Latest PUBLICATIONS

  • Aldehyde suppression of copepod recruitment in blooms of a ubiquitous planktonic diatom.
    Publication Date: 27/05/2004, on Nature
    by Ianora A, Miralto A, Poulet SA, Carotenuto Y, Buttino I, Romano G, Casotti R, Pohnert G, Wichard T, Colucci-D'Amato L, Terrazzano G, Smetacek V
    DOI: 10.1038/nature02526

    The growth cycle in nutrient-rich, aquatic environments starts with a diatom bloom that ends in mass sinking of ungrazed cells and phytodetritus. The low grazing pressure on these blooms has been attributed to the inability of overwintering copepod populations to track them temporally. We tested an alternative explanation: that dominant diatom species impair the reproductive success of their grazers. We compared larval development of a common overwintering copepod fed on a ubiquitous, early-blooming diatom species with its development when fed on a typical post-bloom dinoflagellate. Development was arrested in all larvae in which both mothers and their larvae were fed the diatom diet. Mortality remained high even if larvae were switched to the dinoflagellate diet. Aldehydes, cleaved from a fatty acid precursor by enzymes activated within seconds after crushing of the cell, elicit the teratogenic effect. This insidious mechanism, which does not deter the herbivore from feeding but impairs its recruitment, will restrain the cohort size of the next generation of early-rising overwinterers. Such a transgenerational plant-herbivore interaction could explain the recurringly inefficient use of a predictable, potentially valuable food resource--the spring diatom bloom--by marine zooplankton.

  • Genetic characterization of Pompeii and Herculaneum Equidae buried by Vesuvius in 79 AD.
    Publication Date: 01/05/2004, on Journal of cellular physiology
    by Di Bernardo G, Galderisi U, Del Gaudio S, D'Aniello A, Lanave C, De Robertis MT, Cascino A, Cipollaro M
    DOI: 10.1002/jcp.10461

    DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post-mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals-donkeys, horses, or crossbreeds-were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth.

  • Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis.
    Publication Date: 01/05/2004, on Protein science : a publication of the Protein Society
    by Casbarra A, Birolo L, Infusini G, Dal Piaz F, Svensson M, Pucci P, Svanborg C, Marino G
    DOI: 10.1110/ps.03474704

    A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the beta-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of alpha-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.

  • Stability and structure of telomeric DNA sequences forming quadruplexes containing four G-tetrads with different topological arrangements.
    Publication Date: 27/04/2004, on Biochemistry
    by Petraccone L, Erra E, Esposito V, Randazzo A, Mayol L, Nasti L, Barone G, Giancola C
    DOI: 10.1021/bi0300985

    Telomeres are DNA-protein structures at the ends of eukaryotic chromosomes, the DNA of which comprise noncoding repeats of guanine-rich sequences. Telomeric DNA plays a fundamental role in protecting the cell from recombination and degradation. Telomeric sequences can form quadruplex structures stabilized by guanine quartets. These structures can be constructed from one, two, or four oligonucleotidic strands. Here, we report the thermodynamic characterization of the stability, analyzed by differential scanning calorimetry, of three DNA quadruplexes of different molecularity, all containing four G-tetrads. The conformational properties of these quadruple helices were studied by circular dichroism. The investigated oligomers form well-defined G-quadruplex structures in the presence of sodium ions. Two have the truncated telomeric sequence from Oxytricha, d(TGGGGT) and d(GGGGTTTTGGGG), which form a tetramolecular and bimolecular quadruplex, respectively. The third sequence, d(GGGGTTGGGGTGTGGGGTTGGGG) was designed to form a unimolecular quadruplex. The thermodynamic parameters of these quadruplexes have been determined. The tetramolecular structure is thermodynamically more stable than the bimolecular one, which, in turn, is more stable than the unimolecular one. The experimental data were discussed in light of the molecular-modeling study.

  • Lycopene in association with alpha-tocopherol or tomato lipophilic extracts enhances acyl-platelet-activating factor biosynthesis in endothelial cells during oxidative stress.
    Publication Date: 15/04/2004, on Free radical biology & medicine
    by Balestrieri ML, De Prisco R, Nicolaus B, Pari P, Moriello VS, Strazzullo G, Iorio EL, Servillo L, Balestrieri C
    DOI: 10.1016/j.freeradbiomed.2004.01.014

    Lipophilic compounds contained in tomato can prevent cardiovascular diseases by modulating the atherogenic processes in vascular endothelium mediated by oxidized low-density lipoproteins (LDLs). We investigated the effects of lycopene on the metabolism of platelet-activating factor (PAF) and its much less biologically active acyl analog, acyl-PAF, known to prevent LDL oxidation. Lycopene, or lycopene in association with alpha-tocopherol, or whole tomato lipophilic extracts (containing more than 80% lycopene) were used in experiments in which endothelial cells (ECs) are known to synthesize PAF following H(2)O(2)-induced oxidative stress. The results indicated that in each case H(2)O(2)-stimulated PAF biosynthesis in ECs, which is catalyzed by acetyl-CoA acetyltransferase (AT), appeared strongly inhibited. However, acyl-PAF biosynthesis, which also occurs through the PAF-dependent transacetylase (TA), was significantly increased by lycopene only when it was in association with alpha-tocopherol or with the minor compounds present in the whole lipophilic tomato extract. These findings suggest that alpha-tocopherol or lipophilic compounds present in tomato juice potentiate the effects of lycopene on the modulation of PAF and acyl-PAF biosynthesis in ECs during oxidative stress.

  • Sporadic Creutzfeldt-Jakob disease with MM1-type prion protein and plaques.
    Publication Date: 13/04/2004, on Neurology
    by Puoti G, Limido L, Cotrufo R, Di Fede G, Tagliavini F
    DOI:

  • Tuber borchii fruit body: 2-dimensional profile and protein identification.
    Publication Date: 01/04/2004, on Phytochemistry
    by Pierleoni R, Buffalini M, Vallorani L, Guidi C, Zeppa S, Sacconi C, Pucci P, Amoresano A, Casbarra A, Stocchi V
    DOI: 10.1016/j.phytochem.2004.02.012

    The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified.

  • Fibrillogenesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F.
    Publication Date: 26/03/2004, on The Journal of biological chemistry
    by Sirangelo I, Malmo C, Iannuzzi C, Mezzogiorno A, Bianco MR, Papa M, Irace G
    DOI: 10.1074/jbc.M308207200

    The apomyoglobin mutant W7FW14F forms amyloid-like fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing beta-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

  • Association of a homozygous nonsense mutation in the ABCA4 (ABCR) gene with cone-rod dystrophy phenotype in an Italian family.
    Publication Date: 01/03/2004, on Ophthalmic research
    by Simonelli F, Testa F, Zernant J, Nesti A, Rossi S, Rinaldi E, Allikmets R
    DOI: 10.1159/000076886

    Genetic variation in the ABCA4 (ABCR) gene has been associated with several distinct retinal phenotypes, including Stargardt disease/fundus flavimaculatus (STGD/FFM), cone-rod dystrophy (CRD), retinitis pigmentosa (RP) and age-related macular degeneration. The current model of genotype/phenotype association suggests that patients harboring deleterious mutations in both ABCR alleles would develop RP-like retinal pathology. Here we describe ABCA4-associated phenotypes, including a proband with a homozygous nonsense mutation in a family from Southern Italy. The proband had been originally diagnosed with STGD. Ophthalmologic examination included kinetic perimetry, electrophysiological studies and fluorescein angiography. DNA of the affected individual and family members was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray. A homozygous nonsense mutation 2971G>T (G991X) was detected in a patient initially diagnosed with STGD based on funduscopic evidence, including bull's eye depigmentation of the fovea and flecks at the posterior pole extending to the mid-peripheral retina. Since this novel nucleotide substitution results in a truncated, nonfunctional, ABCA4 protein, the patient was examined in-depth for the severity of the disease phenotype. Indeed, subsequent electrophysiological studies determined severely reduced cone amplitude as compared to the rod amplitude, suggesting the diagnosis of CRD. ABCR400 microarray is an efficient tool for determining causal genetic variation, including new mutations. A homozygous protein-truncating mutation in ABCA4 can cause a phenotype ranging from STGD to CRD as diagnosed at an early stage of the disease. Only a combination of comprehensive genotype/phenotype correlation studies will determine the proper diagnosis and prognosis of ABCA4-associated pathology.

  • Modulation of nurr1 gene expression in mesencephalic dopaminergic neurones.
    Publication Date: 01/03/2004, on Journal of neurochemistry
    by Volpicelli F, Perrone-Capano C, Da Pozzo P, Colucci-D'Amato L, di Porzio U
    DOI:

    The transcription factor/nuclear receptor Nurr1 is essential for the differentiation of midbrain dopaminergic neurones. Here we demonstrate that, during the ontogeny of rat ventral mesencephalon, nurr1 gene expression is developmentally regulated and its levels show a sharp peak between embryonic day E13 and E15, when most dopaminergic neurones differentiate. In addition, in primary cultures from embryonic rat mesencephalon, nurr1 gene follows a temporal pattern of expression comparable to that observed in vivo. We also report that exposure of embryonic mesencephalic cultures to depolarizing stimuli leads to a robust increase in nurr1 mRNA and protein. The depolarizing effect is also detected in mesencephalic cultures enriched in dopaminergic neurones by using a combination of bFGF and Sonic hedgehog. The latter further increases the number of dopaminergic neurones in these 'expanded' cultures, an effect abolished in the presence of anti-Sonic hedgehog antibodies. Our data show that nurr1 gene is highly expressed in midbrain dopaminergic neurones in a sharp temporal window and that its expression is plastic, both in vivo and in vitro. In addition we show that Sonic hedgehog can direct dopaminergic differentiation in proliferating dopaminergic neuroblasts in vitro.

  • Preconditioning-induced cytoprotection in hepatocytes requires Ca(2+)-dependent exocytosis of lysosomes.
    Publication Date: 01/03/2004, on Journal of cell science
    by Carini R, Castino R, De Cesaris MG, Splendore R, Démoz M, Albano E, Isidoro C
    DOI: 10.1242/jcs.00923

    A short period of hypoxia reduces the cytotoxicity produced by a subsequent prolonged hypoxia in isolated hepatocytes. This phenomenon, termed hypoxic preconditioning, is mediated by the activation of adenosine A2A-receptor and is associated with the attenuation of cellular acidosis and Na+ overload normally occurring during hypoxia. Bafilomycin, an inhibitor of the vacuolar H+/ATPase, reverts the latter effects and abrogates the preconditioning-induced cytoprotection. Here we provide evidence that the acquisition of preconditioning-induced cytoprotection requires the fusion with plasma membrane and exocytosis of endosomal-lysosomal organelles. Poisons of the vesicular traffic, such as wortmannin and 3-methyladenine, which inhibit phosphatydilinositol 3-kinase, or cytochalasin D, which disassembles the actin cytoskeleton, prevented lysosome exocytosis and also abolished the preconditioning-associated protection from acidosis and necrosis provoked by hypoxia. Preconditioning was associated with the phosphatydilinositol 3-kinase-dependent increase of cytosolic [Ca2+]. Chelation of free cytosolic Ca2+ in preconditioned cells prevented lysosome exocytosis and the acquisition of cytoprotection. We conclude that lysosome-plasma membrane fusion is the mechanism through which hypoxic preconditioning allows hepatocytes to preserve the intracellular pH and survive hypoxic stress. This process is under the control of phosphatydilinositol 3-kinase and requires the integrity of the cytoskeleton and the rise of intracellular free calcium ions.

  • Targeting duplex DNA with DNA-PNA chimeras? Physico-chemical characterization of a triplex DNA-PNA/DNA/DNA.
    Publication Date: 01/03/2004, on Biopolymers
    by Petraccone L, Erra E, Messere A, Montesarchio D, Piccialli G, De Napoli L, Barone G, Giancola C
    DOI: 10.1002/bip.10599

    Targeting double-stranded DNA with homopyrimidine PNAs results in strand displacement complexes PNA/DNA/PNA rather than PNA/DNA/DNA triplex structures. Not much is known about the binding properties of DNA-PNA chimeras. A 16-mer 5'-DNA-3'-p-(N)PNA(C) has been investigated for its ability to hybridize a complementary duplex DNA by DSC, CD, and molecular modeling studies. The obtained results showed the formation of a triplex structure having similar, if not slightly higher, stability compared to the same all-DNA complex.

  • Glycolipids from sponges. 13. Clarhamnoside, the first rhamnosylated alpha-galactosylceramide from Agelas clathrodes. Improving spectral strategies for glycoconjugate structure determination.
    Publication Date: 20/02/2004, on The Journal of organic chemistry
    by Costantino V, Fattorusso E, Imperatore C, Mangoni A
    DOI: 10.1021/jo034865h

    Reinvestigation of the glycosphingolipid composition of the marine sponge Agelas clathrodes revealed the presence of a new tetraglycosylated alpha-galactoglycosphingolipid (1a), containing an unusual l-rhamnose unit in the sugar head. The structure of the new compound was elucidated using extensive 2D NMR studies. Because of the strong overlapping of the signals of the sugar protons in the (1)H spectrum, (13)C-coupled and (13)C-decoupled phase-sensitive HMQC spectra were used to study the multiplicity of the overlapping signals. In addition, the absolute configuration of sugars was determined using a simple and efficient, yet underutilized CD method.

  • Effects of an 8-bromodeoxyguanosine incorporation on the parallel quadruplex structure [d(TGGGT)]4.
    Publication Date: 07/02/2004, on Organic & biomolecular chemistry
    by Esposito V, Randazzo A, Piccialli G, Petraccone L, Giancola C, Mayol L
    DOI: 10.1039/b314672c

    NMR, molecular dynamics and mechanics calculations, and CD spectroscopy were used to characterise three tetramolecular quadruplex complexes: [d(TG(Br)GGT)](4), [d(TGG(Br)GT)](4) and [d(TGGG(Br)T)](4), where G(Br) indicates an 8-bromoguanine residue. All three quadruplexes are characterised by a 4-fold symmetry with all strands parallel to each other and, differently to what has been observed for other parallel quadruplex structures, with a tetrad (formed by 8-Br-dGs) in a syn conformation. The whole of the data demonstrates that the replacement in turn of different dG residues with 8-Br-dG in the sequence 5[prime or minute]-TGGGT-3[prime or minute] affects the resulting structures in different ways, leading to different CD profiles and thermal stabilities. Particularly, [d(TG(Br)GGT)](4) and [d(TGG(Br)GT)](4) are more stable than the unmodified sequence, whereas [d(TGGG(Br)T)](4) is much less stable than the natural counterpart. The conformational features found in the three quadruplexes might, in principle, amplify the range of applicability of synthetic oligonucleotides as aptamers or catalysts, by providing novel structural motifs with different molecular recognition capabilities from those of native DNA sequences.

  • The regions of the sequence most exposed to the solvent within the amyloidogenic state of a protein initiate the aggregation process.
    Publication Date: 06/02/2004, on Journal of molecular biology
    by Monti M, Garolla di Bard BL, Calloni G, Chiti F, Amoresano A, Ramponi G, Pucci P
    DOI:

    Formation of misfolded aggregates is an essential part of what proteins can do. The process of protein aggregation is central to many human diseases and any aggregating event needs to be prevented within a cell and in protein design. In order to aggregate, a protein needs to unfold its native state, at least partially. The conformational state that is prone to aggregate is difficult to study, due to its aggregating potential and heterogeneous nature. Here, we use a systematic approach of limited proteolysis, in combination with electrospray ionisation mass spectrometry, to investigate the regions that are most flexible and solvent-exposed within the native, ligand-bound and amyloidogenic states of muscle acylphosphatase (AcP), a protein previously shown to form amyloid fibrils in the presence of trifluoroethanol. Seven proteases with different degrees of specificity have been used for this purpose. Following exposure to the aggregating conditions, a number of sites along the sequence of AcP become susceptible to proteolytic digestion. The pattern of proteolytic cleavages obtained under these conditions is considerably different from that of the native and ligand-bound conformations and includes a portion within the N-terminal tail of the protein (residues 6-7), the region of the sequence 18-23 and the position 94 near the C terminus. There is a significant overlap between the regions of the sequence found to be solvent-exposed from the present study and those previously identified to be critical in the rate-determining steps of aggregation from protein engineering approaches. This indicates that a considerable degree of solvent exposure is a feature of the portions of a protein that initiate the process of aggregation.