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2. RANDOMIZED CONTROLLED TRIAL OF STREPTOKINASE, ASPIRIN, AND COMBINATION OF BOTH IN TREATMENT OF ACUTE ISCHEMIC STROKE

Author

CANDELISE L, ARITZU E, CICCONE A, RICCI S, WARDLAW J, TOGNONI G, RONCAGLIONI MC, NEGRI E, COLOMBO F, BOCCARDI E, DEGRANDI C, SCIALFA G, ARGENTINO C, BERTELE V, MAGGIONI AP, PERRONE P, BARNETT HJM, BOGOUSSLAVSKY J, DELFAVERO A, LOI U, PETO R, WARLOW C, CANZI S, COMPARETTI S, CLERICI F, PALUMBO A, SGARONI G, POLONARA S, REGINELLI R, CERAVOLO MG, PROVINCIALI L, DELGOBBO M, SCARPINO O, BOTTACCHI E, DALESSANDRO G, DIGIOVANNI M, BLANC S, ROVEYAZ L, RALLI L, VANNI D, REFI C, FEDERICO F, CONTE C, INCHINGOLO V, INSABATO R, SALSA F, LORIZIO A, ROTTOLI MR, BRUNI L, DEFANTI CA, FERA L, CAMERLINGO M, CASTO L, CENSORI B, MAMOLI A, PORAZZI D, GRAMPA G, LASPINA I, GIGLIA L, AVENIA V, GUELI S, LOLLI V, MIELE V, SANTANGELO M, COPPOLA G, TRIANNI G, MARRA M, GRECO E, TONTI D, PRETOLANI E, STELLIO M, ARNABOLDI M, CIOLA R, DANIELI G, REZZONICO M, GUIDOTTI M, PELLEGRINI G, RAUDINO F, DELFAVERO C, FRATTINI T, RICCARDI T, LEVIMINZI C, LOCATELLI F, PASSERI F, LOMBARDO G, COCCO F, PRATESI M, SANTINI S, CARDOPATRI F, TAFANI O, LANDINI GC, PIERAGNOLI E, BELLESI R, BAGNOLI L, GHETTI A, MARRAZZA OB, MENEGAZZO P, SPOLVERI S, CAPPELLETTI C, CANDELIERE G, COSTANTINO G, DACUNO F, LOMUZIO T, RIZZITELLI FP, BOVIO G, GRILLI GP, ZOCCHI M, MAZZANTI I, PISAPIA G, NUZZI R, RUSSO G, LAMA G, BALDASSARRI G, BETTINI R, CANI E, CERIOLI E, DEGIORGI M, GARAGNANI A, PASQUALI S, CHIOMA V, FINI C, MARZARA G, BALOTTA A, BERTUZZI D, MASINA M, MATACENA C, MICHELINI M, PIRAZZOLI G, SACCHET C, MAROTTA P, CALVI L, SCACCABAROZZI C, MENOZZI C, SASANELLI F, BASCELLI C, PONTRELLI V, SCHIERONI F, BELVEDERE D, DECAPITANI E, DICOSTANZO M, RASCAROLI M, CITTERIO O, MILANI R, CAPIALBI R, ARENA G, MUSOLINO R, DIPERRI R, BONAVENTI C, FINZI F, MESSINA A, ROMANAZZI V, STERZI R, BOTTINI G, SANTILLI I, BRUCATO A, DEJULI E, PALMIERI G, RAGAINI S, THIELLA G, ALLI C, CAROSELLA L, RIZZATO G, BIANCHI M, MOTTO C, PINARDI G, SCARLATO G, BETTINELLI A, PORRO F, RANDAZZO A, MARINI U, LATTUADA P, PIETRA A, FRANCESCHI M, VOLOUTE MA, MALFERRARI G, BONDI M, GARUTI W, MELINI L, COLOMBO A, ALBORINI G, PANZETTI P, SCARPA M, SORGATO P, CRISI G, BONASERA N, BASSO F, NATALI E, MONACO P, GASPARRO AM, TRIOLO F, LECHI A, COLONNA F, DASCOLA I, GIORGI C, SCODITTI U, BASSI P, MENOZZI R, PIAZZA P, DEGLIANTONI G, DESIMONI M, GIANNINI A, MICILI G, BOSONE D, CAVALLINI A, MARTELLI A, BARZIZZA F, POLI M, BIAGINI S, CAPOCCHI G, CELANI MG, PICCHIARELLI A, RIGHETTI E, ZAMPOLINI M, CAPUTO N, BARTOLINI S, SANTONI M, SITA D, TAGLIOLI M, VOLPI G, ROSSI F, ALOY F, CASTELLANO AE, MINOTTA F, GATTA A, VINATTIERI A, CANTINI A, REBUCCI GG, BISSI G, DEBLASI F, PASCARELLI E, TURIAMO F, GUIDETTI D, BARATTI M, FERRARINI G, GHIDONI E, GRECO G, MARCELLO N, MOTTI L, SOLIME F, TERENZIANI S, ZUCCO R, TROISO A, VENEZIANI M, IORI I, CURATOLA L, BOLLETTINI G, CARBONI T, GOBBATO R, INFRICCIOLO P, SABATINI D, SFRAPPINI M, FANCELLU A, CASU G, DELEDDA MG, SPANU MA, PIRISI A, MARRAS FA, ROSATI G, BRAMBILLA A, ERLI GC, FELICE B, GRANDI R, MIRABILE D, ZADRA M, CAVESTRI R, GORI D, LONGHINI E, MIELE N, DIVIZIA G, GRASSELLI S, PEZZELLA F, ZUCCARI F, SALLUSTO L, LINCESSO F, SCARPATI C, IANNONE G, BARTOCCI A, COSTANTINI F, DESANTIS L, LANCIA G, MOSCHINI E, PACI A, SENSIDONI A, TRENTA A, BARTOLINI N, OTTAVIANO P, BISCOTTINI B, ALUNNI G, BARTOLINI I, BELLADONNA D, BOCCALI A, CRUCIANI M, IBBA R, LUCCIOLI R, MARCECA A, PACINI M, PALLONE M, GIRARDI P, ORRICO D, LANZA E, BALLINI A, GRANDI FC, MUSCO G, STEIDL L, SANTORO G, VEMCO A, DUDINE P, MILONE FF, MUNARI L, PERRETI A, PORTA M, LONGONI C, FERANI R, WATT M, SANDERCOCK P, FENNETRY A, BANNISTER P, CLARKE CE, BAMFORD J, VENABLES G, CORREIA M, CANDELISE L, ARITZU E, CICCONE A, RICCI S, WARDLAW J, TOGNONI G, RONCAGLIONI MC, NEGRI E, COLOMBO F, BOCCARDI E, DEGRANDI C, SCIALFA G, ARGENTINO C, BERTELE V, MAGGIONI AP, PERRONE P, BARNETT HJM, BOGOUSSLAVSKY J, DELFAVERO A, LOI U, PETO R, WARLOW C, CANZI S, COMPARETTI S, CLERICI F, PALUMBO A, SGARONI G, POLONARA S, REGINELLI R, CERAVOLO MG, PROVINCIALI L, DELGOBBO M, SCARPINO O, BOTTACCHI E, DALESSANDRO G, DIGIOVANNI M, BLANC S, ROVEYAZ L, RALLI L, VANNI D, REFI C, FEDERICO F, CONTE C, INCHINGOLO V, INSABATO R, SALSA F, LORIZIO A, ROTTOLI MR, BRUNI L, DEFANTI CA, FERA L, CAMERLINGO M, CASTO L, CENSORI B, MAMOLI A, PORAZZI D, GRAMPA G, LASPINA I, GIGLIA L, AVENIA V, GUELI S, LOLLI V, MIELE V, SANTANGELO M, COPPOLA G, TRIANNI G, MARRA M, GRECO E, TONTI D, PRETOLANI E, STELLIO M, ARNABOLDI M, CIOLA R, DANIELI G, REZZONICO M, GUIDOTTI M, PELLEGRINI G, RAUDINO F, DELFAVERO C, FRATTINI T, RICCARDI T, LEVIMINZI C, LOCATELLI F, PASSERI F, LOMBARDO G, COCCO F, PRATESI M, SANTINI S, CARDOPATRI F, TAFANI O, LANDINI GC, PIERAGNOLI E, BELLESI R, BAGNOLI L, GHETTI A, MARRAZZA OB, MENEGAZZO P, SPOLVERI S, CAPPELLETTI C, CANDELIERE G, COSTANTINO G, DACUNO F, LOMUZIO T, RIZZITELLI FP, BOVIO G, GRILLI GP, ZOCCHI M, MAZZANTI I, PISAPIA G, NUZZI R, RUSSO G, LAMA G, BALDASSARRI G, BETTINI R, CANI E, CERIOLI E, DEGIORGI M, GARAGNANI A, PASQUALI S, CHIOMA V, FINI C, MARZARA G, BALOTTA A, BERTUZZI D, MASINA M, MATACENA C, MICHELINI M, PIRAZZOLI G, SACCHET C, MAROTTA P, CALVI L, SCACCABAROZZI C, MENOZZI C, SASANELLI F, BASCELLI C, PONTRELLI V, SCHIERONI F, BELVEDERE D, DECAPITANI E, DICOSTANZO M, RASCAROLI M, CITTERIO O, MILANI R, CAPIALBI R, ARENA G, MUSOLINO R, DIPERRI R, BONAVENTI C, FINZI F, MESSINA A, ROMANAZZI V, STERZI R, BOTTINI G, SANTILLI I, BRUCATO A, DEJULI E, PALMIERI G, RAGAINI S, THIELLA G, ALLI C, CAROSELLA L, RIZZATO G, BIANCHI M, MOTTO C, PINARDI G, SCARLATO G, BETTINELLI A, PORRO F, RANDAZZO A, MARINI U, LATTUADA P, PIETRA A, FRANCESCHI M, VOLOUTE MA, MALFERRARI G, BONDI M, GARUTI W, MELINI L, COLOMBO A, ALBORINI G, PANZETTI P, SCARPA M, SORGATO P, CRISI G, BONASERA N, BASSO F, NATALI E, MONACO P, GASPARRO AM, TRIOLO F, LECHI A, COLONNA F, DASCOLA I, GIORGI C, SCODITTI U, BASSI P, MENOZZI R, PIAZZA P, DEGLIANTONI G, DESIMONI M, GIANNINI A, MICILI G, BOSONE D, CAVALLINI A, MARTELLI A, BARZIZZA F, POLI M, BIAGINI S, CAPOCCHI G, CELANI MG, PICCHIARELLI A, RIGHETTI E, ZAMPOLINI M, CAPUTO N, BARTOLINI S, SANTONI M, SITA D, TAGLIOLI M, VOLPI G, ROSSI F, ALOY F, CASTELLANO AE, MINOTTA F, GATTA A, VINATTIERI A, CANTINI A, REBUCCI GG, BISSI G, DEBLASI F, PASCARELLI E, TURIAMO F, GUIDETTI D, BARATTI M, FERRARINI G, GHIDONI E, GRECO G, MARCELLO N, MOTTI L, SOLIME F, TERENZIANI S, ZUCCO R, TROISO A, VENEZIANI M, IORI I, CURATOLA L, BOLLETTINI G, CARBONI T, GOBBATO R, INFRICCIOLO P, SABATINI D, SFRAPPINI M, FANCELLU A, CASU G, DELEDDA MG, SPANU MA, PIRISI A, MARRAS FA, ROSATI G, BRAMBILLA A, ERLI GC, FELICE B, GRANDI R, MIRABILE D, ZADRA M, CAVESTRI R, GORI D, LONGHINI E, MIELE N, DIVIZIA G, GRASSELLI S, PEZZELLA F, ZUCCARI F, SALLUSTO L, LINCESSO F, SCARPATI C, IANNONE G, BARTOCCI A, COSTANTINI F, DESANTIS L, LANCIA G, MOSCHINI E, PACI A, SENSIDONI A, TRENTA A, BARTOLINI N, OTTAVIANO P, BISCOTTINI B, ALUNNI G, BARTOLINI I, BELLADONNA D, BOCCALI A, CRUCIANI M, IBBA R, LUCCIOLI R, MARCECA A, PACINI M, PALLONE M, GIRARDI P, ORRICO D, LANZA E, BALLINI A, GRANDI FC, MUSCO G, STEIDL L, SANTORO G, VEMCO A, DUDINE P, MILONE FF, MUNARI L, PERRETI A, PORTA M, LONGONI C, FERANI R, WATT M, SANDERCOCK P, FENNETRY A, BANNISTER P, CLARKE CE, BAMFORD J, VENABLES G, and CORREIA M

Abstract

In ischaemic stroke, thrombolytic drugs speed the recanalisation of intracerebral arteries. The effects of aspirin are not known. A trial was conducted to determine whether, separately or together, streptokinase and aspirin have clinical benefits in acute ischaemic stroke similar to those in acute myocardial infarction. 622 patients with acute ischaemic stroke within 6 hours of symptom onset were randomised with a 2x2 factorial design to (i) a 1-hour intravenous infusion of 1 . 5 MU streptokinase, (ii) 300 mg/day buffered aspirin for 10 days, (iii) both active treatments, or (iv) neither. Early results raised a question whether the trial should be continued. Streptokinase (alone or with aspirin) was associated with an excess of 10-day case fatality (odds ratio 2 . 7; 95% confidence interval 1 . 7-4 . 3; 2p

Published
1995

4. Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma

Author

Fini C., Talamo F., Cherri S., Coli M., Floridi A., Ferrara L., and Scaloni A.

Abstract

Ecto-5'-nucleotidase (ecto-5'-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5'-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln(547) and/or Phe(548). Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn(403). Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5'-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

Published
2003

9. Mass spectrometry study of ecto-5'-nucleotidase from bull seminal plasma

Author

Fini C, Amoresano A, Andolfo A, D’Auria S, Floridi A, Paolini S, and Pucci P.

Subjects
Male, Glycosylation, Glycosylphosphatidylinositols, Fatty Acids, Molecular Sequence Data, Oligosaccharides, Gas Chromatography-Mass Spectrometry, Molecular Weight, Carbohydrate Sequence, Semen, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Animals, Cattle, Amino Acid Sequence, 5'-Nucleotidase
Abstract

The structure of ecto-5'-nucleotidase from bull seminal plasma, containing a glycosyl-phosphatidylinositol anchor, was studied using mass spectrometry. MALDI-MS analysis of intact protein indicated a mass of 65 568.2 Da for the monomeric form, and it also showed a heterogeneous population of glycoforms with the glycosidic moiety accounting for approximately 6000 Da. MALDI-MS analysis showed that Asn53, Asn311, Asn333 and Asn403 were four sites of N-glycosylation. GC-MS analysis provided information on the glycosidic structures linked to the four asparagines. Asn53, Asn311 and Asn333 were linked to high-mannose saccharide chains, whereas the glycan chains linked to Asn403 contained a heterogeneous mixture of oligosaccharides, the high-mannose type structure being the most abundant and hybrid or complex type glycans being minor components. By combining enzymatic and/or chemical hydrolysis with GC-MS analysis, detailed characterization of the glycosyl-phpsphatidylinositol anchor was obtained. MALDI spectral analysis indicated that the glycosyl-phosphatidylinositol core contained EtN(P)Man3GlcNH2-myo-inositol(P)-glycerol, principally modified by stearoyl and palmitoyl residues or by stearoyl and myristoyl residues to a minor extent. Moreover, 1-palmitoylglycerol and 1-stearoylglycerol outweighed 2-palmitoylglycerol and 2-stearoylglycerol. The combination of chemical and enzymatic digestions of the protein with the mass spectral analysis yielded a complete pattern of S-S bridges. The protein does not contain free thiols and its eight cysteines are linked by intramolecular disulfide bonds, the pairs being: Cys51-Cys57, Cys353-Cys358, Cys365-Cys387 and Cys476-Cys479. This work resolves details of the structure of ecto-5'-nucleotidase, with particular regard to the localization and composition of the glycidic moiety, number and localization of the disulfide bridges and characterization of the glycosyl-phosphatidylinositol anchor.

Published
2000

10. RANDOMIZED CONTROLLED TRIAL OF STREPTOKINASE, ASPIRIN, AND COMBINATION OF BOTH IN TREATMENT OF ACUTE ISCHEMIC STROKE

Author

CANDELISE, L, ARITZU, E, CICCONE, A, RICCI, S, WARDLAW, J, TOGNONI, G, RONCAGLIONI, M, NEGRI, E, COLOMBO, F, BOCCARDI, E, DEGRANDI, C, SCIALFA, G, ARGENTINO, C, BERTELE, V, MAGGIONI, A, PERRONE, P, BARNETT, H, BOGOUSSLAVSKY, J, DELFAVERO, A, LOI, U, PETO, R, WARLOW, C, CANZI, S, COMPARETTI, S, CLERICI, F, PALUMBO, A, SGARONI, G, POLONARA, S, REGINELLI, R, CERAVOLO, M, PROVINCIALI, L, DELGOBBO, M, SCARPINO, O, BOTTACCHI, E, DALESSANDRO, G, DIGIOVANNI, M, BLANC, S, ROVEYAZ, L, RALLI, L, VANNI, D, REFI, C, FEDERICO, F, CONTE, C, INCHINGOLO, V, INSABATO, R, SALSA, F, LORIZIO, A, ROTTOLI, M, BRUNI, L, DEFANTI, C, FERA, L, CAMERLINGO, M, CASTO, L, CENSORI, B, MAMOLI, A, PORAZZI, D, GRAMPA, G, LASPINA, I, GIGLIA, L, AVENIA, V, GUELI, S, LOLLI, V, MIELE, V, SANTANGELO, M, COPPOLA, G, TRIANNI, G, MARRA, M, GRECO, E, TONTI, D, PRETOLANI, E, STELLIO, M, ARNABOLDI, M, CIOLA, R, DANIELI, G, REZZONICO, M, GUIDOTTI, M, PELLEGRINI, G, RAUDINO, F, DELFAVERO, C, FRATTINI, T, RICCARDI, T, LEVIMINZI, C, LOCATELLI, F, PASSERI, F, LOMBARDO, G, COCCO, F, PRATESI, M, SANTINI, S, CARDOPATRI, F, TAFANI, O, LANDINI, G, PIERAGNOLI, E, BELLESI, R, BAGNOLI, L, GHETTI, A, MARRAZZA, O, MENEGAZZO, P, SPOLVERI, S, CAPPELLETTI, C, CANDELIERE, G, COSTANTINO, G, DACUNO, F, LOMUZIO, T, RIZZITELLI, F, BOVIO, G, GRILLI, G, ZOCCHI, M, MAZZANTI, I, PISAPIA, G, NUZZI, R, RUSSO, G, LAMA, G, BALDASSARRI, G, BETTINI, R, CANI, E, CERIOLI, E, DEGIORGI, M, GARAGNANI, A, PASQUALI, S, CHIOMA, V, FINI, C, MARZARA, G, BALOTTA, A, BERTUZZI, D, MASINA, M, MATACENA, C, MICHELINI, M, PIRAZZOLI, G, SACCHET, C, MAROTTA, P, CALVI, L, SCACCABAROZZI, C, MENOZZI, C, SASANELLI, F, BASCELLI, C, PONTRELLI, V, SCHIERONI, F, BELVEDERE, D, DECAPITANI, E, DICOSTANZO, M, RASCAROLI, M, CITTERIO, O, MILANI, R, CAPIALBI, R, ARENA, G, MUSOLINO, R, DIPERRI, R, BONAVENTI, C, FINZI, F, MESSINA, A, ROMANAZZI, V, STERZI, R, BOTTINI, G, SANTILLI, I, BRUCATO, A, DEJULI, E, PALMIERI, G, RAGAINI, S, THIELLA, G, ALLI, C, CAROSELLA, L, RIZZATO, G, BIANCHI, M, MOTTO, C, PINARDI, G, SCARLATO, G, BETTINELLI, A, PORRO, F, RANDAZZO, A, MARINI, U, LATTUADA, P, PIETRA, A, FRANCESCHI, M, VOLOUTE, M, MALFERRARI, G, BONDI, M, GARUTI, W, MELINI, L, COLOMBO, A, ALBORINI, G, PANZETTI, P, SCARPA, M, SORGATO, P, CRISI, G, BONASERA, N, BASSO, F, NATALI, E, MONACO, P, GASPARRO, A, TRIOLO, F, LECHI, A, COLONNA, F, DASCOLA, I, GIORGI, C, SCODITTI, U, BASSI, P, MENOZZI, R, PIAZZA, P, DEGLIANTONI, G, DESIMONI, M, GIANNINI, A, MICILI, G, BOSONE, D, CAVALLINI, A, MARTELLI, A, BARZIZZA, F, POLI, M, BIAGINI, S, CAPOCCHI, G, CELANI, M, PICCHIARELLI, A, RIGHETTI, E, ZAMPOLINI, M, CAPUTO, N, BARTOLINI, S, SANTONI, M, SITA, D, TAGLIOLI, M, VOLPI, G, ROSSI, F, ALOY, F, CASTELLANO, A, MINOTTA, F, GATTA, A, VINATTIERI, A, CANTINI, A, REBUCCI, G, BISSI, G, DEBLASI, F, PASCARELLI, E, TURIAMO, F, GUIDETTI, D, BARATTI, M, FERRARINI, G, GHIDONI, E, GRECO, G, MARCELLO, N, MOTTI, L, SOLIME, F, TERENZIANI, S, ZUCCO, R, TROISO, A, VENEZIANI, M, IORI, I, CURATOLA, L, BOLLETTINI, G, CARBONI, T, GOBBATO, R, INFRICCIOLO, P, SABATINI, D, SFRAPPINI, M, FANCELLU, A, CASU, G, DELEDDA, M, SPANU, M, PIRISI, A, MARRAS, F, ROSATI, G, BRAMBILLA, A, ERLI, G, FELICE, B, GRANDI, R, MIRABILE, D, ZADRA, M, CAVESTRI, R, GORI, D, LONGHINI, E, MIELE, N, DIVIZIA, G, GRASSELLI, S, PEZZELLA, F, ZUCCARI, F, SALLUSTO, L, LINCESSO, F, SCARPATI, C, IANNONE, G, BARTOCCI, A, COSTANTINI, F, DESANTIS, L, LANCIA, G, MOSCHINI, E, PACI, A, SENSIDONI, A, TRENTA, A, BARTOLINI, N, OTTAVIANO, P, BISCOTTINI, B, ALUNNI, G, BARTOLINI, I, BELLADONNA, D, BOCCALI, A, CRUCIANI, M, IBBA, R, LUCCIOLI, R, MARCECA, A, PACINI, M, PALLONE, M, GIRARDI, P, ORRICO, D, LANZA, E, BALLINI, A, GRANDI, F, MUSCO, G, STEIDL, L, SANTORO, G, VEMCO, A, DUDINE, P, MILONE, F, MUNARI, L, PERRETI, A, PORTA, M, LONGONI, C, FERANI, R, WATT, M, SANDERCOCK, P, FENNETRY, A, BANNISTER, P, CLARKE, C, BAMFORD, J, VENABLES, G, and CORREIA, M

Published
1995

13. Rationale and methodology of the ICAI study, a randomised clinical trial of alprostadil in the treatment of chronic critical leg ischemia

Author

Belgrano, Ea, Nardella, L., Guala, A., Mazzucchetti, S., Marinoni, V., Calzoni, D., Bedoni, P., Confalonieri, Ma, Agus, Gb, Mondani, P., Deangelis, R., Biasi, Gm, Piglionica, MR, Abbritti, F., Agrifoglio, G., Costantini, A., DellaVedova, MR, Miglierina, L., Marrocu, R., Bragherio, G., Zanoni, Ce, Borin, F., Alderi, G., Emanuelli, G., Flandoli, C., Colzani, M., Ponti, Gb, Berra, S., Bevilacqua, A., Bocca, M., Invernizzi, C., Deangelis, E., Tacconi, A., Dangelo, F., Vaghi, M., Arzini, A., Boccalon, L., Losapio, Gm, Ambrosi, R., Briolini, F., Inzoli, MR, Lombardi, G., Tarantola, P., Zocca, N., Tenchini, P., Bruni, T., Fontanili, M., Guidetti, D., Pedeferri, G., Bordoni, Mc, Catalano, A., Visconti, W., Vedovato, F., Zucchella, M., Bittolo, Bg, Busetto, Mt, Zambon, C., Carlassara, Gb, Barbato, O., Zambelli, V., Mazzilli, G., Lino, M., Pavan, S., Pagnan, A., Visona, A., Perissinotto, C., Tonietto, G., Michelet, I., Agresta, F., Favretti, F., Burigo, E., Delazzer, L., Giansante, C., Fiotti, N., Grego, S., Mozzon, L., Gonano, N., Pfeiffer, P., Petrilli, Gl, Puzzo, A., Giuseppe Baldino, Podesta, A., Guastini, A., Traversaro, A., Zinicola, N., Baglietto, F., Arnuzzo, L., Defabritiis, A., Filippini, M., Ferrari, F., Martini, L., Testoni, P., Accorsi, F., Maurizi, P., Evangelisti, G., Roffi, A., Marzara, G., Fini, C., Coppi, G., Camparini, S., Tusini, N., Tuscano, G., Lonardi, R., Rozza, A., Botta, Gc, Villani, Lg, Pavarini, E., Campanella, P., Moratti, A., Ieran, M., Bertini, D., Pratesi, C., Narcetti, S., Corsi, C., Pollastri, M., Marrapodi, E., Melillo, E., Iabichella, Ml, Setacci, C., Sozio, G., Cao, P., Verzini, F., Mannarino, E., Pasqualini, L., Vaudo, G., Alo, F., Ioannidis, G., Spartera, C., Marino, G., Bafile, G., Anselmi, E., Maniscalco, G., Longo, P., Digiovanni, V., Colli, R., Fabbri, Mc, Bracale, G., Bernardo, B., Perretti, B., Valitutti, P., Vigliotti, G., Cimino, G., Rolli, F., Pascali, M., Sabella, G., Grilli, M., Correra, M., Palese, E., Florena, M., Cassina, I., Cumbo, P., Comande, C., Notarbartolo, A., Novo, S., Belvedere, M., Caruso, R., Verghi, F., Cavallaro, S., Martello, G., Romeo, S., Cormaci, Of, Binaghi, F., Fronteddu, P., Cannas, F., Degaetano, G., Tognoni, G., Avanzini, F., Bertele, V., Digiulio, P., Pangrazzi, J., Roncaglioni, Mc, Colombo, F., Fellin, G., Terzian, E., Coccheri, S., Delfavero, A., Geraci, E., Janzon, L., Vermylen, J., Beghi, E., Coen, D., and Turazza, F.

16. Hydrophobic interactions and ionic networks play an important role in thermal stability and denaturation mechanism of the porcine odorant-binding protein

Author

Mosè Rossi, Carlo Fini, Maria Staiano, Anna Marabotti, Sabato D'Auria, Konstantin K. Turoverov, Irina M. Kuznetsova, Olesya V. Stepanenko, Antonio Varriale, Stepanenko, Ov, Marabotti, A, Kuznetsova, Im, Turoverov, Kk, Fini, C, Varriale, A, Staiano, M, Rossi, Mose', and D’Auria, S.

Subjects
Ions, Circular dichroism, Protein Denaturation, Hot Temperature, Chemistry, Swine, Protein dynamics, Static Electricity, Protein aggregation, Receptors, Odorant, Biochemistry, Hydrophobic effect, Crystallography, Protein structure, Structural Biology, Excited state, Native state, Animals, Denaturation (biochemistry), Molecular Biology, Hydrophobic and Hydrophilic Interactions
Abstract

Despite the fact that the porcine odorant-binding protein (pOBP) possesses a single tryptophan residue (Trp 16) that is characterized by a high density microenvironment (80 atoms in a sphere with radius 7 A) with only one polar group (Lys 120) and three bound water molecules, pOBP displayed a red shifted fluorescence emission spectrum (lambda(max) = 340 nm). The protein unfolding in 5M GdnHCl was accompanied by the red shift of the fluorescence emission spectrum (lambda(max) = 353 nm), by the increase of fluorescence quantum yield, and by the decrease of lifetime of the excited state (from 4.25 ns in native state to 3.15 ns in the presence of 5M GdnHCl). Taken together these data indicate the existence of an exciplex complex (Trp 16 with Lys 120 and/or with bound molecules of water) in the protein native state. Heat-induced denaturation of pOBP resulted in significant red shifts of the fluorescence emission spectra: the value of the ratio (I(320)/I(365)) upon excitation at lambda(ex) = 297 nm (parameter A) decreases from 1.07 to 0.64 passing from 60 to 85 degrees C, and the calculated midpoint of transition was centered at 70 degrees C. Interestingly, even at higher temperature, the values of the parameter A both in the absence and in the presence of GdnHCl did not coincide. This suggests that a portion of the protein structure is still preserved upon the temperature-induced denaturation of the protein in the absence of GdnHCl. CD experiments performed on pOBP in the absence and in the presence of GdnHCl and at different temperatures were in agreement with the fluorescence results. In addition, the obtained experimental data were corroborated by the analysis of the 3D structure of pOBP which revealed the amino acid residues that contribute to the protein dynamics and stability. Finally, molecular dynamics simulation experiments pointed out the important role of ion pair interactions as well as the molecular motifs that are responsible for the high thermal stability of pOBP, and elucidated the reasons of the protein aggregation that occurred at high temperature.

Published
2008

17. Time-resolved fluorescence spectroscopy and molecular dynamics simulations point out the effects of pressure on the stability and dynamics of the porcine odorant-binding protein

Author

Zygmunt Grycznyski, Antonietta Parracino, Michele Saviano, Sabato D'Auria, Petr Herman, Antonio Varriale, Ane Bisgaard Kold, Mosè Rossi, Carlo Fini, Maria Staiano, Staiano, M., Saviano, M., Herman, P., Grycznyski, Z., Fini, C., Varriale, A., Parracino, A., Kold, A. B., Rossi, Mose', and D’Auria, S.

Subjects
Models, Molecular, Time Factors, Molecular Structure, Chemistry, Swine, Organic Chemistry, Hydrostatic pressure, Biophysics, General Medicine, Ligand (biochemistry), Receptors, Odorant, Biochemistry, Fluorescence, Fluorescence spectroscopy, Biomaterials, Molecular dynamics, Spectrometry, Fluorescence, Pyrazines, Pressure, Molecule, Animals, Computer Simulation, Time-resolved spectroscopy, Spectroscopy
Abstract

The effects of hydrostatic pressure on the structure and stability of porcine odorant-binding protein (pOBP) in the presence and absence of the odorant molecule 2-isobutyl-3-methoxypyrazine (IBMP) were studied by steady-state and time-resolved fluorescence spectroscopy as well as by molecular dynamics simulation. The authors found that the application of moderate values of hydrostatic pressure to pOBP solutions perturbed the microenvironment of Trp(16) and disrupted its highly quenched complex with Met(39). In addition, compared with the protein in the absence of IBMP, the MD simulations experiments carried out at different pressures highlighted the role of this ligand in stabilizing the Trp(16)/Met(39) interaction even at 2000 bar. The obtained results will assist for the tailoring of this protein as specific sensing element in a new class of fluorescence-based biosensors for the detection of explosives.

Published
2008

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