Latest PUBLICATIONS

  • Gene expression and morphological changes in surgically injured carotids of spontaneously hypertensive rats.
    Publication Date: 01/03/2002, on Journal of vascular research
    by Forte A, Di Micco G, Galderisi U, De Feo M, Esposito F, Esposito S, Renzulli A, Berrino L, Cipollaro M, Agozzino L, Cotrufo M, Rossi F, Cascino A
    DOI: 10.1159/000057760

    The expression profiles of genes involved in cell proliferation, differentiation and programmed death were investigated in carotids of spontaneously hypertensive rats (SHR) treated with a model of surgical injury that mimics events occurring during arterial grafts, endarterectomy and organ transplantation. The mRNA level of the c-myc, angiotensin II receptor 1 (AT1), Rb/p105, Rb2/p130, Bcl-2 and Bax-alpha genes was assessed by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique at different times up to 48 h after injury, while the morphological changes were evaluated 30 days after injury. The proliferation marker c-myc increases almost immediately, peaks after 4 h and returns to basal levels after 24 h; the AT1 receptor mRNA reaches its maximal level 48 h after injury. The level of cell cycle exit markers Rb/p105 and Rb2/p130 gradually decreases after injury. The apoptosis marker Bcl-2/Bax-alpha ratio shows a significant reduction only 4 h after injury, resuming the initial value after 24 and 48 h. Morphological analysis reveals that surgical injury in SHR induces adventitial and medial constrictive remodeling changes rather than intima proliferation as in balloon angioplasty. Both molecular and histological data show substantial differences with respect to normotensive rats.

  • Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas.
    Publication Date: 20/02/2002, on International journal of cancer
    by Castino R, Pace D, Démoz M, Gargiulo M, Ariatta C, Raiteri E, Isidoro C
    DOI:

    Neuroblastoma is the most common type of cancer in infants. In children this tumor is particularly aggressive; despite various new therapeutic approaches, it is associated with poor prognosis. Given the importance of endosomal-lysosomal proteolysis in cellular metabolism, we hypothesized that inhibition of lysosomal protease would impact negatively on neuroblastoma cell survival. Treatment with E-64 or CA074Me (2 specific inhibitors of cathepsin B) or with pepstatin A (a specific inhibitor of cathepsin D) was cytotoxic for 2 neuroblastoma cell lines having different degrees of malignancy. Cell death was associated with condensation and fragmentation of chromatin and externalization of plasma membrane phosphatidylserine, 2 hallmarks of apoptosis. Concomitant inhibition of the caspase cascade protected neuroblastoma cells from cathepsin inhibitor-induced cytotoxicity. These data indicate that prolonged inhibition of the lysosomal proteolytic pathway is incompatible with cell survival, leading to apoptosis of neuroblastoma cells, and that the cathepsin-mediated and caspase-mediated proteolytic systems are connected and cooperate in the regulation of such an event. Since modern antitumor chemotherapy is aimed at restoring the normal rate of apoptosis in neoplastic tissues, the demonstration that endosomal-lysosomal cathepsins are involved in this process may constitute a basis for novel strategies that include cathepsin inhibitors in the therapeutic regimen.

  • Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.
    Publication Date: 15/02/2002, on Nucleic acids research
    by Di Bernardo G, Del Gaudio S, Cammarota M, Galderisi U, Cascino A, Cipollaro M
    DOI:

    Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

  • Environmental mimic of receptor interaction: conformational analysis of CCK-15 in solution.
    Publication Date: 14/02/2002, on Journal of medicinal chemistry
    by Albrizio S, Carotenuto A, Fattorusso C, Moroder L, Picone D, Temussi PA, D'Ursi A
    DOI:

    CCK-15, a peptide derived from the 115-membered CCK preprohormone, was the object of a comparative conformational analysis by NMR spectroscopy and molecular modeling methods. NMR data in several solvents demonstrate that the propensity of the peptide to fold into a helical conformation is intrinsic, not merely a consequence of the interaction with phosphatidylcholine micelles or with a putative receptor, as suggested by a previous study on CCK-8 (Pellegrini, M.; Mierke, D. Biochemistry 1999, 38, 14775-14783.). The prevailing CCK-15 conformer in a mixture 1,1,1,3,3,3-hexafluoroacetone/water reveals that the residues common to CCK-15 and CCK-8 assume very similar conformations. Our CCK-15 structure is consistent with the model of receptor interaction proposed by Pellegrini and Mierke and discloses possible novel interactions that involve a larger area of the putative receptor. The consensus structure between CCK-15 and CCK-8 shows a good superposition of the side chains of residues 12-14 with crucial moieties of two non-peptidic CCK-A antagonists.

  • Detergent-resistant membrane microdomains and apical sorting of GPI-anchored proteins in polarized epithelial cells.
    Publication Date: 01/02/2002, on International journal of medical microbiology : IJMM
    by Paladino S, Sarnataro D, Zurzolo C
    DOI:

    Detergent-insoluble microdomains or rafts play a crucial role in many cellular functions: membrane traffic, cell signalling and human diseases. In this work we investigate the role of rafts in the sorting of GPI-anchored proteins in polarized epithelial cells. In contrast to MDCK cells, the majority of endogenous GPI-anchored proteins are sorted to the basolateral surface of Fischer rat thyroid cells (Zurzolo et al., J. Cell Biol. 121, 1031-1039, 1993). We analyzed a set of transfected GPI proteins in order to understand the role of the GPI anchor and of association with rafts for apical sorting. We found that the GPI moiety is necessary but not sufficient for apical sorting of GPI proteins and that the ectodomain has a major role. We propose a new model in which the stabilization of proteins into rafts, probably mediated by interactions between protein ectodomains and a putative receptor, plays a crucial role in apical sorting.

  • 17 beta-estradiol induces spermatogonial proliferation through mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activity in the lizard (Podarcis s. sicula).
    Publication Date: 01/02/2002, on Molecular reproduction and development
    by Chieffi P, Colucci D'Amato L, Guarino F, Salvatore G, Angelini F
    DOI: 10.1002/mrd.1151

    There are always more evidences indicating that 17beta-estradiol (E(2)) is necessary for normal male fertility. We have used a nonmammalian vertebrate model (the lizard Podarcis s. sicula) to investigate the regulation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity in the testis during the annual sexual cycle and to study whether E(2) exerts a role in the spermatogenesis through ERK1/2 activity. Immunocytochemistry analysis shows that ERK1/2 proteins are present in the nucleus of the spermatogonia (SPG), and in primary (I) spermatocytes (SPC). The annual E(2) profile shows a progressive increase during the active spermatogenesis (from April to June) and a peak in the month of August (spermatogonial mitosis). In parallel, ERK1/2 (molecular weight 44 and 42 kDa, respectively) are highly phosphorylated during the period of active spermatogenesis and in post-refractory period (August) compared with the winter stasis (from November to March). Present results demonstrate that E(2) treatment induces spermatogonial proliferation, possibly via the activation of ERK1/2, and this effect is counteracted by the antiestrogen ICI 182-780.

  • Unusual manifestation of vertebral osteoid osteoma: case report.
    Publication Date: 01/01/2002, on European radiology
    by Scuotto A, Accardo C, Rotondo M, Lus G, La Marca P, Natale M, Agozzino L, Cotrufo R
    DOI: 10.1007/s003300100893

    We report the case of a 64 year-old man with a clinical history suggesting a low thoracic-cord involvement, in which an unexpected vertebral osteoid osteoma was discovered. The patient underwent MRI of the thoraco-lumbar spine, which included sagittal and axial T1-weighted images, and sagittal double-echo T2-weighted images. Subsequently, CT scan was carried out with 2-mm-thick axial sections, aimed at T10 vertebra. Magnetic resonance imaging disclosed an extra-axial mass at T10 level. Computed tomography scan suggested an osteoid osteoma of the tenth thoracic vertebra, involving the lamina with marked sclerosis and prevalently endocanalar extension. Histology following surgical resection confirmed the diagnosis. In the reported case CT scan provided the correct pre-operative diagnosis of osteoid osteoma despite its unusual clinical--anamnestic presentation. Magnetic resonance imaging was useful in establishing the relationship of the neoplasm with the spinal cord.

  • Coordinate and categorical judgements in spatial imagery. An fMRI study.
    Publication Date: 01/01/2002, on Neuropsychologia
    by Trojano L, Grossi D, Linden DE, Formisano E, Goebel R, Cirillo S, Elefante R, Di Salle F
    DOI:

    We aimed at verifying whether the hemispheric specialisation for categorical/coordinate spatial judgements also applies to the spatial imagery domain by the use of whole-brain fMRI. In a block-design experiment we used the "coordinate" mental clock test, contrasting it with a "categorical" task applied to the same clock stimuli; as a control task we used a syllable counting task requiring a verbal-phonological judgement on the same material of the two imagery tasks. Our results showed that categorical and coordinate spatial judgements on imagined stimuli rely on the activation of a set of cortical areas, centred upon the superior parietal lobule (SPL) bilaterally. These regions, together with other parietal and prefrontal areas, showed a pattern of relative lateralization, with the left hemisphere being mainly activated during the categorical task and the right in the coordinate task. These data confirm the strong involvement of the SPL in spatial processing. Moreover, our findings suggest that different interconnected neural networks are activated to comply with specific test requirements, giving rise to functional imaging patterns compatible with psychological theories on hemispheric specialization.

  • Messenger RNAs in synaptosomal fractions from rat brain.
    Publication Date: 30/12/2001, on Brain research. Molecular brain research
    by Crispino M, Capano CP, Aiello A, Iannetti E, Cupello A, Giuditta A
    DOI:

    Synaptosomal fractions from rat brain have been analyzed with semi-quantitative RT-PCR methods to determine their content of mRNAs coding for presynaptic, postsynaptic, glial, and neuronal proteins. Each mRNA was determined with reference to the standard HPRT mRNA. In our analyses, mRNAs were considered to be associated with synaptosomes only if their relative amounts were higher than in microsomes prepared in a polysome stabilizing medium, rich in Mg(++) and K(+) ions, or in the homogenate. According to this stringent criterion, the following synaptosomal mRNAs could not be attributed to microsomal contamination and were assumed to derive from the subcellular structures known to harbor their translation products, i.e. GAT-1 mRNAs from presynaptic terminals and glial processes, MAP2 mRNA from dendrites, GFAP mRNA from glial processes, and TAU mRNA from neuronal fragments. This interpretation is in agreement with the involvement of extrasomatic mRNAs in local translation processes.

  • RB2/p130 ectopic gene expression in neuroblastoma stem cells: evidence of cell-fate restriction and induction of differentiation.
    Publication Date: 15/12/2001, on The Biochemical journal
    by Jori FP, Galderisi U, Piegari E, Peluso G, Cipollaro M, Cascino A, Giordano A, Melone MA
    DOI:

    The activity of the RB2/p130 gene, which is a member of the retinoblastoma gene family, is cell-cycle-regulated and plays a key role in growth inhibition and differentiation. We used neuroblastoma cell lines as a model for studies on neural crest progenitor cell differentiation. We show that Rb2/p130 ectopic protein expression induces morphological and molecular modifications, promoting differentiation of intermediate (I) phenotype SK-N-BE(2)-C neuroblastoma cells towards a neuroblastic (N) rather than a Schwann/glial/melanocytic (S) phenotype. These modifications are stable as they persist even after treatment with an S-phenotype inducer. Rb2/p130 ectopic expression also induces a more differentiated phenotype in N-type SH-SY-5Y cells. Further, this function appears to be independent of cell-cycle withdrawal. The data reported suggest that the Rb2/p130 protein is able to induce neuronal lineage specification and differentiation in neural crest stem and committed neuroblastoma cells, respectively. Thus, the Rb2/p130 protein seems to be required throughout the full neural maturation process.

  • Thermodynamic and computational studies of DNA triple helices containing a nucleotide or a non-nucleotide linker in the third strand.
    Publication Date: 11/12/2001, on Biophysical chemistry
    by Giancola C, Petraccone L, Pieri M, Barone G
    DOI:

    In this paper we report a thermodynamic characterisation of stability and melting behaviour of four different triple helices at pH 6.0. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)(m)(pyrimidine)(m)-3'. The four triplexes are formed by targeting the 16-mer duplex with an all pyrimidine 16-mer or 15-mer or 14-mer third strand. The 16-mer oligonucleotide contains a 3'-3' phosphodiester junction and corresponding triplex was named 16-mer P. The 14-mer oligonucleotide contains a non-nucleotide linker, the 1,2,3 propanetriol residue and the corresponding triplex was named 14-mer PT. For the 15-mer oligonucleotide both junctions were alternatively used and the relative triplexes were named 15-mer P and 15-mer PT, respectively. These linkers introduce the appropriate polarity inversion and let the third strand switch from one oligopurine strand of the duplex to the other. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex. Transition enthalpies, entropies and free energies were derived from differential scanning calorimetric measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region and a propanetriol residue as linker was used. The thermodynamic data were discussed in light of molecular mechanics and dynamics calculations.

  • On selective left neglect during walking in a child.
    Publication Date: 01/12/2001, on Brain and cognition
    by Grossi D, Lepore M, Napolitano A, Trojano L
    DOI: 10.1006/brcg.2001.1460

    In this article we describe a child affected by right parieto-occipital lesion due to head injury. The patient showed left hemianopia, but not unilateral spatial neglect on traditional paper-and-pencil tests and on "ecological" tests. However, his parents reported frequent collisions with obstacles on the left side. A specific test was set up: The patient had to kick down skittles put on both sides of a route traced on the floor. He kicked down 89% of skittles on the right, but only 38% on the left side. These findings are discussed in light of recent theories on unilateral neglect.

  • Ontogeny of AMPA receptor gene expression in the developing rat midbrain and striatum.
    Publication Date: 30/11/2001, on Brain research. Molecular brain research
    by Lilliu V, Pernas-Alonso R, Trelles RD, di Porzio U, Zuddas A, Perrone-Capano C
    DOI:

    AMPA receptors mediate most of the fast excitatory synaptic transmission in the mammalian CNS. Their ontogeny during embryonic (E) and postnatal (P) development is still poorly understood. We have studied the expression of the genes encoding for AMPA glutamate receptor subunits (GlurA, GlurB, GlurC and GlurD) in the rat ventral mesencephalon (MES) and striatum (STR) and in fetal midbrain primary cultures. Each receptor subunit shows unique area- and temporal-expression pattern. In MES, GluRA, GlurB and GlurC mRNA are detectable from the earliest embryonic stage studied (E13) and raise thereafter between E15 and E17, to plateau at E19 to adult values. Differently, GlurD mRNA increases throughout embryonic and postnatal development reaching its highest levels in the adult MES. The pattern of AMPA proteins corresponded to the mRNA levels for all subunits. In the STR, GlurA gene expression increases between E15 and E19, GlurB mRNA levels are sustained from the first embryonic stages analyzed (E15) until E19 and gradually decrease thereafter toward adult levels, GlurC gene expression increases gradually throughout ontogeny to reach its highest levels in the adult. STR GlurD transcripts remain at constant levels in all stages studied. In embryonic MES primary cultures, every subunit show a characteristic expression profile similar to that observed in vivo. They all decrease significantly during the second week in vitro. Thus, all the AMPA receptor subunit transcripts appear independently regulated during development, probably depending on the tissue-specific environment, which seems preserved in MES cultures.

  • Apolipoprotein E polymorphisms in age-related macular degeneration in an Italian population.
    Publication Date: 01/11/2001, on Ophthalmic research
    by Simonelli F, Margaglione M, Testa F, Cappucci G, Manitto MP, Brancato R, Rinaldi E
    DOI:

    Apolipoprotein E (apoE) is an important regulator of cholesterol and lipid transport during compensatory synaptogenesis. Our purpose was to investigate the role of apoE gene polymorphisms in Italian patients with age-related macular degeneration (AMD).

  • Oligonucleotides containing a lysine residue as 3'-3' junction for alternate strand triple helix formation.
    Publication Date: 01/11/2001, on Bioorganic & medicinal chemistry
    by Barone G, De Napoli L, Di Fabio G, Giancola C, Messere A, Montesarchio D, Petraccone L, Piccialli G
    DOI:

    Oligonucleotides with a 3'-3' inversion of polarity site assured by one lysine residue have been synthesized, characterized and used as third strands in alternate strand triple helix formation. UV melting studies and molecular mechanics calculations have been carried out to investigate the stability and the geometry of these new triplexes.