Latest PUBLICATIONS

  • Interaction of porphyrin with G-quadruplex structures.
    Publication Date: 01/01/2005, on Nucleosides, nucleotides & nucleic acids
    by Erra E, Petraccone L, Esposito V, Randazzo A, Mayol L, Ladbury J, Barone G, Giancola C
    DOI:

    Isothermal titration calorimetry (ITC) is a sensitive technique for probing bimolecular processes and can provide direct information about the binding affinity and stoichiometry and the key thermodynamic parameters involved. ITC has been used to investigate the interaction of the ligand H2TMPyP to the two DNA quadruplexes, [d(AGGGT)]4 and [d(TGGGGT)]. Analysis of the ITC data reveals that porphyrin/quadruplex binding stoichiometry under saturating conditions is 1:2 for [d(AGGGT)]4 and 2:1 for [d(TGGGGT)], respectively.

  • Protein oligomerization modulates raft partitioning and apical sorting of GPI-anchored proteins.
    Publication Date: 22/11/2004, on The Journal of cell biology
    by Paladino S, Sarnataro D, Pillich R, Tivodar S, Nitsch L, Zurzolo C
    DOI: 10.1083/jcb.200407094

    An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein-protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.

  • The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1.
    Publication Date: 19/11/2004, on The Journal of biological chemistry
    by Viggiani A, Siani L, Notomista E, Birolo L, Pucci P, Di Donato A
    DOI: 10.1074/jbc.M406243200

    Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)-C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of (H199N)-C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.

  • 2000 Year-old ancient equids: an ancient-DNA lesson from pompeii remains.
    Publication Date: 15/11/2004, on Journal of experimental zoology. Part B, Molecular and developmental evolution
    by Di Bernardo G, Del Gaudio S, Galderisi U, Cipollaro M
    DOI: 10.1002/jez.b.21017

    Ancient DNA extracted from 2000 year-old equine bones was examined in order to amplify mitochondrial and nuclear DNA fragments. A specific equine satellite-type sequence representing 3.7%-11% of the entire equine genome, proved to be a suitable target to address the question of the presence of aDNA in ancient bones. The PCR strategy designed to investigate this specific target also allowed us to calculate the molecular weight of amplifiable DNA fragments. Sequencing of a 370 bp DNA fragment of mitochondrial control region allowed the comparison of ancient DNA sequences with those of modern horses to assess their genetic relationship. The 16S rRNA mitochondrial gene was also examined to unravel the post-mortem base modification feature and to test the status of Pompeian equids taxon on the basis of a Mae III restriction site polymorphism.

  • Relationships between constructional and visuospatial abilities in normal subjects and in focal brain-damaged patients.
    Publication Date: 01/11/2004, on Journal of clinical and experimental neuropsychology
    by Trojano L, Fragassi NA, Chiacchio L, Izzo O, Izzo G, Di Cesare G, Cristinzio C, Grossi D
    DOI: 10.1080/13803390490515522

    We tested 125 normal subjects and 24 right and 22 left focal brain-damaged patients (RBD and LBD) on the Rey figure copying test and on a battery of perceptual and representational visuospatial tasks, in search of relationships between constructional and visuospatial abilities. Selected RBD and LBD were not affected by severe aphasia, unilateral spatial neglect or general intellectual defects. Both RBD and LBD showed defective performances on the constructional task with respect to normal subjects. As regards visuospatial tasks, both patient groups scored lower than normal subjects in judging angle width and mentally assembling geometrical figures; moreover, RBD, but not LBD, achieved scores significantly lower than healthy controls in judging line orientation and analyzing geometrical figures. Post-hoc comparisons did not reveal any significant differences between RBD and LBD. Multiple regression analysis showed that visuospatial abilities correlate with accuracy in copying geometrical drawings in normal subjects and in RBD, but not in LBD. From a theoretical perspective, these findings support the idea that visual perceptual and representational abilities do play a role in constructional skills.

  • Spatial transpositions across tasks and response modalities: exploring representational allochiria.
    Publication Date: 01/10/2004, on Neurocase
    by Lepore M, Conson M, Ferrigno A, Grossi D, Trojano L
    DOI: 10.1080/13554790490892275

    We describe a neglect patient who showed systematic transpositions of left-sided items onto the right side in clock drawing. When the patient had to write single hours on blank clock dials he again showed allochiria, while he copied single spatial locations without transpositions. The patient also showed a variable number of spatial transpositions on imaginal tasks with well known and novel material acquired through visual modality and on controlled constructional tasks, independently from response modality (verbal, graphic or motor). From this basis, we argued that spatial transpositions may derive from an impairment of the mental representation of space. Moreover, we speculated that such errors may result from cognitive conflict between different sources of information.

  • Cathepsin D released by lactating rat mammary epithelial cells is involved in prolactin cleavage under physiological conditions.
    Publication Date: 01/10/2004, on Journal of cell science
    by Lkhider M, Castino R, Bouguyon E, Isidoro C, Ollivier-Bousquet M
    DOI: 10.1242/jcs.01396

    The 16 kDa prolactin fragment arises from partial proteolysis of the native 23 kDa prolactin pituitary hormone. The mammary gland has been involved in this processing, although it has not been clarified whether it occurs in stroma or epithelial cells or extracellularly. Also, the processing enzyme has not been defined yet. Here we show that the incubation medium of stroma-deprived mammary acini from lactating rat contains an enzymatic activity able to cleave, in a temperature- and time-dependent fashion, the 23 kDa prolactin to generate a 16 kDa prolactin detectable under reducing conditions. This cleavage was not impaired in the presence of hirudin, a thrombin inhibitor, but strongly weakened in the presence of pepstatin A, a cathepsin D inhibitor. Cathepsin D immuno-depletion abolished the capability of acini-conditioned medium to cleave the 23 kDa prolactin. Brefeldin A treatment of acini, a condition that largely abolished the apical secretion of milk proteins, did not impair the secretion of the enzymatically active single chain of cathepsin D. These results show that mature cathepsin D from endosomes or lysosomes is released, likely at the baso-lateral site of mammary epithelial cells, and that a cathepsin D-dependent activity is required to effect, under physiological conditions, the cleavage of 23 kDa prolactin in the extracellular medium. This is the first report demonstrating that cathepsin D can perform a limited proteolysis of a substrate at physiological pH outside the cell.

  • Local Synthesis of Presynaptic RNA in Squid Optic Lobe Slices.
    Publication Date: 01/10/2004, on The Biological bulletin
    by Giuditta A, Eyman M, Cefaliello C, Ferrara E, Kaplan BB, Lavina ZS, De Stefano R
    DOI: 10.1086/BBLv207n2p156a

  • PrP(C) association with lipid rafts in the early secretory pathway stabilizes its cellular conformation.
    Publication Date: 01/09/2004, on Molecular biology of the cell
    by Sarnataro D, Campana V, Paladino S, Stornaiuolo M, Nitsch L, Zurzolo C
    DOI: 10.1091/mbc.E03-05-0271

    The pathological conversion of cellular prion protein (PrP(C)) into the scrapie prion protein (PrP(Sc)) isoform appears to have a central role in the pathogenesis of transmissible spongiform encephalopathies. However, the identity of the intracellular compartment where this conversion occurs is unknown. Several lines of evidence indicate that detergent-resistant membrane domains (DRMs or rafts) could be involved in this process. We have characterized the association of PrP(C) to rafts during its biosynthesis. We found that PrP(C) associates with rafts already as an immature precursor in the endoplasmic reticulum. Interestingly, compared with the mature protein, the immature diglycosylated form has a different susceptibility to cholesterol depletion vs. sphingolipid depletion, suggesting that the two forms associate with different lipid domains. We also found that cholesterol depletion, which affects raft-association of the immature protein, slows down protein maturation and leads to protein misfolding. On the contrary, sphingolipid depletion does not have any effect on the kinetics of protein maturation or on the conformation of the protein. These data indicate that the early association of PrP(C) with cholesterol-enriched rafts facilitates its correct folding and reinforce the hypothesis that cholesterol and sphingolipids have different roles in PrP metabolism.

  • Role of RB and RB2/P130 genes in marrow stromal stem cells plasticity.
    Publication Date: 01/08/2004, on Journal of cellular physiology
    by Jori FP, Napolitano MA, Melone MA, Cipollaro M, Cascino A, Giordano A, Galderisi U
    DOI: 10.1002/jcp.20026

    Marrow stromal cells (MSCs) are stem-like cells having a striking somatic plasticity. In fact, besides differentiating into mesenchymal lineages (bone, cartilage, and fat), they are capable of differentiating into neurons and astrocytes in vitro and in vivo. The RB and RB2/P130 genes, belonging to the retinoblastoma gene family, play a key role in neurogenesis, and for this reason, we investigated their role in neural commitment and differentiation of MSCs. In MSCs that were either uncommitted or committed toward neural differentiation, we ectopically expressed RB and RB2/P130 genes and analyzed their role in regulating the cell cycle, apoptosis and differentiation. In uncommitted MSCs, the activity of RB and RB2/P130 appeared limited to negatively regulating cell cycle progression, having no role in apoptosis and differentiation (toward either mesenchymal or neural lineages). On the other hand, in MSCs committed toward the neural phenotype, both RB and RB2/P130 reduced cell proliferation rate and affected the apoptotic process. RB protected differentiating cells from programmed cell death. On the contrary, RB2/P130 increased the percentage of cells in apoptosis. All of these activities were accomplished mainly in an HDAC-independent way. The retinoblastoma genes also influenced differentiation in neural committed MSCs. RB2/P130 contributes mainly to the induction of generic neural properties, while RB triggers cholinergic differentiation. These differentiating activities are HDAC-dependent. Our research shows that there is a critical temporal requirement for the RB genes during neuronal differentiation of MSCs: they are not required for cell commitment but play a role in the maturation process. For the above reasons, RB and RB2/P130 may have a role in neural differentiation but not in neural determination.

  • Nerve terminals of squid photoreceptor neurons contain a heterogeneous population of mRNAs and translate a transfected reporter mRNA.
    Publication Date: 01/08/2004, on The European journal of neuroscience
    by Gioio AE, Lavina ZS, Jurkovicova D, Zhang H, Eyman M, Giuditta A, Kaplan BB
    DOI: 10.1111/j.1460-9568.2004.03538.x

    It is now well established that the distal structural/functional domains of the neuron contain 2a diverse population of mRNAs that program the local synthesis of protein. However, there is still a paucity of information on the composition and function of these mRNA populations in the adult nervous system. To generate empirically, hypotheses regarding the function of the local protein synthetic system, we have compared the mRNAs present in the squid giant axon and its parental cell bodies using differential mRNA display as an unbiased screen. The results of this screen facilitated the identification of 31 mRNAs that encoded cytoskeletal proteins, translation factors, ribosomal proteins, molecular motors, metabolic enzymes, nuclear-encoded mitochondrial mRNAs, and a molecular chaperone. Results of cell fractionation and RT-PCR analyses established that several of these mRNAs were present in polysomes present in the presynaptic nerve terminal of photoreceptor neurons, indicating that these mRNAs were being actively translated. Findings derived from in vitro transfection studies established that these isolated nerve terminals had the ability to translate a heterologous reporter mRNA. Based upon these data, it is hypothesized that the local protein synthetic system plays an important role in the maintenance/remodelling of the cytoarchitecture of the axon and nerve terminal, maintenance of the axon transport and mRNA translation systems, as well as contributing to the viability and function of the local mitochondria.

  • Chemical diversity of bioactive marine natural products: an illustrative case study.
    Publication Date: 01/07/2004, on Current medicinal chemistry
    by Costantino V, Fattorusso E, Menna M, Taglialatela-Scafati O
    DOI:

    The marine environment contains a number of plants, animals and micro organisms, which, due to the unique adaptations to their habitat, elaborate a wide diversity of natural products with specific bioactivities. These products provide a rich source of chemical diversity that can be used to design and develop new potentially useful therapeutic agents. The huge variety of the structures present in marine organisms has been illustrated through the case study of the sponge Plakortis simplex, whose chemical analysis, started in our laboratories about ten years ago, revealed an incredible variety and abundance of secondary metabolites. The obtained results have been presented with the intention of drawing some conclusions of general relevance. Particularly, the problem of the limited availability of natural compounds for both structural and preliminary pharmacological studies has been discussed, this issue becoming a serious obstacle when the pharmacological research reaches a more advanced stage. Furthermore, the origin of the chemodiversity in Plakortis simplex and, in general, in marine invertebrates has been discussed; in this respect, the possible cooperative role of symbiotic micro-organisms in the biosynthesis of the varied metabolic content typical of these organisms has been considered.

  • The Shp-1 and Shp-2, tyrosine phosphatases, are recruited on cell membrane in two distinct molecular complexes including Ret oncogenes.
    Publication Date: 01/07/2004, on Cellular signalling
    by Incoronato M, D'Alessio A, Paladino S, Zurzolo C, Carlomagno MS, Cerchia L, de Franciscis V
    DOI: 10.1016/j.cellsig.2004.01.002

    The Shp-2 and Shp-1 non-transmembrane tyrosine phosphatases display different and even opposing effects on downstream signaling events initiated by Ret activation. By using rat pheochromocytoma-derived PC12 cells, here we studied the interactions of Shp-2 and Shp-1 with two activated mutants of Ret receptor, Ret(C634Y) and Ret(M918T). Each of these mutated receptors causes inheritance of distinct cancer syndromes, multiple endocrine neoplasia (MEN) type 2A and type 2B, respectively. We show that: (i) both Shp-1 and Shp-2 are associated to a multiprotein complex that includes Ret mutants; (ii) the Shp-1-Ret complexes are distinct from Shp-2-Ret complexes, and these complexes are differently distributed inside and outside lipid rafts; (iii) constitutively activated Ret proteins neither directly bind to nor are substrates of these phosphatases. Our results well support the evidence that Ret complexes within and outside rafts mediate distinct biological functions, and indicate that the presence of either Shps participates to determine such functions.

  • Linkage of proton binding to the thermal dissociation of triple helix complex.
    Publication Date: 01/07/2004, on Biophysical chemistry
    by Petraccone L, Erra E, Mattia CA, Fedullo V, Barone G, Giancola C
    DOI: 10.1016/j.bpc.2004.01.004

    The effects of cytosine protonation on the thermodynamic properties of parallel pyrimidine motif DNA triplex were investigated and characterized by different techniques, such as circular dichroism (CD), ultraviolet spectroscopy (UV) and differential scanning calorimetry (DSC). A thermodynamic model was developed which, by linking the cytosine ionization equilibrium to the dissociation process of the triplex, is able to rationalize the experimental data and to reproduce the pH dependence of the free energy, enthalpy and entropy changes associated with the triplex formation. The results are useful to systematically introduce the effect of pH in a more general model able to predict the stability of DNA triplexes on the basis of the sequence alone.

  • Left on the right or viceversa: a case of "alternating" constructional allochiria.
    Publication Date: 01/06/2004, on Cortex; a journal devoted to the study of the nervous system and behavior
    by Grossi D, Di Cesare G, Trojano L
    DOI:

    We describe a patient with an ischemic right frontal lesion and mild left neglect who showed a systematic tendency to transpose drawings on one side of the page, which varied depending on the starting point (left or right) of his graphic productions. When not specifically cued, the patient started to draw in the ipsilesional (right) side and tended to show allochiria on the right, but occasionally, or under specific instructions, the patient started drawing from the left side and then showed a complete reversion of his spatial transpositions. To clarify the basic mechanisms underlying such a peculiar constructional phenomenon, we performed a series of experimental investigations, including extended copying tasks, a clock-marking test (to mark the position of single hours on a clock-face), and a line bisection task with progressive left-toright or right-to-left stimulus presentation. Findings suggested that "alternating" allochiria in copying and drawing from memory tasks is an epiphenomenon of a basic inability to move attention and action away from the starting point of graphic productions. The present case study, contrasted with observations on other brain-damaged patients, demonstrates that allochiria may have different neuro-cognitive bases and offers new insights for theoretical interpretations of unilateral spatial neglect.