Objective:Avidly phagocytosed hemozoin(malarial pigment) alters several functions of human monocytes and stimulates generation of several cytokines.Recently,we showed that phagocytosis of hemozoin by human monocytes i...Objective:Avidly phagocytosed hemozoin(malarial pigment) alters several functions of human monocytes and stimulates generation of several cytokines.Recently,we showed that phagocytosis of hemozoin by human monocytes increases expression and activity of matrix metalloproteinase-9,a proteolytic enzyme available in specific gelatinase granules,which contain several enzymes including lysozyme.Present work investigated active lysozyme release after phagocytosis of hemozoin and its dependence on production of tumor necrosis factor alpha. Methods:After phagocytosis of hemozoin,hemozoin-containing trophozoites or control meals(opsonized nonparasitized red blood cells and latex particles),monocyte supematants were monitored for 2 hours,in presence of blocking anti-human tumor necrosis factor alpha antibodies or recombinant human tumor necrosis factor alpha cytokine in selected experiments.Lysozyme release was evaluated by a specific spectrometric assay measuring lysozyme activity after coincubation of cell supematants with suspensions of Mycrococcus Lysodeikticus,while levels of soluble tumor necrosis factor alpha were analyzed by specific enzyme-linked immunodsorbent assay. Results:Levels of lysozyme activity and soluble tumor necrosis factor alpha protein were increased in hemozoin in-or trophozoites-laden monocytes supematants.Phagocytosis per se(control meals) also increased lysozyme release,but levels were significantly lower than those obtained after phagocytosis of hemozoin or trophozoites. Interestingly,all effects on lysozyme release observed after phagocytosis were abrogated by blocking anti-human tumor necrosis factor alpha antibodies,while they were mimicked by recombinant human tumor necrosis factor alpha cytokine.Conclusions:Present work shows that phagocytosis of hemozoin promotes monocyte degranulation and enhances active lysozyme release.The effect requires tumor necrosis factor alpha mediation.展开更多
Objective:To investigate the regulation of matrix metalloproteinases(MMPs) and tissue inhibitors of melalloproleinases(TIMPs) in human microvascular endothelium(HMEC-1) exposed to erythrocytes infected by different st...Objective:To investigate the regulation of matrix metalloproteinases(MMPs) and tissue inhibitors of melalloproleinases(TIMPs) in human microvascular endothelium(HMEC-1) exposed to erythrocytes infected by different strains of Plasmodium falciparum(P.falciparum).Methods: HMEC—1 eells were co—incubated for 72 h with erythrocytes infected by late stage trophozoite of D10(chloroquine-sensilive) or W 2(chloroquine-resistant) P.falciparum strains.Cell supernatants were then collected and the levels of pro- or active gelatinases MMP-9 and MMP-2 were evaluated by gelatin zymograpln and densitometry.The release of pro-MMP-9,MMP-3.MMP-1 and TIMP-1 proteins was analyzed by western blotting and densitometry.Results:Infected erythrocytes induced de novo proMMP-9 and MMP-9 release.Neither basal levels of proMMP-2 were altered,nor active MMP-2 was found.MMP-3 and MMP-1 secretion was significant!) enhanced,whereas basal TIMP-1 was unaffected.All effects were similar for both strains. Conclusions:P.falciparum parasites,either chloroquine-sonsitive or -resistant,induce the release of active MMP-9 protein from human microvascular endothelium,by impairing balances between proMMP-9 and its inhibitor,and by enhancing the levels of its activators.This work provides new evidence on MMP involvement in malaria,pointing at MMP-9 as a possible target in adjuvant therapy.展开更多
Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play important roles in the formation and regression of corpus luteum (CL). This study is to investigate the expression of gelatinas...Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play important roles in the formation and regression of corpus luteum (CL). This study is to investigate the expression of gelatinases (MMP-2, -9) and TIMPs in the rhesus monkey CL in both early and late luteal phases and during the early stages of pregnancy. Ovaries were collected from regularly cycling rhesus monkey at D5 and D15 following ovulation and at D12, D18 and D26 of pregnancy. In situ hybridization revealed that in the CL MMP-2 niRNA was expressed during both formation and regression, while MMP-9 mRNA was mainly localized in the late luteal phase. Reduction of MMP-2, -9 transcripts in the CL was observed during pregnancy. MMP-2 mRNA in the CL reduced to an undetectable level at D26 of pregnancy. TIMP-1 mRNA was highly expressed in the CL in both early and late luteal phases and persisted throughout the early stages of pregnancy. Strong signal for TIMP-2 mRNA was also detected in both luteal phases, and the展开更多
To ascertain the molecular basis of Ca2+-mediated activation of matrix metalloproteinase-9 (MMP-9), we determined the accessibility of tryptophan residues to externally added acrylamide as quencher in the absence and ...To ascertain the molecular basis of Ca2+-mediated activation of matrix metalloproteinase-9 (MMP-9), we determined the accessibility of tryptophan residues to externally added acrylamide as quencher in the absence and presence of the metal ion. The steady-state and time resolved fluorescence data revealed that MMP-9 possesses two classes of tryptophan residues, “exposed” and “buried” which are quenched by the collisional rate constants (kq) of 3.2′ 109M-1.s-1 and 7.5′ 108M-1.s-1, respectively. These values are impaired by approximately two and three-fold, respectively, in the presence of 10 mM Ca2+. The Stern-Volmer constants (Ksv values) predicted from the time resolved fluorescence data (in the absence of Ca2+ ) satisfied the dynamic quenching model of the enzyme’s tryptophan residues. This was not the case in the presence of Ca2+;the steady-state acrylamide quenching data could only be explained by a combination of “dynamic” and “static” quenching models. A cumulative account of these data led to the suggestion that the binding of Ca2+ modulated the tertiary structure of the protein by decreasing the dynamic flexibility of the enzyme, which is manifested in further structuring of the enzyme’s active site pocket toward facilitating catalysis. Arguments are presented that the binding of Ca2+ at distal sites “dynamically” communicates with the active site residues of MMP-9 during catalysis.展开更多
基金supported in the context of the Italian Malaria Network by grants from Compagnia di San Paolo-IMIthe University of Torino Intramural FundsRegione Piemonte,Ricerca Sanitaria Finalizzata 2007 to PA
文摘Objective:Avidly phagocytosed hemozoin(malarial pigment) alters several functions of human monocytes and stimulates generation of several cytokines.Recently,we showed that phagocytosis of hemozoin by human monocytes increases expression and activity of matrix metalloproteinase-9,a proteolytic enzyme available in specific gelatinase granules,which contain several enzymes including lysozyme.Present work investigated active lysozyme release after phagocytosis of hemozoin and its dependence on production of tumor necrosis factor alpha. Methods:After phagocytosis of hemozoin,hemozoin-containing trophozoites or control meals(opsonized nonparasitized red blood cells and latex particles),monocyte supematants were monitored for 2 hours,in presence of blocking anti-human tumor necrosis factor alpha antibodies or recombinant human tumor necrosis factor alpha cytokine in selected experiments.Lysozyme release was evaluated by a specific spectrometric assay measuring lysozyme activity after coincubation of cell supematants with suspensions of Mycrococcus Lysodeikticus,while levels of soluble tumor necrosis factor alpha were analyzed by specific enzyme-linked immunodsorbent assay. Results:Levels of lysozyme activity and soluble tumor necrosis factor alpha protein were increased in hemozoin in-or trophozoites-laden monocytes supematants.Phagocytosis per se(control meals) also increased lysozyme release,but levels were significantly lower than those obtained after phagocytosis of hemozoin or trophozoites. Interestingly,all effects on lysozyme release observed after phagocytosis were abrogated by blocking anti-human tumor necrosis factor alpha antibodies,while they were mimicked by recombinant human tumor necrosis factor alpha cytokine.Conclusions:Present work shows that phagocytosis of hemozoin promotes monocyte degranulation and enhances active lysozyme release.The effect requires tumor necrosis factor alpha mediation.
基金supported by Universita di Milano(PUR.2009) to Nicoletta Basilico and Charity Funds from Mrs.Franca Squazza to Mauro PratoMauro Prato holds a professorshipgranted by Universita Torino and Azienda Sanitaria Locale-19(ASL-19)
文摘Objective:To investigate the regulation of matrix metalloproteinases(MMPs) and tissue inhibitors of melalloproleinases(TIMPs) in human microvascular endothelium(HMEC-1) exposed to erythrocytes infected by different strains of Plasmodium falciparum(P.falciparum).Methods: HMEC—1 eells were co—incubated for 72 h with erythrocytes infected by late stage trophozoite of D10(chloroquine-sensilive) or W 2(chloroquine-resistant) P.falciparum strains.Cell supernatants were then collected and the levels of pro- or active gelatinases MMP-9 and MMP-2 were evaluated by gelatin zymograpln and densitometry.The release of pro-MMP-9,MMP-3.MMP-1 and TIMP-1 proteins was analyzed by western blotting and densitometry.Results:Infected erythrocytes induced de novo proMMP-9 and MMP-9 release.Neither basal levels of proMMP-2 were altered,nor active MMP-2 was found.MMP-3 and MMP-1 secretion was significant!) enhanced,whereas basal TIMP-1 was unaffected.All effects were similar for both strains. Conclusions:P.falciparum parasites,either chloroquine-sonsitive or -resistant,induce the release of active MMP-9 protein from human microvascular endothelium,by impairing balances between proMMP-9 and its inhibitor,and by enhancing the levels of its activators.This work provides new evidence on MMP involvement in malaria,pointing at MMP-9 as a possible target in adjuvant therapy.
基金This work was supported by the "95" National"Climbing Project" and the Knowledge Innovation Project of the Chinese Academy of Sciences.
文摘Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play important roles in the formation and regression of corpus luteum (CL). This study is to investigate the expression of gelatinases (MMP-2, -9) and TIMPs in the rhesus monkey CL in both early and late luteal phases and during the early stages of pregnancy. Ovaries were collected from regularly cycling rhesus monkey at D5 and D15 following ovulation and at D12, D18 and D26 of pregnancy. In situ hybridization revealed that in the CL MMP-2 niRNA was expressed during both formation and regression, while MMP-9 mRNA was mainly localized in the late luteal phase. Reduction of MMP-2, -9 transcripts in the CL was observed during pregnancy. MMP-2 mRNA in the CL reduced to an undetectable level at D26 of pregnancy. TIMP-1 mRNA was highly expressed in the CL in both early and late luteal phases and persisted throughout the early stages of pregnancy. Strong signal for TIMP-2 mRNA was also detected in both luteal phases, and the
文摘To ascertain the molecular basis of Ca2+-mediated activation of matrix metalloproteinase-9 (MMP-9), we determined the accessibility of tryptophan residues to externally added acrylamide as quencher in the absence and presence of the metal ion. The steady-state and time resolved fluorescence data revealed that MMP-9 possesses two classes of tryptophan residues, “exposed” and “buried” which are quenched by the collisional rate constants (kq) of 3.2′ 109M-1.s-1 and 7.5′ 108M-1.s-1, respectively. These values are impaired by approximately two and three-fold, respectively, in the presence of 10 mM Ca2+. The Stern-Volmer constants (Ksv values) predicted from the time resolved fluorescence data (in the absence of Ca2+ ) satisfied the dynamic quenching model of the enzyme’s tryptophan residues. This was not the case in the presence of Ca2+;the steady-state acrylamide quenching data could only be explained by a combination of “dynamic” and “static” quenching models. A cumulative account of these data led to the suggestion that the binding of Ca2+ modulated the tertiary structure of the protein by decreasing the dynamic flexibility of the enzyme, which is manifested in further structuring of the enzyme’s active site pocket toward facilitating catalysis. Arguments are presented that the binding of Ca2+ at distal sites “dynamically” communicates with the active site residues of MMP-9 during catalysis.
