Latest PUBLICATIONS

  • Bacterial expression and conformational analysis of a chemosensory protein from Schistocerca gregaria.
    Publication Date: 01/09/2001, on European journal of biochemistry
    by Picone D, Crescenzi O, Angeli S, Marchese S, Brandazza A, Ferrara L, Pelosi P, Scaloni A
    DOI:

    Chemosensory proteins (CSPs) are a class of small, soluble proteins present at high concentrations in chemosensory organs of different insect species. Several pieces of evidence suggest their involvement in carrying chemical messages from the environment to chemosensory receptors. However, a structural description of the mechanism of delivery has not been reported. In order to provide the first detailed conformational characterization of these molecules, we cloned a specific isoform (CSP-sg4) from Schistocerca gregaria and expressed it in Escherichia coli. The product was obtained with yields of more than 20 mg per L of culture, all in its soluble form. The recombinant protein was identical to the native one with respect to pairing of the disulfide bridges, aggregative state and secondary structure elements. Structural investigations revealed a significantly stable polypeptide with respect to variations in temperature and acidity. CD analysis, preliminary NMR data and secondary structure prediction pointed to a correctly folded structure where helical regions and loops are alternated in a similar fashion as that observed for other classes of odorant- and pheromone-binding proteins presenting no sequence similarity to CSPs.

  • Regionalized neurofilament accumulation and motoneuron degeneration are linked phenotypes in wobbler neuromuscular disease.
    Publication Date: 01/08/2001, on Neurobiology of disease
    by Pernas-Alonso R, Perrone-Capano C, Volpicelli F, di Porzio U
    DOI: 10.1006/nbdi.2001.0403

    Abnormal neurofilament aggregates are pathological hall-mark of most neurodegenerative diseases, although their pathogenic role remains unclear. Increased expression of medium neurofilament (NFM) is an early molecular marker of wobbler mouse, an animal model of motoneuron disease. In the wr/wr, a vacuolar neuronal degeneration (VND) starts at 15 days postnatally, selectively in cervical spinal cord and brain stem motoneurons. Here we show that nfm gene hyperexpression is restricted to the aforementioned motoneurons and is specific for wr mutation. NF proteins accumulate in wr/wr before VND. wr/+ mice, which are asymptomatic, show intermediate NF accumulation between wr/wr and +/+ littermates, suggesting a gene dosage dependence of the wobbler pathology. Altogether our data indicate that NF hyperexpression and regionalized motoneuron degeneration are linked to the wr mutation, although with a still unknown relationship to the mutant gene activity.

  • Expression of protein kinase C beta1 confers resistance to TNFalpha- and paclitaxel-induced apoptosis in HT-29 colon carcinoma cells.
    Publication Date: 15/07/2001, on International journal of cancer
    by Cesaro P, Raiteri E, Démoz M, Castino R, Baccino FM, Bonelli G, Isidoro C
    DOI: 10.1002/ijc.1314

    The expression of different protein kinase C (PKC) isoenzymes has been shown to vary with proliferation rates, differentiation or apoptosis in normal colon crypts. In addition, the activity of some PKC isoenzymes appears to be reduced in colorectal cancer. The aim of the present work was to determine whether modulation of PKC expression would affect the susceptibility of a p53-defective colon carcinoma cell line to different apoptotic treatments. HT-29 cells exhibited sensitivity to paclitaxel (Taxol) and tumor necrosis factor alpha (TNFalpha) in a dose- and time-dependent manner but were relatively resistant to etoposide. Inhibition of PKC activity augmented the susceptibility of HT-29 cells to apoptosis, and phorbol ester induction of PKC reduced such susceptibility. Transfected HT-29(PKC) cells, hyper-expressing the beta1 isoform of PKC, were less sensitive to TNFalpha and paclitaxel than the normal counterpart. The present data 1) indicate that the expression of PKC influences the susceptibility of HT-29 colon cancer cells to apoptotic drugs apparently regardless of their mechanism of action, and 2) suggest paclitaxel as a potential candidate for the treatment of colon cancer, possibly in association with inhibitors of PKC (alpha and beta) at doses not cytotoxic per se.

  • Carnitine protects the molecular chaperone activity of lens alpha-crystallin and decreases the post-translational protein modifications induced by oxidative stress.
    Publication Date: 01/07/2001, on FASEB journal : official publication of the Federation of American Societies for Experimental Biology
    by Peluso G, Petillo O, Barbarisi A, Melone MA, Reda E, Nicolai R, Calvani M
    DOI:

  • 1H and 15N sequential assignment and secondary structure of the monomeric N67D mutant of bovine seminal ribonuclease.
    Publication Date: 01/07/2001, on Journal of biomolecular NMR
    by Crescenzi O, Carotenuto A, D'Ursi AM, Tancredi T, D'Alessio G, Avitabile F, Picone D
    DOI:

  • Physico-chemical studies on DNA triplexes containing an alternate third strand with a non-nucleotide linker.
    Publication Date: 12/06/2001, on International journal of biological macromolecules
    by Giancola C, Petraccone L, Pieri M, De Napoli L, Montesarchio D, Piccialli G, Barone G
    DOI:

    Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)m(pyrimidine)m-3'. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine-pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson-Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3'-3' phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P = phosphate and X = 1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.

  • Assignment of the complete disulphide bridge pattern in the human recombinant follitropin beta-chain.
    Publication Date: 01/06/2001, on Biological chemistry
    by Amoresano A, Orrù S, Siciliano RA, De Luca E, Napoleoni R, Sirna A, Pucci P
    DOI: 10.1515/BC.2001.120

    The chemical assessment of the complete disulphide bridge pattern in the beta-chain of human recombinant follicotropin (betaFSH) was accomplished by integrating classical biochemical methodologies with mass spectrometric procedures. A proteolytic strategy consisting of a double digestion of native betaFSH using the broad-specificity protease subtilisin first, followed by trypsin, was employed. The resulting peptide mixture was directly analysed by FAB-MS, leading to the assignment of the first three disulphide bridges. The remaining S-S bridges were determined by HPLC fractionation of the proteolytic digest followed by ESMS analysis of the individual fractions. The pattern of cysteine couplings in betaFSH was determined as: Cys3-Cys5l, Cys17-Cys66, Cys20-Cys104, Cys28-Cys82, Cys32-Cys84 and Cys87-Cys94, confirming the arrangement inferred from the crystal structure of the homologous betaCG. A subset of the S-S bridge pattern comprising Cys3-Cys51, Cys28-Cys82 and Cys32-Cys84 constitutes a cysteine knot motif similar to that found in the growth factor superfamily.

  • Local synthesis of nuclear-encoded mitochondrial proteins in the presynaptic nerve terminal.
    Publication Date: 01/06/2001, on Journal of neuroscience research
    by Gioio AE, Eyman M, Zhang H, Lavina ZS, Giuditta A, Kaplan BB
    DOI: 10.1002/jnr.1096

    One of the central tenets in neuroscience has been that the protein constituents of distal compartments of the neuron (e.g., the axon and nerve terminal) are synthesized in the nerve cell body and are subsequently transported to their ultimate sites of function. In contrast to this postulate, we have established previously that a heterogeneous population of mRNAs and biologically active polyribosomes exist in the giant axon and presynaptic nerve terminals of the photoreceptor neurons in squid. We report that these mRNA populations contain mRNAs for nuclear-encoded mitochondrial proteins to include: cytochrome oxidase subunit 17, propionyl-CoA carboxylase (EC 6.4.1.3), dihydrolipoamide dehydrogenase (EC 1.8.1.4), and coenzyme Q subunit 7. The mRNA for heat shock protein 70, a chaperone protein known to be involved in the import of proteins into mitochondria, has also been identified. Electrophoretic gel analysis of newly synthesized proteins in the synaptosomal fraction isolated from the squid optic lobe revealed that the large presynaptic terminals of the photoreceptor neuron contain a cytoplasmic protein synthetic system. Importantly, a significant amount of the cycloheximide resistant proteins locally synthesized in the terminal becomes associated with mitochondria. PCR analysis of RNA from synaptosomal polysomes establishes that COX17 and CoQ7 mRNAs are being actively translated. Taken together, these findings indicate that proteins required for the maintenance of mitochondrial function are synthesized locally in the presynaptic nerve terminal, and call attention to the intimacy of the relationship between the terminal and its energy generating system. J. Neurosci. Res. 64:447-453, 2001. Published 2001 Wiley-Liss, Inc.

  • Trains of sleep sequences are indices of learning capacity in rats.
    Publication Date: 08/04/2001, on Behavioural brain research
    by Piscopo S, Mandile P, Montagnese P, Cotugno M, Giuditta A, Vescia S
    DOI:

    In previous work dealing with the identification of four sleep sequences (SS-->W, SS-->PS, SS-->TS-->W and SS-->TS-->PS) in the baseline session of adult male Wistar rats [Mandile P, Vescia S, Montagnese P, Romano F, Giuditta A. Characterization of transition sleep episodes in baseline EEG recordings of adults rats, Physiol Behav 1996;60:1435-1439], we have shown that those containing an intervening episode of transition sleep (TS) strongly correlate with the number of avoidances scored the following day [Vescia S, Mandile P, Montagnese P, Romano F, Cataldo G, Cotugno M, Giuditta A. Baseline transition sleep and associated sleep episodes are related to the learning ability of rats, Physiol Behav 1996;60:1513-152]. More recently, clusters of sleep sequences (trains) separated by waking intervals longer than 60 s have been identified in the baseline session of the same rats [Piscopo S, Mandile P, Montagnese P, Cotugno M, Giuditta A, Vescia S. Identification of trains of sleep sequences in adult rats, Behav Brain Res, this volume], and distinguished in homogeneous or mixed trains according to the presence of a single sleep sequence or more than one sequence. Mixed trains have been further separated into trains containing the SS-->TS-->W sequence (+TSW trains) and trains lacking it (-TSW trains). Analysis of the distribution of variables of baseline trains (and of their sleep sequences and components) among fast learning (FL), slow learning (SL), or non-learning (NL) rats, indicates that variables of +TSW trains prevail in FL rats, while variables of -TSW trains prevail in NL rats. In addition, variables of +TSW trains correlate with the number of avoidances of the training session, while variables of -TSW trains do not significantly correlate, or show inverse correlations. Interestingly, sleep sequences such as SS-->W or SS-->TS-->W show direct or inverse correlations with avoidances depending on whether they are included in +TSW trains or in -TSW trains. The data are interpreted to suggest that the outcome of brain operations performed during a sleep sequence may selectively condition the appearance of later sequences within a time interval shorter than a given threshold. An analogous mechanism may be responsible for the aggregation of sleep components in sleep sequences.

  • Clinical trials of a new class of therapeutic agents: antisense oligonucleotides.
    Publication Date: 01/04/2001, on Expert opinion on emerging drugs
    by Galderisi U, Cipollaro M, Cascino A
    DOI: 10.1517/14728214.6.1.69

    Antisense oligodeoxynucleotides (ODNs) are short stretches of DNA complementary to a target mRNA. The ODNs selectively hybridise to their complementary RNA by Watson-Crick base pairing rules. In theory, the use of antisense ODNs provides a method to specifically inhibit the intracellular expression of any disorder whose genetic aetiology is well known. For this reason, researchers thought that if antisense drugs proved to be so specific there would be no side effects. However, toxicity-related problems arose in initial animal studies of antisense drugs in the early 1990s and since then companies have been using these compounds cautiously. In order to be useful therapeutically, an ODN must (a) exhibit reasonable stability in the physiological environment, (b) be taken up and retained in adequate quantities by the target cells, (c) specifically bind target mRNA with high affinity, (d) have an acceptable therapeutic ratio, free of unwanted toxic and non-specific side effects and (e) be easily synthesised in sufficient quantities to allow clinical use. Most of these criteria have already been met by ODNs recently used in this way. This review describes certain therapeutic applications of antisense techniques currently under investigation in oncology, haematopathology and inflammatory diseases.

  • Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteins.
    Publication Date: 01/04/2001, on Journal of peptide science : an official publication of the European Peptide Society
    by Picone D, Rivieccio A, Crescenzi O, Caliendo G, Santagada V, Perissutti E, Spisani S, Traniello S, Temussi PA
    DOI: 10.1002/psc.320

    Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.

  • Genetics of diabetic retinopathy.
    Publication Date: 01/03/2001, on Seminars in ophthalmology
    by Simonelli F, Testa F, Bandello F
    DOI:

  • pRb2/p130 gene overexpression induces astrocyte differentiation.
    Publication Date: 01/03/2001, on Molecular and cellular neurosciences
    by Galderisi U, Melone MA, Jori FP, Piegari E, Di Bernardo G, Cipollaro M, Cascino A, Peluso G, Claudio PP, Giordano A
    DOI: 10.1006/mcne.2000.0949

    There are many data on the activity of the RB gene in neural differentiation and apoptosis, but the role of pRb2/p130 in neuronal and glial maturation has been far less investigated. To elucidate the role of pRb2/p130 in astrocyte development we overexpressed this protein in astrocytoma and normal astrocyte cultures by adenoviral-mediated gene transfer. In astrocytoma cells, p130/RB2 overexpression resulted in a significant reduction of cell growth and in an increased G(0)/G(1) cell population. We did not observe any induction of programmed cell death as determined by TUNEL reaction. Interestingly, pRb2/p130 overexpression induced astrocyte differentiation. Astrocyte cell cycle arrest and differentiation seemed to proceed through a way distinct from the p53 pathway.

  • 17-beta estradiol elicits an autocrine leiomyoma cell proliferation: evidence for a stimulation of protein kinase-dependent pathway.
    Publication Date: 01/03/2001, on Journal of cellular physiology
    by Barbarisi A, Petillo O, Di Lieto A, Melone MA, Margarucci S, Cannas M, Peluso G
    DOI: 10.1002/1097-4652(2000)9999:999<000::AID-JCP1040>3.0.CO;2-E

    The mechanism by which estradiol (E2) acts on cell proliferation is still unclear. In this paper, we report the results of a series of experiments in an attempt to elucidate the effector pathway(s) involved in coupling the E2 receptors binding to cellular growth response in leiomyoma cells (LSMC). Under conditions of E2-dependent growth, E2 treatment of LSMC triggers rapid and transient activation of the MAP-kinase pathway. Interestingly, we demonstrate that the early downstream signal transduction events determined by E2-stimulation in quiescent LSMC, including the rapid protein tyrosine phosphorylation of a subset of intracellular proteins, such GAP, PI-3-K, and PLCgamma, and the concomitant activation of ancillary protein kinases, are related to E2-induced PDGF secretion. Moreover, we identify the PDGF, alone or in association with other growth factors, as the main growth factor involved in the proliferation response of LSMC to E2 stimulation. The addition of neutralizing antibodies anti-PDGF was able to inhibit the mitogenic activity present in LSMC conditioned media samples. On the other hand, E2 did not affect the constitutive expression as well as the ligand affinity of PDGF receptors on LSMC plasmamembrane. Cell treatment with the antiestrogen ICI 182780 correlate both with a perturbation of E2-induced transductional circuit and with the disappearance of the mitogenic factor, PDGF, in LSMC conditioned media; the latter therefore, represents the main autocrine mediator of cell growth modulation, upregulated by E2 and down-regulated by antiestrogenic compound. Our experiments suggest that growth factor secretion is an initial and integral part of the signaling events mediated by the estradiol receptors, not related, at least in part, to E2 transcriptional modulation.

  • Differential regulation of three catalytic activities of platelet-activating factor (PAF)-dependent transacetylase.
    Publication Date: 01/03/2001, on Archives of biochemistry and biophysics
    by Lee T, Malone B, Longobardi L, Balestrieri ML
    DOI:

    We have previously established that PAF-dependent transacetylase (TA) purified to apparent homogeneity from rat kidney membranes and cytosol contains three separate catalytic activities, namely PAF lysophospholipid transacetylase (TAL), PAF sphingosine transacetylase (TAS), and PAF acetylhydrolase (AH). In the present investigation, we studied the biochemical factors and mechanism(s) that differentially regulate these three TA activities of the purified enzymes. We found that only the TAS activity of the TA purified from the membranes was stimulated by phosphatidyl-serine (PS) with optimal concentration of activation occurring at 25 microM. Other acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP), are partially effective, while diacylglycerol and free fatty acids had no effect on the TAS activity. PS exerted its effect on the TAS activity through the increases of both Km and Vmax. In addition, N-ethylmalimide (NEM) and dithiobis-(2-nitro-5-thiobenzoic acid) (DTNB) strongly inhibited the TAS activity and partially decreased the TAL and AH activities of the purified membrane enzyme in a dose-dependent manner. The addition of PS, but not by its substrate, sphingosine, could prevented the inhibition by NEM on the basal level of TAS. On the other hand, the inhibition of TAL by NEM and DTNB were partially protected by the substrate, lysoplasmalogens. Furthermore, PAF fully protects the inhibition of AH, partially protects the inhibition of TAL, and does not protect the inhibition of TAS by NEM. These results suggested that the three individual catalytic activities of TA have different dependencies on the thiol-containing residue(s) of the enzyme, i.e., cysteine. Furthermore, the nonresponsiveness of the purified cytosolic TAS to PS activation is consistent with our previous notions that membrane and cytosolic TA are posttranslationally distinct.