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D. Barra - Peptide effectors of innate immunity with each other and found that the isomers A and B, which are only weakly active on Gram-negative bacteria, could synergize when combined separately with temporin L, overcoming the bacterial resistance imposed by the LPS protective layer. This effect depends on the length of the LPS sugar chain on the target microorganism. To understand the underlying mechanism, we investigated the effect of LPS from two strains of E. coli with different LPS moiety on the structural organization of temporins, alone and when mixed one with each other. Our data have indicated that the synergistic effect is related to the ability of temporin L to prevent the oligomerization of the A and B isomers, thus allowing their translocation across the bacterial cell wall into the target cytoplasmic membrane. Overall such studies have revealed two important findings: (i) temporins A and B are not active on Gram-negative bacteria, because of their oligomerization when in contact with the outer membrane: their larger size should interfere with the peptide diffusion through the cell wall into the target inner membrane; (ii) the synergistic activity between temporins on Gram-negative bacteria is related to the ability of temporin L to assist A and B in traversing the LPS layer by preventing their oligomerization. This effect is highly dependent on the LPS structure. In addition, we have shown that the same temporin combinations suppress the endotoxin effects of LPS, by inhibiting TNF-α release (considered to be a primary mediator of endotoxemia) from macrophages. Furthermore, by means of spectroscopic and thermodynamic studies, we have demonstrated that the synergism of temporins in the anti-endotoxin activity relies on the peptide’s ability to dissociate LPS aggregates to smaller sized vesicles and that this property is inversely related to the length of the polysaccharide region of LPS. Another interesting family of frog skin AMPs is given by bombinins H, isolated from amphibia of Bombina genus and containing isomers with a single D-amino acid, resulting from a post-translational epimerization of the corresponding L-residue. Indepth studies on the biological activity of these molecules and the underlying mode/s of action have shown that bombinin H2 (IIGPVL- GLVGSALGGLLKKI-NH 2 ) and H4, differing by 114 only the configuration of the second amino acid (an L-isoleucine in H2 and a D-alloisoleucine in H4) display antibacterial and anti-parasitic activities, with a stronger potency and faster killing kinetic of the Damino acid-containing bombinin H4. This peptide was also found to have a higher haemolytic capacity on human erythrocytes. In all cases, the isoforms H2 and H4 were able to perturb the membrane of the target cells, causing leakage of large cytosolic components (e.g. proteins) or diffusion of smaller molecules through local membrane disruptions. We have therefore demonstrated the importance of a single L- to D-epimerization as a new approach developed by nature to modulate not only the bioavailability (e.g. higher solubility) and biostability (i.e. protection from proteolytic degradation) of AMPs, but also their biophysical properties (peptide structure and organization within membranes) and antimicrobial activities. These studies help getting insight into the molecular basis accounting for the quantitative difference in the biological activities of H2 and H4 epimers and the role of the D-amino acid in their different behaviour, and in general to have a deeper understanding of the mechanisms of action of these new potentially useful weapons to fight infections. Selected publications Mangoni ML, Maisetta G, Di Luca M, Marcellini HGL, Esin S, Florio W, Brancatisano FL, Barra D, Campa M, Batoni G. Comparative analysis of bactericidal activity of amphibian peptide analogues against multidrug-resistant nosocomial bacterial strains. Antimicrob Agents Chemother. 2007, 52:85-91. Mangoni ML, Marcellini HGL, Simmaco M. Biological characterization and modes of action of temporins and bombinins H, multiple forms of short and mildly cationic anti-microbial peptides from amphibian skin. J Pept Sci. 2007, 13:603-13. Mangoni ML, Epand RF, Rosenfeld Y, Peleg A, Barra D, Epand RM, Shai Y. Lipopolysaccharide, a key molecule involved in the synergism between temporins in inhibiting bacterial growth and in endotoxin neutralization. J Biol Chem. 2008, 283:22907-17.
P a r t i c i p a n t s : Gustavo Portalone, professor; Cinzia Conti, Paola Goldoni, researchers. C o l l a b o r a t i o n s : <strong>Istituto</strong> Superiore di Sanità, Roma (Prof. Maria Giovanna Quaglia, Dr. Elena Bossù). Report of activity Picornaviruses, in particular enteroviruses (EVs) and rhinoviruses (HRVs), are responsible for several human viral diseases ranging from mild upper respiratory infections to fatal neurological or cardiacbased illnesses. Due to the widespread nature of the diseases associated with picornaviruses and the difficulty of vaccine development for the majority of these viruses, extensive efforts have been expended in the search for effective anti-picornavirus agents. However, despite the in vitro activity of several specific compounds, to date only few drugs have shown efficacy in humans and none have been approved for clinical use. The target of this search is to identify new lead compounds with potent activity, low cytotoxicity and broad spectrum of antipicornavirus activity. The study of the mechanism of action of compounds selected during the antiviral screening is a further aim of the project. In continuation of our search on antipicornavirus activity of flavanoids and related classes of inhibitors, we designed and synthesized new series of (Z)-3-benzylidenechromans, 3-benzylchromans and 3-benzyl-2H-chromenes related to the most active synthetic 3(2H)-isoflavenes and homoisoflavones previously studied by the participant of this project. The antiviral potency of the new synthesized compounds was evaluated against HRVs in a plaque reduction assay, starting from the maximum non-cytotoxic concentration (MNTC). The exis- Principal investigator: Nicoletta Desideri Professor of Drug Analysis Dipartimento di Chimica e Tecnologie del Farmaco Tel: (+39) 06 49913892; Fax: (+39) 06 491491 nicoletta.desideri@uniroma1.it 115 New antimicrobial and antiviral agents - AREA 6 New antipicornavirus flavanoids: synthesis, biological evaluation and structure-activity relationship studies tence of two groups (A and B) of HRVs with contrasting susceptibility for antiviral compounds suggests the selection of at least one serotype from each antiviral group. Group B contains twice as many serotypes as group A, and accounts for five times as many colds as group A serotypes. We utilized HRV 1B and 14 as representative serotypes for group B and A, respectively. With the exception of 3-benzyl-2H-chromene, all chromenes and chromans showed high antiviral activity against HRV 1B within micro or submicromolar range (IC 50s ranging from 0.11 µM to 6.62 µM). Generally, the potent inhibitory activity on HRV 1B coupled with the low cytotoxicity resulted in compounds with high therapeutic index (TI). In contrast, only a modest inhibition of HRV 14 replication was observed up to the MNTC. (Z)-3-(4-chlorobenzylidene)chroman (1) On the basis of both high anti-HRV1B activity and therapeutic index (IC 50 = 0.12 µM and TI = 625), (Z)-3-(4-chlorobenzylidene)chroman (1) was chosen to clarify the mechanism of antiviral action by evaluating the effects produced either on virus particles or multiplication. Initially, the direct effect of 1 on HRV1B infectivity and stability was investigated. Afterwards, the antiviral activity of 1 (12 µM or 36 µM) towards different stages of HRV1B multiplication was investigated under one-step growth conditions. Compound was continuously present during the entire time of virus replication, during virus binding to the cell membrane only or added or removed at different time intervals after virus adsorption in the cold. The overall analysis of data from inactivation and stabilization studies of HRV infectivity are consistent
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Istituto Pasteur Fondazione Cenci B
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Contents Forward by Paolo Amati, P
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Research area 6: New antimicrobial
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REPORT OF ACTIVITY 2007-2008 Boards
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REPORT OF ACTIVITY 2007-2008 Dario
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REPORT OF ACTIVITY 2007-2008 Meetin
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AREA1 Molecular biology of microorg
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P. Amati - Role of the early phases
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A. Boffi - Escherichia coli strains
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D. De Biase - The glutamate-based a
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A. Faggioni - Control of latency an
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Paola Londei - Translational regula
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A structural biology study of schis
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P a r t i c i p a n t s : Luigi Lem
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P a r t i c i p a n t s : Gianni Pr
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P a r t i c i p a n t s : Lucia Nen
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P a r t i c i p a n t s : Alessandr
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P a r t i c i p a n t s : Maurizio
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AREA3 Molecular genetics of eukaryo
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F. Ascenzioni - Development and ana
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P. Ballario - Molecular machines an
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I. Bozzoni - RNA-RNA and RNA-protei
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P. Caiafa - Crosstalk between poly(
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G. Camilloni - DNA topoisomerases a
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P. Costantino - The role of the DAG
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M. d’Erme - Epigenetic modificati
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P. Dimitri - Drosophila melanogaste
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L. Fabiani - DNA replication mechan
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C. Falcone - New tools in decipheri
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L. Frontali - New complex mitochond
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M. Gatti - The role of membrane tra
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A. Giacomello - Biology and physiol
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A. Gulino - Regulation of the Hedge
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M. Levrero - Histone Acetyltransfer
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F. Mangia - Molecular regulation of
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A. Musarò - Study of the molecular
Page 76 and 77: R. Negri - Role of the presumptive
Page 78 and 79: S. Pimpinelli - Heterochromatin, ge
Page 80 and 81: M. Stefanini - Biological character
Page 82 and 83: M. Tripodi - Molecular mechanisms o
Page 84 and 85: C. Turano - Roles of ERp57 in DNA r
Page 86 and 87: G. Zardo - Identification of novel
Page 89 and 90: P a r t i c i p a n t s : Carlo Tra
Page 91 and 92: P a r t i c i p a n t s : Giulia De
Page 93 and 94: P a r t i c i p a n t s : Giovanna
Page 95 and 96: P a r t i c i p a n t s : Francesco
Page 97 and 98: P a r t i c i p a n t s : Bruno Bot
Page 99 and 100: P a r t i c i p a n t s : Sergio Fu
Page 101 and 102: P a r t i c i p a n t s : Maria Egl
Page 103 and 104: P a r t i c i p a n t s : Stefano C
Page 105: AREA5 Cellular and molecular immuno
Page 108 and 109: V. Barnaba - Innovative strategies
Page 110 and 111: R. Foà - Potential role of miRNAS
Page 112 and 113: E. Piccolella - Analysis of the mol
Page 114 and 115: A. Santoni - Molecular mechanism un
Page 116 and 117: I. Screpanti - Analysis of the role
Page 118 and 119: R. Sorrentino - The role of HLA-B27
Page 120 and 121: L. Tuosto - CD28 co-stimulatory mol
Page 122 and 123: E. Ziparo - The role of Toll Like R
Page 125: Peptide effectors of innate immunit
Page 129 and 130: P a r t i c i p a n t s : Roberta C
Page 131 and 132: P a r t i c i p a n t s : Giovanna
Page 133 and 134: P a r t i c i p a n t s : Livia Di
Page 135 and 136: P a r t i c i p a n t s : Marino Ar
Page 137: AREA7 Biology of malaria and other
Page 140 and 141: Della Torre - Petrarca - Bionomical
Page 142 and 143: A. Tramontano - Computational Analy
Page 145 and 146: Year 2007 Barile L, Chimenti I, Gae
Page 147 and 148: Puglisi R, Bevilacqua A, Carlomagno
Page 149 and 150: BIBLIOGRAPHY Conigliaro A, Colletti
Page 151 and 152: BIBLIOGRAPHY Muscolini M, Cianfrocc
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