A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus / Atividade anti-helmíntica in vitro de uma protease cisteínica do látex de Ficus benjamina contra Haemonchus contortus

Abstract Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Resumo Haemonchus contortus é um nematoide gastrintestinal, responsável por altas taxas de mortalidade em rebanhos de pequenos ruminantes. A resistência dos nematoides aos anti-helmínticos sintéticos está generalizada e requer uma busca contínua por novos compostos bioativos, como as proteínas. O objetivo deste trabalho foi avaliar o potencial anti-helmíntico da protease purificada do látex de Ficus benjamina contra H. contortus . O látex fresco foi coletado das plantas por pequenas incisões nas hastes verdes e o extrato proteico de látex (EPL) foi obtido. Após o fracionamento do EPL com sulfato de amônio e cromatografia da fração contendo a maior atividade proteolítica da CM-Celulose, uma protease cisteínica (FbP) foi purificada. A FbP tem massa molecular de cerca de 23,97 kDa, a atividade proteolítica foi estável entre pH 6,0 e pH 10 e ao longo de uma ampla faixa de temperatura, com atividade ótima a 60 °C. A FbP inibiu tanto o desenvolvimento quanto o desembainhamento das larvas de H. contortus, com 50% de inibição nas concentrações de 0,26 e 0,79 mg/mL, respectivamente. Concluímos que esta protease cisteínica do látex de F. benjamina, com ação anti-helmíntica contra H. contortus, pode ser uma alternativa promissora para o desenvolvimento de produtos a serem utilizados em programas de controle de parasitos.

Drought-Induced effects and recovery of nitrate assimilation and nodule activity in cowpea plants inoculated with Bradyrhizobium spp. under moderate nitrate level

This study was carried out to establish comparative effects of drought and recovery on the nitrate assimilation and nodule activity related to N2 fixation in cowpea plants [Vigna unguiculata L. (Walp.)] previously inoculated with Bradyrhizobium spp. BR-3256 (CB-756) strain in the presence of 5 mol m-3 NO-3. Twenty-eight-day-old nodulated plants were submitted to water deprivation during 4 consecutive days and afterwards resupplied with nutrient solution during 2 days. The water deprivation caused a rapid increase in the nitrate content in root and a marked reduction in leaf nitrate reductase (NR) activity. In contrast nodule NR activity was slightly increased by water deprivation. Concomitantly, in nodules of water stressed plants, leghemoglobin and glutamine synthetase (GS) activity declined and a progressive reduction in ureide-N concentration in xylem sap was observed. Leaf-NR activity increased rapidly after rehydration while leaf nitrate content declined. In contrast both GS activity and soluble protein content in the nodule continued to decline in rewatered plants. In addition the concentration of leghemoglobin recovered well, while the xylem ureide-N content experienced a slight increase after rehydration. Despite the nitrate assimilation in leaves and the nodule activity had been both severely affected by water stress, the rapid recovery of nitrate reductase activity suggests that the nitrate assimilation process is less sensitive to drought/rehydration cycle when cowpea plants are nodulated in presence of moderate nitrate level.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similiar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConsA looked very similar. However, doking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, reveled conformational changes in side chains of the animo acid residues invlved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConsA requires conformational chances of this monosaccharide-binding site.

Plant lectins, chemical and biological aspects

Lectins, carbohydrate-binding proteins of non-immune origin, that agglutinate cells or precipitate polysaccharides and glycoconjugates, are well distributed in nature, mainly in the Plant Kingdom. The great majority of the plante lectins are present in seed cotyledons where they are found in the cytoplasm or int he protein bodies, although they have also been found in roots, stems and leaves. Due to their peculiar properties, the lectins are used as a tool both for analytical and preparative purposes in biochemistry, cellular biology, immunology and related areas. In agriculture and medicine the use of lectins greatly improved in the last few years. The lextins, with few exceptions, are glycoproteins, need divalent cations to display full activity and are, in general, oligomers with variable molecular weight. Although the studies on lectins have completed a century, their role in nature is yet ynknown . Several hypotheses on their physiological functions have been suggested. Thus, lectins could play important roles in defense against pathogens, plant-microorganism symbiosis, cell organization, embryo morphogenesis, phagocytosis, cell wall elongation, pollen recognition and as reserve proteins. A brief review on the general properties and roles of the lectins is given.