Extracellular ATP triggers proteolysis and cytosolic Ca²⁺ rise in Plasmodium berghei and Plasmodium yoelii malaria parasites.

BACKGROUND: Plasmodium has a complex cell biology and it is essential to dissect the cell-signalling pathways underlying its survival within the host. METHODS: Using the fluorescence resonance energy transfer (FRET) peptide substrate Abz-AIKFFARQ-EDDnp and Fluo4/AM, the effects of extracellular ATP on triggering proteolysis and Ca²⁺ signalling in Plasmodium berghei and Plasmodium yoelii malaria parasites were investigated. RESULTS: The protease activity was blocked in the presence of the purinergic receptor blockers suramin (50 μM) and PPADS (50 μM) or the extracellular and intracellular calcium chelators EGTA (5 mM) and BAPTA/AM (25, 100, 200 and 500 μM), respectively for P. yoelii and P. berghei. Addition of ATP (50, 70, 200 and 250 μM) to isolated parasites previously loaded with Fluo4/AM in a Ca²⁺-containing medium led to an increase in cytosolic calcium. This rise was blocked by pre-incubating the parasites with either purinergic antagonists PPADS (50 μM), TNP-ATP (50 μM) or the purinergic blockers KN-62 (10 μM) and Ip5I (10 μM). Incubating P. berghei infected cells with KN-62 (200 μM) resulted in a changed profile of merozoite surface protein 1 (MSP1) processing as revealed by western blot assays. Moreover incubating P. berghei for 17 h with KN-62 (10 μM) led to an increase in rings forms (82% ± 4, n = 11) and a decrease in trophozoite forms (18% ± 4, n = 11). CONCLUSIONS: The data clearly show that purinergic signalling modulates P. berghei protease(s) activity and that MSP1 is one target in this pathway

Effects of 'casoparan', a peptide isolated from casein hydrolysates with mastoparan-like properties.

Casein, a protein found in milk of several species, is divided into different chains from 19 to 25 kDa. Casein is also considered as a source of amino acids and generating peptides with biological activities such as opiate, immunostimulating, antibacterial, peptidase inhibitors, among others. In this work, Sephadex G-10 chromatography followed by high-performance liquid chromatography isolation purified NZCase TT, an industrial culture media for tetanus toxin production. In the first step, four pools were isolated and tested in different assays: isolated smooth muscle assay (guinea pig ileum, rat uterus), phagocytosis in vitro of opsonized sheep red blood cells, and hydrogen peroxide (H2O2) release from mouse peritoneal macrophages. Pool III was the main active pool being able to potentiate bradykinin action in guinea pig ileum, stimulating phagocitic activity by resident macrophages and increasing H2O2 release from macrophages previously activated with bacille Calmette Guérin. Using mass spectra the primary structure of the main peptide from pool III was obtained--INKKI, which corresponds to beta-casein fragment 26-30. The immunostimulating action is probably related to a direct action in macrophage cells

Cysteine 904 is required for maximal insulin degrading enzyme activity and polyanion activation

Cysteine residues in insulin degrading enzyme have been reported as non-critical for its activity. We found that converting the twelve cysteine residues in rat insulin degrading enzyme (IDE) to serines resulted in a cysteine-free form of the enzyme with reduced activity and decreased activation by polyanions. Mutation of each cysteine residue individually revealed cysteine 904 as the key residue required for maximal activity and polyanion activation, although other cysteines affect polyanion binding to a lesser extent. Based on the structure of IDE, Asn 575 was identified as a potential hydrogen bond partner for Cys904 and mutation of this residue also reduced activity and decreased polyanion activation. The oligomerization state of IDE did not correlate with its activity, with the dimer being the predominant form in all the samples examined. These data suggest that there are several conformational states of the dimer that affect activity and polyanion activation

Characterization of thimet oligopeptidase and neurolysin activities in B16F10-Nex2 tumor cells and their involvement in angiogenesis and tumor growth

<p>Abstract</p> <p>Background</p> <p>Angiogenesis is a fundamental process that allows tumor growth by providing nutrients and oxygen to the tumor cells. Beyond the oxygen diffusion limit from a capillary blood vessel, tumor cells become apoptotic. Angiogenesis results from a balance of pro- and anti-angiogenic stimuli. Endogenous inhibitors regulate enzyme activities that promote angiogenesis. Tumor cells may express pro-angiogenic factors and hydrolytic enzymes but also kinin-degrading oligopeptidases which have been investigated.</p> <p>Results</p> <p>Angiogenesis induced by B16F10-Nex2 melanoma cells was studied in a co-culture with HUVEC on Matrigel. A stimulating effect on angiogenesis was observed in the presence of B16F10-Nex2 lysate and plasma membrane. In contrast, the B16F10-Nex2 culture supernatant inhibited angiogenesis in a dose-dependent manner. This effect was abolished by the endo-oligopeptidase inhibitor, JA-2. Thimet oligopeptidase (TOP) and neurolysin activities were then investigated in B16F10-Nex2 melanoma cells aiming at gene sequencing, enzyme distribution and activity, influence on tumor development, substrate specificity, hydrolytic products and susceptibility to inhibitors. Fluorescence resonance energy transfer (FRET) peptides as well as neurotensin and bradykinin were used as substrates. The hydrolytic activities in B16F10-Nex2 culture supernatant were totally inhibited by <it>o</it>-phenanthrolin, JA-2 and partially by Pro-Ile. Leupeptin, PMSF, E-64, Z-Pro-Prolinal and captopril failed to inhibit these hydrolytic activities. Genes encoding M3A enzymes in melanoma cells were cloned and sequenced being highly similar to mouse genes. A decreased proliferation of B16F10-Nex2 cells was observed in vitro with specific inhibitors of these oligopeptidases. Active rTOP but not the inactive protein inhibited melanoma cell development in vivo increasing significantly the survival of mice challenged with the tumor cells. On Matrigel, rTOP inhibited the bradykinin – induced angiogenesis. A possible regulation of the homologous tumor enzyme in the perivascular microenvironment is suggested based on the observed rTOP inhibition by an S-nitrosothiol NO donor.</p> <p>Conclusion</p> <p>Data show that melanoma cells secrete endo-oligopeptidases which have an important role in tumor proliferation in vitro and in vivo. rTOP inhibited growth of subcutaneously injected B16F10-Nex2 cells in mice. TOP from tumor cells and bradykinin in endothelial cells are two antagonist factors that may control angiogenesis essential for melanoma growth. A regulatory role of NO or S-nitrosothiols is suggested.</p

Analysis of the Specificity and Biochemical Characterization of Metalloproteases Isolated from Eupenicillium javanicum Using Fluorescence Resonance Energy Transfer Peptides

Enzymes have important features that may facilitate their application in industrial processes and have been used as alternatives to chemical catalysts. In particular, proteases can be isolated from microorganisms, which provide important sources of advantageous enzymes for industrial processes. For example, Eupenicillium javanicum is a filamentous fungus that has been shown to express industrially applicable enzymes and chemical components, such as antifungal compounds. The biotechnological potential of E. javanicum and proteases made us search a novel protease from this microorganism. The macromolecule was isolated, the main biochemical properties was evaluated, and the specificity of the protease subsites was determined. The protease was produced under solid-state bioprocess with wheat bran and isolated by two chromatography steps with yield of 27.5% and 12.4-fold purification. The molecular mass was estimated at 30 kDa. The N-terminal sequence of the first 20 amino acid residues was AVGAGYNASVALALEKALNN. The enzyme presented higher proteolytic activity at pH 6.0 and 60 degrees C. The protease is stable at wide range of pH values and temperatures and in the presence of surfactants. The primed side of the catalytic site showed the highest catalytic efficiency of the enzyme isolated from E. javanicum. The S'(1) subsite is responsible for catalyzing the protease reaction with substrates with tyrosine in P'(1). These findings provide important insights into the biochemical characterization of a highly active protease from E. javanicum and may facilitate the development of industrial processes involving this protease.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2012/24703-8, 2011/06986-0]Univ Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Pharmaceut Sci, Ribeirao Preto, BrazilUniv Fed Sao Paulo, Paulista Med Sch, Dept Biophys, Sao Paulo, BrazilUniv Sao Paulo, Sch Pharmaceut Sci Ribeirao Preto, Dept Phys & Chem, Ribeirao Preto, BrazilUniv Fed Sao Paulo, Paulista Med Sch, Dept Biophys, Sao Paulo, BrazilFAPESP: 2012/24703-8FAPESP: 2011/06986-0Web of Scienc

Results of nutritional intervention in children and adolescents with cystic fibrosis

Objective: few studies have verified longitudinally the evolution of the nutritional status of patients with cystic fibrosis. The objective of this study is to follow the evolution of the nutritional status, body composition and energy consumption, macronutrients and micronutrients ingested by children and adolescents by means of nutritional interventions at the Clinic of Cystic Fibrosis/Pediatric Pneumology of the Department of Pediatrics of Universidade Federal de São Paulo (UNIFESP). Methods: 18 patients were involved in this study, thirteen males and five females with ages ranging from 0.3 to 18.4 years. We performed three evaluations: evaluation 1 (M1- prenutritional intervention), M2 after 6 months, and M3 after 12 months. In these three instances we verified: the z score for weight/age, weight/height and height/age and the calculation of a 3-day diet record. We verified the body composition (anthropometry) in M1 and M3. The nutritional interventions were hypercaloric, hyperproteic, with adequate amount of ingested macronutrients and micronutrients.Results: we observed an increase in the z score for height/age (M1=-1.07; M2=-0.69; M3=-0.50) and fat-free mass after the nutritional interventions, without improvement in the z score for weight/height and fat mass. We verified an increase in the energy intake during M2 (139%) and M3 (132%) compared to M1 (106%). Remarkable increase in the intake of protein, calcium, iron and vitamin C by the patients was found. The occurrence of anemia was found in 44% (8/18) of the patients.Conclusion: the improvement of the z score in height/age and fat-free mass was probably due to the increase in energy consumption after the nutritional intervention. A significant improvement in the z score for weight/height and fat mass was not found, probably due to a gain in height and fat-free mass.Objetivo: poucos estudos têm verificado longitudinalmente a evolução do estado nutricional de pacientes com fibrose cística. O objetivo deste estudo foi acompanhar a evolução do estado nutricional, composição corporal e consumo de energia, macro e micronutrientes ingeridos por crianças e adolescentes, mediante intervenção nutricional, no Ambulatório de Fibrose Cística/Pneumologia Pediátrica, do Departamento de Pediatria da UNIFESP.Métodos: a casuística constituiu-se de 18 pacientes, sendo 13 do sexo masculino e 5 do feminino, faixa etária de 0,3 a 18,4 anos. Realizaram-se 3 avaliações: no momento 1 (M1: pré-intervenção nutricional), no M2, após 6 meses, e no M3, após 12 meses. Foram analisados nos 3 momentos: o escore Z de peso/idade (P/I), peso/estatura (P/E) e estatura/idade (E/I) e o cálculo do registro alimentar de 3 dias. No M1 e M3, verificou-se a composição corporal (antropometria). A conduta nutricional foi hipercalórica, hiperprotéica e houve adequação de macro e micronutrientes.Resultados: observou-se aumento significante do escore Z de E/I (M1=-1,07; M2=-0,69; M3=-0,50) e de massa magra após a intervenção nutricional, sem melhora no escore Z de P/E e massa gorda. Verificou-se aumento no consumo energético nos M2 (139%) e M3 (132%) em relação ao M1 (106%). Houve considerável aumento no consumo de proteína, cálcio, ferro e vitamina C pelos pacientes. A presença de anemia ocorreu em 44,4% (8/18) dos pacientes.Conclusão: o aumento no escore Z de E/I e massa magra ocorreu devido a adequação no consumo de energia, após a intervenção nutricional. Não houve melhora significante no escore Z de P/E e massa gorda, em função do ganho de estatura e de massa magra.UNIFESP-EPM Dep. de PediatriaHospital São Paulo Ambulatório de Fibrose CísticaUNISA Faculdade de MedicinaUNISA Dep. de PediatriaUNIFESP, EPM, Dep. de PediatriaHospital São Paulo Ambulatório de Fibrose CísticaSciEL