RESUMO
In mammals, haem degradation to biliverdin (BV) through the action of haem oxygenase (HO) is a critical step in haem metabolism. The malaria parasite converts haem into the chemically inert haemozoin to avoid toxicity. We discovered that the knock-out of HO in P. berghei is lethal; therefore, we investigated the function of biliverdin (BV) and haem in the parasite. Addition of external BV and haem to P. falciparum-infected red blood cell (RBC) cultures delays the progression of parasite development. The search for a BV molecular target within the parasites identified P. falciparum enolase (Pf enolase) as the strongest candidate. Isothermal titration calorimetry using recombinant full-length Plasmodium enolase suggested one binding site for BV. Kinetic assays revealed that BV is a non-competitive inhibitor. We employed molecular modelling studies to predict the new binding site as well as the binding mode of BV to P. falciparum enolase. Furthermore, addition of BV and haem targets the phosphorylation of Plasmodium falciparum eIF2α factor, an eukaryotic initiation factor phosphorylated by eIF2α kinases under stress conditions. We propose that BV targets enolase to reduce parasite glycolysis rates and changes the eIF2α phosphorylation pattern as a molecular mechanism for its action.
Assuntos
Biliverdina/metabolismo , Eritrócitos/parasitologia , Fator de Iniciação 2 em Eucariotos/antagonistas & inibidores , Fosfopiruvato Hidratase/antagonistas & inibidores , Plasmodium falciparum/metabolismo , Sequência de Aminoácidos , Biliverdina/farmacologia , Eritrócitos/metabolismo , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Malária Falciparum/metabolismo , Modelos Moleculares , Proteínas de Protozoários/antagonistas & inibidores , Alinhamento de SequênciaRESUMO
Hybrid scaffolds made of xanthan and magnetite nanoparticles (XCA/mag) were prepared by dipping xanthan membranes (XCA) into dispersions of magnetic nanoparticles for different periods of time. The resulting hybrid scaffolds presented magnetization values ranging from 0.25 emu g(-1) to 1.80 emu g(-1) at 70 kOe and corresponding iron contents ranging from 0.25% to 2.3%, respectively. They were applied as matrices for in vitro embryoid body adhesion and neuronal differentiation of embryonic stem cells; for comparison, neat XCA and commercial plastic plates were also used. Adhesion rates were more pronounced when cells were seeded on XCA/mag than on neat XCA or plastic dishes; however, proliferation levels were independent from those of the scaffold type. Embryonic stem cells showed similar differentiation rates on XCA/mag scaffolds with magnetization of 0.25 and 0.60 emu g(-1), but did not survive on scaffolds with 1.80 emu g(-1). Differentiation rates, expressed as the number of neurons obtained on the chosen scaffolds, were the largest on neat XCA, which has a high density of negative charge, and were smallest on the commercial plastic dishes. The local magnetic field inherent of magnetite particles present on the surface of XCA/mag facilitates synapse formation, because synaptophysin expression and electrical transmission were increased when compared to the other scaffolds used. We conclude that XCA/mag and XCA hydrogels are scaffolds with distinguishable performance for adhesion and differentiation of ESCs into neurons.
Assuntos
Células-Tronco Embrionárias/citologia , Nanopartículas de Magnetita/química , Neurônios/citologia , Polissacarídeos Bacterianos/química , Alicerces Teciduais , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Células-Tronco Embrionárias/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Regeneração Tecidual Guiada/instrumentação , Humanos , Nanopartículas de Magnetita/ultraestrutura , Teste de Materiais , Neurogênese/fisiologia , Neurônios/fisiologia , Tamanho da Partícula , Engenharia Tecidual/instrumentaçãoRESUMO
The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle-like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake by P. falciparum-infected erythrocytes shows that at R and S stages, a time-increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time-increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.
Assuntos
Eritrócitos/parasitologia , Plasmodium falciparum/metabolismo , Protoporfirinas/metabolismo , Biliverdina/metabolismo , Eritrócitos/metabolismo , Heme Oxigenase (Desciclizante)/classificação , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Hemeproteínas/metabolismo , Hemina/metabolismo , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
There is a growing body of evidence that melatonin and its oxidation product, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle.