Role of Synadenium grantii latex proteases in nematicidal activity on Meloidogyne incognita and Panagrellus redivivus / Papel das proteases do látex de Synadenium grantii na atividade nematicida sobre Meloidogyne incognita e Panagrellus redivivus

Abstract Synadenium grantii is a Euphorbiaceae plant commonly found in Brazil, known as Janaúba or Leitosinha, whose latex is traditionally used for several purposes. However, it is not known whether the nematicidal action of this plant latex occurs due to the action of proteases. The present work aims to evaluate the nematicidal activity of proteases from Synadenium grantii latex on Meloidogyne incognita and Panagrellus redivivus. S. grantii latex used in the present study was collected from specimens found in Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. The drained latex was collected in Eppendorf microtubes and immediately stored on ice at 4 °C. After this extraction, the latex was frozen (-20 °C) during 2 hours, thawed at room temperature (25 °C) and centrifuged at 10,000 g at 4 °C for 30 minutes to remove larger particles and concentrate the proteases. After the centrifugation, assays of enzymatic activity were performed in order to know in which of the phases the enzymes were found. S. grantii latex presented protease, but no chitinase activity. The results show that there was a significant difference (p <0.01) between the treated and control groups, with 100% mortality of Meloidogyne incognita and 72% average mortality of Panagrellus redivivus. In addition, it was demonstrated that the nematicidal action occurred due to the action of the proteases, since the control was only differentiated from the treatment by the presence of the enzymes with biological activity.

Resumo Synadenium grantii é uma planta Euphorbiaceae comumente encontrada no Brasil, conhecida como Janaúba ou Leitosinha, e tem seu látex usado tradicionalmente para diferentes propósitos. Entretanto, não se conhece se a atividade nematicida da planta ocorre devido à ação de proteases. O presente trabalho tem como objetivo avaliar a atividade nematicida das proteases do látex de Synadenium grantii sobre Meloidogyne incognita e Panagrellus redivivus. O látex de S. grantii utilizado no presente trabalho foi coletado a partir de espécimes encontradas na Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. O látex foi coletado em microtubos Eppendorf e imediatamente armazenado em gelo a 4 °C. Após esta extração, o látex foi congelado (-20 °C) durante 2 horas, descongelado à temperatura ambiente (25 °C) e centrifugado a 10000 g a 4 °C durante 30 minutos para a remoção de partículas e concentração das proteases. Após a centrifugação, foram realizados ensaios de atividade enzimática para saber em qual das fases as enzimas foram encontradas. O látex de S. grantii apresentou atividade de protease, mas nenhuma atividade de quitinase. Os resultados mostram que houve diferença significativa (p <0,01) entre os grupos tratados e controle, com 100% de mortalidade de Meloidogyne incognita e 72% de mortalidade média de Panagrellus redivivus. Além disso, foi demonstrado que a ação nematicida ocorreu devido à ação das proteases, uma vez que o grupo controle só foi diferenciado do tratamento pela presença das enzimas com atividade biológica.

Enzymatic cleavage of cell surface proteins of pig and cow erythrocytes and its effect on concanavalin-mediated agglutinability.

Study was carried out to understand and compare architecture of the proteins of erythrocyte cell surface of some mammals viz., Homo sapiens (human), Sus scorfa domestica (pig) and Bos taurus domestica (cow). In this study, we investigated the action of proteinases viz., trypsin and chymotrypsin and neuraminidase on the erythrocyte surface proteins and erythrocyte agglutination tendency with a lectin (concanavalin A). The electrophoretic pattern of membrane proteins and glycophorins (analyzed by SDS-PAGE and visualized by Coomassie brilliant blue and periodic acid-schiff stains, respectively) and concanavalin A (Con A) agglutinability revealed that: (i) There were variations in the number and molecular weights of glycophorins in human, pig and cow, (ii) trypsin action on pig and cow erythrocyte membrane proteins was similar, unlike human, (iii) glycophorins degradation by trypsin and chymotrypsin was not similar in pig, as compared to that of human and cow, (iv) erythrocytes agglutination with Con A was significantly different due to differences in membrane composition and alterations in the surface proteins after enzyme treatment, (v) a direct correlation was found between degradation of glycophorins and Con A agglutinability, and (vi) removal of erythrocyte surface sialic acids by neuraminidase specifically indicated an increase in Con A agglutinability of pig and cow erythrocytes, similar to human.

Proteolytic enzyme mediated antagonistic potential of Pseudomonas aeruginosa against Macrophomina phaseolina.

A new antagonistic bacterial strain PGPR2 was isolated from the mungbean rhizosphere and documented for the production of hydrolytic enzymes with antifungal activity. Based on the phylogenetic analysis of the 16S rRNA gene sequence and phenotyping, this strain was identified as Pseudomonas aeruginosa. Maximum protease activity (235 U/mL) was obtained at 24 h of fermentation. The protease was purified to homogeneity in three steps: ammonium sulphate precipitation, anion exchange chromatography on DEAE- cellulose resin and gel filtration chromatography using P6 column. The purified enzyme had a molecular weight of ~33 kDa. The purified protease exhibited maximum activity at pH 6.0 and retained 80% of activity in a pH range of 5.0 - 9.0. Proteolytic activity was maximum in a temperature range of 40–70 °C. However, the enzyme was stable at 40 °C for 60 min. Among the metals tested, Mg2+ enhanced the protease activity. Internal amino acid sequence of the protease obtained by MALDI -ToF and subsequent Mascot database search showed maximum similarity to the HtpX protease of P. aeruginosa strain PA7. Thus, by virtue of its early production time, thermostability and effective antifungal ability, the protease purified and characterized from P. aeruginosa PGPR2 has several potential applications as fungicidal agents in agriculture.

Effect of vitamin C on endothelial dysfunction during N-alpha-tosyl L-arginine methyl ester [TAME]-esterase induced contractions in rat aorta in vitro.

Contractions induced by TAME-esterase on rat aorta strips mounted in vitro were significantly inhibited in presence of Vitamin C. The work lends support to the role of ascorbic acid in preventing endothelial dysfunction through release of nitric oxide. It is suggested that conclusions TAME-esterase could be an important biological marker associated with onset of vascular discases such as hypertension.

Experimental evidence for a non-renin mediated pathway during TAME-esterase induced contractions in rat aorta in vitro.

Possible pharmacological effects of N-alpha-tosyl L-arginine methyl ester [TAME] were studied on rat aorta strips in vitro. Results showed that [TAME]-esterase was an endothelium dependent component that involved a nitric oxide cyclic-GMP mediated pathway. Furthermore, during activation of Kinin-Kallikrein system, TAME-esterase induced contractions involve degradation of kinins by kininases.

Efecto de enzimas proteolíticas y de la abstinencia sexual en la cuantificación bioquímica de alfa glucosidasa en plasma seminal humano / Effects of proteolytic enzymes and sexual abstinence on alfa glucosidase levels in human seminal plasma

Background: a-glucosidase is found in human seminal plasma as an acid form, located in accessory glands, and as a neutral form secreted almost exclusively by the epididymis. Quantification of a-glucosidase activity is a marker of the secretory function of the epididymis and indemnity of the sperm transport pathway Aim: To obtain reference values for a-glucosidase in normal samples of seminal plasma, to evaluate its behavior in serial samples and to determine the effect of proteolytic enzymes. Material and methods: Fifty donors, with normal semen analysis according to the criteria of the World Health Organization, were evaluated. For the study with alpha-quimotrypsin, 0.1 to 10 mg/ml of the enzyme was added to the seminal plasma from a group of donors. a-glucosidase was also measured in semen obtained from nine patients at different time intervals. Results: Normal a-glucosidase values ranged from 14.52 to 25.69 µU/ml. Concentrations up to 10 mg/ml of alpha-quimotrypsin (10 times of that usually used in the liquefaction of the semen) did not alter the quantification of a-glucosidase. Serial determinations revealed oscillations in their magnitude, which stayed in each patient's characteristic range. However a subgroup presented a marked reduction of the activity of a-glucosidase as the abstinence diminished (40 percent). Conclusions: Evaluation of a-glucosidase in seminal plasma gives reliable information of the secretor state of the epididymis and especially replaces invasive methods used to evaluate the indemnity of the spermatic transport from the epididymis to the anterior urethra