Purification, sequencing and structural characterization of the phospholipase A(1) from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae)

The biochemical and functional characterization of wasp venom toxins is an important prerequisite for the development of new tools both for the therapy of the toxic reactions due to envenomation caused by multiple stinging accidents and also for the diagnosis and therapy of allergic reactions caused by this type of venom. PLA(1) was purified from the venom of the neotropical social wasp Polybia paulista by using molecular exclusion and cation exchange chromatographies; its amino acid sequence was determined by using automated Edman degradation and compared to the sequences of other vespid venom PLA(1)'s. The enzyme exists as a 33,961.40 da protein, which was identified as a lipase of the GX class, liprotein lipase superfamily, pancreatic lipases (ab20.3) homologous family and RP2 sub-group of phospholipase. P. paulista PLA(1) is 53-82% identical to the phospholipases from wasp species from Northern Hemisphere. The use restrained-based modeling permitted to describe the 3-D structure of the enzyme, revealing that its molecule presents 23% alpha-helix, 28% beta-sheet and 49% coil. The protein structure has the alpha/beta fold common to many lipases; the core consists of a tightly packed beta-sheet constituted of six-stranded parallel and one anti-parallel beta-strand, surrounded by four alpha-helices. P. paulista PLA(1) exhibits direct hemolytic action against washed red blood cells with activity similar to the Cobra cardiotoxin from Naja naja atra. In addition to this, PLA(1) was immunoreactive to specific IgE from the sera of P. paulista-sensitive patients. (c) 2007 Elsevier Ltd. All rights reserved.

Development of rapidly fermenting strains of saccharomyces-diastaticus for direct conversion of starch and dextrins to ethanol

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

A molecular mechanism for Lys(49)-phospholipase A(2) activity based on ligand-induced conformational change

Agkistrodon contortrix laticinctus myotoxin is a Lys(49)- phospholipase A(2) (EC 3.1.1.4) isolated from the venom of the serpent A contortrix laticinctus (broad-banded copperhead). We present here three monomeric crystal structures of the myotoxin, obtained under different crystallization conditions. The three forms present notable structural differences and reveal that the presence of a ligand in the active site (naturally presumed to be a fatty acid) induces the exposure of a hydrophobic surface (the hydrophobic knuckle) toward the C terminus. The knuckle in A contortrix laticinctus myotoxin involves the side chains of Phe(121) and Phe(124) and is a consequence of the formation of a canonical structure for the main chain within the region of residues 118-125. Comparison with other Lys(49)-phospholipase A(2) myotoxins shows that although the knuckle is a generic structural motif common to all members of the family, it is not readily recognizable by simple sequence analyses. An activation mechanism is proposed that relates fatty acid retention at the active site to conformational changes within the C-terminal region, a part of the molecule that has long been associated with Ca2+-independent membrane damaging activity and myotoxicity. This provides, for the first time, a direct structural connection between the phospholipase active site and the C-terminal myotoxic site, justifying the otherwise enigmatic conservation of the residues of the former in supposedly catalytically inactive molecules.

A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity

A, M. Soares, V, M, Rodrigues, M. I. Homsi-Brandeburgo, M. H. Toyama, F, R, Lombardi, K. Arni and J. R, Giglio. A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: Biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon 36, 503-514, 1998.-Bothrops moojeni snake venom was fractionated on a CM-Sepharose column which was previously equilibrated with 0.05 M ammonium bicarbonate buffer at pH 8.0 and subsequently eluted with an ammonium bicarbonate concentration gradient from 0.05 to 0.5 M at constant pH (8.0) and temperature (25 degrees C). The fraction which eluted last (M-VI) showed, after direct lyophilization, a single band by polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE, indicating an approximate M,. of 14 000 and 77 000, in the presence and absence of dithiothreitol, respectively. Its amino acid composition revealed a high level of hydrophobic and basic amino acids as well as 13 half-cystine residues. Its isoelectric point and extinction coefficient (E-1.0cm(1.0mg/ml) at 278 nm and pH 7.0) were 8.2 and 1.170, respectively. M-VI was devoid of phospholipase A(2) (PLA(2)) activity on egg yolk, as well as of hemorrhagic, anticoagulant and coagulant activities, but could induce drastic necrosis on skeletal muscle fibres as well as rapid and transient edema on the rat paw. Its N-terminal sequence: SLFELGKMILQETGKNPAKSYGVYGCNCGVGGRGKPKDATDRCCYVHKCCYK.... revealed high homology with other Lys 49 PLA(2)-like myotoxins from other bothropic venoms. Orthorhombic crystals of M-VI? which diffracted to a maximal resolution of 1.6 Angstrom. were obtained and indicated the presence of a dimer in the asymmetrical unit. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Anticlastogenic activity of aqueous extract of Agaricus blazei in drug-metabolizing cells (HTCs) during cell cycle

The mushroom Agaricus blazei has been extensively investigated because of evidence of its antimutagenic, antitumor, and anticarcinogenic activities. This study investigated the clastogenic and/or anticlastogenic activity of aqueous extract of Agaricus blazei (10% w/v) in drug-metabolizing rat hepatoma tissue cells (HTCs), with continuous treatment and treatment during different phases of the cell cycle. DNA damage was induced utilizing two directacting agents-methyl methane sulfonate and ethyl methane sulfonate-and two indirect-acting agents-2-aminoanthracene and cyclophosphamide. The aqueous extract of A. blazei with either continuous treatment or treatment during different phases of the cell cycle showed clastogenic activity. The results with continuous treatment showed that A. blazei does not protect against DNA damage-inducing agents that are direct acting. Meanwhile, when combined with indirect-acting agents, a protective effect was demonstrated. A protective effect was also found during different phases of the cell cycle when cells were treated with indirect-acting agents. The protective effects against indirect-acting agents (continuous treatment and during the different phases of the cell cycle) suggest that A. blazei may provide some health benefits to the public when used as a functional food.

Gas exchange in lisianthus plants (Eustoma grandiflorum) submitted to different doses of nitrogen

The effect of different doses of nitrogen (N) on gas exchange, relative chlorophyll (Chl) amount, and the content of N in the aerial biomass of lisianthus was evaluated. The treatments consisted of six different concentrations of N (50, 100, 150, 200, 250, and 300 g m(-3) noted as N-50, N-100, N-150, N-200, N-250, and N-300, respectively), applied through the fertirrigation technique. N-250 and N-300 induced increase in the contents of foliar Chl and N in the aerial biomass, that in turn contributed to an increase of photosynthetic activity in lisianthus.

Direct determination of phosphite in fertilizers by flow-injection amperometry

A sensor based on graphite electrode modified with palladium-platinum-palladium film is proposed for phosphite determination by flow-injection amperometry. The modified electrode was prepared by a sequential cathodic deposition of Pd, Pt and Pd on a graphite electrode from 0.5% m/v PdCl2 + 28% m/v NH4OH and 2% m/v H2PtCl6 + 10% v/v H2SO4 solutions. After suitable conditioning, the electrode showed catalytic activity for phosphite oxidation when 0. 15 V was applied. The proposed system handles approximately 50 samples per hour (0.0.1 - 0.05 mol L-1 Na-2 HPO3; R-2 = 0.9997), consuming ca. 70 mu L of sample per determination. The limit of detection and amperometric sensibility were 5 X 10(-4) mol L-1 and 1.5 mA L mol(-1), respectively. The proposed method was applied to analysis of fertilizer samples without pre-treatment. Results are in agreement with those obtained by spectrophotometry and titrimetry at 95% confidence level and good recoveries (96-109%) of spiked samples were found. Relative standard deviation (n=12) of a 0.01 mol L-1 Na2HPO3 sample was 2%. The useful lifetime of modified electrode was around 220 determinations. For routine purposes it means that this electrode can be continuously used for 5 hours.

(5,10,15,20-Tetra(4-pyridil)porphinato)manganes(III) acetate modified by four mu(3)-oxo-triruthenium acetate clusters: synthesis, characterization, electrochemical behavior and catalytic activity

A novel supermolecule constituted by four mu(3)-oxo-triruthenium acetate clusters coordinated to manganese(III)-meso -tetra(4-pyridil)porphyrin acetate ([MnTPyP]CH3COO) has been synthesized. Characterization has been performed by UV-Vis and H-1 NMR spectroscopy. The electrochemical behavior (cyclic voltammetry and spectroelectrochemistry) in N,N'-dimethylformamide has been analyzed in terms of five redox processes: three related to peripheral clusters (Ru-IV,Ru-III,Ru-III/Ru-III,Ru-III,Ru-III/Ru-III,Ru-II,Ru-II) and two centered on the Mn-porphyrin core ((MnP)-P-III/(MnP)-P-II/(MnP2-)-P-II). A direct comparison has been performed between MnTCP and MnTPyP as catalysts for the cyclooctene and cyclohexane oxidation reactions. The improved selectivity exhibited by the supramolecular catalyst for cyclohexane oxidation has been ascribed to electronic effects on the oxomanganese(V) porphyrin species induced by the four peripheral clusters, in the formal (RuRuRuIII)-Ru-IV-Ru-III oxidation state. (C) 2000 Elsevier B.V. S.A. All rights reserved.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

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

Biological activity of Gomphrena elegans Mart. Var. Elegans (Amaranthaceae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Irradiance stress responses of gas exchange and antioxidant enzyme contents in pariparoba [Pothomorphe umbellata (L.) Miq.] plants

We evaluated the growth and development of the medicinal species Pothomorphe umbellata ( L.) Miq. under different shade levels ( full sun and 30, 50, and 70 % shade, marked as I(100), I(70), I(50), and I(30), respectively) and their effects on gas exchange and activities of antioxidant enzymes. Photosynthetically active radiation varied from 1 254 mu mol m(-2) s(-1) at I(100) to 285 mu mol m(-2) s(-1) at I(30). Stomatal conductance, net photosynthetic rate, and relative chlorophyll (Chl) content were maximal in I(70) plants. Plants grown under I(100) produced leaves with lower Chl content and signs of chlorosis and necrosis. These symptoms indicated Chl degradation induced by the generation of reactive oxygen species. Stress related antioxidant enzyme activities ( Mn-SOD, Fe-SOD, and Cu/Zn-SOD) were highest in I(100) plants, whereas catalase activity was the lowest. Hence P. umbellata is a shade species ( sciophyte), a feature that should be considered in reforestation programs or in field plantings for production of medicinal constituents.

Foraging activity of Trachymyrmex fuscus (hymenoptera: formicidae) in a degraded area

Two Trachymyrmex fuscus nests were observed between 2001 and 2002 in order to analyze the ant's foraging activity and the substrate collected by workers in an area modified by human activity. The analysis of results demonstrated alterations in the diel activity patterns of workers during the several months studied; however, we could not establish direct relations with variations in temperature and relative humidity. The materials transported to grow the symbiotic fungus were, for the most part, recently fallen or dry leaves, flowers, twigs, and seeds. The distances traveled by the ants until the foraging area and their collected material preferences revealed an opportunistic behavior, in which workers use plant parts according to the vegetation's phenology.

Modulatory effect of Byrsonima basiloba extracts on the mutagenicity of certain direct and indirect-acting mutagens in Salmonella typhimurium assays

Byrsonima basiloba A. Juss. species is a native arboreal type from the Brazilian cerrado (tropical American savanna), and the local population uses it to treat diseases, such as diarrhea and gastric ulcer. It belongs to the Malpighiaceae family, and it is commonly known as murici. Considering the popular use of B. basiloba derivatives and the lack of pharmacological potential studies regarding this vegetal species, the mutagenic and antimutagenic effect of methanol (MeOH) and chloroform extracts were evaluated by the Ames test, using strains TA97a, TA98, TA100, and TA102 of Salmonella typhimurium. No mutagenic activity was observed in any of the extracts. To evaluate the antimutagenic potential, direct and indirect mutagenic agents were used: 4 nitro-o-phenylenediamine, sodium azide, mitomycin C, aflatoxin B1, benzo[a]pyrene, and hydrogen peroxide. Both the extracts evaluated showed antimutagenic activity, but the highest value of inhibition level (89%) was obtained with the MeOH extract and strain TA100 in the presence of aflatoxin B1. Phytochemical analysis of the extracts revealed the presence of n-alkanes, lupeol, ursolic and oleanolic acid, (+)-catechin, quercetin-3-O-α-L-arabinopyranoside, gallic acid, methyl gallate, amentoflavone, quercetin, quercetin-3-O-(2″-O-galloyl)-β-D- galactopyranoside, and quercetin-3-O-(2″-O-galloyl)-α-L- arabinopyranoside. © 2008 Mary Ann Liebert, Inc.

Preliminary Overview of the Economies of Latin America and the Caribbean 2006

Includes bibliography

Preliminary Overview of the Economies of Latin America and the Caribbean 2007

Includes bibliography