55 research outputs found
Kinetics of cyanide and carbon monoxide dissociation from ferrous human haptoglobin:hemoglobin(II) complexes
No full textHaptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) trapping the alpha beta dimers of Hb. In turn, the Hp:Hb complexes display heme-based reactivity. Here, the kinetics of cyanide and carbon monoxide dissociation from ferrous-ligated Hp:Hb complexes are reported at pH 7.0 and 20.0 degrees C. Cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb-CN- has been followed upon the dithionite-mediated conversion of ferric to ferrous-ligated Hp:Hb complexes. Values of k(on) for the dithionite-mediated reduction of Hp1-1:Hb(III)-CN- and Hp2-2:Hb(III)-CN- are (7.3 +/- 1.1) x 10(6) M-1 s(-1) and (6.2 +/- 1.0) x 10(6) M-1 s(-1), respectively. Values of the first-order rate constant (i.e., h) for cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb(II)-CN- are (1.2 +/- 0.2) x 10(-1) s(-1) and (1.3 +/- 0.2) x 10(-1) s(-1), respectively. CO dissociation from Hp:Hb(II)-CO complexes has been followed by replacing CO with NO. Values of the first-order rate constant (i.e., l) for CO dissociation from Hp1-1:Hb(II)-CO are (1.4 +/- 0.2) x 10(-2) s(-1) and (6.2 +/- 0.8) x 10(-3) s(-1), and those from Hp2-2:Hb(II)-CO are (1.3 +/- 0.2) x 10(-2) s(-1) and (7.3 +/- 0.9) x 10(-3) s(-1). Values of k(on), h, and l correspond to those reported for the R-state of tetrameric Hb and isolated alpha and beta chains. This highlights the view that the conformation of the Hb alpha beta-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. Furthermore, unlike ferric Hb(III), ligated ferrous Hb(II) does not show an assembly-linked structural change. Graphic abstrac
Kinetics of cyanide and carbon monoxide dissociation from ferrous human haptoglobin:hemoglobin(II) complexes
No full textHaptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) trapping the αβ dimers of Hb. In turn, the Hp:Hb complexes display heme-based reactivity. Here, the kinetics of cyanide and carbon monoxide dissociation from ferrous-ligated Hp:Hb complexes are reported at pH 7.0 and 20.0 °C. Cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb-CN- has been followed upon the dithionite-mediated conversion of ferric to ferrous-ligated Hp:Hb complexes. Values of kon for the dithionite-mediated reduction of Hp1-1:Hb(III)-CN- and Hp2-2:Hb(III)-CN- are (7.3 ± 1.1) × 106 M-1 s-1 and (6.2 ± 1.0) × 106 M-1 s-1, respectively. Values of the first-order rate constant (i.e., h) for cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb(II)-CN- are (1.2 ± 0.2) × 10-1 s-1 and (1.3 ± 0.2) × 10-1 s-1, respectively. CO dissociation from Hp:Hb(II)-CO complexes has been followed by replacing CO with NO. Values of the first-order rate constant (i.e., l) for CO dissociation from Hp1-1:Hb(II)-CO are (1.4 ± 0.2) × 10-2 s-1 and (6.2 ± 0.8) × 10-3 s-1, and those from Hp2-2:Hb(II)-CO are (1.3 ± 0.2) × 10-2 s-1 and (7.3 ± 0.9) × 10-3 s-1. Values of kon, h, and l correspond to those reported for the R-state of tetrameric Hb and isolated α and β chains. This highlights the view that the conformation of the Hb αβ-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. Furthermore, unlike ferric Hb(III), ligated ferrous Hb(II) does not show an assembly-linked structural change
Hydroxylamine-induced oxidation of ferrous nitrobindins
No full textHemoglobin and myoglobin are generally taken as molecular models of all-α-helical heme-proteins. On the other hand, nitrophorins and nitrobindins (Nb), which are arranged in 8 and 10 β-strands, respectively, represent the molecular models of all-β-barrel heme-proteins. Here, kinetics of the hydroxylamine- (HA-) mediated oxidation of ferrous Mycobacterium tuberculosis, Arabidopsis thaliana, and Homo sapiens nitrobindins (Mt-Nb(II), At-Nb(II), and Hs-Nb(II), respectively), at pH 7.0 and 20.0 °C, are reported. Of note, HA displays antibacterial properties and is a good candidate for the treatment and/or prevention of reactive nitrogen species- (RNS-) linked aging-related pathologies, such as macular degeneration. Under anaerobic conditions, mixing the Mt-Nb(II), At-Nb(II), and Hs-Nb(II) solutions with the HA solutions brings about absorbance spectral changes reflecting the formation of the ferric derivative (i.e., Mt-Nb(III), At-Nb(III), and Hs-Nb(III), respectively). Values of the second order rate constant for the HA-mediated oxidation of Mt-Nb(II), At-Nb(II), and Hs-Nb(II) are 1.1 × 104 M-1 s-1, 6.5 × 104 M-1 s-1, and 2.2 × 104 M-1 s-1, respectively. Moreover, the HA:Nb(II) stoichiometry is 1:2 as reported for ferrous deoxygenated and carbonylated all-α-helical heme-proteins. A comparative look of the HA reduction kinetics by several ferrous heme-proteins suggests that an important role might be played by residues (such as His or Tyr) in the proximity of the heme-Fe atom either coordinating it or not. In this respect, Nbs seem to exploit somewhat different structural aspects, indicating that redox mechanisms for the heme-Fe(II)-to-heme-Fe(III) conversion might differ between all-α-helical and all-β-barrel heme-proteins
Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study
No full textNitrobindins (Nbs) are all-β-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3−. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2− to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M−1 s−1, 9.3 M−1 s−1, 1.4 × 101 M−1 s−1, and 5.8 M−1 s−1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2−-based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-β-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-β-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged
Ragionare per diventare filosofi. Tra Kant e Habermas
No full textIl saggio analizza la fonte kantiana dell'etica del discorso di Habermas. Attraverso la mediazione della filosofia postdeontica, la crisi delle prospettive universalistiche, la parzialità delle validità concettuali e assiologiche guarda alla reinterpretazione habermasiana che corrisponde alla pluralizzazione delle letture della realtà e della normatività del pratico
Il cosmopolitismo. Vincoli morali e progettualità politica.
No full textL'ampio saggio ricostruisce i luoghi e i modi del formarsi di una tradizione teorica definita cosmopolitica lungo il pensiero antico e la modernità. Si confronta con la Globalizzazione, la scienza-tecnologia, la rete delle interdipendenze. Indaga le nuove vie del pensiero cosmopolitico
Role of the chitin-binding domain (CBD) of a grapevine class IV chitinase against Botrytis cinerea
No full textDuring plant infection, fungal chitin deposition at the hyphal tip is counteracted by host chitinases. We studied the interaction between Botrytis cinerea and a class IV chitinase, that is constitutively expressed in mature grapevine berries. Early during fungal growth, the fungus proteolytically removes the chitin-binding domain (CBD). This removal results in a 50% reduction in the enzymatic activity of the chitinase and a complete loss of its botryticidal activity. Despite this, the pattern of chitin degradation by the native and the cleaved chitinase remains similar. We also detected the expression of this class IV chitinase in grapevine leaves during B. cinerea infection. Analysis of chitinase activity in leaf samples at various infection stages revealed that while this chitinase is not the most active among all chitinases, it is the most abundant at the protein level, with its expression correlating with infection progression. MALDI-TOF analysis of specific bands after SDS-PAGE confirmed the presence of both the native and CBD-deprived forms of the chitinase in the infected leaves, indicating that CBD removal observed in vitro also occurs during infection. Given the relatively low activity of this chitinase, as shown by zymogram, we further investigated the role of the CBD against B. cinerea. We transiently expressed the CBD in Nicotiana tabacum and heterologously expressed it in Pichia pastoris to assess its function. These findings highlight the significant role of the CBD in the chitinase's antifungal activity and its potential application in enhancing plant resistance to fungal infections
Preliminary Design and Assessment of a Waste Heat Recovery System for a Hybrid-Electric Heavy-Duty Vehicle
No full textFerric nitrosylated myoglobin catalyzes peroxynitrite scavenging
No full textMyoglobin (Mb), generally taken as the molecular model of monomeric globular heme-proteins, is devoted: (i) to act as an intracellular oxygen reservoir, (ii) to transport oxygen from the sarcolemma to the mitochondria of vertebrate heart and red muscle cells, and (iii) to act as a scavenger of nitrogen and oxygen reactive species protecting mitochondrial respiration. Here, the first evidence of ·NO inhibition of ferric Mb- (Mb(III)) mediated detoxification of peroxynitrite is reported, at pH 7.2 and 20.0 °C. ·NO binds to Mb(III) with a simple equilibrium; the value of the second-order rate constant for Mb(III) nitrosylation (i.e., ·NOkon) is (6.8 ± 0.7) × 104 M-1 s-1 and the value of the first-order rate constant for Mb(III)-NO denitrosylation (i.e., ·NOkoff) is 3.1 ± 0.3 s-1. The calculated value of the dissociation equilibrium constant for Mb(III)-NO complex formation (i.e., ·NOkoff/·NOkon = (4.6 ± 0.7) × 10-5 M) is virtually the same as that directly measured (i.e., ·NOK = (3.8 ± 0.5) × 10-5 M). In the absence of ·NO, Mb(III) catalyzes the conversion of peroxynitrite to NO3-, the value of the second-order rate constant (i.e., Pkon) being (1.9 ± 0.2) × 104 M-1 s-1. However, in the presence of ·NO, Mb(III)-mediated detoxification of peroxynitrite is only partially inhibited, underlying the possibility that also Mb(III)-NO is able to catalyze the peroxynitrite isomerization, though with a reduced rate (Pkon* = (2.8 ± 0.3) × 103 M-1 s-1). These data expand the multiple roles of ·NO in modulating heme-protein actions, envisaging a delicate balancing between peroxynitrite and ·NO, which is modulated through the relative amount of Mb(III) and Mb(III)-NO
Botrytis cinerea cleaves a grapevine chitinase reducing its enzymatic activity and its detrimental effects on fungal growth
No full textChitin represents the main fibrillary component of the cell wall in filamentous fungi. During plant infection, chitin apposition to the fungal cell wall is counteracted by host chitinases. Following Botrytis cinerea infection, a chitinase IV is highly expressed in grapevine leaves and is also abundantly contained in grapevine berries. The fungus decreases the molecular size of this protein (Favaron et al., 2009, Journal of Plant Pathology, 91, 579-588) by its proteolytic activity (Marcato et al., 2017, Physiological and Molecular Plant Pathology, 99, 7-15). The cleavage of the chitinase occurs early during the in vitro fungal growth. The N-terminal sequencing of the cleaved chitinase shows that the fragment removed is the chitin-binding domain (CBD). Without the CBD, the chitinase decreases its activity by about 50%. To investigate the possible effects of the native and cleaved chitinase on B. cinerea, the two purified proteins were administered at 100 μg/ml to the fungal spores providing the proper conditions to avoid the chitinase cleavage. The native chitinase significantly decreased the conidia germination rate and the length of the germination tube while the cleaved chitinase did not. Protease inhibition assays provided evidence that metalloprotease activity is involved in the chitinase cleavage.
To ascertain whether the native and cleaved chitinase can differently affect the expression of genes involved in B. cinerea cell wall modeling, we analyzed the expression of five fungal chitin synthase (Chs) and four chitin deacetylase (Cda) genes. By quantitative PCR we observed that only one Chs gene decreased its expression in presence of the native chitinase and three Cda genes were slightly down-regulated by both native and cleaved chitinase.
In conclusion, the removal of the CBD by B. cinerea proteases appears as a mechanism preserving fungal growth from plant chitinase activity as highlighted also with other fungi (Jashni et al. 2015, Frontiers of plant science, 6, 1-7). Further experiments will better clarify the type of B. cinerea protease activity capable to disarm the plant chitinases
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