Potential tuberculostatic agents: Micelle-forming copolymer poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid

With the objective of obtaining slow-acting isoniazid derivatives, of potential use as chemoprophylactics or chemotherapeutics in tuberculosis, the micelle-forming copolymer of poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid was synthesized. The derivative obtained was found to be active in Mycobacterium Il(tuberculosis culture, with a minimal inhibitory concentration (MIC) 5.6 times lower than that of the tuberculostatic drug.

Crystallization, preliminary X-ray analysis and Patterson search of a new aspartic protease isolated from human urine

Aspartic protease (EC 3.4.23) make up a widely distributed class of enzymes in animals, plants, microbes and, viruses. In animals these enzymes perform diverse functions, which range from digestion of food proteins to very specific regulatory roles. In contrast the information about the well-characterized aspartic proteases, very little is known about the corresponding enzyme in urine. A new aspartic protease isolated from human urine has been crystallized and X-ray diffraction data collected to 2.45 Angstrom resolution using a synchrotron radiation source. Crystals belong to the space group P2(1)2(1)2(1) the cell parameters obtained were a=50.99, b=75.56 and c=89.90 Angstrom. Preliminary analysis revealed the presence of one molecule in the asymmetric unit. The structure was determined using the molecular replacement technique and is currently being refined using simulated annealing and conjugate gradient protocols.

Aspartic acid racemization dating of Holocene brachiopods and bivalves from the southern Brazilian shelf, South Atlantic

The extent of racemization of aspartic acid (Asp) has been used to estimate the ages of 9 shells of the epifaunal calcitic brachiopod Bouchardia rosea and 9 shells of the infaunal aragonitic bivalve Semele casali. Both taxa were collected concurrently from the same sites at depths of 10 m and 30 m off the coast of Brazil. Asp D/L values show an excellent correlation with radiocarbon age at both sites and for both taxa (r(Site)(2) (9) (B. rosea) = 0.97 r(Site)(2) (1) (B.) (rosea) = 0.997, r(Site)(2) (9) (S.) (casali) = 0.9998, r(2) (Site) (1) (S.casali) = 0.93). The Asp ratios plotted against reservoir-corrected AMS radiocarbon ages over the time span of multiple millennia can thus be used to develop reliable and precise geochronologies not only for aragonitic mollusks (widely used for dating previously), but also for calcitic brachiopods. At each collection site, Bouchardia specimens display consistently higher D/L values than specimens of Semele. Thermal differences between sites are also notable and in agreement with theoretical expectations, as extents of racemization for both taxa are greater at the warmer, shallower site than at the cooler, deeper one. In late Holocene marine settings, concurrent time series of aragonitic and calcitic shells can be assembled using Asp racemization dating, and parallel multi-centennial to multi-millennial records can be developed simultaneously for multiple biomineral systems. (c) 2006 University of Washington. All rights reserved.

Effect of the aspartic acid D2 on the affinity of Polybia-MP1 to anionic lipid vesicles

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa

Based on the genetic analysis of the phytopathogen Xylella fastidiosa genome, five media with defined composition were developed and the growth abilities of this fastidious prokaryote were evaluated in liquid media and on solid plates. All media had a common salt composition and included the same amounts of glucose and vitamins but differed in their amino acid content. XDM1 medium contained amino acids threonine, serine, glycine, alanine, aspartic acid and glutamic acid, for which complete degradation pathways occur in X fastidiosa; XDM2 included serine and methionine, amino acids for which biosynthetic enzymes are absent, plus asparagine and glutamine, which are abundant in the xylem sap; XDM3 had the same composition as XDM2 but with asparagine replaced by aspartic acid due to the presence of complete degradation pathway for aspartic acid; XDM4 was a minimal medium with glutamine as a sole nitrogen source; XDM5 had the same composition as XDM4, plus methionine. The liquid and solidified XDM2 and XDM3 media were the most effective for the growth of X. fastidiosa. This work opens the opportunity for the in silico design of bacterial defined media once their genome is sequenced. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier B.V. B.V. All rights reserved.

Digestibilidade do extrato de leveduras em frangos de corte

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Potential tuberculostatic agent: Micelle-forming pyrazinamide prodrug

Pyrazinamide was condensed with the poly(ethylene glycol)-poly(aspartic acid) copolymer (PEG-PASP), a micelle-forming derivative was obtained that was characterized in terms of its critical micelle concentration (CMC) and micelle diameter. The CMC was found by observing the solubility of Sudan III in Poly(ethylene glycol)-poly(pyrazinamidomethyl aspartate) copolymer (PEG-PASP-PZA) solutions. The mean diameter of PEG-PASP-PZA micelles, obtained by analyzing the dynamic light-scattering data, was 78.2 nm. The PEG-PASP-PZA derivative, when assayed for anti-Mycobacterium activity, exhibited stronger activity than the simple drug.

Preparation of polymeric micelles for use as carriers of tuberculostatic drugs

Purpose: This paper focuses on the characterization of polymeric micelle-forming tuberculostatic prodrugs and the antimycobacterial activity of these prodrugs.Method: By the condensation of hydroxymethylpyrazinamide, isoniazid and rifampin with free carboxyl groups on the copolymer poly(ethyleneglycol)-poly(aspartic acid), micelle-forming carrier-drug conjugates were obtained. These micelles were characterized by dynamic light scattering, to measure the micelle diameter; by acid-base titration, to determine the percentage of carboxylic groups occupied by the tuberculostatic; by Sudan III solubility tests, to estimate the critical micelle concentration (CMC); and visual control and spectrophotometric measurement, to determine the stability of micelles. These micelles were tested in vitro against several Mycobacterium strains.Results: As expected, the size and distribution of the micelle-forming tuberculostatic prodrugs found to be small (78.2nm, 84.2nm and 98.9 nm) while the level of the drug conjugated was high (65.02-85.7%). Furthermore, the micelles were stable in vitro, exhibiting a low level of CMC and stronger antimycobacterial activity than the original drugs.Conclusion: the results demonstrate that polymeric micelles can be used as efficient carriers for drugs, which alone, exhibit undesired pharmacokinetics, poor solubility, and low stability. The synthesized micelle-forming tuberculostatic prodrugs opens a perspective of alternative prodrugs that prolong action and decrease the toxicity of the tuberculostatic drugs of choice.

A first case of protease codon 35 amino acid insertion in a HIV-1 subtype B sequence detected in the Bauru region, state of São Paulo, Brazil: case report

Inserções de aminoácidos na protease têm sido raramente descritas em pacientes infectados pelo HIV. Uma destas inserções foi, recentemente, descrita no codon 35, embora seu impacto na resistência mantém-se pouco conhecido. Este trabalho apresenta um caso de uma variante viral com inserção no codon 35 da protease, descrita pela primeira vez em Bauru, São Paulo, Brasil, circulante em um homem, caucasiano, com 38 anos, o qual apresenta infecção assintomática pelo HIV desde 1997. A variante isolada mostrou uma inserção no codon 35 da protease de dois aminoácidos: uma treonina e um ácido aspártico, resultando na sequência de aminoácidos E35E_TD.

Digestibilidade aparente do triticale para a tilápia-do-nilo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phylogenetic analysis of PRP8 intein in Paracoccidioides brasiliensis species

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Giardia duodenalis: protein substrates degradation by trophozoite proteases

The present investigation was undertaken to identify and characterize trophozoite proteases of five axenic strains of Giardia duodenalis isolated in Brazil and the reference strain Portland 1 isolated in the United States. Trophozoite cell lysates of each strain were analysed for the pattern of proteins and for proteolytic activity. Samples were tested in SDS-polyacrylamide gel electrophoresis for the protein profiles, and the detection of proteases in cell lysates was performed using substrate gel electrophoresis [gelatin, collagen, bovine serum albumin (BSA) and haemoglobin] and azocasein assays. Indeed, synthetic inhibitors were included in the assays to characterize the protease classes. Differences on the hydrolysis patterns of protein substrates were observed in relation to the substrate composition as much as the Giardia trophozoite strain. The substrate-containing gels revealed hydrolysis bands with molecular masses ranging from > 97 to 20-15 kDa, and most zones were common to the five strains. However, some pronounced differences could be detected in the BTU-11 pattern. Azocasein was also degraded; however, depending on the lysate assayed, the degree of substrate degradation was variable. It was observed that inhibitory effects are substrate-dependent since the activity was predominantly due to cysteine proteases against gelatin, collagen, BSA and azocasein substrates and due to serine against haemoglobin. The presence of aspartic protease and aminopeptidase activity in the lysates was also indicated.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

O presente estudo consiste em uma caracterização preliminar da atividade proteolítica de frações de proteínas purificadas a partir de lisados de trofozoítos de cepa isolada e axenizada no Brasil. Frações obtidas por cromatografia líquida (FPLC) foram analisadas quanto ao perfil eletroforético em géis de poliacrilamida (SDS-PAGE) e a atividade proteolítica foi avaliada em géis contendo gelatina como substrato. A caracterização das enzimas foi realizada a partir da análise do efeito de inibidores sintéticos de cisteína-proteases (E-64, IAA), serina-proteases (PMSF), serina e cisteína-proteases (TPCK, TLCK, elastatinal), metalo-proteases (EDTA) e aspartil proteases (pepstatina) sobre a degradação do substrato. Entre 30 frações eluídas, bandas de proteínas foram observadas em oito delas, entretanto, atividade proteolítica foi detectada apenas nas frações 23, 24, 25 e 26. O perfil eletroforético das proteínas revelou poucas bandas distribuídas na faixa de 45 a 18 kDa. Os zimogramas revelaram zonas de proteólise na faixa de aproximadamente 62 a 35 kDa, entretanto destacaram-se as bandas de hidrólise de 62, 55, 53, 50, 46 e 40 kDa. Nos ensaios de inibição, a proteólise foi marcantemente inibida por E-64, TPCK, TLCK e elastatinal. Redução discreta da proteólise foi observada com IAA e PMSF, enquanto que EDTA e pepstatina não promoveram alteração dos perfis de hidrólise. Estas observações são relevantes, especialmente se considerarmos que para elucidar o envolvimento das proteases na relação parasita-hospedeiro, a purificação dessas moléculas é um requisito importante.

Quantitative comparisons and models of time-averaging in bivalve and brachiopod shell accumulations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)