Vicilinas de sementes de leguminosas selvagens:purificação, caracterização, efeito de deletério e mecanismo de ação para bruquídeos e para fungos fitopatogênicos

Globulins fractions of legume seeds of Crotalaria pallida, Erytrina veluntina and Enterolobium contortisiliquum were isolated and submitted to assays against serine, cysteine and aspartic proteinases, as also amylase present in midgut of C. maculatus and Z. subfasciatus. Hemagglutination assays indicated presence of a lectin in E. veluntina globulin fractions. This lectin had affinity to human erythrocytes type A, B and O. Vicilins were purified by chromatography on Sephacryl S-300 followed of a chromatography on Sephacryl S-200, which was calibrated using protein markers. Vicilins from C. pallida (CpV) and E. veluntina (EvV) seeds had a molecular mass of 124.6 kDa and E. contortisiliquum a molecular mass of 151kDa. Eletrophoresis in presence of SDS showed that CpV was constituted by four subunities with apparent molecular mass of 66, 63, 57 and 45 kDa, EvV with three subunities with apparent molecular mass of 45kDa and EcV four subunities, two with 37.1 kDa and two with 25.8 kDa. Non denaturantig eletrophoresis displayed single bands with high homogeneity, where CpV had lower acidic behavior. All vicilins are glycoproteins with carbohydrate contents at 1 to1.5%. Bioassays were done to detect deleterious effects of vicilins against C. maculatus and Z. subfasciatus larvae. CpV, EvV and EcV exhibited a WD50 of 0.28, 0.19 and 1.03%; LD50 0.2, 0.26, and 1.11% respectively to C. maculatus. The dose responses of CpV, EvV and EcV to Z. subfasciatus were: WD50 of 0.12, 0.14, 0.65% and LD50 of 0.09, 0.1, and 0.43% respectively. The mechanism of action of these proteins to bruchids should be based on their properties of bind to chitin present in mid gut of larvae associated with the low digestibility of vicilin. In assays against phytopatogenous fungus, only EcV was capable of inhibit F. solani growth at concentrations of 10 and 20 µg and its action mechanism should be also based in the affinity of EcV to chitin present in the fungi wall

Ação de polissacarídeos sulfatados de Fucus Vesiculosus na Hemostasia e no sistema complemento

Fucans are a family of sulfated homo and teropolysaccharides respectively, composed mainly of a- (1®2) and a- (1®3) linked by L-fucose residues. Properties such as the ability to act as an anti-contraceptive, to reduce cholesterol levels, and to act as an anti-tumor agent are much related. We have focused our attention on the anticoagulant properties, platelet aggregation, hemorrhagic activity and complement system in vitro of commercial fucoidan (F) and their purified fractions (F1, F2 and F3) from Fucus vesiculosus obtained from fractionation of the fucoidan with different concentrations of acetone 1, 2 and 3v. These compounds were chemically characterized and the fucoidan (F) was modified by desulfation. The anticoagulant activity of the compounds was assessment by activated partial thromboplastin time (APTT) and prothrombine time assay (PT) using citrated normal human plasma. The results of APPT test showed that F, F1 and F2 have high anticoagulants activities 240.0 s (5 µg). The F3 showed 73.7 s in the same concentrations. The results obtained with PT test to F, F1, F2 and F3 were 81.5 s, 120.0 s, 57.1 and 32.5 s respectively with 50 µg. The dessulfated polymer showed a decrease in the anticoagulant activity in these two tests. Platelet aggregation assay was measured turbidimetrically with platelet aggregometer by method of Born. The aggregation platelet with F and fractions F1, F2 and F3 exhibited a two-phase answer in the concentration of 5 mg/mL with maximum aggregation of 76.36 ± 10.3% ; 69.54 ± 9.40%; 75.94 ± 9.01%; 51.13 ± 9.59% respectively. However, was observed a hipoaggregate profile F (15.17 ± 5.2%), F1 (7.40 ± 3.04 %), F2 (19.1 ± 5.41%) and F3 (5.09 ± 3.02%) at 0.1 mg/mL. The hemorrhagic activity assay was carried in Wistar rats and showed that these compounds have low hemorrhagic effect when compared to heparin. The complement system ( alternative pathway was made using non-sensibilized rabbit red blood cells The results of complement system essay showed that F , F2 and F3 have action inhibitory in relation to the group control 0.544, 0.697, 0.622 and 0.958 respectively The results showed that these compounds have action on this system. Interaction of the polisaccharides with proteins C3 and C4 showed that the fraction F1 stimulated the activity assay hemolytic using red blood cells

Polissacarídeos sulfatados de interesse farmacológico no camarão litopenaeus schimitti

Sulfated Polysaccharides with unique chemical structures and important biological activities has been found in a diversity of sea invertebrates. For that, to exist a huger interest on the biotechnology field in the research theses sulfated compounds isolated from sea organisms. Despite the privileged brazilian position for these compounds attainment, there are still a few scientific informations about the isolated substances and their biological activities. A head the displayed, the present work has for objectives, to evaluate the pharmacological properties of the glycosaminoglycans isolated from the sea shrimp Litopenaeus schimitti on homeostasis, blood coagulation, leukocytes migration and platelet/leukocyte adhesion. For this, yhe glycosaminoglycans were extracted from crustacean tissues by proteolysis, fractionation with acetone and later submitted to pharmacological assays. The crustacean tissues showed compounds heparin-like, with anticoagulant activity of 45 IU/mg and 90 IU/mg, respectively. These molecules showed low residual hemorrhagic effects in the tested concentration (100 µg/mL), when compared to unfractionated commercial heparin (UFH). Another dermatan sulfate-like compound, predominately constituted for disulfated disaccharides, was isolated from crustacean abdomen. This compound showed an efficient effect on leukocytes migration inhibition, in the concentration of 15 µg/mL, reducing the cellular infiltration in 65% when compared to the controlled animals. In this same concentration, the DS reduced in 60% the protein concentration of the peritoneal exudates. In the concentration, this compound of 0.5 mg/mL, it was capable to reduce in 40% platelet/leukocytes adhesion. Our data demonstrate that these sulfated polysaccharides isolated from the shrimp L. schimitti will can be used as bioactive compounds, appearing as active principles for pharmacological development, anticoagulants and inflammatory response regulators

La discusión de escenarios clínicos en equipos colaborativos promueve el aprendizaje de la bioquímica

Introducción. La bioquímica es una asignatura difícil para los médicos porque se enseña de manera ajena al quehacer médico. Por ello es necesario desarrollar estrategias que vinculen su aprendizaje con la solución de problemas médicos y permitan el desarrollo de competencias. Sujetos y métodos. Se desarrollaron dos ejercicios consistentes en viñetas clínicas que relacionan temas bioquímicos con problemas de salud prevalentes en el país, los cuales se analizaron y discutieron de manera dirigida en tres grupos de intervención de dos generaciones subsecuentes de alumnos de primer año de medicina. El aprendizaje obtenido en estos grupos se comparó con el de tres grupos control, quienes recibieron exposiciones orales de los profesores. Los grupos fueron equivalentes respecto al número de estudiantes por grupo y distribución por sexo. El aprendizaje se evaluó por exámenes de opción múltiple, y el desarrollo de trabajo colaborativo y comunicación en los grupos de intervención, por rúbricas ya reportadas. Los resultados se analizaron estadísticamente mediante una prueba t de Student y un ANOVA de una variable usando el programa SIGMA STATT. Resultados. En general, los grupos de intervención mostraron un mejor aprendizaje de bioquímica que los grupos control, aunque esto no siempre fue consistente, probablemente por la influencia del profesor y cierta apatía de los estudiantes. Asimismo, mejoraron su comunicación oral y escrita y evolucionaron en el trabajo colaborativo. Conclusión. El uso de viñetas clínicas permite un mejor aprendizaje y vinculación de los temas de bioquímica con el quehacer médico y permite el desarrollo de competencias.

Ação Sacietogênica de um inibidor de tripsina da semente de tamarindo (Tamarindus indica L.)

The seeds are excellent sources of proteinase inhibitors and have been highlighted owing to various applications. Among these applications are those in effect on food intake and weight gain that stand out because of the increasing number of obese individuals. This study evaluated the effects of trypsin inhibitor present in the seed of tamarind (Tamarindus indica L.) reduction in weight gain, biochemical and morphological alterations in Wistar rats. For this, we partially purified a trypsin inhibitor tamarind seed. This inhibitor, ITT2 at a concentration of 25 mg / kg body weight, over a period of 14 days was able to reduce food intake in rats (n = 6) by approximately 47%, causing a reduction in weight gain approximately 70% when compared with the control group. With the evaluation of the in vivo digestibility was demonstrated that the animals lost weight due to satiety, presented by the reduction of food intake, since there were significant differences between true digestibility for the control group (90.7%) and the group treated with inhibitor (89.88%). Additionally, we checked the deeds of ITT2 on biochemical parameters (glucose, triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, glutamic-pyruvic transaminase, glutamic oxaloacetic transaminase, gamma glutamyl transferase albumin, globulin, total protein and C-reactive protein) and these, when assessed in the study groups showed no statistically significant variations. We also evaluate the histology of some organs, liver, stomach, intestine, and pancreas, and showed no changes. And to evaluate the effect of trypsin inhibitor on food intake due to the satiety is regulated by cholecystokinin (CCK) were measured plasma levels, and it was observed that the levels of CCK in animals receiving ITT2 were significantly higher ( 20 + 1.22) than in animals receiving only solution with casein (10.14 + 2.9) or water (5.92 + 1.15). Thus, the results indicate that the effect caused ITT2 satiety, reducing food intake, which in turn caused a reduction in weight gain in animals without causing morphological and biochemical changes, this effect caused by the elevation of plasma levels CCK

Efeito anti-inflamatório do heparinóide isolado do camarão Litopenaeus vannamei sobre a peritonite aguda

In recent years the heparin has been the subject of several studies that aim to expand its use as a therapeutic agent, due to its ability to modulate the activity of various proteins that play important roles in the regulation of pathophysiological processes. In several experiments and preclinical trials, heparin has demonstrated an anti-inflammatory role. However, its clinical use is limited, due to its strong anticoagulant activity and hemorrhagic complications. For this reason, considerable efforts have been employed in discovery of heparin analogous (heparinoid) with reduced side effects, that retain the anti-inflammatory properties of heparin. In this context, a heparinoid obtained from the head of Litopenaeus vannamei shrimp, which presents a structural similarity to heparin, showed, in previous studies, anti-inflammatory activity in a model of acute peritonitis with reduced anticoagulant effect in vitro and low hemorrhagic activity. Thus, the present work had as objective to evaluate the effect the heparinoid of the cephalothorax of gray shrimp on the acute inflammatory response in different times (3 or 6 hours after the induction of inflammatory stimulus), using the model of acute peritonitis induced in mice. It was also analyzed the HL effect over the activity of elastase, an enzyme involved in leukocyte recruitment. Furthermore to check if the different doses of heparin and heparinoid change the hemostatic balance in vivo, was assessed the effect of these compounds on the plasma clotting time in animals submitted to inflammation. The results show that in 3 hours, all doses of heparinoid were able to prevent efficiently in the acute inflammatory process without any anticoagulant effects, unlike the extrapolation dose of heparin, which has induced a large hemorrhage due its high anticoagulant activity. However, 6 hours after induction of inflammation, only the dosages of 0.1 and 1.0 μg/Kg of heparin and 1.0 μg/Kg of heparinoid kept anti-migratory effect, without changing of the hemostatic balance. These results indicate that the anti-migratory effect of theses compounds depends on the dosage and time of inflammatory stimulus. The HL and heparin were also able to inhibit the activity of the enzyme elastase. The discovery of this bioactive compound in the cephalothorax of shrimps can arouse great interest in biotechnology, since this compound could be useful as a structural model interesting for the development of new therapeutic agents for peritonitis

Atividade antioxidante e antiproliferativa de extratos de folhas de Plukenetia volubilis L. (EUPHORBIACEAE)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Vicilina de sementes da leguminosa selvagem Anadenanthera macrocarpa: purificação, caracterização, efeito deletério e mecanismo de ação para Callosobruchus maculatus

Grains and legume seeds are foods that form the basis of the diets of many cultures around the world, winch contritbute to the daily nutrient requirements of humans. Vicilins (7S globulin) are storage proteins found in legume seeds, and may have an additional function constitutive defense of the embryo against pests and pathogens. In this work the vicilin from Anadenanthera macrocarpa - AmV (red-angico), was purified and partially characterized, its effect on development and larval survival and adult emergence of Callosobruchus maculatus was evaluated by determination of LD50, WD50 and ED50 in system bioassay. Purification of vicilin was initiated by the chitin affinity chromatography and then gel filtration (Superdex 75 Tricorn 10x300 mm) FPLC system followed by reverse phase chromatography (C8 phenomenex) on HPLC system. Bioassays WD50 and LD50 for larvae were 0.32% and 0.33% (w:w) respectively, since the ED50 for adults was 0.096%. The probable mechanism of action was evaluated by testing digestibility of AmV in vitro, and observed for the involvement of two fragments vicilins immunoreactive against polyclonal Anti-vicilin from Erythrina velutina (Anti-EvV) about of 22 and 13 kDa chitin binding. The AmV in its native form has been recognized by the anti-EvV, indicating that there is a conserved region in the vicilin and is probably corresponding to the chitin binding domains. These results point to a new vicilin chitin binding that can subsequently be used as a possible biopesticide protein source, in order to control insect pest C. maculatus and confirm literature findings that demonstrate vicilin in the presence of different kinds of ligands to conserved regions chitin not yet characterized

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Estudo do genoma do vírus causador da mionecrose infecciosa em camarões e desenvolvimento de métodos para detecção de polimorfismos

Shrimp farming is one of the activities that contribute most to the growth of global aquaculture. However, this business has undergone significant economic losses due to the onset of viral diseases such as Infectious Myonecrosis (IMN). The IMN is already widespread throughout Northeastern Brazil and affects other countries such as Indonesia, Thailand and China. The main symptom of disease is myonecrosis, which consists of necrosis of striated muscles of the abdomen and cephalothorax of shrimp. The IMN is caused by infectious myonecrosis virus (IMNV), a non-enveloped virus which has protrusions along its capsid. The viral genome consists of a single molecule of double-stranded RNA and has two Open Reading Frames (ORFs). The ORF1 encodes the major capsid protein (MCP) and a potential RNA binding protein (RBP). ORF2 encodes a probable RNA-dependent RNA polymerase (RdRp) and classifies IMNV in Totiviridae family. Thus, the objective of this research was study the IMNV complete genome and encoded proteins in order to develop a system differentiate virus isolates based on polymorphisms presence. The phylogenetic relationship among some totivirus was investigated and showed a new group to IMNV within Totiviridae family. Two new genomes were sequenced, analyzed and compared to two other genomes already deposited in GenBank. The new genomes were more similar to each other than those already described. Conserved and variable regions of the genome were identified through similarity graphs and alignments using the four IMNV sequences. This analyze allowed mapping of polymorphic sites and revealed that the most variable region of the genome is in the first half of ORF1, which coincides with the regions that possibly encode the viral protrusion, while the most stable regions of the genome were found in conserved domains of proteins that interact with RNA. Moreover, secondary structures were predicted for all proteins using various softwares and protein structural models were calculated using threading and ab initio modeling approaches. From these analyses was possible to observe that the IMNV proteins have motifs and shapes similar to proteins of other totiviruses and new possible protein functions have been proposed. The genome and proteins study was essential for development of a PCR-based detection system able to discriminate the four IMNV isolates based on the presence of polymorphic sites

Efeitos da dieta hipoprotéica na formação e composição de estruturas dentárias: estudo experimental em ratos Wistar

Protein and caloric malnutrition has been considered one of the most concerned endemic diseases in Brazil and in the world. It has been known that depletion or reduction of proteins as far as meals are concerned can steer irreversible damages upon several organic systems. This study had as aim evaluate the effects the low-protein diet had over the formation and composition of the teeth components. 18 females and 6 males were used for the experiment. 12 from the 18 females had undertaken the low-protein diet (DH) for 03 weeks and the other 6, which remained, and those males had undertaken a controlled diet (DC) for the same period. All animals had the diets during their mating, pregnancy and lactation cycle. As soon as the offsprings had been born, 10 young males and females of each group faced a disease hood analysis to check the teeth germs of their lower fore teeth. The rest of the group had their lactation cycle normally 60 days. Then they were put to death and had their lower fore teeth removed both to be analyzed through a scanning electronic microscopy (SEM) of the structure alterations and to have their calcium checked by an atomic absorption of the phosphorus vanadate-molibdate method and by other minerals EDX method. The animals livers were removed to have their hepatic proteins analyzed as well. The histopatologic study showed that at first day of birth, all animals had their lower fore teeth come out. It was verified that 90% of the animals teeth were in an apposition and calcification period and it was possible to observe the dentin formation from 60% of the 90% already mentioned. Through the SEM method it could be realized that 90% of the animals of the DH group had their lower fore teeth easily broken and no definite shape. In this same group itself, it was also observed long micro fissures 369,66 nm ± 3,45 while the DC group had fissures of 174 nm ± 5,72. Now regarding the calcium and phosphorus concentration, it could be noticed that there was a great reduction of these components and other minerals in the DH group. Almost all minerals, except for the Cl and K, presented higher levels in the DC group enamel.The reduction of the protein input greatly influenced the offsprings´ weight and height. However the hepatic proteins had no important difference between the groups what can make one believe that those animals suffered from protein malnutrition of marasmic kind

Avaliação da ação bioinseticida de SBTI e vicilina de Erythrina velutina em enzimas digestivas e membrana peritrófica de larvas de Plodia interpunctella (Lepidoptera: Pyralidae)

Plodia interpunctella (Indian meal moth) is a cosmopolitan pest that attacks not only a wide range of stored grain as well other food products. Due to its economic importance several researches have focused in a method with ability to control this pest with few or no damage to the environment. The study of digestive enzymes inhibitors, lectins and chitin-binding proteins, has often been proposed as an alternative to reduce insect damage. In this study we report the major classes of digestive enzymes during larval growth in P. Interpunctella, being those proteinases actives at pH 9.5 and optimum temperature of 50 oC to both larvae of the 3rd instar and pre-pupal stage of development. In vitro and zymogram assays presented the effects of several inhibitors, such as SBTI, TLCK and PMSF to intestinal homogenate of 3rd instar larvae of 62%, 92% and 87% of inhibition and In pre-pupal stage of 87%, 62 % and 55% of inhibition, respectively. Zymograms showed inhibition of two low molecular masses protein bands by TLCK and that in presence of SBTI were retarded. These results are indicative of predominance of digestive serine proteinases in gut homogenate from Plodia interpunctella larvae. This serine proteinase was then used as a target to evaluate the effect of SBTI on larvae in in vivo assay. Effect of SBTI on mortality and larval mass was not observed at until 4% of concentration (w/w) in diets. Chitin, another target to insecticidal proteins, was observed by chemical method. Moreover, optic microscopy confirmed the presence of a peritrophic membrane. Established this target, in vivo effect of EvV, a chitin binding vicilin, evaluated during the larval development of P. interpunctella and was obtained a LD50 of 0,23% and WD50 of 0,27% to this protein. Mechanism of action was proposed through of the in vivo digestibility of EvV methodology. During the passage through the larval digestive tract was observed that EvV was susceptible to digestive enzymes and a reactive fragment, visualized by Western blotting, produced by digestion was recovered after dissociation of the peritrophic membrane. The bound of EvV to peritrophic membrane was confirmed by immunohystochemical assays that showed strong immunofluorescent signal of EvV-FITC binding and peritrophic membrane. These results are a indicative that vicilins could be utilized as potential insecticide to Plodia interpunctella and a control methods using EvV as bioinsecticide should be studied to reduce lost caused by storage insect pests

Multi-­‐dimensional Protein Identification Technology

Before the rise of the Multidimentional Protein Identification Technology (MudPIT), protein and peptide mixtures were resolved using traditional proteomic technologies like the gel-­‐ based 2D chromatography that separates proteins by isoelectric point and molecular weight. This technique was tedious and limited, since the characterization of single proteins required isolation of protein gel spots, their subsequent proteolyzation and analysis using Matrix-­‐ assisted laser desorption/ionization-­‐time of flight (MALDI-­‐TOF) mass spectrometry.