http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Sat, 25 May 2013 20:37:44 GMT 2013-05-25T20:37:44Z http://hdl.handle.net/2067/1842 Title: Proteomics and Transcriptomics Investigation on longissimus Muscles in Large White and Casertana Pig Breeds Authors: Murgiano, Leonardo; D'Alessandro, Angelo; Egidi, Maria Giulia; Crisa, Alessandra; Prosperini, Gianluca; Timperio, Anna Maria; Zolla, Lello Abstract: Consumer complaints against the blandness of modern lean meat and the frequent reference to the more strongly flavored meat that was available years ago have prompted reconsideration of high fatdepositing typical pig breeds. Casertana and Large White pig breeds are characterized by a different tendency toward fat accumulation as they exhibit opposite genetic and physiological traits with respect to the energy metabolism. These physiological differences were investigated in longissimus lumborum muscles through proteomics (2-DE, MS/MS) and microarray approaches. Data were analyzed for pathway and network analyses, as well as GO term enrichment of biological functions. As a result, Casertana showed a greater amount of proteins involved in glycolitic metabolism and mainly rely on fast-mobilizable energy sources. Large White overexpressed cell cycle and skeletal muscle growth related genes. Metabolic behavior and other implications are discussed. Description: L'articolo è disponibile sul sito dell'editore http://pubs.acs.org/ Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1842 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1479 Title: Changes in neuronal response to ischemia in retinas with genetic alterations of somatostatin receptor expression Authors: Catalani, Elisabetta; Cervia, Davide; Martini, Davide; Bagnoli, Paola; Simonetti, Elisa; Timperio, Anna Maria; Casini, Giovanni Abstract: Ischemia is a primary cause of neuronal death in retinal diseases. The repertoire of expressed transmitter receptors would determine the neurons’ responses to ischemic damage, and peptidergic receptors may be involved. With a new in vitro model of the ischemic mouse retina, we investigated whether an altered expression of somatostatin receptors could modulate retinal responses to ischemia. We used retinas of somatostatin receptor 1 (sst1) knock out (KO) mice, where sst2 are over-expressed and over-functional, and of sst2 KO mice. TUNEL analysis of ischemic retinas showed a marked reduction of cell death in sst1 KO retinas, while there were no differences between wild-type (WT) and sst2 KO retinas. In addition, caspase-3 mRNA expression was also reduced in sst1 KO as compared to WT retinas. An immunohistochemical analysis demonstrated that different cell populations responded differently to the ischemic insult, and that the persistence of some immunohistochemical markers was greater in sst1 KO than in WT or in sst2 KO retinas. In particular, rod bipolar cell survival was markedly improved in sst1 KO retinas, while it was dramatically decreased in sst2 KO retinas. Furthermore, consistent with a role of glutamate excitotoxicity in ischemia-induced neuronal death, retinal glutamate release was observed to increase under ischemic conditions, but this increase was significantly reduced in sst1 KO retinas. These observations demonstrate that an increased presence of functional sst2 protects against retinal ischemia, thus implementing the background for the use of sst2 analogs in therapies of retinal diseases such as glaucoma or diabetic retinopathy. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Sun, 31 Dec 2006 23:00:00 GMT http://hdl.handle.net/2067/1479 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1488 Title: Modulation of the neuronal response to ischemia by somatostatin analogues in wild-type and knock-out mouse retinas Authors: Cervia, Davide; Martini, Davide; Ristori, Davide; Catalani, Elisabetta; Timperio, Anna Maria; Bagnoli, Paola; Casini, Giovanni Abstract: Somatostatin acts at five G protein-coupled receptors, sst1-sst5. In mouse ischemic retinas, the overexpression of sst2 (as in sst1 knock-out mice) results in reduction of cell death and glutamate release. Here, we reported that, in wild-type retinas, somatostatin, the multireceptor ligand pasireotide and the sst2 agonist octreotide decreased ischemia-induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst2 with cyanamide. In sst2 over-expressing ischemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti-ischemic effect of somatostatin agonists in the presence of sst2 over-expression, we tested sst2 desensitisation due to internalisation or altered receptor function. We observed that: i) sst2 was not internalised, ii) among G protein-coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischemia, iii) both GRK1 and RGS1 were downregulated by octreotide in wild-type ischemic retinas, iv) octreotide down-regulated GRK1 but not RGS1 in sst2 over-expressing ischemic retinas. These results demonstrate that sst2 activation protects against retinal ischemia. However, in the presence of sst2 over-expression sst2 is functionally desensitised by agonists, possibly due to sustained RGS1 levels. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1488 2007-12-31T23:00:00Z http://hdl.handle.net/2067/1848 Title: Comparative proteomics and transcriptomics analyses of livers from two different Bos taurus breeds: “Chianina and Holstein Friesian” Authors: Timperio, Anna Maria; D'Alessandro, Angelo; Pariset, Lorraine; D'Amici, Gian Maria; Valentini, Alessio; Zolla, Lello Abstract: The Holstein Friesian and Chianina cattle breeds are representative of extreme selection for milk and meat traits, respectively, with significant changes in metabolism resulting from human selection over the past centuries. In the present study, we wanted to assess whether selection for different purposes has had a measurable effect on liver metabolism through a comparison of the protein and gene expression profiles of the two breeds. We applied 2-DE in order to identify proteins which were differentially expressed in the livers of the two breeds and relate them to different liver functions. We expected to find that only a small number of proteins would be differentially expressed, due to the relatively short phylogenetic distance between these cattle breeds. Nonetheless, thirty nine differentially-expressed proteins were characterized between Chianina and Holstein Friesian, out of a total of 560 ± 57 spots that matched. Microarray analyses evidenced the differential expression of 167 genes (148 for the Holstein Friesian and 19 for the Chianina). Despite being closely related at the genetic level, the disparity of the proteomic and transcriptomic profiles of these two breeds allows us to perform pathway analysis thus to pinpoint proteins whose expression might render the latter capable of greater milk production, or proteins involved in altered thermoregulatory ability or hormone production. On the other hand, we found proteins and gene transcripts in Chianina, not expressed in Holstein, which, upon interaction pathway analysis, were mainly involved in anabolic pathways. In brief, our integrated study provides molecular evidences to support the physiological differences between Holstein and Chianina cattle breeds. Description: L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1848 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1843 Title: Recombinant clotting factor VIII concentrates: Heterogeneity and high-purity evaluation Authors: D'Amici, Gian Maria; Timperio, Anna Maria; Gevi, Federica; Grazzini, Giuliano; Zolla, Lello Abstract: Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advates, Helixate NexGens and Refactos), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGens showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refactos showed glutathione S-transferase and b-lactamase, whereas Advates apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants. Description: L'artcoo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1843 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1887 Title: Comparative proteomics and transcriptomics analyses of livers from two different Bos taurus breeds: “Chianina and Holstein Friesian” Authors: Timperio, Anna Maria; D'Alessandro, Angelo; Pariset, Lorraine; D'Amici, Gian Maria; Valentini, Alessio; Zolla, Lello Abstract: The Holstein Friesian and Chianina cattle breeds are representative of extreme selection for milk and meat traits, respectively, with significant changes in metabolism resulting from human selection over the past centuries. In the present study, we wanted to assess whethe selection for different purposes has had a measurable effect on liver metabolism through a comparison of the protein and gene expression profiles of the two breeds. We applied 2-DE in order to identify proteins which were differentially expressed in the livers of the two breeds and relate them to different liver functions. We expected to find that only a small number of proteins would be differentially expressed, due to the relatively short phylogenetic distance between these cattle breeds. Nonetheless, thirty nine differentially expressed proteins were characterized between Chianina and Holstein Friesian, out of a total of 560 ± 57 spots that matched. Microarray analyses evidenced the differential expression of 167 genes (148 for the Holstein Friesian and 19 for the Chianina). Despite being closely related at the genetic level, the disparity of the proteomic and transcriptomic profiles of these two breeds allows us to perform pathway analysis thus to pinpoint proteins whose expression might render the latter capable of greater milk production, or proteins involved in altered thermoregulatory ability or hormone production. On the other hand, we found proteins and gene transcripts in Chianina, not expressed in Holstein, which, upon interaction pathway analysis, were mainly involved in anabolic pathways. In brief, our integrated study provides molecular evidences to support the physiological differences between Holstein and Chianina cattle breeds. Tue, 05 Apr 2011 22:00:00 GMT http://hdl.handle.net/2067/1887 2011-04-05T22:00:00Z http://hdl.handle.net/2067/1844 Title: Comparison among plasma-derived clotting Factor VIII by using Authors: Timperio, Anna Maria; Gevi, Federica; Grazzini, Giuliano; Vaglio, Stefania; Zolla, Lello Abstract: Background. Deficiency or dysfunction of coagulation factor VIII (FVIII) is the underlying cause of haemophilia A. Haemophilic patients are at present treated with plasma-derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) in order to correct their clotting deficiency. pdFVIII concentrates are exclusively produced from human plasma upon pooling from multiple donors. It is not know whether the presence of excess of other plasma proteins, in addition to von Willebrand factor, could stimulate untoward immune responses in the recipient. Thus, information regarding the presence of contaminants in commercial products is of concern. Materials and Methods. Two commercially available pdFVIII concentrates were characterized through SDS-PAGE and mass spectrometry Emoclot® and Beriate® . Results. The components of two pdFVIII products considered in this study were well identified by mass spectrometry analysis, in both cases we found abundant components coming from blood plasma, and some other contaminants. Only in Beriate® we also found truncated form of pdFVIII. Conclusion. The two pdFVIII examined showed the presence of vWF, Fibrinogen in excess, and other substances that could be considered as contaminants or impurities. Description: L'articolo è disponibile sul sito dell'editore http://www.bloodtransfusion.it Thu, 31 Dec 2009 23:00:00 GMT http://hdl.handle.net/2067/1844 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1845 Title: Functional effect of somatostatin receptor 1 activation on synaptic transmission in the mouse hyppocampus Authors: Cammalleri, Maurizio; Martini, Davide; Timperio, Anna Maria; Bagnoli, Paola Abstract: Somatostatin-14 (SRIF) co-localizes with GABA in the hippocampus and regulates neuronal excitability. A role of SRIF in the control of hippocampal activity has been proposed, although the exact contribution of each SRIF receptor (sst1– sst5) in mediating SRIF action requires some clarification. We used hippocampal slices of wild-type and sst1 knockout (KO) mice and selective pharmacological tools to provide conclusive evidence for a role of sst1 in mediating SRIF inhibition of synaptic transmission. With single- and double-label immunohistochemistry, we determined the distribution of sst1 in hippocampal slices and we quantified sst1 colocalization with SRIF. With electrophysiology, we found that sst1 activation with CH-275 inhibited both the NMDA- and the a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated responses. Results from sst1 KO slices confirmed the specificity of CH-275 effects; sst1 activation did not affect the inhibitory transmission which was in contrast increased by sst4 activation with L-803,087 in both wild-type and sst1 KO slices. The AMPA-mediated responses were increased by L-803,087. Functional interaction between sst1 and sst4 is suggested by the finding that their combined activation prevented the CH-275-induced inhibition of AMPA transmission. The involvement of pre-synaptic mechanisms in mediating inhibitory effects of sst1 on excitatory transmission was demonstrated by the finding that CH-275 (i) increased the paired-pulse facilitation ratio, (ii) did not influence the AMPA depolarization in the presence of tetrodotoxin, and (iii) inhibited glutamate release induced by epileptiform treatment. We conclude that SRIF control of excitatory transmission through an action at sst1 may represent an important contribution to the regulation of hippocampal activity. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Wed, 31 Dec 2008 23:00:00 GMT http://hdl.handle.net/2067/1845 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1880 Title: Comparison of Milk Fat Globule Membrane (MFGM) Proteins of Chianina and Holstein Cattle Breed Milk Samples Through Proteomics Methods Authors: Murgiano, Leonardo; Timperio, Anna Maria; Zolla, Lello; Bongiorni, Silvia; Valentini, Alessio; Pariset, Lorraine Abstract: Identification of proteins involved in milk production is important to understand the biology of lactation. Many studies have advanced the understanding of mammary function and milk secretion, but the critical molecular mechanisms implicated in milk fat secretion is still incomplete. Milk Fat Globules are secreted from the apical surface of the mammary cells, surrounded by a thin membrane bilayer, the Milk Fat Globule Membrane (MFGM), formed by proteins which have been suggested to be holesterolemia-lowering factors, inhibitors of cancer cell growth, vitamin binders, bactericidal, suppressors of multiple sclerosis. Using a proteomic approach, we compared MFGM from milk samples of individuals belonging to two different cattle breeds, Chianina and Holstein,representative of selection for milk and meat traits, respectively. We were able to isolate some of the major MFGM proteins in the examined samples and to identify differences between the protein fractions of the two breeds. We detected differences in the amount of proteins linked to mammary gland development and lipid droplets formation, as well as host defence mechanisms. We have shown that proteomics is a suitable, unbiased method for the study of milk fractions proteins and a powerful tool in nutritional genomics. Tue, 05 Apr 2011 22:00:00 GMT http://hdl.handle.net/2067/1880 2011-04-05T22:00:00Z http://hdl.handle.net/2067/1415 Title: Comparative Proteomic Analysis of Hemocyanins in Dinocras cephalotes and Perla marginata (Plecoptera) Authors: Fochetti, Romolo; Amore, Valentina; Ubero-Pascal, Nicolás; Egidi, Maria Giulia; Timperio, Anna Maria Abstract: Hemocyanins are large oligomeric respiratory proteins found in many arthropods and mollusks. The overall expression of hemocyanin mRNA, revealed by studies on Plecoptera hemocyanin sequencing, has raised the question of whether the protein is expressed or not. In fact, the presence of expressed hemocyanin has only been reported in the literature for one species, Perla marginata (Panzer, 1799). In this paper, we report the presence of hemocyanin and hexamerin proteins in Dinocras cephalotes (Curtis, 1827), a species closely related to P. marginata. To assess the presence of hemocyanin, we used a reproducible and highly sensitive method based on liquid chromatography tandem mass spectrometry. We conclude that regardless of its putative function (respiratory, immune defense, storage protein), the hemocyanin is actually expressed in species in which its mRNA is present. Description: L'articolo è disponibile sul sito dell'editore http://www.entsoc.org Fri, 31 Dec 2010 23:00:00 GMT http://hdl.handle.net/2067/1415 2010-12-31T23:00:00Z http://hdl.handle.net/2067/1470 Title: Modulation of the neuronal response to ischemia by somatostatin analogues in wild-type and knock-out mouse retinas Authors: Cervia, Davide; Martini, Davide; Ristori, Chiara; Catalani, Elisabetta; Timperio, Anna Maria; Bagnoli, Paola; Casini, Giovanni Abstract: Somatostatin acts at five G protein-coupled receptors, sst1-sst5. In mouse ischemic retinas, the over-expression of sst2 (as in sst1 knock-out mice) results in reduction of cell death and glutamate release. Here, we reported that, in wild-type retinas, somatostatin, the multireceptor ligand pasireotide and the sst2 agonist octreotide decreased ischemia-induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst2 with cyanamide. In sst2 over-expressing ischemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti-ischemic effect of somatostatin agonists in the presence of sst2 over-expression, we tested sst2 desensitisation due to internalisation or altered receptor function. We observed that: i) sst2 was not internalised, ii) among G protein-coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischemia, iii) both GRK1 and RGS1 were down-regulated by octreotide in wild-type ischemic retinas, iv) octreotide down-regulated GRK1 but not RGS1 in sst2 over-expressing ischemic retinas. These results demonstrate that sst2 activation protects against retinal ischemia. However, in the presence of sst2 over-expression sst2 is functionally desensitised by agonists, possibly due to sustained RGS1 levels. Description: L'articolo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/ Mon, 31 Dec 2007 23:00:00 GMT http://hdl.handle.net/2067/1470 2007-12-31T23:00:00Z