Latest PUBLICATIONS

  • Hb J-Cape Town [alpha92(FG4)Arg-->Gln (alpha1), CGG-->CAG] in Southern Italy found in a patient with erythrocytosis.
    Publication Date: 01/01/2007, on Hemoglobin
    by Pagano L, Flagiello A, Tedesco R, Ammirabile M, Pollio F, Prossomariti L, Giambona A, Passarello C, Pucci P
    DOI: 10.1080/03630260701277487

    A high oxygen affinity hemoglobin (Hb) variant, Hb J-Cape Town [alpha92(FG4)Arg-->Gln (alpha1), CGG-->CAG] was identified in a 30-year-old woman patient from Cosenza (Southern Italy) who had previously been diagnosed with juvenile polycythemia in other hospitals. The occurrence of the variant Hb was assessed by both cation exchange chromatography and liquid chromatography-mass spectrometry (LC-MS) analyses. A detailed structural and functional characterization of the variant was performed at both the protein and DNA level. Structural investigation of the Hb variant by mass spectrometric methodologies and peptide sequencing identified the amino acid replacement as Arg-->Gln at alpha92. The corresponding DNA mutation CGGCAG was assigned to codon 92 of the alpha1 gene by DNA sequencing. These findings highlight the importance of investigating the hypothesis of a high affinity variant in the presence of a polycythemia so as to avoid unnecessary bone marrow examination or radioactive treatment. This report represents the first observation of the Hb J-Cape Town variant in Italy.

  • Thermodynamic analysis of quadruplex DNA-drug interaction.
    Publication Date: 01/01/2007, on Nucleosides, nucleotides & nucleic acids
    by Pagano B, Mattia CA, Virno A, Randazzo A, Mayol L, Giancola C
    DOI: 10.1080/15257770701499069

    This work studies the binding properties of distamycin and its carbamoyl analog, containing four pyrrole units, with the [d(TGGGGT)](4) quadruplex by means of isothermal titration calorimetry (ITC). Analysis of the ITC data reveals that drug/quadruplex binding stoichiometry is 1:1 for both interactions and that distamycin analog gives approximately a 10-fold increase in the quadruplex affinity.

  • Effect of the incorporation of 2'-deoxy-8-(hydroxyl)adenosine on the stability of quadruplexes formed by modified human telomeric DNA.
    Publication Date: 01/01/2007, on Nucleosides, nucleotides & nucleic acids
    by Petraccone L, Duro I, Erra E, Randazzo A, Virno A, Giancola C
    DOI: 10.1080/15257770701490597

    Differential scanning calorimetry (DSC) and circular dichroism (CD) techniques were used to investigate the physico-chemical properties of the quadruplexes formed by the two different truncations of human telomeric sequence d(TAGGGT) and d(AGGGT), where the adenines were substituted by 2'-deoxy-8-(hydroxyl)adenosine (A --> A OH). CD spectra show that the modified sequences are able to form parallel-stranded quadruplex structure. Analysis of the thermodynamic parameters reveals that the introduction of the modified adenine affects in different way the thermal stability of the [d(TAGGGT)]4 and [d(AGGGT)]4 quadruplexes.

  • Biophysical properties of quadruplexes containing two or three 8-bromodeoxyguanosine residues.
    Publication Date: 01/01/2007, on Nucleosides, nucleotides & nucleic acids
    by Petraccone L, Duro I, Randazzo A, Virno A, Mayol L, Giancola C
    DOI: 10.1080/15257770701490589

    A physico-chemical characterization, based on NMR and CD spectroscopy, of quadruplexes formed by the oligonucleotide d(TGGGT), where two or three Gs are substituted by 8-bromo-2'-deoxyguanosine residues (dGBr), is reported. The oligonucleotidic sequences d(TGBr GBr GT), d(TGBr GGBr T), d(TGGBr GBr T), and d(TGBr GBr GBr T) have been synthesized. Only sequences d(TGBr GGBr T) and d(TGBr GBr GT) were able to fold into a well defined quadruplex structure, and their CD profiles and thermal stabilities turned out to be very different from those observed for the natural counterpart, indicating that the 8-Br-dG residues dramatically affect the structure of the quadruplex.

  • Synthesis and biophysical characterization of G-rich oligonucleotides conjugated with sugar-phosphate tails.
    Publication Date: 01/01/2007, on Nucleosides, nucleotides & nucleic acids
    by D'Onofrio J, Erra E, Di Fabio G, Iadonisi A, Petraccone L, De Napoli L, Barone G, Balzarini J, Giancola C, Montesarchio D
    DOI: 10.1080/15257770701528164

    A number of 5'-and 3'-glycoconjugates of the oligonucleotide (5')d(TGGGAG)(3') have been synthesized, exploiting fully automated, online phosphoramidite-based solid phase strategy, as potential anti-HIV-1 agents. The thermodynamic stability of the resulting quadruplexes has been investigated by thermal denaturation studies, via a detailed CD Q1 analysis.

  • Local synthesis of axonal and presynaptic RNA in squid model systems.
    Publication Date: 01/01/2007, on The European journal of neuroscience
    by Eyman M, Cefaliello C, Ferrara E, De Stefano R, Lavina ZS, Crispino M, Squillace A, van Minnen J, Kaplan BB, Giuditta A
    DOI: 10.1111/j.1460-9568.2007.05304.x

    The presence of active systems of protein synthesis in axons and nerve endings raises the question of the cellular origin of the corresponding RNAs. Our present experiments demonstrate that, besides a possible derivation from neuronal cell bodies, axoplasmic RNAs originate in periaxonal glial cells and presynaptic RNAs derive from nearby cells, presumably glial cells. Indeed, in perfused squid giant axons, delivery of newly synthesized RNA to the axon perfusate is strongly stimulated by axonal depolarization or agonists of glial glutamate and acetylcholine receptors. Likewise, incubation of squid optic lobe slices with [3H]uridine leads to a marked accumulation of [3H]RNA in the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons. As the cell bodies of these neurons lie outside the optic lobe, the data demonstrate that presynaptic RNA is locally synthesized, presumably by perisynaptic glial cells. Overall, our results support the view that axons and presynaptic regions are endowed with local systems of gene expression which may prove essential for the maintenance and plasticity of these extrasomatic neuronal domains.

  • Intrafamilial clinical heterogeneity associated with a novel mutation of the retinal degeneration slow/peripherin gene.
    Publication Date: 01/01/2007, on Ophthalmic research
    by Simonelli F, Testa F, Marini V, Interlandi E, Rossi S, Pognuz DR, Virgili G, Garrè C, Bandello F
    DOI: 10.1159/000108118

    To identify the phenotypic variations in 6 related individuals affected by a novel mutation in the retinal degeneration slow/peripherin gene.

  • Energetic aspects of locked nucleic acids quadruplex association and dissociation.
    Publication Date: 15/12/2006, on Biopolymers
    by Petraccone L, Erra E, Randazzo A, Giancola C
    DOI: 10.1002/bip.20591

    The design of modified nucleic acid aptamers is improved by considering thermodynamics and kinetics of their association/dissociation processes. Locked Nucleic Acids (LNA) is a promising class of nucleic acid analogs. In this work the thermodynamic and kinetic properties of a LNA quadruplex formed by the TGGGT sequence, containing only conformationally restricted LNA residues, are reported and compared to those of 2'-OMe-RNA (O-RNA) and DNA quadruplexes. The thermodynamic analysis indicates that the sugar-modified quadruplexes (LNA and O-RNA) are stabilized by entropic effects. The kinetic analysis shows that LNA and O-RNA quadruplexes are characterized by a slower dissociation and a faster association with respect to DNA quadruplex. Interestingly, the LNA quadruplex formation process shows a second-order kinetics with respect to single strand concentration and has a negative activation energy. To explain these data, a mechanism for tetramer formation with two intermediate states was proposed.

  • Recombinant amyloidogenic domain of ApoA-I: analysis of its fibrillogenic potential.
    Publication Date: 08/12/2006, on Biochemical and biophysical research communications
    by Di Gaetano S, Guglielmi F, Arciello A, Mangione P, Monti M, Pagnozzi D, Raimondi S, Giorgetti S, Orrù S, Canale C, Pucci P, Dobson CM, Bellotti V, Piccoli R
    DOI: 10.1016/j.bbrc.2006.10.026

    A variety of amyloid diseases are associated with fibrillar aggregates from N-terminal fragments of ApoA-I generated through a largely unexplored multi-step process. The understanding of the molecular mechanism is impaired by the lack of suitable amounts of the fibrillogenic polypeptides that could not be produced by recombinant methods so far. We report the production and the conformational analysis of recombinant ApoA-I 1-93 fragment. Similarly to the polypeptide isolated ex vivo, a pH switch from 7 to 4 induces a fast and reversible conformational transition to a helical state and leads to the identification of a key intermediate in the fibrillogenesis process. Limited proteolysis experiments suggested that the C-terminal region is involved in helix formation. The recombinant polypeptide generates fibrils at pH 4 on a time scale comparable with that of the native fragment. These findings open the way to studies on structural, thermodynamic, and kinetic aspects of ApoA-I fibrillogenesis.

  • Rethinking primary prevention of atherosclerosis-related diseases.
    Publication Date: 05/12/2006, on Circulation
    by Napoli C, Lerman LO, de Nigris F, Gossl M, Balestrieri ML, Lerman A
    DOI: 10.1161/CIRCULATIONAHA.105.570358

  • Autologous bone marrow cell therapy and metabolic intervention in ischemia-induced angiogenesis in the diabetic mouse hindlimb.
    Publication Date: 01/12/2006, on Cell cycle (Georgetown, Tex.)
    by Sica V, Williams-Ignarro S, de Nigris F, D'Armiento FP, Lerman LO, Balestrieri ML, Maione C, Palagiano A, Rossiello L, Ignarro LJ, Napoli C
    DOI: 10.4161/cc.5.24.3568

    Peripheral arterial disease (PAD) is a major health problem especially when associated to diabetes. Administration of autologous bone marrow cells (BMC) is emerging as a novel intervention to induce therapeutic angiogenesis in experimental ischemic limb models and in patients with PAD. Since tissue ischemia and diabetes are associated with an overwhelming generation of oxygen radicals and detrimental effects due to formation of glycosylation end-products, metabolic intervention with antioxidants and L-arginine can confer beneficial effects beyond those achieved by BMC alone. The effects of cotreatment with intravenous BMCs and metabolic vascular protection (1.0% vitamin E, 0.05% vitamin C, and 6% L-arginine) were examined in the ischemic hindlimb of diabetic and non diabetic mice. BMC therapy increased blood flow and capillary densities and Ki67 proliferative marker, and decreased interstitial fibrosis. This effect was amplified by metabolic cotreatment, an intervention inducing vascular protection, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress, and macrophage activation.

  • Genetically modified pigs produced with a nonviral episomal vector.
    Publication Date: 21/11/2006, on Proceedings of the National Academy of Sciences of the United States of America
    by Manzini S, Vargiolu A, Stehle IM, Bacci ML, Cerrito MG, Giovannoni R, Zannoni A, Bianco MR, Forni M, Donini P, Papa M, Lipps HJ, Lavitrano M
    DOI: 10.1073/pnas.0604938103

    Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.

  • Raf plus TGFbeta-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin.
    Publication Date: 16/11/2006, on Oncogene
    by Janda E, Nevolo M, Lehmann K, Downward J, Beug H, Grieco M
    DOI: 10.1038/sj.onc.1209701

    Oncogenic Ras interferes with adhesive functions of epithelial cells, but requires tumor growth factor beta (TGFbeta) signaling to cause epithelial-mesenchymal transition (EMT) and tumor progression in model systems. To investigate the mechanisms by which Ras and TGFbeta pathways cooperate in EMT induction, we introduced a tamoxifen-inducible version of Raf-1 (RafER) into fully polarized, mammary epithelial cells (EpH4). EMT characterized by loss of E-cadherin expression and upregulation of invasiveness-promoting genes was induced by TGFbeta plus 4-hydroxytamoxifen (4HT) activation of RafER. Downregulation of E-cadherin by RafER plus TGFbeta was detectable in total cell lysates after 48 h and much earlier in detergent-insoluble fractions of E-cadherin. Both pathways cooperated to strongly enhance endocytosis of E-cadherin, mainly via the clathrin-dependent route. Pulse-chase experiments showed decreased E-cadherin protein stability in cells stimulated with TGFbeta and 4HT and increased E-cadherin half-life in the presence of monensin. Monensin and chloroquine prevented E-cadherin degradation to different extent, but only monensin effectively blocked the loss of E-cadherin from the junctional complexes. Both lysosome inhibitors caused accumulation of E-cadherin vesicles, some of which were positive for Cathepsin D and lysosome-associated membrane protein 1 (LAMP-1). In addition, TGFbeta and mitogen-activated protein kinase hyperactivation synergistically induced E-cadherin ubiquitination, suggesting that the cooperation of Raf and TGFbeta favors lysosomal degradation of E-cadherin instead of its recycling. Our data indicate that early stages of EMT involve cooperative, post-translational downregulation of E-cadherin, whereas loss of E-cadherin via transcriptional repression is a late event in EMT.

  • Analysis of detergent-resistant membranes associated with apical and basolateral GPI-anchored proteins in polarized epithelial cells.
    Publication Date: 16/10/2006, on FEBS letters
    by Tivodar S, Paladino S, Pillich R, Prinetti A, Chigorno V, van Meer G, Sonnino S, Zurzolo C
    DOI: 10.1016/j.febslet.2006.09.022

    Detergent-resistant membranes (DRMs) represent specialized membrane domains resistant to detergent extraction, which may serve to segregate proteins in a specific environment in order to improve their function. Segregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in DRMs has been shown to be involved in their sorting to the apical membrane in polarized epithelial cells. Nonetheless, we have shown that both apical and basolateral GPI-APs associate with DRMs. In this report we investigated the lipid composition of DRMs associated with an apical and a basolateral GPI-AP. We found that apical and basolateral DRMs contain the same lipid species although in different ratios. This specific lipid ratio is maintained after mixing the cells before lysis indicating that DRMs maintain their identity after Triton extraction.

  • Independent component model of the default-mode brain function: Assessing the impact of active thinking.
    Publication Date: 16/10/2006, on Brain research bulletin
    by Esposito F, Bertolino A, Scarabino T, Latorre V, Blasi G, Popolizio T, Tedeschi G, Cirillo S, Goebel R, Di Salle F
    DOI: 10.1016/j.brainresbull.2006.06.012

    The "default-mode" network is an ensemble of cortical regions, which are typically deactivated during demanding cognitive tasks in functional magnetic resonance imaging (fMRI) studies. Using functional connectivity, this network can be conceptualized and studied as a "stand-alone" function or system. Regardless of the task, independent component analysis (ICA) produces a picture of the "default-mode" function even when the subject is performing a simple sensori-motor task or just resting in the scanner. This has boosted the use of default-mode fMRI for non-invasive research in brain disorders. Here, we studied the effect of cognitive load modulation of fMRI responses on the ICA-based pictures of the default-mode function. In a standard graded working memory study based on the n-back task, we used group-level ICA to explore the variability of the default-mode network related to the engagement in the task, in 10 healthy volunteers. The analysis of the default-mode components highlighted similarities and differences in the layout under three different cognitive loads. We found a load-related general increase of deactivation in the cortical network. Nonetheless, a variable recruitment of the cingulate regions was evident, with greater extension of the anterior and lesser extension of the posterior clusters when switching from lower to higher working memory loads. A co-activation of the hippocampus was only found under no working memory load. As a generalization of our results, the variability of the default-mode pattern may link the default-mode system as a whole to cognition and may more directly support use of the ICA model for evaluating cognitive decline in brain disorders.