Latest PUBLICATIONS

  • The alpha-to-beta conformational transition of Alzheimer's Abeta-(1-42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding.
    Publication Date: 01/02/2006, on Chembiochem : a European journal of chemical biology
    by Tomaselli S, Esposito V, Vangone P, van Nuland NA, Bonvin AM, Guerrini R, Tancredi T, Temussi PA, Picone D
    DOI: 10.1002/cbic.200500223

    Current views of the role of beta-amyloid (Abeta) peptide fibrils range from regarding them as the cause of Alzheimer's pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of Abeta peptides in soluble form constitutes a basic approach to the design of molecules with "antiamyloid" activity. We have experimentally investigated the conformational path that can lead the Abeta-(1-42) peptide from the native state, which is represented by an alpha helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by beta-sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, beta conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to alpha helices simply by adding the appropriate amount of HFIP. The transition of Abeta-(1-42) from alpha to beta conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N-terminal helix is essentially retained, whereas the shorter C-terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular-dynamics simulations in explicit solvent, which led to the localization of residues that might seed beta conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes.

  • The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting.
    Publication Date: 01/01/2006, on Nucleic acids research
    by Cobucci-Ponzano B, Conte F, Benelli D, Londei P, Flagiello A, Monti M, Pucci P, Rossi M, Moracci M
    DOI: 10.1093/nar/gkl574

    The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.

  • Stem cells and brain cancer.
    Publication Date: 01/01/2006, on Cell death and differentiation
    by Galderisi U, Cipollaro M, Giordano A
    DOI: 10.1038/sj.cdd.4401757

    An increasing body of research is showing that cancers might contain their own stem cells. In fact, cancer cells, like stem cells, can proliferate indefinitely through a deregulated cellular self-renewal capacity. This raises the possibility that some features of tumor cells may be due to cancer stem cells. Stem cell-like cancer cells were isolated from several solid tumors. Now, evidence has shown that brain cancers, such as glioblastomas, medulloblastomas and astrocytomas, also contain cells that may be multipotent neural stem cell-like cells. In this review, we discuss the results of these studies, along with the molecular pathways that could be involved in cancer stem cell physiopathology.

  • Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation.
    Publication Date: 01/01/2006, on Rapid communications in mass spectrometry : RCM
    by Amoresano A, Monti G, Cirulli C, Marino G
    DOI: 10.1002/rcm.2461

    Protein phosphorylation regulates many cellular processes and pathways, such as cell cycle progression, signal transduction cascades and gene expression. Selective detection of phosphopeptides from proteolytic digests is a challenging and highly relevant task in many proteomics applications. Often phosphopeptides are present in small amounts and need selective isolation or enrichment before identification. Here we report a novel approach to label selectively phospho-Ser/-Thr residues by exploiting the features of a novel linear ion trap mass spectrometer. Using dansyl labelling and MS3 fragmentation, we developed a method useful for the large-scale proteomic profiling of phosphorylation sites. The new residues in the sequence were stable and easily identifiable under general conditions for tandem mass spectrometric sequencing.

  • Categorical and coordinate spatial processing in the imagery domain investigated by rTMS.
    Publication Date: 01/01/2006, on Neuropsychologia
    by Trojano L, Conson M, Maffei R, Grossi D
    DOI: 10.1016/j.neuropsychologia.2006.01.017

    Using repetitive transcranical magnetic stimulation (rTMS), we investigated the functional relevance of posterior parietal cortex for categorical and coordinate judgements in the spatial imagery domain. In the coordinate task, subjects were asked to imagine two analogue clock faces based on acoustically presented pairs of times, and to judge at which of the two times the clock hands form the greater angle (mental clock task); in the categorical task subjects were again asked to imagine an analogue clock face showing the time verbally presented by the examiner, but in this case they had to judge whether both hands lay in the half of the clock face cued by an auditorily presented label. We matched the performance of three groups of subjects, two of which received rTMS stimulation over left and right posterior parietal cortex, respectively, while the third group received a sham stimulation. The results showed that right parietal stimulation interfered with the execution of the coordinate task, while left parietal stimulation mainly affected the categorical task, but also reduced the learning effect on the coordinate task. The present findings support the hemispheric specialization of the posterior parietal cortex in different spatial information processing in the imagery domain.

  • Glycolipids from sponges. Part 17. Clathrosides and isoclathrosides, unique glycolipids from the Caribbean sponge Agelas clathrodes.
    Publication Date: 01/01/2006, on Journal of natural products
    by Costantino V, Fattorusso E, Imperatore C, Mangoni A
    DOI: 10.1021/np050331v

    Two families of unique glycolipids, clathrosides A-C (2a-4a) and isoclathrosides A-C (5a-7a) were isolated from the Caribbean sponge Agelas clathrodes. Clathrosides and isoclathrosides are glycosides of a very-long-chain alcohol derived from fatty acids, a new class of glycolipids that appears to be characteristic of marine sponges. The six compounds differ in configuration and in the branching of alkyl chains. Stereostructures of the clathrosides were determined by NMR and CD spectroscopy, mass spectrometry, and chemical degradation. Location of the methyl branch on the proper alkyl chain required an exceptional 1-D TOCSY experiment, in which coherence was transferred through as many as 13 vicinal couplings.

  • High yield synthesis and characterization of phosphorylated recombinant human procathepsin D expressed in mammalian cells.
    Publication Date: 01/01/2006, on Protein expression and purification
    by Démoz M, Castino R, Follo C, Hasilik A, Sloane BF, Isidoro C
    DOI: 10.1016/j.pep.2005.07.024

    We used a vaccinia virus expression system for the production of recombinant human cathepsin D (CD), a lysosomal protease implicated in various patho-physiological processes including cancer, neurodegeneration, and development. The recombinant protein was successfully expressed in various human and non-human cells. It was correctly synthesized as a glycosylated 53 kDa precursor (proCDrec) that reacted with a polyclonal antibody against residues 7-21 of the propeptide sequence. In contrast to the control, in cells infected with the recombinant virus proCDrec was largely secreted into the culture medium, although it contained high-mannose oligosaccharides with uncovered mannose-6-phosphate residues. Intracellular proCDrec was processed into the 48 kDa intermediate single-chain and the 31 plus 13 kDa double-chain forms, however, the processing was slower than in normal cells. A method based on Pepstatin A-affinity chromatography allowed to isolate the recombinant protein from the medium of infected cells. Based on its latency in activity assay at acid pH and on its reactivity with antibodies specific for the N-terminus, the purified protein was judged to be in the inactive precursor form. During incubation at acid pH the purified proCDrec underwent autocatalytic processing and acquired pepstatin A-sensitive enzyme activity, as expected for correctly folded proCD. Antiserum raised in rabbits against proCDrec specifically reacted with human, but not with mouse proCD under non-denaturing conditions. We conclude that our vaccinia virus-directed proCDrec displays structural and functional features resembling those of native human proCD. This system can therefore be exploited for the synthesis of large quantities of human proCD, allowing further studies on the structure and function of this interesting protein.

  • A new modified thrombin binding aptamer containing a 5'-5' inversion of polarity site.
    Publication Date: 01/01/2006, on Nucleic acids research
    by Martino L, Virno A, Randazzo A, Virgilio A, Esposito V, Giancola C, Bucci M, Cirino G, Mayol L
    DOI: 10.1093/nar/gkl915

    The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5'-5' inversion of polarity site, namely d(3'GGT5'-5'TGGTGTGGTTGG3'), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D 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(3'GGT5'-5'TGGTGTGGTTGG3') is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4 degrees C higher than the corresponding unmodified TBA. d(3'GGT5'-5'TGGTGTGGTTGG3') continues to display an anticoagulant activity, even if decreased with respect to the TBA.

  • Carotid arteriotomy induces different temporal gene expression profiles in normotensive and hypertensive rat strains.
    Publication Date: 01/12/2005, on International journal of molecular medicine
    by Forte A, Galderisi U, De Feo M, Quarto C, Renzulli A, Berrino L, Rossi F, Cascino A, Cipollaro M
    DOI:

    Analysis of gene expression profiles in patients or in animal models affected by cardiovascular diseases may provide insight into therapeutic strategies. In this study, 3 rat strains, Wistar Kyoto (WKY), spontaneously hypertensive rats (SHR) and the Milan hypertensive rat strain (MHS), have been investigated to assess the influence of genetic background and/or of hypertension on gene expression in arteriotomy-injured carotid arteries (CAs). Expression profiles of genes, c-myc, AT1, AT2, ETA, ETB, Bcl-2, Bax and Bcl-X, were determined by reverse transcription-polymerase chain reaction (RT-PCR) in the acute phase, from 1 to 48 h, following CA arteriotomy. WKY, SHR and MHS show significant differences in gene expression profiles after CA arteriotomy. c-Myc mRNA is activated earlier and/or to a greater extent in hypertensive strains than in WKY (p<0.05). AT1 mRNA increases in WKY after injury, while it decreases in both SHR and MHS (p<0.05). AT2 shows the opposite behaviour, decreasing in WKY and increasing in hypertensive strains (p<0.05). ETA mRNA decreases in all strains although with different timing and levels, associated with a replacement by ETB mRNA (p<0.05). Bcl-2/Bax ratio gradually decreases in WKY, while it decreases only transiently in SHR and MHS 4 h after injury (p<0.05). Overall data indicate that therapeutic strategies for stenosis prevention should carefully consider the gene expression profile after injury, the genetic background, the kind of vascular trauma and the diseases affecting the animal model or the patient.

  • Subacute demyelinating polyneuropathy in B-cell lymphoma with IgM antibodies against glycolipid GD1b.
    Publication Date: 01/12/2005, on Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology
    by Marfia GA, Pachatz C, Terracciano C, Leone G, Bernardini S, Bernardi G, Massa R
    DOI: 10.1007/s10072-005-0500-z

    Peripheral neuropathy associated with IgM monoclonal gammopathy of unknown significance is a common disorder, while the association of paraproteinaemic neuropathies with haematological malignancies is far less frequent. We report a 76-year-old patient with a subacute and rapidly progressive sensorimotor demyelinating polyneuropathy causing sensory ataxia, painful paraesthesias and marked motor and sensory deficit in four limbs. Monoclonal gammopathy of IgM type associated with a rectal low-grade B-cell non-Hodgkin lymphoma was detected. Research for anti-MAG and antiganglioside autoantibodies including anti-GM1 and anti-GQ1b evidenced a high titre of IgM antibodies against the disialosyl group of GD1b. This is the first report on a paraproteinaemic polyneuropathy with IgM autoantibodies against glycolipid GD1b associated with B-cell lymphoma. The IgM type of these autoantibodies suggests that they represent all or part of the paraprotein produced by lymphoma cells.

  • Thermodynamics and kinetics of PNA-DNA quadruplex-forming chimeras.
    Publication Date: 23/11/2005, on Journal of the American Chemical Society
    by Petraccone L, Pagano B, Esposito V, Randazzo A, Piccialli G, Barone G, Mattia CA, Giancola C
    DOI: 10.1021/ja0545923

    PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to complementary single-strand (DNA or RNA), and the resistance to nuclease and protease degradation. At the same time, the limitations of an oligomer containing all PNA residues, such as low water solubility, self-aggregation, and low cellular uptake, are effectively overcome. Further, PNA-DNA chimeras possess interesting biological properties as antisense agents. We have explored the ability of PNA-DNA chimeric strands to assemble in quadruplex structures. The rate constant for association of the quadruplexes and their thermodynamic properties have been determined by CD spectroscopy and differential scanning calorimetry (DSC). Thermal denaturation experiments indicated higher thermal and thermodynamic stabilities for chimeric quadruplexes in comparison with the corresponding unmodified DNA quadruplex. Singular value decomposition analysis (SVD) suggests the presence of kinetically stable intermediate species in the quadruplex formation process. The experimental results have been discussed on the basis of molecular dynamic simulations. The ability of PNA-DNA chimeras to form stable quadruplex structures expands their potential utility as therapeutic agents.

  • Damicoside from Axinella damicornis: the influence of a glycosylated galactose 4-OH group on the immunostimulatory activity of alpha-galactoglycosphingolipids.
    Publication Date: 17/11/2005, on Journal of medicinal chemistry
    by Costantino V, D'Esposito M, Fattorusso E, Mangoni A, Basilico N, Parapini S, Taramelli D
    DOI: 10.1021/jm050506y

    Alpha-galactoglycosphingolipids (alpha-GalGSLs) are unique immunostimulatory glycosphingolipids from marine sponges. Analysis of the glycosphingolipid composition of the marine sponge Axinella damicornis revealed the presence of a new alpha-GalGSL, damicoside (3a), which is the first alpha-GalGSL with a glycosylated galactose 4-OH group. Structure elucidation of damicoside was performed using spectroscopic and chemical methods. When tested in a spleen cell proliferation assay, 3a exhibited a stimulatory activity comparable to that of agelasphin (2), showing that a free galactose 4-OH group is not essential for the immunostimulatory activity of alpha-GalGSLs and providing a further step toward the complete understanding of their structure-activity relationship.

  • Proteomics of beta2-microglobulin amyloid fibrils.
    Publication Date: 10/11/2005, on Biochimica et biophysica acta
    by Stoppini M, Mangione P, Monti M, Giorgetti S, Marchese L, Arcidiaco P, Verga L, Segagni S, Pucci P, Merlini G, Bellotti V
    DOI: 10.1016/j.bbapap.2005.07.019

    Knowledge on the chemical structure of beta2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of beta2-microglobulin extracted from non-osteotendinous tissues. beta2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to beta2-microglobulin derived from fibrils but is not observed in the beta2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar beta2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1-2% of the intact molecule.

  • Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states.
    Publication Date: 10/11/2005, on Biochimica et biophysica acta
    by Monti M, Amoresano A, Giorgetti S, Bellotti V, Pucci P
    DOI: 10.1016/j.bbapap.2005.09.004

    Amyloid fibrils of patients treated with regular haemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native beta2-m and the truncated DeltaN6beta2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant DeltaN3beta2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native beta2-m and DeltaN3beta2-m shared essentially the same conformation, significative structural differences exist between the native and the DeltaN6beta2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of DeltaN6beta2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the beta2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been proposed.

  • Galphas protein C-terminal alpha-helix at the interface: does the plasma membrane play a critical role in the Galphas protein functionality?
    Publication Date: 01/10/2005, on Journal of peptide science : an official publication of the European Peptide Society
    by Albrizio S, Caliendo G, D'Errico G, Novellino E, Rovero P, D'Ursi AM
    DOI: 10.1002/psc.677

    The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, Galphabetagamma) mediate the signalling process of a large number of receptors, known as G protein-coupled receptors. The C-terminal domain of the heterotrimeric G protein alpha-subunit plays a key role in the selective activation of G proteins by their cognate receptors. The interaction of this domain can take place at the end of a cascade including several successive conformational modifications. Galpha(s)(350-394) is the 45-mer peptide corresponding to the C-terminal region of the Galpha(s) subunit. In the crystal structure of the Galpha(s) subunit it encompasses the alpha4/beta6 loop, the beta6 beta-sheet segment and the alpha5 helix region. Following a previous study based on the synthesis, biological activity and conformational analysis of shorter peptides belonging to the same Galpha(s) region, Galpha(s)(350-394) was synthesized and investigated. The present study outlines the central role played by the residues involved in the alpha4/beta6 loop and beta6/alpha5 loops in the stabilization of the C-terminal Galpha(s)alpha-helix. H(2)O/(2)H(2)O exchange experiments, and NMR diffusion experiments show interesting evidence concerning the interaction between the SDS micelles and the polypeptide. These data prompt intriguing speculations on the role of the intracellular environment/cellular membrane interface in the stabilization and functionality of the C-terminal Galpha(s) region.