Produção de etanol a partir de torta de mamona (Ricinus communis L.) e avaliação da letalidade da torta hidrolisada para camundongos

The castor bean cake is rich in starch (48 ± 0.53%) and bears a problem linked to the occurrence of a toxic protein (ricin). The chemical hydrolysis (ratio solid:liquid = 1:6; H2SO4= 0.1 mol L-1; 120 °C; 40 min) generated a medium with 27 g L-1 of reducing sugars (hydrolysis efficiency= 32%). The hydrolyzed product was fermented and produced 11 g L-1 of ethanol (volumetric productivity=1.38 g L-1 h-1 and ethanol yield on substrate consumed=0.45 g g-1). In vivo experiments (DL50) revealed a reduction of roughly 240 times in the CBC toxicity (2.11 µg g-1).

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds

Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.

Obtenção e caracterização de um compósito de poliuretano de mamona e pó de vidro para aplicações em insolantes térmicos

Thermal insulation is used to protect the heated or cooled surfaces by the low thermal conductivity materials. The rigid ricin polyurethane foams (PURM) are used for thermal insulation and depend on the type and concentration of blowing agent. Obtaining PURM occurs by the use of polyol, silicone, catalyst and blowing agent are pre -mixed, reacting with the isocyanate. The glass is reusable, returnable and recyclable heat insulating material, whose time of heat dissipation determines the degree of relaxation of its structure; and viscosity determines the conditions for fusion, operating temperatures, annealing, etc. The production of PURM composites with waste glass powder (PV) represents economical and renewable actions of manufacturing of thermal insulating materials. Based on these aspects, the study aimed to produce and characterize the PURM composites with PV, whose the mass percentages were 5, 10, 20, 30, 40 and 50 wt%. PURM was obtained commercially, while the PV was recycled from the tailings of the stoning process of a glassmaking; when the refining process was applied to obtain micrometer particles. The PURM + PV composites were studied taking into account the standard sample of pure PURM and the influence of the percentage of PV in this PURM matrix. The results of the chemical, physical and morphological characterization were discussed taking into account the difference in the microstructural morphology of the PURM+PV composites and the pure PURM, as well the results of the physicochemical, mechanical e thermophysical tests by values obtained of density, hardness, compressive strength, specific heat, thermal conductivity and diffusivity. In general, the structure of pure PURM showed large, elongated and regular pores, while PURM+PV composites showed irregular, small and rounded pores with shapeless cells. This may have contributed to reducing their mechanical strength, especially for PURM - PV50. The hardness and density were found to have a proportional relationship with the PV content on PURM matrix. The specific heat, thermal diffusivity and thermal conductivity showed proportional relationship to each other. So, this has been realized that the increasing the PV content on PURM matrix resulted in the rise of diffusivity and thermal conductivity and the decrease of the specific heat. However, the values obtained by the PURM composites were similar the values of pure PURM, mainly the PURM-PV5 and PURM-PV10. Therefore, these composites can be applied like thermal insulator; furthermore, their use could reduce the production costs and to preserve the environment

Estudo comparativo da atividade antiinflamatória e anti-tumoral de proteínas inativadoras de ribossomos extraídas de plantas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Metodologia para quantificar ricina em sementes de mamona com o uso de Caenorhabditis elegans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems

Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244,239 and 236 residues (rPAC(244), rPAC(239) and rPAC(236)), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure-area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC(236)), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly alpha-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC(236) the alpha-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity. (C) 2011 Elsevier B.V. All rights reserved.

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

Extraction of Cholesterol with Methyl-β-Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles

The importance of cholesterol for endocytosis has been investigated in HEp-2 and other cell lines by using methyl-β-cyclodextrin (MβCD) to selectively extract cholesterol from the plasma membrane. MβCD treatment strongly inhibited endocytosis of transferrin and EGF, whereas endocytosis of ricin was less affected. The inhibition of transferrin endocytosis was completely reversible. On removal of MβCD it was restored by continued incubation of the cells even in serum-free medium. The recovery in serum-free medium was inhibited by addition of lovastatin, which prevents cholesterol synthesis, but endocytosis recovered when a water-soluble form of cholesterol was added together with lovastatin. Electron microscopical studies of MβCD-treated HEp-2 cells revealed that typical invaginated caveolae were no longer present. Moreover, the invagination of clathrin-coated pits was strongly inhibited, resulting in accumulation of shallow coated pits. Quantitative immunogold labeling showed that transferrin receptors were concentrated in coated pits to the same degree (approximately sevenfold) after MβCD treatment as in control cells. Our results therefore indicate that although clathrin-independent (and caveolae-independent) endocytosis still operates after removal of cholesterol, cholesterol is essential for the formation of clathrin-coated endocytic vesicles.

SQV-7, a protein involved in Caenorhabditis elegans epithelial invagination and early embryogenesis, transports UDP-glucuronic acid, UDP-N- acetylgalactosamine, and UDP-galactose

Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility. The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi membrane. A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner. These nucleotide sugars are competitive, alternate, noncooperative substrates. The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities. SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDP-mannose. SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi. These results demonstrate that unlike most nucleotide sugar transporters, SQV-7 can transport multiple distinct nucleotide sugars. We propose that SQV-7 translocates multiple nucleotide sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.

Effect of castor cake and elephant grass composting on edaphic fauna

Elephant grass and castor cake when combined can make a promising organic fertilizer. However, castor cake contains potentially toxic chemicals, such as ricin and ricinine. To test potential effects of these chemicals, compost piles of elephant grass ( Pennisetum purpureum Schum.) with castor cake were prepared with different C:N ratios (T1 = 40, T2 = 30, T3 = 20; T4 = 30 [control, elephant grass + crotalaria]) to evaluate colonization by edaphic fauna and any suppressive effects of castor cake. Soil organisms were collected with Berlese-Tullgren funnels. There were temporal differences between the treatments, and the epigeous fauna was mainly represented by members of the Acari and Entomobryomorpha. Elapsed time is the major factor in determining the composition of the epigeous fauna community associated with composting, indicating that castor cake has no suppressive effect.