Locomotor and metabolic activities of Gymnostreptus olivaceus (Diplopoda, Spirostreptida) at different photoperiod conditions

In the present investigation the locomotor and the metabolic activity of Gymnostreptus olivaceus were studied, using 24-hr cycles at different photoperiods and constant temperature and RH. Locomotor activity was studied by the actography method and was reported as coefficients of nocturnalism [CN (N/N + D). 100]. The results showed a nocturnalism coefficient of 98,71% under normal photoperiod conditions and of 29,58% under inverted photoperiod conditions. In constant illumination, the CN of G. olivaceus was 88,22%, and in constant darkness, its rhythm was close to that of the normal photoperiod (CN = 94,92%). The metabolic activity was studied by manometric Warburg respirometry and lit was reported as mu l O-2 . g(-1). h(-1). The respiratory rate of G. olivaceus under normal photoperiod condition was higher at night than during the day (52,52 x 28,76), coinciding with the pattern of nocturnal locomotor activity of the animal. However, under conditions of inverted photoperiod, the millipede maintained its tendency toward a more intense nocturnal respiratory rate (50,35 x 39,14). Similar behaviours were observed under constant illumination and constant darkness, in which G. olivaceus again presented higher nocturnal respiratory rates than diurnal ones(85,84 x 53,48 and 73,18 x 57,0, respectively). The present experimental data suggests the persistence of an endogenous rhythm where the light may not be an important exogenous synchronizer of the activity of G. olivaceus, because it was insufficient to block the start of the biological clock and the natural tendency of higher nocturnal activities of millipedes, principally when the tests were performed in constant illumination or darkness (free-running tests).

Comparative studies of the anti-leishmanial activity of three Crotalus durissus ssp venoms

In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus-Cdt, Crotalus durissus cascavella-Cdca, and Crotalus durissus collilineatus-Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70 +/- 1.72 mu g/ml) in comparison with that of Cdca venom (IC50 value of 9.41 +/- 1.21 mu g/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30 +/- 2.18 mu g/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00 +/- 9.50 mu g/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80 +/- 0.52 mu g/ml and 19.95 +/- 4.21 mu g/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80 +/- 2.21 mu g/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.

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.

Leukotoxic activity of Actinobacillus actinomycetemcomitans isolated from Brazilian periodontal patients

The leukotoxic activity of 31 Actinobacillus actinomycetemcomitans isolates from Brazilian periodontal patients [nine from Localized Juvenile Periodontitis (LJP) patients, 22 from patients with AIDS-associated Necrotizing Ulcerative Periodontitis (AIDS/NUP)], and from the reference strain A. actinomycetemcomitans ATCC43718, were analyses for their cytotoxicity on human monocytes. A cytotoxicity inhibitory assay of the isolate P35 and the reference strain ATCC 43718 with sera from ten LJP patients and ten healthy subjects was also performed and leukotoxin reactivity was evaluated with serum from rabbits immune to leukotoxin from A. actinomycetemcomitans ATCC 43718. The cytotoxicity results were not statistically different among groups of A. actinomycetemcomitans isolates from LJP and AIDS/NUP patients, but the individual analysis of each isolate showed two isolates (P24 and P35) from LJP patients with high leukotoxic activity (P<0.05). Also, a high leucotoxic inhibitory effect with LJP patients' sera compared with healthy subjects with sonic extract from isolate P35 (P<0.05) and the reactivity of rabbit antiserum to leukotoxin were observed. Both leukotoxic and non-leukotoxic activity is more frequent in PJL than AIDS/NUP patients. Even though A. actinomycetemcomitans exhibits leukotoxic activity, there is an immune response to the leucotoxin in LJP patients. (C) 2000 Academic Press.

Whydration effects on DNA double helix stability modulates ligand binding to natural DNA in response to changes in water activity

In this work we present evidence that water molecules are actively involved on the control of binding affinity and binding site discrimination of a drug to natural DNA. In a previous study, the effect of water activity (a(w)) on the energetic parameters of actinomycin-D intercalation to natural DNA was determined using the osmotic stress method (39). This earlier study has shown evidence that water molecules act as an allosteric regulator of ligand binding to DNA via the effect of water activity on the long-range stability of the DNA secondary structure. In this work we have carried out DNA circularization experiments using the plasmid pUC18 in the absence of drugs and in the presence of different neutral solutes to evaluate the contribution of water activity to the energetics of DNA helix unwinding. The contribution of water to these independent reactions were made explicit by the description of how the changes in the free energy of ligand binding to DNA and in the free energy associated with DNA helix torsional deformation are linked to a(w) via changes in structural hydration. Taken together, the results of these studies reveal an extensive linkage between ligand binding affinity and site binding discrimination, and long range helix conformational changes and DNA hydration, This is strong evidence that water molecules work as a classical allosteric regulator of ligand binding to the DNA via its contribution to the stability of the double helix secondary structure, suggesting a possible mechanism by which the biochemical machinery of DNA processing takes advantage of the low activity of water into the cellular milieu.

EVALUATION OF DIFFERENT TECHNIQUES FOR THE DIFFERENTIATION OF PATHOGENIC AND NON PATHOGENIC STRAINS OF YERSINIA-ENTEROCOLITICA

Different methods and tests have been used to evaluate the pathogenic potential of distinct Y. enterocolitica serotypes and biotypes. We tested a total of 60 Y. enterocolitica strains, being 25 of human origin (serotype O3 biotype 4 and serotype O5 biotype 1); 6 of animal origin (serotype O3 biotype 4); 19 isolated from the environment (serotype O5.27 biotypes 1 and 2); and 8 isolated from food (serotype O5 biotype 1 and serotype 05.27 biotype 1). The methods used were based on plasmid gene expression (autoagglutination, calcium-dependence at 37 degrees C and Congo Red absorption tests), chromosomal gene expression (assays for pyrazinamidase activity, salicin fermentation and esculin hydrolysis), and invasion of HEp-2 cells. All but one of the Y. enterocolitica O3 strains, were found to be potentially pathogenic when submitted to the pyrazinamidase-salicin-esculin tests. In contrast, the results obtained with the assays related to plasmidial gene expression were not so uniform, probably due to plasmid loss. The least homogeneous results were obtained with the HEp-2 cell invasion test. Y. enterocolitica O5 behaved in a uniform manner when tested with the first two groups of tests (based on chromosomal and plasmidial gene expression), but not when tested with the HEp-2 invasion assay. The strains of serotype O5.27 biotype 1 presented a uniform behavior hen submitted to the chromosomic-related tests, showing no pathogenicity. However, they did not provide conclusive results with the tests related to plasmidial gene expression or HEp-2 cell invasion. We conclude that the tests related to chromosomal gene expression (esculin-salicin-pyrazinamidase) are simple and highly effective for the detection of potentially pathogenic Y. enterocolitica isolated from clinical cases.

Squalene does not exhibit a chemopreventive activity and increases plasma cholesterol in a Wistar rat hepatocarcinogenesis model

The eventual chemopreventive effect of squalene (SQ), a triterpene present in olive oil, was evaluated when administered to Wistar rats during a period comprising the initiation and selection/promotion of the resistant hepatocyte (RH) model of hepatocarcinogenesis. During 8 consecutive wk, animals received by gavage SQ (100 or 150 mg/100 g body weight) dissolved in corn oil (CO) daily. Animals treated with only CO and submitted to the RH model were used as controls. Treatments with SQ did not result in inhibition of macroscopically visible hepatocyte nodules (P > 0.05) or of hepatic placental glutathione S-transferase-positive preneoplastic lesions (PNL; P > 0.05). Hepatic cell proliferation and apoptosis indexes were not different (P > 0.05) among the different experimental groups, both regarding PNL and surrounding normal tissue areas. There were no significant differences (P > 0.05) among comets presented by rats treated with the two SQ doses or with CO. on the other hand, SQ increased total plasma cholesterol levels when administered at both doses (P < 0.05). This indicates that the isoprenoid was absorbed. Thus, SQ did not present chemopreventive activity during hepatocarcinogenesis and had a hypercholesterolemic effect, suggesting caution when considering its use in chemoprevention of cancer.

Influence of condensed tannins from Brazilian semi-arid legumes on ruminal degradability, microbial colonization and ruminal enzymatic activity in Saanen goats

The present study aimed at determining the influence of condensed tannins present in the Brazilian legume species Mimosa hostilis, Mimosa caesalpinifolia and Bauhinia cheilantha on ruminal degradability, microbial colonization and enzymatic activity. Polyethylene glycol (PEG) was used to reduce the astringency and concentration of soluble condensed tannins. Four ruminally-cannulated Saanen goats (60 +/- 8 kg BW) were fed, in two experimental periods, with a hay diet based on the studied legumes treated or non-treated with PEG. Voluntary intake, microbial colonization, DM, CP, NDF, and ruminal degradability of PEG treated and non-treated forage leaves, as well as pH, ammonia and 1,4 P-endoglucanase activity of the rumen content were evaluated. Astringency and soluble tannin concentration of the studied legumes were reduced by approximately 70% and 50%, respectively, with PEG treatment. Average DM intake was higher for the treated diet (16.76 g DM/kg BW/day against 13.06 g DM/kg BW/day). Percentile values for degradation parameters and for potential and effective degradabilities of DM, CP and NDF were also affected by the tannins, but at different intensities. Electron microscopic observations of ruminally-incubated legume leaves showed a more effective microbial colonization of PEG-treated leaves for all legume species. A decrease in pH and an increase in ammonia concentration and in endoglucanase activity in the ruminal content was also observed for PEG-treated diets at all sampling periods. Condensed tannins of the studied legume species have influenced the adhesion conditions, colonization and enzymatic activity of the microbial ecosystem, and consequently the ruminal degradation of the different dietary fractions. For this reason, the reduction in condensed tannin would be of great importance to improve the nutrition of ruminant feeding of these species. (c) 2005 Elsevier B.V. All rights reserved.

Electrophoretic variants of intracellular catalase of different Candida species

Intracellular and extracellular catalases of different species of Candida were investigated using different culture media. All the Candida strains produced intracellular catalase, whose enzymatic activity was detected by non-denaturating polyacrylamide gradient (4-30%) gel electrophoresis. The cell extracts presented a major 230 kDa catalase band and in some strains variants of catalase with different molecular weights were detected. Candida catalase activity was not affected by heating at 50degreesC and incubation with beta-mercaptoethanol, but treatment with sodium dodecyl sulphate inhibited or reduced enzymatic activity. Extracellular enzyme activity was not detected in any of the culture filtrate extracts tested.