Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.

Evaluation of GFP Tag as a Screening Reporter in Directed Evolution of a Hyperthermophilic beta-Glucosidase

By applying a directed evolution methodology specific enzymatic characteristics can be enhanced, but to select mutants of interest from a large mutant bank, this approach requires high throughput screening and facile selection. To facilitate such primary screening of enhanced clones, an expression system was tested that uses a green fluorescent protein (GFP) tag from Aequorea victoria linked to the enzyme of interest. As GFP`s fluorescence is readily measured, and as there is a 1:1 molar correlation between the target protein and GFP, the concept proposed was to determine whether GFP could facilitate primary screening of error-prone PCR (EPP) clones. For this purpose a thermostable beta-glucosidase (BglA) from Fervidobacterium sp. was used as a model enzyme. A vector expressing the chimeric protein BglA-GFP-6XHis was constructed and the fusion protein purified and characterized. When compared to the native proteins, the components of the fusion displayed modified characteristics, such as enhanced GFP thermostability and a higher BglA optimum temperature. Clones carrying mutant BglA proteins obtained by EPP, were screened based on the BglA/GFP activity ratio. Purified tagged enzymes from selected clones resulted in modified substrate specificity.

Antioxidant response of Nicotiana tabacum cv. Bright Yellow 2 cells to cadmium and nickel stress

Plant cell cultures are a suitable model system for investigation of the physiological mechanisms of tolerance to environmental stress. We have determined the effects of Cd (0.1 and 0.2 mM CdCl(2)) and Ni (0.075 and 0.75 mM NiCl(2)) on Nicotiana tabacum L. cv. Bright Yellow (TBY-2) cell suspension cultures over a 72-h period. Inhibition of growth, loss of cell viability and lipid peroxidation occurred, in general, only when the TBY-2 cells were grown at 0.2 mM CdCl(2) and at 0.75 mM NiCl(2). At 0.1 mM CdCl(2), a significant increase in growth was determined at the end of the experiment. Increases in the activities of all of the four enzymatic antioxidant defence systems tested, were induced by the two concentrations of Cd and Ni, but at different times during the period of metal exposure. Overall, the cellular antioxidant responses to Cd and Ni were similar and were apparently sufficient to avoid oxidative stress at the lower concentrations of Cd and Ni. The activities of glutathione reductase and glutathione S-transferase increased early but transiently, whereas the activities of catalase and guaiacol peroxidase increased in the latter half of the experimental period. Therefore it is likely that the metabolism of reduced glutathione was enhanced during the initial onset of the stress, while catalase and guaiacol-type peroxidase appeared to play a more important role in the antioxidant response once the stress became severe.

Diversity and biotechnological potential of culturable bacteria from Brazilian mangrove sediment

Mangrove ecosystems are environments subject to substantial degradation by anthropogenic activities. Its location, in coastal area, interfacing the continents and the oceans makes it substantially important in the prospection for biotechnological applications. In this study, we assessed the diversity of culturable bacteria present over the seasons at two depths (0-10 and 30-40 cm) in a mangrove sediment and in a transect area from the land to the sea. In total, 238 bacteria were isolated, characterized by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and further identified, by Fatty Acid Methyl Esther (FAME-MIDI), into the orders of Vibrionales, Actinomycetales and Bacillales. Also the ability of the isolates in producing economically important enzymes (amylases, proteases, esterases and lipases) was evaluated and the order Vibrionales was the main enzymatic source.

Changes in the genetic structure of Bacteria and microbial activity in an agricultural soil amended with tannery sludge

The application of tannery sludge to soils is a form of recycling; however, few studies have examined the impacts of this practice on soil microbial properties. We studied effects of two applications (2006 and 2007) of tannery sludge (with a low chromium content) on the structure of the bacterial community and on the microbial activity of soils. We fertilized an agricultural area in Rolandia, Parana state, Brazil with different doses of sludge based on total N content, which ranged from 0 to 1200 kg N ha(-1). Sludge remained on the soil surface for three months before being plowed. Soils were sampled seven times during the experiment. Bacterial community structure, assessed by denaturing gradient gel electrophoresis (DGGE), was modified by the application of tannery sludge. Soon after the first application, there was clear separation between the bacterial communities in different treatments, such that each dose of sludge was associated with a specific community. These differences remained until 300 days after application and also after the second sludge application, but 666 days after the beginning of the experiment no differences were found in the bacterial communities of the lowest doses and the control. The principal response curve (PRC) analysis showed that the first sludge application strongly stimulated biological activity even 300 days after application. The second application also stimulated activity, but at a lower magnitude and for a shorter time, given that 260 days after the second application there was no difference in biological activity among treatments. PRC also showed that the properties most influenced by the application of tannery sludge were enzymatic activities related to N cycling (asparaginase and urease). The redundancy analysis (RDA) showed that tannery sludge`s influence on microbial activity is mainly related to increases in inorganic N and soil pH. Results showed that changes in the structure of the bacterial community in the studied soils were directly related to changes of their biological activity. (C) 2010 Elsevier Ltd. All rights reserved.

LC-MSn analysis of the cis isomers of chlorogenic acids

The behaviour of cis isomers of selected mono- and di-acyl chlorogenic acids produced by UV-irradiation has been investigated by LC-MSn. cis Isomers fragment identically to the more common trans isomers. cis-5-Acyl chlorogenic acids are more hydrophobic and elute later than their mono- or di-trans counterparts whereas the reverse is true for cis-3-acyl and cis-4-acyl chlorogenic acids. The cis isomers of 1,3-dicaffeoylquinic acid, the only I-acyl chlorogenic acid investigated, are also more hydrophobic than the di-trans isomer. Coffee leaves had a proportionately greater content of cis isomers relative to trans isomers compared with coffee beans suggesting that UV-irradiation in vivo may also cause geometric isomerisation. (c) 2007 Elsevier Ltd. All rights reserved.

Antioxidant Status in Humans after Consumption of Blackberry (Rubus fruticosus L.) Juices With and Without Defatted Milk

The present study was designed to evaluate the possible effect of the consumption of blackberry juices (BJ) prepared with water (BJW) and defatted milk (BJM) on the plasma antioxidant capacity and the. enzymatic and nonenzymatic antioxidants. A significant (p < 0.05) increase in the ascorbic acid content in the plasma was observed after intake of both BJs. However, no changes were observed in the plasma urate and a-tocopherol levels. An increase on the plasma antioxidant capacity, by ORAC assay, was observed only after consumption of BJW but not statistically significant. Plasma antioxidant capacity had a good positive correlation with ascorbic acid (r = 0.93) and a negative correlation with urate level (r = -0.79). No correlation was observed between antioxidant capacity and total cyanidin or total ellagic acid contents. Further, it was observed that plasma catalase increased following intake of BJ`s. No change was observed on the plasma and erythrocyte CAT and glutathione peroxidase activities. A significant decrease (p < 0.05) in the urinary antioxidant capacity between 1 and 4 h after intake of both BJs was observed. A good correlation was observed between total antioxidant capacity and urate and total cyanidin levels. These results suggested association between anthocyanin levels and CAT and a good correlation between antioxidant capacity and ascorbic acid in the human plasma after intake of BJs. Follow-up studies investigating the antioxidant properties and health benefits are necessary to demonstrate the health benefits of polyphenols.

Secretory phospholipases A(2) isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E-2 and D-2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A(2) (sPLA(2)) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA(2)s promoted the synthesis of PGD(2) and PGE(2), with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA(2)s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. inhibition of cPLA(2) by AACOCF(3), but not of iPLA(2) by PACOCF(3) or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs` synthesis are triggered by these snake venom sPLA(2)s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA(2)s in vivo. (C) 2008 Elsevier Ltd. All rights reserved.

Ascorbic acid metabolism in fruits: activity of enzymes involved in synthesis and degradation during ripening in mango and guava

BACKGROUND: Ascorbic acid is a very important compound for plants. It has essential functions, mainly as an antioxidant and growth regulator. Ascorbic acid biosynthesis has been extensively studied, but studies in fruits are very limited. In this work we studied the influence of five enzymes involved in synthesis (L-galactono-1,4-lactone dehydrogenase, GalLDH, EC 1.3.2.3), oxidation (ascorbate oxidase, EC 1.10.3.3, and ascorbate peroxidase, APX, EC and recycling (monodehydroascorbate reductase, EC 1.6.5.4, and dehydroascorbate reductase, DHAR, EC 1.8.5.1) on changes in ascorbic acid content during development and ripening of mangoes (Mangifera indica L. cv. Keitt) and during the ripening of white pulp guavas (Psidium guayava L. cv. Paloma). RESULTS: It was found that there was a balance between the activities of GalLDH, APX and DHAR, both in mangoes and guavas. CONCLUSIONS: Equilibrium between the enzymatic activities of synthesis, catabolism and recycling is important for the regulation of ascorbic acid content in mango and guava. These results have contributed to understanding some of the changes that occur in ascorbic acid levels during fruit ripening. (C) 2008 Society of Chemical Industry.

Hydrolysis Influence on Phytochemical Composition, Antioxidant Activity, Plasma Concentration, and Tissue Distribution of Hydroethanolic Ilex paraguariensis Extract Components

The infusion of aerial parts of Ilex paraguariensis is widely consumed. Its antioxidant activity suggests an important role of this plant in the treatment/prevention of oxidative stress related diseases. Plant extract active compounds are frequently found in esterified form that may be poorly absorbed. Hydrolysis of the extract is a possible approach to increase its bioavailability. The aim of this study was to perform a phytochemical analysis and evaluate in rats the plasma concentration and tissue distribution of antioxidant compounds in the hydroethanolic extract of Ilex paraguariensis, before and after enzymatic hydrolysis. Both extracts presented high antioxidant activity and phenolic content. Rats given single or repeated doses of the hydrolyzed extract showed increased plasma antioxidant activity and higher plasma levels of caffeic acid. However, no changes of endogenous antioxidants were observed. In conclusion, hydrolysis of the extract of Ilex paraguariensis is a strategy to improve its bioavailability and in vivo antioxidant activity.

Synthesis of 5 `-seleno-xylofuranosides

The synthesis of selenium derivatives of naturally occurring chiral molecules is becoming increasingly important in recent years. In this context, we describe herein an easy, straightforward synthetic route for the preparation of a series of chiral seleno-furanosides, starting from the readily available carbohydrate D-xylose. In addition, selected compounds were screened as inhibitors of the delta-aminolevulinate dehyclratase (delta-ALA-D) enzyme. Diselenide 4 was found to reduce significantly the enzymatic activity, while seleno-furanoside la increased delta-ALA-D activity. (C) 2010 Elsevier Ltd. All rights reserved.

Biochemical and biophysical characterization of lysozyme modified by PEGylation

PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH(2) modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 degrees C, and demonstrated resistance to proteolytic degradation. (C) 2010 Elsevier B.V. All rights reserved.

Structured lipids obtained by chemical interesterification of olive oil and palm stearin

Interesterification of palm stearin (PS) with liquid vegetable oils could yield a good solid fat stock that may impart desirable physical properties, because PS is a useful source of vegetable hard fat, providing beta` stable solid fats Dietary ingestion of olive oil (OO) has been reported to have physiological benefits such as lowering serum cholesterol levels Fat blends, formulated by binary blends of palm stearin and olive oil in different ratios, were subjected to chemical interesterification with sodium methoxide The original and interesterified blends were examined for fatty acid and triacylglycerol composition, melting point, solid fat content (SFC) and consistency. Interestenfication caused rearrangement of triacylglycerol species, reduction of trisaturated and triunsaturated triacylglycerols content and increase in diunsaturated-monosaturated triacylglycerols of all blends, resulting in lowering of melting point and solid fat content The incorporation of OO to PS reduced consistency, producing more plastic blends The mixture and chemical interesterification allowed obtaining fats with various degrees of plasticity, increasing the possibilities for the commercial use of palm stearin and olive oil (C) 2009 Elsevier Ltd All rights reserved

Effects of chemical interesterification on physicochemical properties of blends of palm stearin and palm olein

Chemical interesterification is an important technological option for the production of fats targeting commercial applications. Fat blends, formulated by binary blends of palm stearin and palm olein in different ratios, were subjected to chemical interesterification. The following determinations, before and after the interesterification reactions, were done: fatty acid composition, softening point, melting point, solid fat content and consistency. For the analytical responses a multiple regression statistical model was applied. This study has shown that blending and chemical interesterifications are an effective way to modify the physical and chemical properties of palm stearin, palm olein and their blends. The mixture and chemical interesterification allowed obtaining fats with various degrees of plasticity, increasing the possibilities for the commercial use of palm stearin and palm olein. (C) 2009 Elsevier Ltd. All rights reserved.