Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG aginst T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytothropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Organic Matter Fractions and Quality of the Surface Layer of a Constructed and Vegetated Soil After Coal Mining. II - Physical Compartments and Carbon Management Index

Soils constructed after mining often have low carbon (C) stocks and low quality of organic matter (OM). Cover crops are decisive for the recovery process of these stocks, improving the quality of constructed soils. Therefore, the goal of this study was to evaluate the effect of cover crops on total organic C (TOC) stocks, C distribution in physical fractions of OM and the C management index (CMI) of a soil constructed after coal mining. The experiment was initiated in 2003 with six treatments: Hemarthria altissima (T1), Paspalum notatum (T2), Cynodon dactylon (T3), Urochloa brizantha (T4), bare constructed soil (T5), and natural soil (T6). Soil samples were collected in 2009 from the 0.00-0.03 m layer, and the TOC and C stocks in the physical particle size fractions (carbon in the coarse fraction - CCF, and mineral-associated carbon - MAC) and density fractions (free light fraction - FLF; occluded light fraction - OLF, and heavy fraction - HF) of OM were determined. The CMI components: carbon pool index (CPI), lability (L) and lability index (LI) were estimated by both fractionation methods. No differences were observed between TOC, CCF and MAC stocks. The lowest C stocks in FLF and OLF fractions were presented by T2, 0.86 and 0.61 Mg ha-1, respectively. The values of TOC stock, C stock in physical fractions and CMI were intermediate, greater than T5 and lower than T6 in all treatments, indicating the partial recovery of soil quality. As a result of the better adaptation of the species Hemarthria and Brizantha, resulting in greater accumulation of labile organic material, the CPI, L, LI and CMI values were higher in these treatments, suggesting a greater potential of these species for recovery of constructed soils.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8% inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

Spatial variability of particle size fractions of an Oxisol cultivated with conilon coffee

Information on the spatial distribution of particle size fractions is essential for use planning and management of soils. The aim of this work to was to study the spatial variability of particle size fractions of a Typic Hapludox cultivated with conilon coffee. The soil samples were collected at depths of 0-0.20 and 0.20-0.40 m in the coffee canopy projection, totaling 109 georeferentiated points. At the depth of 0.2-0.4 m the clay fraction showed average value significantly higher, while the sand fraction showed was higher in the depth of 0-0.20 m. The silt showed no significant difference between the two depths. The particle size fractions showed medium and high spatial variability. The levels of total sand and clay have positive and negative correlation, respectively, with the altitude of the sampling points, indicating the influence of landscape configuration.

Determination of reactivity rates of silicate particle-size fractions

The efficiency of sources used for soil acidity correction depends on reactivity rate (RR) and neutralization power (NP), indicated by effective calcium carbonate (ECC). Few studies establish relative efficiency of reactivity (RER) for silicate particle-size fractions, therefore, the RER applied for lime are used. This study aimed to evaluate the reactivity of silicate materials affected by particle size throughout incubation periods in comparison to lime, and to calculate the RER for silicate particle-size fractions. Six correction sources were evaluated: three slags from distinct origins, dolomitic and calcitic lime separated into four particle-size fractions (2, 0.84, 0.30 and <0.30-mm sieves), and wollastonite, as an additional treatment. The treatments were applied to three soils with different texture classes. The dose of neutralizing material (calcium and magnesium oxides) was applied at equal quantities, and the only variation was the particle-size material. After a 90-day incubation period, the RER was calculated for each particle-size fraction, as well as the RR and ECC of each source. The neutralization of soil acidity of the same particle-size fraction for different sources showed distinct solubility and a distinct reaction between silicates and lime. The RER for slag were higher than the limits established by Brazilian legislation, indicating that the method used for limes should not be used for the slags studied here.

Comparison of different methods for the determination of total organic carbon and humic substances in Brazilian soils

ABSTRACTAiming to compare three different methods for the determination of organic carbon (OC) in the soil and fractions of humic substances, seventeen Brazilian soil samples of different classes and textures were evaluated. Amounts of OC in the soil samples and the humic fractions were measured by the dichromate-oxidation method, with and without external heating in a digestion block at 130 °C for 30 min; by the loss-on-ignition method at 450 °C during 5 h and at 600 °C during 6 h; and by the dry combustion method. Dry combustion was used as reference in order to measure the efficiency of the other methods. Soil OC measured by the dichromate-oxidation method with external heating had the highest efficiency and the best results comparing to the reference method. When external heating was not used, the mean recovery efficiency dropped to 71%. The amount of OC was overestimated by the loss-on-ignition methods. Regression equations obtained between total OC contents of the reference method and those of the other methods showed relatively good adjustment, but all intercepts were different from zero (p < 0.01), which suggests that more accuracy can be obtained using not one single correction factor, but considering also the intercept. The Walkley-Black method underestimated the OC contents of the humic fractions, which was associated with the partial oxidation of the humin fraction. Better results were obtained when external heating was used. For the organic matter fractions, the OC in the humic and fulvic acid fractions can be determined without external heating if the reference method is not available, but the humin fraction requires the external heating.

Aggregatory behaviour of platelets incubated with subcellular fractions of normal and chagasic human syncytiotrophoblast

The surface of human syncytiotrophoblast does not induce maternal blood platelet aggregation even though it is not an endothelium. It can be surmised that as occurs in endothelial injury the subcellular components of the syncytiotrophoblast may have pro-or antiaggregatory activity. During congenital Chagas' disease which is associated to trophoblast lesions, platelets may play a role in the development of T. cruzi-induced placentitis. In the present work the aggregatory behaviour of normal human blood platelets was recorded after their challenging with subcellular fractions of syncytiotrophoblast isolated from normal and chagasic women. Nuclear, Mitochondrial, Microsomal and Supernatant fractions isolated from normal and chagasic syncytiotrophoblast failed to induce per se any aggregatory reaction on platelets. When samples of platelet-rich plasma (PRP) were preincubated with normal and chagasic nuclear fractions and then stimulated with collagen at threshold level (CT-PRP) an inhibition of the aggregatory response was observed. Treatment of CT-PRP with normal and chagasic mitochondrial fractions induced inhibition of platelet aggregation whereas only chagasic fraction reduced latency time. Microsornal fraction from normal placentas showed no significant effects on platelet aggregation. It is concluded that subcellular fractions of normal human syncytiotrophoblast do not exhibit any effect on platelet aggregation, whereas those subcellular fractions enriched in intracellular membrane components isolated from chagasic placentas inhibit platelet aggregation.

Hepatic regeneration after partial hepatectomy in mice infected with Schistosoma mansoni, at the acute and chronic phases of the disease

Outbred male albino mice normal or infected with 30 cercariae of Schistosoma mansoni (LE strain) were submitted to 65% hepatectomy during the acute (70 days) and chronic phase (160 days) phases of the disease. A group of the infected animals was treated with 400 mg/kg of oxamniquine during the acute phase before hepatectomy. Non-infected, infected and treated but not hepatectomized animals were kept as controls. Hepatic regeneration was evaluated by incorporation of tritiated thymidine, intraperitoneally injected into non-hepatectomized and hepatectomized animals, 24 hours after surgery. The results showed that removal of 65% of the hepatic parenchyma, during the acute phase, led to a statistically significant increase of thymidine incorporation, when compared with the uninfected hepatectomized controls. This phenomenon was not observed at the chronic phase. Treatment with oxamniquine administered during the acute phase led to a decrease in thymidine incorporation rate 160 days after infection (90 days after treatment) and 24 hours after hepatectomy. The data suggest that infection with S. mansoni represents a considerable stimulus for the regenerative capacity of the liver during the acute, but not the chronic phase of disease.

Partial chemical characterization of antigenic preparations of chromoblastomycosis agents

Antigenic preparations (saline, methylic, metabolic and exoantigens) of four agents of chromoblastomycosis, Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora (Cladosporium) carrionii and Rhinocladiella aquaspersa were obtained. Partial chemical characterization of these antigenic preparations was obtained by determination of the levels of total lipids, protein, and carbohydrates, and identification of the main sterols and carbohydrates. Methylic antigens presented the highest lipid contents, whereas metabolic antigens showed the highest carbohydrate content. Total lipid, protein, and carbohydrate levels were in the range of 2.33 to 2.00mg/ml, 0.04 to 0.02 mg/ml and 0.10 to 0.02 mg/ml, respectively, in the methylic antigens and in the range of 0.53 to 0.18mg/ml, 0.44 to 0.26mg/ml, and 1.82 to 1.02 mg/ml, respectively, in saline antigens. Total lipid, protein, and carbohydrate contents were in the range of 0.55 to 0.20mg/ml, 0.69 to 0.57mg/ml and 10.73 to 5.93mg/ml, respectively, in the metabolic antigens, and in the range of 0.55 to 0.15mg/ml, 0.62 to 0.20mg/ml and 3.55 to 0.42mg/ml, respectively, in the exoantigens. Phospholipids were not detected in the preparations. Saline and metabolic antigens and exoantigens presented hexose and the methylic antigen revealed additional pentose units in their composition. The UV light absorption spectra of the sterols revealed squalene and an ergosterol fraction in the antigens. The characterization of these antigenic preparations may be useful for serological evaluation of patients of chromoblastomycosis.

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils

Neutrophils, eosinophils and macrophages are cells that interact with invading parasites and naive hosts have been shown to have anti-parasitic activity. The initial reaction of these leukocytes is the generation of reactive oxygen species (ROS) to play in parasite expulsion. The present work was carried out to study the effect of total extract, scolex and membrane fractions from Cysticercus cellulosae on respiratory burst by pig neutrophils. Hydrogen peroxide (H2O2) production by neutrophils incubated with metacestode fractions from C. cellulosae showed an increase of: 190% (total extract), 120% (scolex) and 44% (membrane). High antioxidant catalatic activity (33%, 28%, 28% by total extract, scolex and membrane, respectively) was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. Scolex and membrane fractions increased the phagocytic capacity of neutrophils (44% and 28%, respectively). On the other hand, total cysticerci did not alter the phagocytosis, possibly due to modifications in membrane function, caused by high ROS production from neutrophils in the presence of total cysticerci. Total fraction from C. cellulosae is toxic for neutrophils as shown by the decrease in phagocytic capacity, probably caused by high levels of ROS formation. The difference in toxicity of total extract, scolex and membrane fractions on neutrophils can be explained by the presence of an antigenic effect of the vesicular fluid in the total extract of C. cellulosae.

MEMBRANE FRACTIONS FROM Strongyloides venezuelensis IN THE IMMUNODIAGNOSIS OF HUMAN STRONGYLOIDIASIS

Strongyloides venezuelensis is a parasitic nematode of rodents frequently used to obtain heterologous antigens for the immunological diagnosis of human strongyloidiasis. The aim of this study was to evaluate membrane fractions from S. venezuelensis for human strongyloidiasis immunodiagnosis. Soluble and membrane fractions were obtained in phosphate saline (SS and SM) and Tris-HCl (TS and TM) from filariform larvae of S. venezuelensis. Ninety-two serum samples (n = 92) were obtained from 20 strongyloidiasis patients (Group I), 32 from patients with other parasitic diseases (Group II), and 40 from healthy individuals (Group III), and were analyzed by enzyme-linked immunosorbent assay (ELISA). Soluble fractions (SS and TS) showed 90.0% sensitivity and 88.9% specificity, whereas the membrane fractions (SM and TM) showed 95.0% sensitivity and 94.4% specificity. The present results suggest the possible use of membrane fractions of S. venezuelensis as an alternative antigen for human strongyloidiasis immunodiagnosis.