Latest PUBLICATIONS

  • Structural and thermodynamic studies of the interaction of distamycin A with the parallel quadruplex structure [d(TGGGGT)]4.
    Publication Date: 26/12/2007, on Journal of the American Chemical Society
    by Martino L, Virno A, Pagano B, Virgilio A, Di Micco S, Galeone A, Giancola C, Bifulco G, Mayol L, Randazzo A
    DOI: 10.1021/ja075710k

    The complex between distamycin A and the parallel DNA quadruplex [d(TGGGGT)]4 has been studied by 1H NMR spectroscopy and isothermal titration calorimetry (ITC). To unambiguously assert that distamycin A interacts with the grooves of the quadruplex [d(TGGGGT)]4, we have analyzed the NMR titration profile of a modified quadruplex, namely [d(TGGMeGGT)]4, and we have applied the recently developed differential frequency-saturation transfer difference (DF-STD) method, for assessing the ligand-DNA binding mode. The three-dimensional structure of the 4:1 distamycin A/[d(TGGGGT)]4 complex has been determined by an in-depth NMR study followed by dynamics and mechanics calculations. All results unequivocally indicate that distamycin molecules interact with [d(TGGGGT)]4 in a 4:1 binding mode, with two antiparallel distamycin dimers that bind simultaneously two opposite grooves of the quadruplex. The affinity between distamycin A and [d(TGGGGT)]4 enhances ( approximately 10-fold) when the ratio of distamycin A to the quadruplex is increased. In this paper we report the first three-dimensional structure of a groove-binder molecule complexed to a DNA quadruplex structure.

  • Novel amphiphilic cyclic oligosaccharides: synthesis and self-aggregation properties.
    Publication Date: 07/12/2007, on The Journal of organic chemistry
    by Coppola C, Saggiomo V, Di Fabio G, De Napoli L, Montesarchio D
    DOI: 10.1021/jo7017087

    Novel amphiphilic cyclic disaccharide analogues, in which the saccharide units are connected through stable phosphodiester linkages (CyPLOS, Cyclic Phosphate-Linked OligoSaccharides) and decorated with long lipophilic tentacles at the 2- and 3-OH moieties, have been synthesized. Their propensity to self-aggregation has been investigated by means of 1H and 31P NMR experiments, making it possible to determine for these macrocycles critical aggregation concentration values in the millimolar range.

  • Functional proteomics: protein-protein interactions in vivo.
    Publication Date: 01/12/2007, on The Italian journal of biochemistry
    by Monti M, Cozzolino M, Cozzolino F, Tedesco R, Pucci P
    DOI:

    Functional proteomics constitutes an emerging research area in the proteomic field focused to two major targets, the elucidation of biological function of unknown proteins and the definition of cellular mechanisms at the molecular level. Understanding protein functions as well as unravelling molecular mechanisms within the cell is then depending on the identification of the interacting protein partners. The association of an unknown protein with partners belonging to a specific protein complex involved in a particular mechanism would in fact be strongly suggestive of its biological function. Furthermore, a detailed description of the cellular signalling pathways might greatly benefit from the elucidation of protein-protein interactions in the cell. Isolation of functional protein complexes essentially rely on affinity-based procedures. The protein of interest and its specific partners can be fished out from the cellular extract by using a suitable ligand as a bait taking advantage of the specific binding properties of the ligand molecule immobilised on agarose-sepharose supports. Alternative strategies essentially relying on immunoprecipitation techniques have been introduced to allow purification of protein complexes formed in vivo within the cell. The gene coding for the bait tagged with an epitope against which good antibodies exist (FLAG, HA, c-myc, etc.), is transfected into the appropriate cell line and expressed in the cognate host. The cell extracts are immunoprecipitated with anti-tag monoclonal antibodies using suitable experimental conditions to avoid dissociation of the complexes. In both cases, protein components specifically recognised by the bait and retained on the agarose beads can then be eluted and fractionated by SDS-PAGE. The protein bands detected on the gel are in situ enzymatically digested and the resulting peptide mixtures analysed by capillary LC-MS/MS techniques leading to the identification of the protein interactors.

  • A neuropsychological longitudinal study in Parkinson's patients with and without hallucinations.
    Publication Date: 01/12/2007, on Movement disorders : official journal of the Movement Disorder Society
    by Santangelo G, Trojano L, Vitale C, Ianniciello M, Amboni M, Grossi D, Barone P
    DOI: 10.1002/mds.21746

    The aim of this work was to determine the progression of cognitive impairment in Parkinson's disease (PD) patients with or without hallucinations. Two years after the first assessment, 36 PD patients were re-evaluated on standardized neuropsychological tests, including the Frontal Assessment Battery (FAB), and on rating scales for overall cognitive functioning, functional autonomy, behavioral disorders. Nine patients had hallucinations at baseline and endpoint assessments; 12 patients developed hallucinations during the follow-up; and 15 patients were hallucination-free throughout the study. Cognitive performance significantly declined in all three groups, but at endpoint assessment PD hallucinators scored significantly lower than nonhallucinators on phonological and semantic fluency tasks, immediate free recall and the go/no-go FAB subtest; moreover, they showed more severe apathy than nonhallucinators. Reduced phonological fluency at baseline (odds ratio [OR], 13.5; 95% CI: 1.34-135.98, P = 0.027) was the only independent predictor of onset of hallucinations after 2 years, whereas hallucinations (OR, 10.1; 95% CI: 1.94-51.54, P = 0.006) and poor phonological fluency (OR, 6.1; 95% CI: 1.04-35.03, P = 0.045) independently predicted development of diffuse cognitive impairment. We concluded that reduced verbal fluency scores may predict the onset of hallucinations, while hallucinations and poor phonological fluency may predict development of dementia in PD patients.

  • Effect of gestational hypercholesterolaemia on omental vasoreactivity, placental enzyme activity and transplacental passage of normal and oxidised fatty acids.
    Publication Date: 01/12/2007, on BJOG : an international journal of obstetrics and gynaecology
    by Liguori A, D'Armiento FP, Palagiano A, Balestrieri ML, Williams-Ignarro S, de Nigris F, Lerman LO, D'Amora M, Rienzo M, Fiorito C, Ignarro LJ, Palinski W, Napoli C
    DOI: 10.1111/j.1471-0528.2007.01510.x

    Maternal hypercholesterolaemia during pregnancy increases lipid peroxidation in mothers and fetuses and programs increased susceptibility to atherosclerosis later in life. The objective of this study was to elucidate the role of the placenta in mediating oxidative stress from mother to offspring.

  • Transcription factor expression, RNA synthesis and NADPH-diaphorase across the rat brain and exposure to spatial novelty.
    Publication Date: 22/11/2007, on Behavioural brain research
    by Romanelli P, Di Matteo L, Cobellis G, Varriale B, Menegazzi M, Gironi Carnevale UA, Ruocco LA, Sadile AG
    DOI: 10.1016/j.bbr.2007.06.021

    The molecular hypothesis of learning and memory processes is based on changes in synaptic weights in neural networks. Aim of this study was to map neural traces of exposure to a spatial novelty were mapped by (i) the transcription factors (TFs) c-fos, c-jun and jun-B using Northern blot and immunocytochemistry (ICC), (ii) RNA synthesis by (3)H-uridine autoradiography and RNA level, (iii) NADPH-diaphorase (NADPH-d) expression by histochemistry. Thus, adult male albino rats were exposed to a Làt-maze and sacrificed at different times. Non-exposed rats served as controls. The latter showed a low constitutive expression of TF, RNA synthesis and NADPH-d across the brain. Northern blots showed a three-fold increase in TFs in exposed versus non-exposed rats in the cerebral cortex. ICC showed in exposed rats several TFs positive cells in the granular and pyramidal layers of the hippocampus and later in all layers of the somatosensory cortex, in the granular layer of the cerebellar cortex. The TF-positivity was stronger in rats exposed for the first time, and was time and NMDA-dependent. Autoradiography for RNA synthesis showed positive cells in the ependyma, hippocampus and cerebellum 6h after testing, and in the somatosensory cortex 24h later. In addition, exposure to novelty induced NADPH-d in the dorsal hippocampus, the caudate-putamen, all the layers of the somatosensory cortex. and the cerebellum. The positivity was absent immediately after exposure, appeared within 2h and disappeared 24h later. A strong neuronal discharge by the convulsant pentylenetetrazol, strongly induced TFs but not din not affect NADPH-d 2h later. Thus, data suggest that the processing of spatial and emotional components of experience activates neural networks across different organization levels of the CNS.

  • A classical Mendelian cross-breeding study of the Naples high and low excitability rat lines.
    Publication Date: 02/11/2007, on Behavioural brain research
    by Gironi Carnevale UA, Vitullo E, Varriale B, Ruocco LA, Sadile AG
    DOI: 10.1016/j.bbr.2007.05.032

    The development of brain and behaviour is controlled by the interaction of genetic determinants and environmental factors. To study genetic determinants, model systems such as the Naples rat lines, i.e. Naples high (NHE) and low excitability (NLE), are useful. They have been selectively bred for divergent behaviour arousal to novelty. Aim of this study was to assess the extent of the genetic control of the selection trait. Thus adult albino rats of NHE and NLE lines have been used throughout. According to a classical Mendelian cross-breeding design, a first experiment was carried out with hybrids obtained from parental lines P1 (NHE) and P2 (NLE) as F1, F2 and related backcrosses B1 (F1xP1) and B2 (F1xP2). Young adults (60-80 days) offspring of both gender were exposed separately for two 10min tests to a spatial novelty (Lát-maze). To verify a possible sex link of the trait, a second experiment was carried out adding to the Mendelian cross design parental gender. Behavioural variables were horizontal (corner-crossings: HA), vertical (rearings on hindlimbs: VA) or total activity (HVA: HA+VA) scores. The heritability of HVA trait was estimated across the 20 generations of selection and Mendelian cross hybrids. Quantitative-genetic analysis on this trait and its HA and VA components, was applied by the Lynch and Walsh joint-scaling test procedure to evaluate underlying genetic mechanism. The correlation between experimental data of hybrids and estimated values from different heritability models were also computed. Results indicated that (i) the activity scores by Mendelian hybrids were intermediate between the two parental lines and were also graded; (ii) there was no sex effect on the heritability of trait but only a general tendency of females to higher activity levels; (iii) the heritability of HVA trait was very high (h2 index=0.824); (iv) heritability model of HVA and HA trait was polygenic with a marked epistatic control where as VA trait was fitted by simpler model with less genes and lower epistatic effect. In conclusion the Naples lines reveal strong genetic determinants for behavioural traits associated with polygenic pattern. Moreover, HA and VA activity components with prevailing cognitive and non-cognitive meaning, respectively, show differential genetic control.

  • Contrast-enhanced sonography in the characterization of small hepatocellular carcinomas in cirrhotic patients: comparison with contrast-enhanced ultrafast magnetic resonance imaging.
    Publication Date: 01/11/2007, on Anticancer research
    by Giorgio A, De Stefano G, Coppola C, Ferraioli G, Esposito V, Di Sarno A, Giorgio V, De Stefano M, Sangiovanni V, Liorre G, Del Viscovo L
    DOI:

    To evaluate the role of low mechanical index (MI) contrast-enhanced sonography (CEUS) for the characterization of small hepatocellular carcinomas (HCC) in cirrhotic patients by comparing the results to ultrafast dynamic gadolinium-enhanced magnetic resonance imaging (MRI) studies.

  • An experimental investigation of the automatic/voluntary dissociation in limb apraxia.
    Publication Date: 01/11/2007, on Brain and cognition
    by Trojano L, Labruna L, Grossi D
    DOI: 10.1016/j.bandc.2007.07.010

    The ability of apraxic patients to perform gestures in everyday life is a controversial issue. In this paper, we aimed to evaluate the automatic/voluntary dissociation (AVD) in four patients affected by clinically relevant limb apraxia. For this purpose, we sampled different kinds of gestures belonging to patients' motor repertoire and then assessed their production in a testing session. Our experimental procedure consisted of two steps: in the first phase, we recorded gestures produced by patients in two natural conditions; in the second phase, we assessed production of correctly produced tool-actions, and of spontaneous non tool-actions and meaningless conversational (cohesive and beats) gestures under different modalities. AVD was observed for all types of gestures, albeit to different degree in single patients. The present findings demonstrate that the context provides strong bottom-up cues for the retrieval of motor patterns, while artificial testing conditions impose an additional cognitive load.

  • Reactive astrocytosis and glial glutamate transporter clustering are early changes in a spinocerebellar ataxia type 1 transgenic mouse model.
    Publication Date: 01/11/2007, on Neuron glia biology
    by Giovannoni R, Maggio N, Rosaria Bianco M, Cavaliere C, Cirillo G, Lavitrano M, Papa M
    DOI: 10.1017/S1740925X08000185

    Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeats within the coding sequence of the ataxin-1 protein. In the present study, we used a conditional transgenic mouse model of SCA1 to investigate very early molecular and morphological changes related to the behavioral phenotype. In mice with neural deficits detected by rotarod performance, and simultaneous spatial impairments in exploratory activity and uncoordinated gait, we observed both significant altered expression and patchy distribution of excitatory amino acids transporter 1. The molecular changes observed in astroglial compartments correlate with changes in synapse morphology; synapses have a dramatic reduction of the synaptic area external to the postsynaptic density. By contrast, Purkinje cells demonstrate preserved structure. In addition, severe reactive astrocytosis matches changes in the glial glutamate transporter and synapse morphology. We propose these morpho-molecular changes are the cause of altered synaptic transmission, which, in turn, determines the onset of the neurological symptoms by altering the synaptic transmission in the cerebellar cortex of transgenic animals. This model might be suitable for testing drugs that target activated glial cells in order to reduce CNS inflammation.

  • The expression of prolactin and its cathepsin D-mediated cleavage in the bovine corpus luteum vary with the estrous cycle.
    Publication Date: 01/11/2007, on American journal of physiology. Endocrinology and metabolism
    by Erdmann S, Ricken A, Merkwitz C, Struman I, Castino R, Hummitzsch K, Gaunitz F, Isidoro C, Martial J, Spanel-Borowski K
    DOI: 10.1152/ajpendo.00280.2007

    In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH(2)-terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH(2) was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis.

  • Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways.
    Publication Date: 01/10/2007, on Journal of cardiovascular pharmacology
    by de Nigris F, Balestrieri ML, Williams-Ignarro S, D'Armiento FP, Lerman LO, Byrns R, Crimi E, Palagiano A, Fatigati G, Ignarro LJ, Napoli C
    DOI: 10.1097/FJC.0b013e31812564e4

    Peripheral arterial disease (PAD) is a major health problem, especially when associated with severe hypertension. Administration of autologous bone marrow cells (BMCs) is emerging as a novel intervention to induce neoangiogenesis in ischemic limb models and in patients with PAD. This study evaluates the neovascularization capacity of BMCs alone or in combination with metabolic cotreatment (0.8% vitamin E, 0.05% vitamin C, and 5% of L-arginine) in a rat model of ischemic hindlimbs of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Molecular mechanisms were investigated in bone marrow-derived endothelial progenitor cells (BM-EPC) derived from rats. BMC therapy increased blood flow and capillary densities and Ki67 proliferative marker, and it decreased interstitial fibrosis. These effects were amplified by metabolic cotreatment, an intervention that induces vascular protection at least partly through the nitric oxide (NO)/endothelial nitric oxide synthase (eNOS) pathway, reduction of systemic oxidative stress, and macrophage activation. In addition, BMC therapy alone and, more consistently, in combination with metabolic treatment, ameliorated BM-EPC functional activity via decreased cellular senescence and improved homing capacity by increasing CXCR4-expression levels. These data suggest potential therapeutic effects of autologous BMCs and metabolic treatment in hypertensive PAD patients.

  • Predictors of remission of hyperprolactinaemia after long-term withdrawal of cabergoline therapy.
    Publication Date: 01/09/2007, on Clinical endocrinology
    by Colao A, Di Sarno A, Guerra E, Pivonello R, Cappabianca P, Caranci F, Elefante A, Cavallo LM, Briganti F, Cirillo S, Lombardi G
    DOI: 10.1111/j.1365-2265.2007.02905.x

    Remission rates of 76, 69.5 and 64.3% have been reported in patients with nontumoural hyperprolactinaemia (NTH), microprolactinoma and macroprolactinoma, respectively, 2-5 years after cabergoline (CAB) withdrawal.

  • Energetics of quadruplex-drug recognition in anticancer therapy.
    Publication Date: 01/09/2007, on Current cancer drug targets
    by Pagano B, Giancola C
    DOI:

    Immortality of tumour cells is strictly correlated to telomerase activity. Telomerase is overexpressed in about 85% of tumour cells and maintains telomere length contributing to cell immortalisation, whereas in somatic cells telomeres progressively shorten until cell death occurs by apoptosis. Different drugs can promote telomeric G-rich overhangs which fold into quadruplex structures that inhibit telomerase activity. Detailed studies on drug-quadruplex complexes are essential to understand quadruplex recognition and address drug design. This review will discuss the energetic aspects of quadruplex-drug interactions with a particular attention to physico-chemical methodologies.

  • A novel thrombin binding aptamer containing a G-LNA residue.
    Publication Date: 01/09/2007, on Bioorganic & medicinal chemistry
    by Virno A, Randazzo A, Giancola C, Bucci M, Cirino G, Mayol L
    DOI: 10.1016/j.bmc.2007.06.008

    In this work, we report the solution structure, thermodynamic studies, and the pharmacological properties of a new modified thrombin binding aptamer (TBA) containing a G-LNA residue, namely d(5'-GGTTGGTGTGGTTGg-3'), where upper case and lower case letters represent DNA and LNA residues, respectively. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the three-dimensional structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT, and TT loops. d(5'-GGTTGGTGTGGTTGg-3') is characterized by the same folding of TBA, being two strands parallel to each other and two strands oriented in opposite manner. This led to a syn-anti-syn-anti and anti-syn-anti-syn arrangements of the Gs in the two tetrads. d(5'-GGTTGGTGTGGTTGg-3') possesses an anticoagulant activity, even if decreased with respect to the TBA.