To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37°C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress. Author keywords AMP-deaminaseEnergy metabolismHuman erythrocytesIMPOxidative stress Indexed keywords EMTREE drug terms: adenosine diphosphateadenosine monophosphate deaminaseadenosine phosphateadenosine triphosphateguanosine triphosphatehydrogen peroxidehypoxanthineinosineinosine phosphatemalonaldehydeuric acidxanthine EMTREE medical terms: articlecell levelcell membrane transportconcentration responsecontrolled studyenergy metabolismenzyme activationenzyme activityerythrocyte metabolismhemolysishumanhuman celllipid peroxidationoxidative stresspriority journalreaction analysis MeSH: Adenine NucleotidesAMP DeaminaseEnergy MetabolismErythrocytesGuanosine TriphosphateHemolysisHumansHydrogen PeroxideInosine MonophosphateLipid PeroxidationMalondialdehydeOxidative Stress Chemicals and CAS Registry Numbers: Adenine Nucleotides; AMP Deaminase, EC 3.5.4.6; Guanosine Triphosphate, 86-01-1; Hydrogen Peroxide, 7722-84-1; Inosine Monophosphate, 131-99-7; Malondialdehyde, 542-78-9 ISSN: 00142956 CODEN: EJBCA Source Type: Journal Original language: English DOI: 10.1046/j.1432-1327.2000.01042.x PubMed ID: 10651804 Document Type: Article References (22) View in search results format All Export Print E-mail Save to PDF Create bibliography 1 Forman, M.B., Virmani, R., Puett, D.W. Mechanisms and therapy of myocardial reperfusion injury (1990) Circulation, 81 (3 SUPPL.), pp. IV69-IV78. Cited 142 times. Locate full-text(opens in a new window) 2 Roof, R.L., Hoffman, S.W., Stein, D.G. Progesterone protects against lipid peroxidation following traumatic brain injury in rats (1997) Molecular and Chemical Neuropathology, 31 (1), pp. 1-11. Cited 240 times. doi: 10.1007/BF02815156 Locate full-text(opens in a new window) View at Publisher 3 De Flora, S., Izzotti, A., Randerath, K., Randerath, E., Bartsch, H., Nair, J., Balansky, R., (...), Lewtas, J. DNA adducts and chronic degenerative diseases. Pathogenetic relevance and implications in preventive medicine (1996) Mutation Research - Reviews in Genetic Toxicology, 366 (3), pp. 197-238. Cited 145 times. doi: 10.1016/S0165-1110(96)00043-7 Locate full-text(opens in a new window) View at Publisher 4 Ravindranath, V., Reed, D.J. Glutathione depletion and formation of glutathione-protein mixed disulfide following exposure of brain mitochondria to oxidative stress (1990) Biochemical and Biophysical Research Communications, 169 (3), pp. 1075-1079. Cited 122 times. doi: 10.1016/0006-291X(90)92004-J Locate full-text(opens in a new window) View at Publisher 5 Tavazzi, B., Lazzarino, G., Di Pierro, D., Giardina, B. Malondialdehyde production and ascorbate decrease are associated to the reperfusion of the isolated postischemic rat heart (1992) Free Radical Biology and Medicine, 13 (1), pp. 75-78. Cited 72 times. doi: 10.1016/0891-5849(92)90167-F Locate full-text(opens in a new window) View at Publisher 6 Di Pierro, D., Tavazzi, B., Lazzarino, G., Giardina, B. Malondialdehyde is a biochemical marker of peroxidative damage in the isolated reperfused rat heart (1992) Molecular and Cellular Biochemistry, 116 (1-2), pp. 193-196. Cited 34 times. doi: 10.1007/BF01270587 Locate full-text(opens in a new window) View at Publisher 7 Maridonneau, I., Braquet, P., Garay, R.P. Na+ and K+ transport damage induced by oxygen free radicals in human red cell membranes (1983) Journal of Biological Chemistry, 258 (5), pp. 3107-3113. Cited 150 times. Locate full-text(opens in a new window) View at Publisher 8 Rohn, T.T., Nelson, L.K., Waeg, G., Quinn, M.T. U-101033E (2,4-diaminopyrrolopyrimidine), a potent inhibitor of membrane lipid peroxidation as assessed by the production of 4-hydroxynonenal, malondialdehyde, and 4-hydroxynonenal- protein adducts (1998) Biochemical Pharmacology, 56 (10), pp. 1371-1379. Cited 35 times. www.elsevier.com/locate/biochempharm doi: 10.1016/S0006-2952(98)00266-4 Locate full-text(opens in a new window) View at Publisher 9 Snyder, L.M., Fortier, N.L., Leb, L., McKenney, J., Trainor, J., Sheerin, H., Mohandas, N. The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes (1988) BBA - Biomembranes, 937 (C), pp. 229-240. Cited 55 times. doi: 10.1016/0005-2736(88)90245-3 Locate full-text(opens in a new window) View at Publisher 10 Davies, K.J., Goldberg, A.L. Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. (1987) Journal of Biological Chemistry, 262 (17), pp. 8220-8226. Cited 344 times. Locate full-text(opens in a new window) View at Publisher 11 Lazzarino, G., Di Pierro, D., Tavazzi, B., Cerroni, L., Giardina, B. Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography (1991) Analytical Biochemistry, 197 (1), pp. 191-196. Cited 107 times. doi: 10.1016/0003-2697(91)90378-7 Locate full-text(opens in a new window) View at Publisher 12 Miki, M., Tamai, H., Mino, M., Yamamoto, Y., Niki, E. Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by α-tocopherol (1987) Archives of Biochemistry and Biophysics, 258 (2), pp. 373-380. Cited 305 times. doi: 10.1016/0003-9861(87)90358-4 Locate full-text(opens in a new window) View at Publisher 13 Stocks, J., Dormandy, T.L. The Autoxidation of Human Red Cell Lipids Induced by Hydrogen Peroxide (1971) British Journal of Haematology, 20 (1), pp. 95-111. Cited 816 times. doi: 10.1111/j.1365-2141.1971.tb00790.x Locate full-text(opens in a new window) View at Publisher 14 Stocchi, V., Biagiarelli, B., Fiorani, M., Palma, F., Piccoli, G., Cucchiarini, L., Dachà, M. Inactivation of rabbit red blood cell hexokinase activity promoted in vitro by an oxygen-radical-generating system (1994) Archives of Biochemistry and Biophysics, 311 (1), pp. 160-167. Cited 25 times. doi: 10.1006/abbi.1994.1221 Locate full-text(opens in a new window) View at Publisher 15 Bontemps, F., Van den Berghe, G., Hers, H.G. Pathways of adenine nucleotide catabolism in erythrocytes (1986) Journal of Clinical Investigation, 77 (3), pp. 824-830. Cited 55 times. doi: 10.1172/JCI112379 Locate full-text(opens in a new window) View at Publisher 16 Paglia, D.E., Valentine, W.N., Nakatani, M., Brockway, R.A. Mechanisms of adenosine 5'-monophosphate catabolism in human erythrocytes (1986) Blood, 67 (4), pp. 988-992. Cited 15 times. Locate full-text(opens in a new window) 17 Giardina, B., Penco, M., Lazzarino, G., Romano, S., Tavazzi, B., Fedele, F., Di Pierro, D., (...), Dagianti, A. Effectiveness of thrombolysis is associated with a time-dependent increase of malondialdehyde in peripheral blood of patients with acute myocardial infarction (1993) The American Journal of Cardiology, 71 (10), pp. 788-793. Cited 20 times. doi: 10.1016/0002-9149(93)90825-W Locate full-text(opens in a new window) View at Publisher 18 Tavazzi, B., Di Pierro, D., Bartolini, M., Marino, M., Distefano, S., Galvano, M., Villani, C., (...), Lazzarino, G. Lipid peroxidation, tissue necrosis, and metabolic and mechanical recovery of isolated reperfused rat heart as a function of increasing ischemia (1998) Free Radical Research, 28 (1), pp. 25-37. Cited 32 times. http://www.tandfonline.com/loi/ifra20#.VtPBubdf27E doi: 10.3109/10715769809097873 Locate full-text(opens in a new window) View at Publisher 19 Sugihara, T., Rawicz, W., Evans, E.A., Hebbel, R.P. Lipid hydroperoxides permit deformation-dependent leak of monovalent cation from erythrocytes (1991) Blood, 77 (12), pp. 2757-2763. Cited 46 times. Locate full-text(opens in a new window) View at Publisher 20 Deuticke, B., Heller, K.B., Haest, C.W.M. Progressive oxidative membrane damage in erythrocytes after pulse treatment with t-butylhydroperoxide (1987) BBA - Biomembranes, 899 (1), pp. 113-124. Cited 46 times. doi: 10.1016/0005-2736(87)90245-8 Locate full-text(opens in a new window) View at Publisher 21 Snyder, L.M., Fortier, N.L., Trainor, J., Jacobs, J., Leb, L., Lubin, B., Chiu, D., (...), Mohandas, N. Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking (1985) Journal of Clinical Investigation, 76 (5), pp. 1971-1977. Cited 189 times. doi: 10.1172/JCI112196 Locate full-text(opens in a new window) View at Publisher 22 Valentine, W.N., Paglia, D.E., Clarke, S., Morimoto, B.H., Nakatani, M., Brockway, R. Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages (1985) Proceedings of the National Academy of Sciences of the United States of America, 82 (19), pp. 6682-6686. Cited 6 times. doi: 10.1073/pnas.82.19.6682 Locate full-text(opens in a new window) View at Publisher Lazzarino, G.; Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Viale A. Doria 6, Italy; email:lazzarig@mbox.unict.it © Copyright 2007 Elsevier B.V., All rights reserved. Back to results Previous 79of114 Next Top of page Metrics Learn more about article metrics in Scopus (opens in a new window) View all metrics 103 Citations in Scopus 88th Percentile 1.39 Field-Weighted Citation Impact PlumX Metrics Usage, Captures, Mentions, Social Media and Citations beyond Scopus.
Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress
TAVAZZI B;
2000-01-01
Abstract
To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37°C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress. Author keywords AMP-deaminaseEnergy metabolismHuman erythrocytesIMPOxidative stress Indexed keywords EMTREE drug terms: adenosine diphosphateadenosine monophosphate deaminaseadenosine phosphateadenosine triphosphateguanosine triphosphatehydrogen peroxidehypoxanthineinosineinosine phosphatemalonaldehydeuric acidxanthine EMTREE medical terms: articlecell levelcell membrane transportconcentration responsecontrolled studyenergy metabolismenzyme activationenzyme activityerythrocyte metabolismhemolysishumanhuman celllipid peroxidationoxidative stresspriority journalreaction analysis MeSH: Adenine NucleotidesAMP DeaminaseEnergy MetabolismErythrocytesGuanosine TriphosphateHemolysisHumansHydrogen PeroxideInosine MonophosphateLipid PeroxidationMalondialdehydeOxidative Stress Chemicals and CAS Registry Numbers: Adenine Nucleotides; AMP Deaminase, EC 3.5.4.6; Guanosine Triphosphate, 86-01-1; Hydrogen Peroxide, 7722-84-1; Inosine Monophosphate, 131-99-7; Malondialdehyde, 542-78-9 ISSN: 00142956 CODEN: EJBCA Source Type: Journal Original language: English DOI: 10.1046/j.1432-1327.2000.01042.x PubMed ID: 10651804 Document Type: Article References (22) View in search results format All Export Print E-mail Save to PDF Create bibliography 1 Forman, M.B., Virmani, R., Puett, D.W. Mechanisms and therapy of myocardial reperfusion injury (1990) Circulation, 81 (3 SUPPL.), pp. IV69-IV78. Cited 142 times. Locate full-text(opens in a new window) 2 Roof, R.L., Hoffman, S.W., Stein, D.G. Progesterone protects against lipid peroxidation following traumatic brain injury in rats (1997) Molecular and Chemical Neuropathology, 31 (1), pp. 1-11. Cited 240 times. doi: 10.1007/BF02815156 Locate full-text(opens in a new window) View at Publisher 3 De Flora, S., Izzotti, A., Randerath, K., Randerath, E., Bartsch, H., Nair, J., Balansky, R., (...), Lewtas, J. DNA adducts and chronic degenerative diseases. Pathogenetic relevance and implications in preventive medicine (1996) Mutation Research - Reviews in Genetic Toxicology, 366 (3), pp. 197-238. Cited 145 times. doi: 10.1016/S0165-1110(96)00043-7 Locate full-text(opens in a new window) View at Publisher 4 Ravindranath, V., Reed, D.J. Glutathione depletion and formation of glutathione-protein mixed disulfide following exposure of brain mitochondria to oxidative stress (1990) Biochemical and Biophysical Research Communications, 169 (3), pp. 1075-1079. Cited 122 times. doi: 10.1016/0006-291X(90)92004-J Locate full-text(opens in a new window) View at Publisher 5 Tavazzi, B., Lazzarino, G., Di Pierro, D., Giardina, B. Malondialdehyde production and ascorbate decrease are associated to the reperfusion of the isolated postischemic rat heart (1992) Free Radical Biology and Medicine, 13 (1), pp. 75-78. Cited 72 times. doi: 10.1016/0891-5849(92)90167-F Locate full-text(opens in a new window) View at Publisher 6 Di Pierro, D., Tavazzi, B., Lazzarino, G., Giardina, B. Malondialdehyde is a biochemical marker of peroxidative damage in the isolated reperfused rat heart (1992) Molecular and Cellular Biochemistry, 116 (1-2), pp. 193-196. Cited 34 times. doi: 10.1007/BF01270587 Locate full-text(opens in a new window) View at Publisher 7 Maridonneau, I., Braquet, P., Garay, R.P. Na+ and K+ transport damage induced by oxygen free radicals in human red cell membranes (1983) Journal of Biological Chemistry, 258 (5), pp. 3107-3113. Cited 150 times. Locate full-text(opens in a new window) View at Publisher 8 Rohn, T.T., Nelson, L.K., Waeg, G., Quinn, M.T. U-101033E (2,4-diaminopyrrolopyrimidine), a potent inhibitor of membrane lipid peroxidation as assessed by the production of 4-hydroxynonenal, malondialdehyde, and 4-hydroxynonenal- protein adducts (1998) Biochemical Pharmacology, 56 (10), pp. 1371-1379. Cited 35 times. www.elsevier.com/locate/biochempharm doi: 10.1016/S0006-2952(98)00266-4 Locate full-text(opens in a new window) View at Publisher 9 Snyder, L.M., Fortier, N.L., Leb, L., McKenney, J., Trainor, J., Sheerin, H., Mohandas, N. The role of membrane protein sulfhydryl groups in hydrogen peroxide-mediated membrane damage in human erythrocytes (1988) BBA - Biomembranes, 937 (C), pp. 229-240. Cited 55 times. doi: 10.1016/0005-2736(88)90245-3 Locate full-text(opens in a new window) View at Publisher 10 Davies, K.J., Goldberg, A.L. Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes. (1987) Journal of Biological Chemistry, 262 (17), pp. 8220-8226. Cited 344 times. Locate full-text(opens in a new window) View at Publisher 11 Lazzarino, G., Di Pierro, D., Tavazzi, B., Cerroni, L., Giardina, B. Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography (1991) Analytical Biochemistry, 197 (1), pp. 191-196. Cited 107 times. doi: 10.1016/0003-2697(91)90378-7 Locate full-text(opens in a new window) View at Publisher 12 Miki, M., Tamai, H., Mino, M., Yamamoto, Y., Niki, E. Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by α-tocopherol (1987) Archives of Biochemistry and Biophysics, 258 (2), pp. 373-380. Cited 305 times. doi: 10.1016/0003-9861(87)90358-4 Locate full-text(opens in a new window) View at Publisher 13 Stocks, J., Dormandy, T.L. The Autoxidation of Human Red Cell Lipids Induced by Hydrogen Peroxide (1971) British Journal of Haematology, 20 (1), pp. 95-111. Cited 816 times. doi: 10.1111/j.1365-2141.1971.tb00790.x Locate full-text(opens in a new window) View at Publisher 14 Stocchi, V., Biagiarelli, B., Fiorani, M., Palma, F., Piccoli, G., Cucchiarini, L., Dachà, M. Inactivation of rabbit red blood cell hexokinase activity promoted in vitro by an oxygen-radical-generating system (1994) Archives of Biochemistry and Biophysics, 311 (1), pp. 160-167. Cited 25 times. doi: 10.1006/abbi.1994.1221 Locate full-text(opens in a new window) View at Publisher 15 Bontemps, F., Van den Berghe, G., Hers, H.G. Pathways of adenine nucleotide catabolism in erythrocytes (1986) Journal of Clinical Investigation, 77 (3), pp. 824-830. Cited 55 times. doi: 10.1172/JCI112379 Locate full-text(opens in a new window) View at Publisher 16 Paglia, D.E., Valentine, W.N., Nakatani, M., Brockway, R.A. Mechanisms of adenosine 5'-monophosphate catabolism in human erythrocytes (1986) Blood, 67 (4), pp. 988-992. Cited 15 times. Locate full-text(opens in a new window) 17 Giardina, B., Penco, M., Lazzarino, G., Romano, S., Tavazzi, B., Fedele, F., Di Pierro, D., (...), Dagianti, A. Effectiveness of thrombolysis is associated with a time-dependent increase of malondialdehyde in peripheral blood of patients with acute myocardial infarction (1993) The American Journal of Cardiology, 71 (10), pp. 788-793. Cited 20 times. doi: 10.1016/0002-9149(93)90825-W Locate full-text(opens in a new window) View at Publisher 18 Tavazzi, B., Di Pierro, D., Bartolini, M., Marino, M., Distefano, S., Galvano, M., Villani, C., (...), Lazzarino, G. Lipid peroxidation, tissue necrosis, and metabolic and mechanical recovery of isolated reperfused rat heart as a function of increasing ischemia (1998) Free Radical Research, 28 (1), pp. 25-37. Cited 32 times. http://www.tandfonline.com/loi/ifra20#.VtPBubdf27E doi: 10.3109/10715769809097873 Locate full-text(opens in a new window) View at Publisher 19 Sugihara, T., Rawicz, W., Evans, E.A., Hebbel, R.P. Lipid hydroperoxides permit deformation-dependent leak of monovalent cation from erythrocytes (1991) Blood, 77 (12), pp. 2757-2763. Cited 46 times. Locate full-text(opens in a new window) View at Publisher 20 Deuticke, B., Heller, K.B., Haest, C.W.M. Progressive oxidative membrane damage in erythrocytes after pulse treatment with t-butylhydroperoxide (1987) BBA - Biomembranes, 899 (1), pp. 113-124. Cited 46 times. doi: 10.1016/0005-2736(87)90245-8 Locate full-text(opens in a new window) View at Publisher 21 Snyder, L.M., Fortier, N.L., Trainor, J., Jacobs, J., Leb, L., Lubin, B., Chiu, D., (...), Mohandas, N. Effect of hydrogen peroxide exposure on normal human erythrocyte deformability, morphology, surface characteristics, and spectrin-hemoglobin cross-linking (1985) Journal of Clinical Investigation, 76 (5), pp. 1971-1977. Cited 189 times. doi: 10.1172/JCI112196 Locate full-text(opens in a new window) View at Publisher 22 Valentine, W.N., Paglia, D.E., Clarke, S., Morimoto, B.H., Nakatani, M., Brockway, R. Adenine ribo- and deoxyribonucleotide metabolism in human erythrocytes, B- and T-lymphocyte cell lines, and monocyte-macrophages (1985) Proceedings of the National Academy of Sciences of the United States of America, 82 (19), pp. 6682-6686. Cited 6 times. doi: 10.1073/pnas.82.19.6682 Locate full-text(opens in a new window) View at Publisher Lazzarino, G.; Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Viale A. Doria 6, Italy; email:lazzarig@mbox.unict.it © Copyright 2007 Elsevier B.V., All rights reserved. Back to results Previous 79of114 Next Top of page Metrics Learn more about article metrics in Scopus (opens in a new window) View all metrics 103 Citations in Scopus 88th Percentile 1.39 Field-Weighted Citation Impact PlumX Metrics Usage, Captures, Mentions, Social Media and Citations beyond Scopus.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
