The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. http://http://dspace.unitus.it:80 2013-05-26T07:58:28Z 2013-05-26T07:58:28Z Buonocore, Francesco Randelli, Elisa Casani, Daniela Costantini, Susan Facchiano, Angelo M. Scapigliati, Giuseppe Stet, Rene http://hdl.handle.net/2067/1357 2011-06-28T14:20:28Z 2006-12-31T23:00:00Z

Title: Molecular cloning, differential expression and 3D structural analysis of the MHC class-II β chain from sea bass (Dicentrarchus labrax L.) Authors: Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Costantini, Susan; Facchiano, Angelo M.; Scapigliati, Giuseppe; Stet, Rene Abstract: The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II b chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28 bp 50-UTR and a 478 bp 30-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II b chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4 h showed very little difference in the Dila-DAB expression, but after 24 h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1b (ranging from 0 to 100 ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

2006-12-31T23:00:00Z Scala, Viviana Casani, Daniela Costantini, Susan Facchiano, Angelo M. Mazzini, Massimo Scapigliati, Giuseppe Buonocore, Francesco http://hdl.handle.net/2067/1663 2011-06-27T10:53:59Z 2007-12-31T23:00:00Z

Title: T cell receptor beta chain from sea bream (Sparus aurata): molecular cloning, expression and modelling of the complexes with MHC class I Authors: Scala, Viviana; Casani, Daniela; Costantini, Susan; Facchiano, Angelo M.; Mazzini, Massimo; Scapigliati, Giuseppe; Buonocore, Francesco Abstract: The T cell receptor is a fundamental mediator of the adaptive immune responses, since TR on T cells recognize foreign structures (peptides derived from processed antigens) bound to the major histocompatibility complex (MHC) on APC cells. In the present study, we report the cloning of six TRB chains cDNA sequences from gilthead sea bream (Sparus aurata), a fish of high economical impact in South Mediterranean aquaculture. The V-BETA domains have the canonical features of known teleost and mammalian TR V-BETA domains and have been divided in four different subgroups. A multiple alignment of the six sea bream TRB chains with other known TRB sequences was assembled and showed the conservation of the four cysteine residues involved in disulphide bonds and of some amino acids with an important role in the assembly and signalling of the TR /CD3 complex. Real-time PCR analysis was used to investigate TRB basal expression, that was maximum in the thymus followed by gut, and TRB in vitro expression after stimulation with LPS or PHA-L at 4 and 24 h (only the 4 h stimulation with LPS gave an significant effect). Moreover, the 3D structures of sea bream TRB chains and MHC-I were predicted by homology modelling with the final aim to investigate the interaction surface in the V-BETA/MHC-I complexes. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

2007-12-31T23:00:00Z Buonocore, Francesco Randelli, Elisa Bird, Steve Secombes, Chris J. Facchiano, Angelo M. Costantini, Susan Scapigliati, Giuseppe http://hdl.handle.net/2067/1535 2011-06-28T14:02:48Z 2006-12-31T23:00:00Z

Title: Interleukin-10 expression by real-time PCR and homology modelling analysis in the European sea bass (Dicentrarchus labrax L.) Authors: Buonocore, Francesco; Randelli, Elisa; Bird, Steve; Secombes, Chris J.; Facchiano, Angelo M.; Costantini, Susan; Scapigliati, Giuseppe Abstract: In this paper we investigate the interleukin-10 (IL-10) expression and 3D modelling of the European sea bass (Dicentrarchus labrax L.). IL-10 is a regulatory cytokine that has been intensively studied in mammals and has been found mainly involved in the suppression or deactivation of activated immune responses. The full-length sea bass IL-10 cDNA consists of 1015 bp and is translated in a predicted molecule of 187 amino acids. A multiple alignment of this peptide with other known sequences showed the conservation of fundamental IL-10 family characteristics. Expression analysis by real-time PCR in control fish showed a high basal expression in the head kidney (HK), followed by gut and brain. In vitro treatment of HK leucocytes with LPS showed an intense increase of IL-10 expression at 4 h and a significant decrease at 24 h, with PHA-L no differences were evidenced. A 3D model for sea bass IL-10 was obtained by accurate homology procedures and putative interaction residues with the IL-10 receptor described. The results suggested that sea bass IL-10 could be involved in anti-inflammatory responses, but further experiments are needed to assess its importance in response to pathogenic agents, vaccinations and immunostimulants. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

2006-12-31T23:00:00Z Bruni, Natalia Di Maro, Antimo Costantini, Susan Chambery, Angela Facchiano, Angelo M. Ficca, Anna Grazia Parente, Augusto Poerio, Elia http://hdl.handle.net/2067/1786 2011-06-30T15:16:15Z 2008-12-31T23:00:00Z

Title: Redesigning the reactive site loop of the wheat subtilisin/chymotrypsin inhibitor (WSCI) by site-directed mutagenesis. A protein–protein interaction study by affinity chromatography and molecular modeling Authors: Bruni, Natalia; Di Maro, Antimo; Costantini, Susan; Chambery, Angela; Facchiano, Angelo M.; Ficca, Anna Grazia; Parente, Augusto; Poerio, Elia Abstract: A site-directed mutagenesis strategy was employed to obtain four mutants of wheat subtilisin/chymotrypsin inhibitor (WSCI), with the aim to produce inactive forms of this protein. The mutants were expressed in Escherichia coli as fusion proteins and, after the tag removal, were purified to homogeneity and characterized, for their interaction with the inhibited enzymes, by affinity chromatography and molecular modeling.; Al fine di ottenere forme inattive di un inibitore proteico della subtilisina e della chimotripsina, è stata impiegata una strategia di mutagenesi sito-specifica. Sono stati così ottenuti quattro prodotti di espressione eterologa (mutanti dell’inibitore WSCI) che dopo purificazione sono stati sottoposti ad ampia caratterizzazione. In particolare, loro interazione con alcuni enzimi proteolitici d’interesse è stata studiata mediante cromatografia di affinità e modellamento molecolare. Description: L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com

2008-12-31T23:00:00Z Costantini, Susan Buonocore, Francesco Facchiano, Angelo M. http://hdl.handle.net/2067/1662 2011-06-28T14:22:03Z 2010-12-31T23:00:00Z

Title: Molecular modelling of co-receptor CD8αα and its complex with MHC class I and T-cell receptor in sea bream (Sparus aurata) Authors: Costantini, Susan; Buonocore, Francesco; Facchiano, Angelo M. Abstract: T-cells are the main actors of cell-mediated immune defence; they recognize and respond to peptide antigens associated with MHC class I and class II molecules. In this paper, we investigated by molecular modelling methods in the teleost sea bream (Sparus aurata) the interaction among the molecules of the tertiary complex CD8/MHC-I/TCR, which determines the T-cell-mediated immunological response to foreign molecules. First, we predicted the three-dimensional structure of CD8αα dimer and MHC-I, and, successively, we simulated the CD8αα/MHC-I complex. Finally, the 3D structure of the CD8/MHC-I/TCR complex was simulated in order to investigate the possible changes that can influence TCR signalling events. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

2010-12-31T23:00:00Z Buonocore, Francesco Randelli, Elisa Casani, Daniela Guerra, Laura Picchietti, Simona Costantini, Susan Facchiano, Angelo M. Zou, Jun Secombes, Chris J. Scapigliati, Giuseppe http://hdl.handle.net/2067/1665 2011-06-27T20:38:24Z 2010-12-31T23:00:00Z

Title: A CD4 homologue in sea bass (Dicentrarchus labrax): Molecular characterization and structural analysis Authors: Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Guerra, Laura; Picchietti, Simona; Costantini, Susan; Facchiano, Angelo M.; Zou, Jun; Secombes, Chris J.; Scapigliati, Giuseppe Abstract: CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell coreceptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4 h and a decrease after 24 h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

2010-12-31T23:00:00Z Casani, Daniela Randelli, Elisa Costantini, Susan Facchiano, Angelo M. Zou, Jun Martin, Sam Secombes, Chris J. Scapigliati, Giuseppe Buonocore, Francesco http://hdl.handle.net/2067/1680 2011-06-28T14:19:13Z 2008-12-31T23:00:00Z

Title: Molecular characterisation and structural analysis of an interferon homologue in sea bass (Dicentrarchus labrax L.) Authors: Casani, Daniela; Randelli, Elisa; Costantini, Susan; Facchiano, Angelo M.; Zou, Jun; Martin, Sam; Secombes, Chris J.; Scapigliati, Giuseppe; Buonocore, Francesco Abstract: The interferons (IFNs) are a large family of soluble cytokines involved in the immune response against viral pathogens. Three families of IFNs have been identified in mammals (type I, type II and type III) and, recently, homologues of type I and type II genes have been found in various teleost fish species. In this paper we report the cloning of a cDNA encoding an type I IFN molecule from sea bass (Dicentrarchus labrax L.), its expression analysis and gene structure and, finally, its 3D structure obtained by template-based modelling. The sea bass IFN cDNA consists of 1047 bp that translates in one reading frame to give the entire molecule containing 185 amino acids. The analysis of the sequence revealed the presence of a putative 22 amino acid signal peptide, two cysteine residues and three potential N-glycosylation sites. The sea bass IFN gene contains four introns as with other type I IFN teleost genes, except medaka that contains three introns. Real time PCR was performed after poly I:C stimulation of DLEC cell line to investigate the expression of sea bass IFN and Mx and an induction was observed for both genes. The predicted 3D structure of sea bass IFN is characterized by an “all-alpha” domain that shows an “up-down bundle” architecture made of six helices (ABB’CDE). The two cysteine residues present in the sequence (i.e. Cys23 and Cys126) are in a position and at a distance that suggest the possible formation of a disulfide bridge that may stabilize the structure. Our results will give the opportunity to investigate more in detail antiviral immune responses in sea bass and add to studies on the evolution of the IFN system in teleosts and Vertebrates more generally. Description: L'articolo è disponibile sul site dell'editore http://www.sciencedirect.com/

2008-12-31T23:00:00Z Buonocore, Francesco Randelli, Elisa Viviana, Scala Belardinelli, Maria Cristina Casani, Daniela Facchiano, Angelo M. Costantini, Susan Scapigliati, Giuseppe http://hdl.handle.net/2067/1681 2011-06-28T10:44:24Z 2008-12-31T23:00:00Z

Title: Evolution of cell-mediated immune defences: cloning and structural characterisation of the T cell receptor beta chain from the icefish Chionodraco hamatus (Perciformes: Channichthyidae) Authors: Buonocore, Francesco; Randelli, Elisa; Viviana, Scala; Belardinelli, Maria Cristina; Casani, Daniela; Facchiano, Angelo M.; Costantini, Susan; Scapigliati, Giuseppe Abstract: Studies on the immune defence system of Antarctic fishes are of interest to investigate the presence of evolutionary adaptations to low temperature seawater. In the present paper, primers designed to conserved regions of TR , the antigen receptor expressed by T lymphocytes and mediator of main adaptive immune responses, were used for the homology cloning of the icefish Chionodraco hamatus TR  chain cDNA. The full length cDNA consists of 1219 nucleotides that translates in a single reading frame to give a predicted 304-amino acid molecule. The sequence shows highest nucleotide and amino acid identity with the sea bream (Sparus aurata) TR  . A multiple alignment with other known TR  molecules evidenced the presence of conserved amino acid residues involved in structural and functional domains within most teleost species. Real-time PCR analysis was used to investigate TR  basal expression, that resulted maximal in the thymus, followed by gills and spleen. Finally, the 3D structure of the icefish TR  was predicted by homology modelling and compared to the closest homologous Sparus aurata, a fish Teleost living in more temperate environmental conditions. The comparison of these two models suggests that the icefish TR  may be more flexible, as a consequence of a less compact structure and a lower number of H-bonds. Description: L'articolo è disponibile sul sito dell'editore http://www.tandfonline.com/

2008-12-31T23:00:00Z Buonocore, Francesco Randelli, Elisa Casani, Daniela Guerra, Laura Picchietti, Simona Costantini, Susan Facchiano, Angelo M. Zou, Jun Secombes, Chris J. Scapigliati, Giuseppe http://hdl.handle.net/2067/1565 2011-06-28T09:57:19Z 2007-12-31T23:00:00Z

Title: CD4 homologue in sea bass (Dicentrarchus labrax): molecular characterization and structural analysis Authors: Buonocore, Francesco; Randelli, Elisa; Casani, Daniela; Guerra, Laura; Picchietti, Simona; Costantini, Susan; Facchiano, Angelo M.; Zou, Jun; Secombes, Chris J.; Scapigliati, Giuseppe Abstract: CD4 is a transmembrane glycoprotein fundamental for cell-mediated immunity. Its action as a T cell coreceptor increases the avidity of association between a T cell and an antigen-presenting cell by interacting with portions of the complex between MHC class II and TR molecules. In this paper we report the cDNA cloning, expression and structural analysis of a CD4 homologue from sea bass (Dicentrarchus labrax). The sea bass CD4 cDNA consists of 2071 bp that translates in one reading frame to give the entire molecule containing 480 amino acids. The analysis of the sequence shows the presence of four putative Ig-like domains and that some fundamental structural features, like a disulphide bond in domain D2 and the CXC signalling motif in the cytoplasmic tail, are conserved from sea bass to mammals. Real-time PCR analysis showed that very high levels of CD4 mRNA transcripts are present in thymus, followed by gut and gills. In vitro stimulation of head kidney leukocytes with LPS and PHA-L gave an increase of CD4 mRNA levels after 4 h and a decrease after 24 h. Homology modelling has been applied to create a 3D model of sea bass CD4 and to investigate its interaction with sea bass MHC-II. The analysis of the 3D complex between sea bass CD4 and sea bass MHC-II suggests that the absence of a disulfide bond in the CD4 D1 domain could make this molecule more flexible, inducing a different conformation and affecting the binding and the way of interaction between CD4 and MHC-II. Our results will add new insights into the sea bass T cell immune responses and will help in the identification of T cell subsets in teleost fishes to better understand the evolution of cell-mediated immunity from fish to mammals. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

2007-12-31T23:00:00Z Costantini, Susan Facchiano, Angelo M. Randelli, Elisa Casani, Daniela Scapigliati, Giuseppe Buonocore, Francesco http://hdl.handle.net/2067/1686 2011-06-28T09:41:33Z 2010-12-31T23:00:00Z

Title: 3D Modelling of three pro-inflammatory molecules in selected fish species. Authors: Costantini, Susan; Facchiano, Angelo M.; Randelli, Elisa; Casani, Daniela; Scapigliati, Giuseppe; Buonocore, Francesco Abstract: The inflammatory response is the reaction of all Metazoan organisms to pathogen invasion that initiates when pathogenderived molecules are recognized by specific pattern recognition receptors expressed mainly on cells of the innate immune system. The successive expression of pro-inflammatory cytokines and chemokines limits pathogen spread, and attracts and activates immune cells to help in the elimination of the invaders. In this paper we focused on the analyses of the 3D structures of three pro-inflammatory molecules (interleukin-1􀀂, tumor necrosis factor-􀀁, interleukin-8) from selected Teleost fish species (Oncorhynchus mykiss, Dicentrarchus labrax, Chionodraco hamatus) generated using as template models those of experimental homologous proteins. These structures were discussed with the aim to investigate the differences between them and mammalian counterparts and, moreover, to verify the presence of the structural requirements for their biological activities, known mainly in mammals. Description: L'articolo è disponibile sul sito dell'editore http://www.benthamscience.com/index.htm

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