Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg(52) in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu(2), Phe(15)) did not modify the toxin`s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. (C) 2011 Elsevier Ltd. All rights reserved.

Total phenolic content and free radical scavenging activities of methanolic extract powders of tropical fruit residues

Methanolic extract powders of acerola, passion fruit and pineapple industrial residues, including pulp, seeds and peel, altogether (except for acerola) devoid of seeds, were screened for antioxidant capacity. The total phenolic contents (TPCs) of the extract powders were compared with their radical-scavenging activities (RSA) against both DPPH(center dot) and superoxide anion (O(2)(center dot-)) radicals, and their protective effect against liposome peroxidation, triggered by peroxyl radical. Lipid peroxidation was followed by the fluorescence decay of the probe, 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C(11)-BODIPY(581/591)). The TPCs of acerola, passion fruit and pineapple extract powders were (94.6 +/- 7.4); (41.2 +/- 4.2) and (9.1 +/- 1.3) mg of gallic acid equivalents g(-1) of dry extract, respectively. Acerola showed the best RSA-DPPH(center dot) scores, whereas passion fruit was more protective on the RSA-O(2)(center dot-) system. Together with the protective effects against lipid peroxidation (rate of BODIPY decay) which, were similar for acerola and passion fruit extracts, these data suggest that the methanolic extracts of acerola and passion fruit residues may be useful as antioxidant supplements, particularly the acerola extract, due to its high phenolic content. (C) 2008 Elsevier Ltd. All rights reserved

The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a beta-glycosidase

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of a beta-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investigated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contribute to stabilize ES double dagger. Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Antioxidant therapy attenuates oxidative stress in the blood of subjects exposed to occupational airborne contamination from coal mining extraction and incineration of hospital residues

Coal mining and incineration of solid residues of health services (SRHS) generate several contaminants that are delivered into the environment, such as heavy metals and dioxins. These xenobiotics can lead to oxidative stress overgeneration in organisms and cause different kinds of pathologies, including cancer. In the present study the concentrations of heavy metals such as lead, copper, iron, manganese and zinc in the urine, as well as several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of lipoperoxidation = TBARS, protein carbonyls = PC, protein thiols = PT, alpha-tocopherol = AT, reduced glutathione = GSH, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of six different groups (n = 20 each) of subjects exposed to airborne contamination related to coal mining as well as incineration of solid residues of health services (SRHS) after vitamin E (800 mg/day) and vitamin C (500 mg/day) supplementation during 6 months, which were compared to the situation before the antioxidant intervention (Avila et al., Ecotoxicology 18:1150-1157, 2009; Possamai et al., Ecotoxicology 18:1158-1164, 2009). Except for the decreased manganese contents, heavy metal concentrations were elevated in all groups exposed to both sources of airborne contamination when compared to controls. TBARS and PC concentrations, which were elevated before the antioxidant intervention decreased after the antioxidant supplementation. Similarly, the contents of PC, AT and GSH, which were decreased before the antioxidant intervention, reached values near those found in controls, GPx activity was reestablished in underground miners, and SOD, CAT and GST activities were reestablished in all groups. The results showed that the oxidative stress condition detected previously to the antioxidant supplementation in both directly and indirectly subjects exposed to the airborne contamination from coal dusts and SRHS incineration, was attenuated after the antioxidant intervention.

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only alpha, alpha`- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has 0312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (0322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity. (C) 2010 Elsevier Ltd. All rights reserved.

Improving the Fluorescence Detectability of Polycyclic Aromatic Hydrocarbons for Evaluation of Workplace Environments of Cement Industries Processing Organic Residues

The burning of organic residues and wastes in furnaces of cement industries has been an attractive and lucrative approach to eliminate stocks of these pollutants. There is a potential risk for producing PAH in the workplace of industries burning organic wastes, so that highly sensitive analytical methods are needed for monitoring the air quality of these environments. An official method for determination of PAH is based on liquid chromatography with fluorescence detection at fixed excitation and emission wavelengths. We demonstrate that a suitable choice of these wavelengths, which are changed during the chromatographic run, significantly improves the detectability of PAH in atmosphere and particulate matter collected in cement industries.

Evaluation of the QuEChERS Method and Gas Chromatography-Mass Spectrometry for the Analysis Pesticide Residues in Water and Sediment

A method for the determination of pesticide residues in water and sediment was developed using the QuEChERS method followed by gas chromatography - mass spectrometry. The method was validated in terms of accuracy, specificity, linearity, detection and quantification limits. The recovery percentages obtained for the pesticides in water at different concentrations ranged from 63 to 116%, with relative standard deviations below 12%. The corresponding results from the sediment ranged from 48 to 115% with relative standard deviations below 16%. The limits of detection for the pesticides in water and sediment were below 0.003 mg L(-1) and 0.02 mg kg(-1), respectively.

Characterization of organic matter from composting of different residues by physicochemical and spectroscopic methods

Chemical and spectroscopic methods were used to characterize organic matter transformations during the composting process. Four different residue mixtures were studied: P1 - garden trimmings (GT) only, P2 - GT plus fresh cattle manure, P3 - GT plus orange pomace and P4 - GT plus filter cake. The thermophilic phase was not reached in PI compost, but the P2, P3 and P4 composts showed all three typical process phases. The thermophilic phase and CEC/C ratio stabilized after 90 days, while C/N ratio and the ash content stabilized after 60 days. The increasing E(4)/E(6) ratio indicated oxidation reactions occurring during the process in the material from P2, P3 and P4. The (13)C NMR and FTIR results suggested extraction of both pectin and lignin in the HA-like fraction. The CEC/C ratio, temperature and E(4)/E(6) ratio showed that within 90 days P2, P3 and P4 composts were humified. However, material from P1 did not show characteristics of humified compost. From these data, it is apparent that C/N ratio and ash content are not reliable methods for monitoring the composting process. (C) 2009 Elsevier Ltd. All rights reserved.

Evaluation of solid residues removed from a mangrove swamp in the Sao Vicente Estuary, SP, Brazil

Mangrove swamps are found in estuaries along the coastal plains of tropical regions and have be subjected to heavy occupation and use pressure due to their privileged locations and abundance of biological resources. The present work evaluated the ecological characteristics and solid wastes accumulated in eight areas along the Santos - Sao Vicente Estuary Complex. The superficially deposited residues at each sampling site were collected and subsequently washed, drained, counted, weighed and separated into classes according to their composition and predominant use. The predominant litter type in terms of density was plastic (62.81%) and, by weight, wood (55.53%). The greatest deposition of residues was associated with areas that were less inclined and that had low plant density levels, indicating that the presence of obstacles was not critical for retaining floating residues in mangrove areas. The presence of the most frequently encountered types of solid waste residues could be explained by local activities. (C) 2010 Elsevier Ltd. All rights reserved.

Soil CO2 emission as related to incorporation of sugarcane crop residues and aggregate breaking after rotary tiller

O preparo do solo é um dos processos que aceleram a decomposição da matéria orgânica, transferindo carbono para atmosfera, principalmente na forma de CO2. Neste trabalho, investigou-se o efeito do preparo com enxada rotativa sobre as emissões de CO2 do solo durante 02 semanas após o preparo do solo, incluindo-se a presença de resíduos vegetais sobre a superfície. As emissões foram avaliadas por 15 dias após preparo em 3 parcelas: 1) sem preparo e sem palha superficial (SPs); 2) preparo com enxada rotativa sem a presença de palha na superfície (ERs), e 3) preparo com enxada rotativa com a presença de palha superficial (ERc). As emissões provenientes da ERc foram superiores às demais (0,777 g CO2 m-2 h-1), sendo as menores emissões registradas na parcela SPs (0,414 g CO2 m-2 h-1). As emissões totais indicaram que a diferença de C-CO2 emitida à atmosfera corresponde a 3% do total de carbono adicional presente na palha, na parcela ERc, quando comparado à parcela ERs. O aumento da emissão da parcela SPs para ERs foi acompanhado de uma modificação na distribuição do tamanho de agregados, especialmente aqueles com diâmetro médio inferior a 2 mm. O aumento da emissão da parcela ERs para ERc esteve relacionado a uma diminuição da massa de palha na superfície, com fragmentação e incorporação da mesma no interior do solo. Quando se analisa a correlação linear entre emissão de CO2 versus temperatura e umidade do solo, somente a emissão da ERc foi significativamente correlacionada (p<0,05) à umidade do solo.

Co-accumulation of microbial residues and particulate organic matter in the surface layer of a no-till Oxisol under different crops

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

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

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

Nitrogen washing from C3 and C4 cover grasses residues by rain

Crop species with the C-4 photosynthetic pathway are more efficient in assimilating N than C-3 plants, which results in different N amounts prone to be washed from its straw by rain water. Such differences may affect N recycling in agricultural systems where these species are grown as cover crops. In this experiment, phytomass production and N leaching from the straw of grasses with different photosynthetic pathways were studied in response to N application. Pearl millet (Pennisetum glaucum) and congo grass (Brachiaria ruziziensis) with the C-4 photosynthetic pathway, and black oat (Arena Strigosa) and triticale (X Triticosecale), with the C-3 photosynthetic pathway, were grown for 47 days. After determining dry matter yields and N and C contents, a 30 mm rainfall was simulated over 8 t ha(-1) of dry matter of each plant residue and the leached amounts of ammonium and nitrate were determined. C-4 grasses responded to higher fertilizer rates, whereas N contents in plant tissue were lower. The amount of N leached from C-4 grass residues was lower, probably because the C/N ratio is higher and N is more tightly bound to organic compounds. When planning a crop rotation system it is important to take into account the difference in N release of different plant residues which may affect N nutrition of the subsequent crop.

Soil Solution as Affected by Plant Residues and Nitrogen Rates

Cation mobility in acidic soils with low organic-matter contents depends not only on sorption intensity but also on the solubility of the species present in soil solution. In general, the following leaching gradient is observed: potassium (K+) magnesium (Mg2+) calcium (Ca2+) aluminum (Al3+). To minimize nutrient losses and ameliorate the subsoil, soil solution must be changed, favoring higher mobility of M2+ (metal ions) forms. This would be theoretically possible if plant residues were kept on the soil surface. An experiment was conducted in pots containing a Distroferric Red Latosol, with soil solution extractors installed at two depths. Pearl millet, black oat, and oilseed radish residues were laid on the soil surface, and nitrogen (as ammonium nitrate) was applied at rates ranging from 0 to 150mgkg-1. Corn was grown for 52 days. Except for K+ and ammonium (NH4 +), nitrogen rates and plant residues had little effect upon the concentrations and forms of the elements in the soil solution. Presence of cover crop residues on soil surface decreased the effect of nitrogen fertilizer on Ca leaching. More than 90% of the Ca2+, Mg2+, and K+ were found as free ions. The Al3+ was almost totally complexed as Al(OH3)0. Nitrogen application increased the concentrations of almost all the ions in soil solution, including Al3+, although there was no modification in the leaching gradient.