Maria Anita Mendes

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (P(i)) and continuous assay of reactions that generate P(i) such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P(i) detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P(i) detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1L cell culture, with a specific activity value of 80 Umg(-1).

Protonectin (1-6): a novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes.

Peptides constitute the largest group of Hymenoptera venom toxins; some of them interact with GPCR, being involved with the activation of different types of leukocytes, smooth muscle contraction and neurotoxicity. Most of these toxins vary from dodecapeptides to tetradecapeptides, amidated at their C-terminal amino acid residue. The venoms of social wasps can also contains some tetra-, penta-, hexa- and hepta-peptides, but just a few of them have been structurally and functionally characterized up to now. Protonectin (ILGTILGLLKGL-NH(2)) is a polyfunctional peptide, presenting mast cell degranulation, release of lactate dehydrogenase (LDH) from mast cells, antibiosis against Gram-positive and Gram-negative bacteria and chemotaxis for polymorphonucleated leukocytes (PMNL), while Protonectin (1-6) (ILGTIL-NH(2)) only presents chemotaxis for PMNL. However, the mixture of Protonectin (1-6) with Protonectin in the molar ratio of 1:1 seems to potentiate the biological activities dependent of the membrane perturbation caused by Protonectin, as observed in the increasing of the activities of mast cell degranulation, LDH releasing from mast cells, and antibiosis. Despite both peptides are able to induce PMNL chemotaxis, the mixture of them presents a reduced activity in comparison to the individual peptides. Apparently, when mixed both peptides seems to form a supra-molecular structure, which interact with the receptors responsible for PMNL chemotaxis, disturbing their individual docking with these receptors. In addition to this, a comparison of the sequences of both peptides suggests that the sequence ILGTIL is conserved, suggesting that it must constitute a linear motif for the structural recognition by the specific receptor which induces leukocytes migration.

One-step purification of 5-enolpyruvylshikimate-3-phosphate synthase enzyme from Mycobacterium tuberculosis

Currently, there are 8 million new cases and 2 million deaths annually from tuberculosis, and it is expected that a total of 225 million new cases and 79 million deaths will occur between 1998 and 2030. The reemergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons, and the proliferation of multi-drug-resistant strains have created a need to develop new antimycobacterial agents. The existence of homologues to the shikimate pathway enzymes has been predicted by the determination of the genome sequence of Mycobacterium tuberculosis. We have previously reported the cloning and overexpression of M. tuberculosis aroA-encoded EPSP synthase in both soluble and active forms, without IPTG induction. Here, we describe the purification of M. tuberculosis EPSP synthase (mtEPSPS) expressed in Escherichia coli BL21(DE3) host cells. Purification of mtEPSPS was achieved by a one-step purification protocol using an anion exchange column. The activity of the homogeneous enzyme was measured by a coupled assay using purified shikimate kinase and purine nucleoside phosphorylase proteins. A total of 53 mg of homogeneous enzyme could be obtained from 1L of LB cell culture, with a specific activity value of approximately 18 Umg(-1). The results presented here provide protein in quantities necessary for structural and kinetic studies, which are currently underway in our laboratory.

Mechanistic implications of zinc(II) ions on the degradation of phenol by the fenton reaction

A study of the interference of Zn2+ ions on phenol degradation by Fenton reaction (Fe2+/Fe3+ + H2O2) is reported. One of the first intermediates formed in the reaction, catechol, can reduce Fe3+ to Fe2+ and, in the presence of H2O2 initiates an efficient catalytic redox cycle. In the initial stages of the reaction, this catechol-mediated cycle becomes the principal route of thermal degradation of phenol and its oxidation products. The Zn2+ ion addition enhances the persistence time of catechol, probably by stabilization of the corresponding semiquinone radical via complexation.

Mechanism of Pyrogallol Red Oxidation Induced by Free Radicals and Reactive Oxidant Species. A Kinetic and Spectroelectrochemistry Study

Pyrogallol red (PGR) presents high reactivity toward reactive (radical and nonradical) species (RS). This property of PGR, together with its characteristic spectroscopic absorption in the visible region, has allowed developing methodologies aimed at evaluating the antioxidant capacity of foods, beverages, and human fluids. These methods are based on the evaluation of the consumption of PGR induced by RS and its inhibition by antioxidants. However, at present, there are no reports regarding the degradation mechanism of PGR, limiting the extrapolation to how antioxidants behave in different systems comprising different RS. In the present study, we evaluate the kinetics of PGR consumption promoted by different RS (peroxyl radicals, peroxynitrite, nitrogen dioxide, and hypochlorite) using spectroscopic techniques and detection of product by HPLC mass spectrometry. The same pattern of oxidation and spectroscopic properties of the products is observed, independently of the RS employed. Mass analysis indicates the formation of only one product identified as a quinone derivative, excluding the formation of peroxides or hydroperoxides and/or chlorinated compounds, in agreement with FOXs assays and oxygen consumption experiments. Cyclic voltammetry, carried out at different pHs, shows an irreversible oxidation of PGR, indicating the initial formation of a phenoxy radical and a second charge transfer reaction generating an ortho-quinone derivative. Spectroelectrochemical oxidation of PGR shows oxidation products with identical UV-visible absorption properties to those observed in RS-induced oxidation.

Comparative study of different matrix/solvent systems for the analysis of crude lyophilized microalgal preparations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

RATIONALE Due to increases in greenhouse gas emissions, it is necessary to explore renewable sources of energy. Interesting alternatives are biofuels derived from microalgae. One challenge is the development of a detailed microalgae database compiling species identifications and characterizations that would facilitate microalgae selection for biomass production. Mass spectrometric (MS) analysis using a matrix-assisted laser desorption/ionization (MALDI) source is an advanced technique that enables advancement in this biological area. In this work a MALDI time-of-flight (TOF)MS method for the rapid identification of proteins in whole cells of selected microalgae species was studied. Furthermore, the efficiency of different matrix and solvent systems was tested. MS analyses were performed using an UltrafleXtreme MALDI-TOF mass spectrometer operating in linear positive ion mode. METHODS Mass spectra were acquired in a mass range from 4000 to 20,000 Da with ions generated from Smartbeam laser irradiation using a frequency of 2000 Hz, a PIE 100 ns and a lens 7 kV. The voltage was 25 kV for the first ion source and 23 kV for the second. Each spectrum was generated by averaging of 10,000 laser shots and the laser irradiance was set at 95-100%. RESULTS Similar mass spectra were obtained for all matrices (SA, HCCA, DHB and sDHB); however, the use of the sDHB matrix resulted in spectrum profiles with a greater amount number of proteins, a better signal/noise (S/N) ratio and higher intensities for the majority of microalgae analyzed. Trifluoroacetic acid (TFA) content was also studied and the best results in terms of S/N ratio, number of proteins and signal intensities were obtained with 0.1% TFA in the matrix solvent. The addition of isopropanol did not produce improvement in the quality of spectrum profiles. CONCLUSIONS Therefore, the optimal matrix for the analysis of protein from intact microalgae cells is sDHB with TA50 as the matrix solvent and without isopropanol. These conditions allow the acquisition of high quality spectrum profiles.

Bioleaching of electronic waste using bacteria isolated from the marine sponge Hymeniacidon heliophila (Porifera)

The bacteria isolated from Hymeniacidon heliophila sponge cells showed bioleaching activity. The most active strain, Hyhel-1, identified as Bacillus sp., was selected for bioleaching tests under two different temperatures, 30°C and 40°C, showing rod-shaped cells and filamentous growth, respectively. At 30°C, the bacteria secreted substances which linked to the leached copper, and at 40°C metallic nanoparticles were produced inside the cells. In addition, infrared analysis detected COOH groups and linear peptides in the tested bacteria at both temperatures. The Hyhel-1 strain in presence of electronic waste (e-waste) induced the formation of crust, which could be observed due to bacteria growing on the e-waste fragment. SEM-EDS measurements showed that the bacterial net surface was composed mostly of iron (16.1% w/w), while a higher concentration of copper was observed in the supernatant (1.7% w/w) and in the precipitated (49.8% w/w). The substances linked to copper in the supernatant were sequenced by MALDI-TOF-ms/ms and identified as macrocyclic surfactin-like peptides, similar to the basic sequence of Iturin, a lipopeptide from Bacillus subtilis. Finally, the results showed that Hyhel-1 is a bioleaching bacteria and cooper nanoparticles producer and that this bacteria could be used as a copper recovery tool from electronic waste.

Multiple bradykinin-related peptides from the capture web of the spider Nephila clavipes (Araneae, tetragnatidae)

Three bradykinin-related peptides (nephilakinins-I to -III) and bradykinin itself were isolated from the aqueous washing extract of the capture web of the spider Nephila clavipes by gel permeation chromatography on a Sephacryl S-100 column, followed by chromatography in a Hi-Trap Sephadex-G25 Superfine column. The novel peptides occurred in low concentrations and were sequenced through ESI-MS/MS analysis: nephilakinin-I (G-P-N-P-G-F-S-P-F-R-NH2), nephilakinin-II (E-A-P-P-G-F-S-P-F-R-NH2) and nephilakinin-III (P-S-P-P-G-F-S-P-F-R-NH2). Synthetic peptides replicated the novel bradykinin-related peptides, which were submitted to biological characterizations. Nephilakinins were shown to cause constriction on isolated rat ileum preparations and relaxation on rat duodenum muscle preparations at amounts higher than bradykinin; apparently these peptides constitute B2-type agonists of ileal and duodenal smooth muscles. All peptides including the bradykinin were moderately lethal to honeybees. These bradykinin peptides may be related to the predation of insects by the webs of N. clavipes.

ESTUDO DO METABOLOMA DE BIOFLUIDOS EM PACIENTES PEDIÁTRICOS NEFROPATAS E SUA ASSOCIAÇÃO COM A DOENÇA PERIODONTAL.

INTRODUCAO: Metabolomica e a tecnica que analisa metabolitos endogenos e exogenos de baixa massa molecular encontrados em biofluidos e tecidos. Diversos estudos tem mostrado a importância da saude bucal em pacientes com doenca renal cronica (DRC). OBJETIVO: Identificar e interpretar a funcao de metabolitos salivares e de urina de pacientes com DRC e sua possivel associacao com a doenca periodontal (DP). METODOS: 30 adolescentes e adultos jovens com DRC, 12-18 anos, cadastrados no CAPE /FOUSP e no ICrFMUSP, fizeram parte deste estudo. O grupo controle foi composto por 30 individuos clinicamente saudaveis. Os especimes clinicos foram coletados e armazenados a -80°C ate a realizacao da analise de cromatografia gasosa acoplada ao espectrometro de massa (GC-MS). no laboratorio Dempster da EPUSP. A analise periodontal foi realizada por meio do indice de higiene oral simplificado (OHI-S), sangramento a sondagem e profundidade de sondagem. RESULTADOS: As analises estatisticas foram realizadas por meio da analise de componentes principais (PCA), testes de Mann-Whitney e Kruskal-Wallis. A quantificacao relativa de cada metabolito mostrou maiores concentracoes entre pacientes com DRC sem DP e com DRC e com DP, de alanina (p<0.0001), glicina (p<0.0001), tirosina (p=0.021),serina (p<0.0001),prolina (p<0.0002),leucina (p<0.0003), citrulina (p<0.0001), arginina (p<0.0002), p-Cresol(p<0.0003) e acetofenona (p<0.0003). Grupos com DRC mostraram concentracoes alteradas de acidos carboxilicos e dimetilarginina. CONCLUSOES: Ambas as patologias estimulam o processo oxidativo sintetizando metabolitos que podem potencializar a severidade da DRC. Verificamos que, apesar do perfil metabolico ser diferente, alguns metabolitos sao compartilhados por todos os grupos.

Identification of microorganisms in biofluids of individuals with periodontitis and chronic kidney disease using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

RATIONALE Chronic kidney disease (CKD) and periodontitis (PD) are important health issues. There is a large variety of microorganisms related to the pathogenesis of periodontitis, and optimising the time and the cost of laboratory assays to detect these organisms is highly valuable in the medical field. METHODS Bacteria were isolated from saliva and oral biofilm of 30 adolescents and young adults with definite medical and dental diagnosis of CKD and PD, respectively, and proteins were extracted for microorganism identification by means of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) technique. RESULTS The results showed that the most incident microorganisms were Actinomyces dentalis (43%), Acinetobacter ursingi (60%), Aggregatibacter actinomycetencomitans (60%), Corynebacterium argentoctens (63%), Staphylococcus aureus (93%), Streptococcus salivarius (97%) and Tannerella forsythensis (43%). The analysis of oral biofilm showed higher incidences for Actinomyces dentalis (33%), Acinetobacter ursingi (50%), Aggregatibacter actinomycetencomitans (50%), Corynebacterium argentoctens (70%), Pseudomonas aeruginosa (40%), Staphylococcus aureus (73%) and Streptococcus salivarius (87%). CONCLUSIONS Based on these results, we concluded that the MALDI Biotyper protocol proves useful as a rapid and reliable assay for distinguishing different microorganisms possibly related to CKD and PD. Copyright