis C virus E1 glycoprotein.
    Publication Date: 01/03/2010, on Biochimica et biophysica acta
    by Spadaccini R, D'Errico G, D'Alessio V, Notomista E, Bianchi A, Merola M, Picone D
    DOI: 10.1016/j.bbamem.2009.10.018

    A detailed knowledge of the mechanism of virus entry represents one of the most promising approaches to develop new therapeutic strategies. However, viral fusion is a very complex process involving fusion glycoproteins present on the viral envelope. In the two hepatitis C virus envelope proteins, E1 and E2, several membranotropic regions with a potential role in the fusion process have been identified. Among these, we have selected the 314-342 E1 region. Circular Dichroism data indicate that the peptide exhibits a clear propensity to adopt a helical folding in different membrane mimicking media, such as mixtures of water with fluorinated alcohols and phospholipids, with a slight preference for negative charged bilayers. The 3D structure of E1(314-342) peptide, calculated by 2D-NMR in a low-polarity environment, consists of two helical stretches encompassing residues 319-323 and 329-338 respectively. The peptide, presenting a largely apolar character, interacts with liposomes, as indicated by fluorescence and electron spin resonance spectra. The strength of the interaction and the deepness of peptide insertion in the phospholipid membrane are modulated by the bilayer composition, the interaction with anionic phospholipids being among the strongest ever observed. The presence of cholesterol also affects the peptide-bilayer interaction, favoring the peptide positioning close to the bilayer surface. Overall, the experimental data support the idea that this region of E1 might be involved in membrane destabilization and viral fusion; therefore it may represent a good target to develop anti-viral molecules.

  • Gene therapy for Leber's congenital amaurosis is safe and effective through 1.5 years after vector administration.
    Publication Date: 01/03/2010, on Molecular therapy : the journal of the American Society of Gene Therapy
    by Simonelli F, Maguire AM, Testa F, Pierce EA, Mingozzi F, Bennicelli JL, Rossi S, Marshall K, Banfi S, Surace EM, Sun J, Redmond TM, Zhu X, Shindler KS, Ying GS, Ziviello C, Acerra C, Wright JF, McDonnell JW, High KA, Bennett J, Auricchio A
    DOI: 10.1038/mt.2009.277

    The safety and efficacy of gene therapy for inherited retinal diseases is being tested in humans affected with Leber's congenital amaurosis (LCA), an autosomal recessive blinding disease. Three independent studies have provided evidence that the subretinal administration of adeno-associated viral (AAV) vectors encoding RPE65 in patients affected with LCA2 due to mutations in the RPE65 gene, is safe and, in some cases, results in efficacy. We evaluated the long-term safety and efficacy (global effects on retinal/visual function) resulting from subretinal administration of AAV2-hRPE65v2. Both the safety and the efficacy noted at early timepoints persist through at least 1.5 years after injection in the three LCA2 patients enrolled in the low dose cohort of our trial. A transient rise in neutralizing antibodies to AAV capsid was observed but there was no humoral response to RPE65 protein. The persistence of functional amelioration suggests that AAV-mediated gene transfer to the human retina does not elicit immunological responses which cause significant loss of transduced cells. The persistence of physiologic effect supports the possibility that gene therapy may influence LCA2 disease progression. The safety of the intervention and the stability of the improvement in visual and retinal function in these subjects support the use of AAV-mediated gene augmentation therapy for treatment of inherited retinal diseases.