Effect of crop residues applied isolated or in combination on the germination and seedling growth of horse purslane (Trianthema portulacastrum)

Soil incorporation of crop residues can lead to weed suppression by posing allelopathic and physical effects. Allelopathic potential of the crops sorghum, sunflower, brassica applied as sole or in combination for horse purslane (Trianthema portulacastrum) suppression was evaluated in a pot investigation. Chopped crop residues alone and in combination were incorporated at 6 g kg-1 soil (12 t ha-1), and a weedy check was maintained. Germination traits time to start germination; time to 50% emergence, mean emergence time, emergence index and final germination percentage were negatively influenced by residue incorporation. Crop residues also exerted a pronounced negative influence on the shoot and root length of horse purslane. Significant suppression in leaf and root score and leaf area per plant was also observed. A combination of sorghum and sunflower residues accounted for maximum (71%) seedling mortality. Soil incorporation of allelopathic crop residues can be employed for horse purslane management.

Integration of sunflower (Helianthus annuus) residues with a pre-plant herbicide enhances weed suppression in broad bean (Vicia faba)

Field trial was conducted with the aim of utilizing allelopathic crop residues to reduce the use of synthetic herbicides in broad bean (Vicia faba) fields. Sunflower residue at 600 and 1,400 g m-2 and Treflan (trifluralin) at 50, 75 and 100% of recommended dose were incorporated into the soil alone or in combination with each other. Untreated plots were maintained as a control. Herbicide application in plots amended with sunflower residue had the least total weed count and biomass, which was even better than herbicide used alone. Integration of recommended dose of Treflan with sunflower residue at 1,400 g m-² produced maximum (987.5 g m-2) aboveground biomass of broad bean, which was 74 and 36% higher than control and recommended herbicide dose applied alone, respectively. Combination of herbicide and sunflower residue appeared to better enhance pod number and yield per unit area than herbicide alone. Application of 50% dose of Treflan in plots amended with sunflower residue resulted in similar yield advantage as was noticed with 100% herbicide dose. Chromatographic analysis of residue-infested field soil indicated the presence of several phytotoxic compounds of phenolic nature. Periodic data revealed that maximum suppression in weed density and dry weight synchronized with peak values of phytotoxins observed 4 weeks after incorporation of sunflower residues. Integration of sunflower residues with lower herbicide rates can produce effective weed suppression without compromising yield as a feasible and environmentally sound approach in broad bean fields.

Early growth of common bean cropped over ruzigrass residues

Ruzigrass (Brachiaria ruziziensis, syn. Urochloa ruziziensis) is used as a cover crop in tropical regions because it has a high yield potential, is widely adapted and has a vigorous root system. However, it may affect early growth of the next crop due to allelopathy and competition for soil nitrate. A greenhouse experiment was conducted in glass-walled pots with soil to determine the effect of ruzigrass residues on the initial growth and mineral nutrition of common bean (Phaseolus vulgaris). Ruzigrass was grown in the pots for 50 days and chemically desiccated. Then, common bean was grown: without ruzigrass residues; with ruzigrass shoots placed on the soil surface; with ruzigrass roots left in the soil; and with ruzigrass shoots and roots left undisturbed. Root growth of common bean was decreased by ruzigrass residues, but shoot biomass was not affected when it was grown in the presence of ruzigrass shoots or roots alone. In pots where ruzigrass residues were undisturbed, common bean biomass yield was decreased. Nitrogen concentration in common bean shoot was not affected by ruzigrass shoot on the soil surface, an evidence that the observed decrease in common bean growth probably was due to allelopathic effects rather than competition for nitrogen.

Allelopathic Influence of Residues from Sphagneticola trilobata on Weeds and Crops

The allelopathic effect studied in many cultures has currently generated great expectations that displayed a natural and environmentally friendly tool for weed management using bioherbicides. The objective of this work was to assess allelopathic influence of residues of S. trilobata on the germination and growth of weeds, as well as their relation with some crops and effects on soil properties. Results show that residues from S. trilobata have inhibited the germination of weeds (31.6 - 72%), increasingly with the applied dose. All residue doses of this specie have inhibited dicotyledonous germination, but only maximum concentration has affected monocotyledons. The residues did not affect onion germination, but stimulated it in radish and tomato, while the dose applied at 50% produced tomato stimulation and inhibition of cabbage. The effects of residues on hypocotyl growth in different crops showed changes in species response. For onion, the three doses had negative effects on the growth of hypocotyl, while tomato was stimulated. For radish, the growth was hindered by any dose applied, and were only different (50 and 100%) compared to control. For cabbage, only hypocotyl length was stimulated, when maximum dose (100%) was applied. For the radicle growth, in onion and radish no differences were found compared to control. While the tomato radicle growth was inhibited, in cabbage, all doses encouraged the elongation of the radicle. The dry mass of weed was affected by increased dose of residue (0.49 - 8.8 g m-2), however the soil microflora was stimulated, while the population of Azotobacter spp. was not affect. Some soil properties were affected, the level of organic material, Na+ and electrical conductivity were increased, while pH (H2O) decreased a bit, however it remained basic.

Impacts of the Belo Monte hydroelectric dam construction on pioneer vegetation formations along the Xingu River, Pará State, Brazil

There is an important pioneer vegetation formation along the Xingu River in the area where the Belo Monte hydroelectric dam is being constructed that is highly adapted to a seasonally fluctuating water levels. The aim of this study was to examine the habitat and flora of the pioneer formations in the Belo Monte area. The area was divided in three sections for study purposes (Reservoir, Low Flow, and Control) that were expected to experience different degrees of impact from the dam project. The calculations of habitat losses were based on satellite imagery classifications, and a total of 111 plots were established in the three areas for vegetation sampling. Habitat losses of the pioneer formations will total 89.7% when the project is fully functional. Forty-five of the 72 recorded species are restricted to single areas. Species richness and diversity were significantly lower in the control area. The completion of the Belo Monte reservoir will result in habitat reductions and will consequently reduce the richness and diversity of pioneer formations. Studies suggest monitoring the populations located in the reduced flow area to determine possible impacts resulting from changes in the regional hydrological cycle caused by the Xingu River dam.

Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis

The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs). Since per os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV). The transfer vector pAcUW2B was used for construction of occluded recombinant viruses. The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus. The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses. Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses. The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

Important amino acid residues of potato plant uncoupling protein (StUCP)

Chemical modifications were used to identify some of the functionally important amino acid residues of the potato plant uncoupling protein (StUCP). The proton-dependent swelling of potato mitochondria in K+-acetate in the presence of linoleic acid and valinomycin was inhibited by mersalyl (Ki = 5 µM) and other hydrophilic SH reagents such as Thiolyte MB, iodoacetate and 5,5'-dithio-bis-(2-nitrobenzoate), but not by hydrophobic N-ethylmaleimide. This pattern of inhibition by SH reagents was similar to that of brown adipose tissue uncoupling protein (UCP1). As with UCP1, the arginine reagent 2,3-butadione, but not N-ethylmaleimide or other hydrophobic SH reagents, prevented the inhibition of StUCP-mediated transport by ATP in isolated potato mitochondria or with reconstituted StUCP. The results indicate that the most reactive amino acid residues in UCP1 and StUCP are similar, with the exception of N-ethylmaleimide-reactive cysteines in the purine nucleotide-binding site.

The 42-kDa coat protein of Andean potato mottle virus acts as a transcriptional activator in yeast

Interactions of viral proteins play an important role in the virus life cycle, especially in capsid assembly. Andean potato mottle comovirus (APMoV) is a plant RNA virus with a virion formed by two coat proteins (CP42 and CP22). Both APMoV coat protein open reading frames were cloned into pGBT9 and pGAD10, two-hybrid system vectors. HF7c yeast cells transformed with the p9CP42 construct grew on yeast dropout selection media lacking tryptophan and histidine. Clones also exhibited ß-galactosidase activity in both qualitative and quantitative assays. These results suggest that CP42 protein contains an amino acid motif able to activate transcription of His3 and lacZ reporter genes in Saccharomyces cerevisiae. Several deletions of the CP42 gene were cloned into the pGBT9 vector to locate the region involved in this activation. CP42 constructions lacking 12 residues from the C-terminal region and another one with 267 residues deleted from the N-terminus are still able to activate transcription of reporter genes. However, transcription activation was not observed with construction p9CP42deltaC57, which does not contain the last 57 amino acid residues. These results demonstrate that a transcription activation domain is present at the C-terminus of CP42 between residues 267 and 374.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Characterization of the interdependency between residues that bind the substrate in a β-glycosidase

The manner by which effects of simultaneous mutations combine to change enzymatic activity is not easily predictable because these effects are not always additive in a linear manner. Hence, the characterization of the effects of simultaneous mutations of amino acid residues that bind the substrate can make a significant contribution to the understanding of the substrate specificity of enzymes. In the β-glycosidase from Spodoptera frugiperda (Sfβgly), both residues Q39 and E451 interact with the substrate and this is essential for defining substrate specificity. Double mutants of Sfβgly (A451E39, S451E39 and S451N39) were prepared by site-directed mutagenesis, expressed in bacteria and purified using affinity chromatography. These enzymes were characterized using p-nitrophenyl β-galactoside and p-nitrophenyl β-fucoside as substrates. The k cat/Km ratio for single and double mutants of Sfβgly containing site-directed mutations at positions Q39 and E451 was used to demonstrate that the effect on the free energy of ES‡ (enzyme-transition state complex) of the double mutations (∆∆G‡xy) is not the sum of the effects resulting from the single mutations (∆∆G‡x and ∆∆G‡y). This difference in ∆∆G‡ indicates that the effects of the single mutations partially overlap. Hence, this common effect counts only once in ∆∆G‡xy. Crystallographic data on β-glycosidases reveal the presence of a bidentate hydrogen bond involving residues Q39 and E451 and the same hydroxyl group of the substrate. Therefore, both thermodynamic and crystallographic data suggest that residues Q39 and E451 exert a mutual influence on their respective interactions with the substrate.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Detection and quantification of Roundup Ready® soybean residues in sausage samples by conventional and real-time PCR

The increasing presence of products derived from genetically modified (GM) plants in human and animal diets has led to the development of detection methods to distinguish biotechnology-derived foods from conventional ones. The conventional and real-time PCR have been used, respectively, to detect and quantify GM residues in highly processed foods. DNA extraction is a critical step during the analysis process. Some factors such as DNA degradation, matrix effects, and the presence of PCR inhibitors imply that a detection or quantification limit, established for a given method, is restricted to a matrix used during validation and cannot be projected to any other matrix outside the scope of the method. In Brazil, sausage samples were the main class of processed products in which Roundup Ready® (RR) soybean residues were detected. Thus, the validation of methodologies for the detection and quantification of those residues is absolutely necessary. Sausage samples were submitted to two different methods of DNA extraction: modified Wizard and the CTAB method. The yield and quality were compared for both methods. DNA samples were analyzed by conventional and real-time PCR for the detection and quantification of Roundup Ready® soybean in the samples. At least 200 ng of total sausage DNA was necessary for a reliable quantification. Reactions containing DNA amounts below this value led to large variations on the expected GM percentage value. In conventional PCR, the detection limit varied from 1.0 to 500 ng, depending on the GM soybean content in the sample. The precision, performance, and linearity were relatively high indicating that the method used for analysis was satisfactory.

Antibiotic residues in Brazilian UHT milk: a screening study

The aim of this research was to carry out a screening study to check the incidence of antimicrobial residues in Brazilian UHT milk according to rapid yoghurt method. Of the 100 (100%) samples analysed, 96 (96%) showed no traces of antibiotic residues while 4 (4%) indicated probable presence of antibiotic residues. The results suggest that the Brazilian Sanitary Surveillance Agency should apply continuous monitoring programs in order to obtain a safe product offering no health risks to consumers.

Physiochemical analysis and centesimal composition of Pleurotus ostreatus mushroom grown in residues from the Amazon

The objective of the present work was to evaluate the nutritional composition of mushrooms produced in alternative substrates in agricultural and agro-industrial residues from the Amazon. C, N, pH, moisture, soluble solids, protein, lipids, total fibers, ashes, carbohydrates and energy were determined. Substrates were formulated from Simarouba amara Aubl. and Ochroma piramidale Cav. ex. Lam. Sawdust and from Bactris gasipaes Kunth and Saccharum officinarum stipe. Results showed that the nutritional composition of P. ostreatus varied according to the cultivation substrate and that it can be considered important food due to its nutritional characteristics such as: high protein content; metabolizable carbohydrates and fiber; and low lipids and calories contents.