Fullerene and omega-3 and omega-6 fatty acids on fish brain antioxidant status

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effects of the medicinal plants Curcuma zedoaria and Camellia sinensis on halitosis control

Volatile sulphur compounds (VSC) are the gases mainly responsible for halitosis (bad breath). The aim of this research was to evaluate the effects of medicinal plants on halitosis control. Two commonly used plants were tested: Curcuma zedoaria and Camellia sinensis (green tea). These plants were prepared as an aqueous solution and used as mouthwashes, compared with a standard mouthwash of 0.12% chlorhexidine gluconate and a placebo (water). The experiment was conducted with 30 volunteers from the School of Dentistry of Sao Jose dos Campos, Univ. Estadual Paulista - UNESP, SP, Brazil. Each volunteer tested the four mouthwashes. The Cysteine Challenge Method, modified for this study, was used for initial breath standardization. Four breath assessments were conducted after volunteers rinsed orally with acetylcysteine: one before the test mouthwash was used; the second, one minute after its use; a third 90 minutes later; and the last 180 minutes later. The results showed that chlorhexidine gluconate lowered VSC production immediately, and that this effect lasted up to 3 hours, while the tested plants had immediate inhibitory effects but no residual inhibitory effects on VSC. We concluded that Curcuma zedoaria and Camellia sinensis, prepared as infusions and used as mouthwashes, did not have a residual neutralizing effect on VSC.

Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins

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

The activity of amino acids produced by Paenibacillus macerans and from commercial sources against the root-knot nematode Meloidogyne exigua

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Níveis plasmáticos de taurina e de seus precursores em pacientes com câncer de esôfago

RACIONAL: O câncer de esôfago tem impacto relevante no metabolismo protéico do hospedeiro, mas pouco se conhece sobre as implicações no metabolismo protéico sulfurado. Deste, destaca-se a taurina, composto participante de várias funções fisiológicas importantes como a manutenção do sistema de defesa celular e possível sobrevida do paciente. OBJETIVO: Estudar as variações plasmáticas da taurina e de seus precursores em pacientes com câncer de esôfago. MÉTODO: em estudo transversal foram triados 16 pacientes (43-73 anos) com câncer de esôfago e 20 voluntários (27-65 anos) controles sadios que preencheram os critérios clínicos e éticos da pesquisa. Para caracterização do estado geral de saúde efetuou-se avaliação antropométrica, hematimétrica (Hb, Ht, glóbulos brancos, linfócitos) e bioquímica (albumina, glicose, lipídios, aminotransferases). Adicionalmente, foram realizadas, no plasma, análises cromatográficas de taurina e seus precursores cisteína e homocisteína. Foi registrado o tempo de sobrevivência dos pacientes, a partir do diagnóstico histopatológico. RESULTADOS: Os pacientes com câncer de esôfago foram predominantemente do sexo masculino, raça branca, classe socioeconômica baixa, tipo carcinoma espinocelular de localização no terço superior, em estádio IV, sobrevida de 7,8 ± 5,5 anos, referindo perda de peso em 16,4% e apresentando hipoalbuminemia em 50%, com massa muscular e adiposa semelhante ao controle. Os pacientes apresentaram valores estatisticamente menores do que os controles para Hb, Ht, colesterol total, HDL-colesterol e cisteína e maiores de AST, ALT, taurina e homocisteína. Dentre os pacientes houve correlação positiva da taurina tanto com a contagem total de linfócitos, como com a sobrevida dos pacientes. CONCLUSÃO: Os níveis reduzidos de cisteína e elevados de homocisteína, taurina e as associações positivas da taurina com os indicadores da imunocompetência celular e da mortalidade sugerem participação efetiva da taurina na sobrevida dos pacientes e, portanto, os cuidados nutricionais específicos com a sua via geradora (cisteína, metionina e vitaminas do complexo B).

Determinação dos parâmetros cinéticos e termodinâmicos da adsorção de L-cisteína em ouro por meio da técnica de microbalança a cristal de quartzo

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

cDNA cloning and 1.75 angstrom crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 +/- 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-lengthamino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 angstrom resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (beta alpha)(8) barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

Synthesis and in vitro evaluation of potential antichagasic dipeptide prodrugs of primaquine

American trypanosomiasis (Chagas' disease) is an endemic parasitic disease afflicting more than 20 million people in Latin America. Currently, therapy is unsatisfactory and only two drugs are available. Primaquine, an antimalarial drug, has trypanocidal activity. Dipeptide derivatives of primaquine, Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ, were synthesized. The choice of the peptides was based on the primary specificity of cruzipain, the major cysteine proteinase from T. cruzi. The prodrugs obtained were tested on the LLC-MK2 cell culture infected with trypomastigotes forms of T. cruzi Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ were active in all stages.

IDENTIFICATION OF A PUTATIVE MEMBRANE-INSERTED SEGMENT IN THE ALPHA-TOXIN OF STAPHYLOCOCCUS-AUREUS

To gain a fuller understanding of the regions of the Staphylococcus aureus alpha-toxin important in pore formation, we have used Forster dipole-dipole energy transfer to demonstrate that a central glycine-rich region of alpha-toxin (the so-called ''hinge'' region) inserts deeply into the bilayer on association of toxin with liposomes. Mutant alpha-toxins with unique cysteine (C) residues at positions 69 and 130 [Palmer, M., et al. (1993) J. Biol. Chem. 268, 11959) were reacted with the C-specific fluorophore acrylodan, which acted as an energy donor. The chosen acceptor was N-(7-nitrobenz-2-oxa-13-diazol-4-yl)-1,2-bis(hexadecanoyl) -sn-glycero-3-phosphoethanolamine (NBD-PE). Measurement of the degree of donor quenching with increasing NBD-PE in the inner bilayer leaflet enables the distance of closest approach between donor and acceptor to be estimated. For toxin labeled with acrylodan at position 130 (in the hinge region), this distance is approximately 5 +/- 2 Angstrom, showing that the probe is close to the inner surface of the liposomes. A second probe labeled at position 69 (in the N-terminal domain) shows negligible energy transfer, indicating a distance of closest approach >40 Angstrom. This implies that this N-terminal region remains ''outside'' the liposome. We propose a model in which the central region of the alpha-toxin inserts into the membrane and possibly participates in forming the wall of the pore.

Dehydromonocrotaline inhibits mitochondrial complex 1. A potential mechanism accounting for hepatotoxicity of monocrotaline

Monocrotaline is a pyrrolizidine alkaloid present in plants of the Crotalaria species, which causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline. We evaluated the effects of monocrotaline and its metabolite on respiration, membrane potential and ATP levels in isolated rat liver mitochondria, and on respiratory chain complex I NADH oxidase activity in submitochondrial particles. Dehydromonocrotaline, but not the parent compound, showed a concentration-dependent inhibition of glutamate/malate-supported state 3 respiration (respiratory chain complex 1), but did not affect succinate-supported respiration (complex II). Only dehydromonocrotaline dissipated mitochondrial membrane potential, depleted ATP, and inhibited complex I NADH oxidase activity (IC50 = 62.06 mu M) through a non-competitive type of inhibition (K-I = 8.1 mu M). Therefore, dehydromonocrotaline is an inhibitor of the activity of respiratory chain complex I NADH oxidase, an action potentially accounting for the well-documented monocrotaline's hepatotoxicity to animals and humans. The mechanism probably involves change of the complex I conformation resulting from modification of cysteine thiol groups by the metabolite. (c) 2007 Elsevier Ltd. All rights reserved.

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear (1)H NMR spectroscopy at 900 MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities. (c) 2005 Elsevier Ltd. All rights reserved.

H-1-N-15 NMR studies of the complex bis(S-allyl-L-cysteinate)palladium(II)

H-1-N-15 2D NMR data for S-allyl-L-cysteine (deoxyalliin) and for bis(S-allyl-L-cysteinate)palladium(II) complex are presented in this manuscript. Large upfield N-15 NMR shift of the amine nitrogen in the spectrum of the complex when compared to the spectrum of the ligand shows clearly coordination of S-allyl-L-cysteine, in the anion form, to palladium(II) through the NH2 group. (c) 2005 Elsevier B.V. All rights reserved.

Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.

Cognitive Deficits in Machado-Joseph Disease Correlate with Hypoperfusion of Visual System Areas

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

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD 147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.