Inhibition of Protease and Egg Hatching of Haemonchus contortus by Soybean Seed Exudates.

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.

Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed.

Proteomics changes during the incompatible interaction between cowpea and Colletotrichum gloeosporioides (Penz.) Penz and Sacc.

Anthracnose represents an important disease of cowpea [Vigna unguiculata L. (Walp.)] caused by the hemibiothrophic fungus Colletotrichum gloeosporioides that drastically reduces cowpea field production. In this study we investigated some biochemical aspects underlying the incompatible interaction between a resistant cowpea genotype and C. gloeosporioides using a proteomic approach. Analyses of two-dimensional gel electrophoresis patterns and protein identification indicate C. gloeosporioides infection-dependent cowpea leaf proteome changes associated with metabolism, photosynthesis, response to stress, oxidative burst and scavenging, defense signaling, and pathogenesis-related proteins. Moreover the C. gloeosporioides responsive proteins interaction network in cowpea revealed the interconnected modulation of key cellular processes involving particularly antioxidants proteins, photosynthetic apparatus forming proteins and proteins of the energetic metabolism that interact with each other suggesting that their expression changes are also important for resistance of cowpea to C. gloeosporioides.

Soybean toxin (SBTX) impairs fungal growth by interfering with molecular transport, carbohydrate/amino acid metabolism and drug/stress responses.

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams.

The present study is focused on the proteome of reproductive tract fluids from tropically-adapted Santa Ines rams. Seminal plasma, cauda epididymal (CEF) and vesicular gland fluid (VGF) proteins were analyzed by 2-D electrophoresis and mass spectrometry. Seminal plasma maps contained 302 ± 16 spots, within the 4-7 pH range. From these maps, 73 spots were identified, corresponding to 41 proteins. Ram Seminal Vesicle Proteins (RSVP) 14 and 22kDa and bodhesins 1 and 2 represented the most abundant seminal components. Other seminal proteins included clusterin, angiotensin-converting enzyme, matrix metalloproteinase-2, tissue-inhibitor of metalloproteinase-2, plasma glutamate carboxypeptidase, albumin, lactoferrin, alpha enolase, peroxiredoxin, leucine aminopeptidase, ß-galactosidase, among others. Later, seminal plasma gels were run within narrow pH intervals (3.9-5.1; 4.7-5.9; 5.5-6.7), allowing the additional identification of 21 proteins not detected in 4-7 pH maps. Major proteins of CEF and VGF were albumin and transferrin, and RSVPs, respectively. Western blots confirmed that RSVPs were mainly present in VGF while bodhesins, in VGF and CEF. Based on RT-PCR, RSVP and bodhesin genes were primarily expressed in the vesicular glands. In summary, the reproductive tract fluids of Brazilian hairy rams contain several categories of proteins, with potential roles in sperm protection, capacitation, acrosome reaction and sperm-oocyte interaction.

WITHDRAWN: Major proteins from the seminal plasma of adult Santa Ines rams.

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Soybean toxin (SBTX), a protein from soybeans that inhibits the life cycle of plant and human pathogenic fungi.

Soybean toxin (SBTX) is a 44 kDa glycoprotein that is lethal to mice (LD(50) = 5.6 mg/kg). This study reports the toxicity of SBTX on pathogenic fungi and yeasts and the mechanism of its action. SBTX inhibited spore germination of Aspergillus niger and Penicillium herguei and was toxic to Candida albicans, Candida parapsilosis, Kluyveromyces marxiannus , Pichia membranifaciens, and Saccharomyces cerevisiae. In addition, SBTX hampered the growth of C. albicans and K. marxiannus and inhibited the glucose-stimulated acidification of the incubation medium by S. cerevisiae, suggesting that SBTX interferes with intracellular proton transport to the external medium. Moreover, SBTX caused cell-wall disruption, condensation/shrinkage of cytosol, pseudohyphae formation, and P. membranifaciens and C. parapsilosis cell death. SBTX is toxic to fungi at concentrations far below the dose lethal to mice and has potential in the design of new antifungal drugs or in the development of transgenic crops resistant to pathogens.

Combination of chemical analyses and animal feeding trials as reliable procedures to assess the safety of heat processed soybean seeds.

This study assessed whether chemical analyses are sufficient to guarantee the safety of heat processing of soybeans (SB) for human/animal consumption. The effects of extrusion and dry-toasting were analyzed upon seed composition and performance of broiler chicks. None of these induced appreciable changes in protein content and amino acid composition. Conversely, toasting reduced all antinutritional proteins by over 85%. Despite that, the animals fed on toasted SB demonstrated a low performance (feed efficiency 57.8 g/100 g). Extrusion gave place to higher contents of antinutrients, particularly of trypsin inhibitors (27.53 g/kg flour), but animal performance was significantly (p < 0.05) better (feed efficiency 63.2 g/100 g). Upon the basis of chemical analyses, dry-toasting represents the treatment of choice. However, considering the results of the feeding trials, extrusion appears to be the safest method. In conclusion, in order to evaluate the reliability of any processing method intended to improve nutritional value, the combination of chemical and animal studies is necessary.

Antinutritional properties of plant lectins.

Lectins are carbohydrate binding (glyco)proteins which are ubiquitous in nature. In plants, they are distributed in various families and hence ingested daily in appreciable amounts by both humans and animals. One of the most nutritionally important features of plant lectins is their ability to survive digestion by the gastrointestinal tract of consumers. This allows the lectins to bind to membrane glycosyl groups of the cells lining the digestive tract. As a result of this interaction a series of harmful local and systemic reactions are triggered placing this class of molecules as antinutritive and/or toxic substances. Locally, they can affect the turnover and loss of gut epithelial cells, damage the luminal membranes of the epithelium, interfere with nutrient digestion and absorption, stimulate shifts in the bacterial flora and modulate the immune state of the digestive tract. Systemically, they can disrupt lipid, carbohydrate and protein metabolism, promote enlargement and/or atrophy of key internal organs and tissues and alter the hormonal and immunological status. At high intakes, lectins can seriously threaten the growth and health of consuming animals. They are also detrimental to numerous insect pests of crop plants although less is presently known about their insecticidal mechanisms of action. This current review surveys the recent knowledge on the antinutritional/toxic effects of plant lectins on higher animals and insects.