N-Acetyl-d-galactosamine prevents soya bean agglutinin-induced intestinal barrier dysfunction in intestinal porcine epithelial cells.

Soya bean agglutinin (SBA) is a glycoprotein and the main anti-nutritional component in most soya bean feedstuffs. It is mainly a non-fibre carbohydrate-based protein and represents about 10% of soya bean-based anti-nutritional effects. In this study, we sought to determine the effects of N-Acetyl-D-galactosamine (GalNAc or D-GalNAc) on the damage induced by SBA on the membrane permeability and tight junction proteins of piglet intestinal epithelium (IPEC-J2) cells. The IPEC-J2 cells were pre-cultured with 0, 0.125 × 10-4 , 0.25 × 10-4 , 0.5 × 10-4 , 1.0 × 10-4 and 2.0 × 10-4  mmol/L GalNAc at different time period (1, 2, 4 and 8 hr) before being exposed to 0.5 mg/ml SBA for 24 hr. The results indicate that pre-incubation with GalNAc mitigates the mechanical barrier injury as reflected by a significant increase in trans-epithelial electric resistance (TEER) value and a decrease in alkaline phosphatase (ALP) activity in cell culture medium pre-treated with GalNAc before incubation with SBA as both indicate a reduction in cellular membrane permeability. In addition, mRNA levels of the tight junction proteins occludin and claudin-3 were lower in the SBA-treated groups without pre-treatment with GalNAc. The mRNA expression of occludin was reduced by 17.3% and claudin-3 by 42% (p < 0.01). Moreover, the corresponding protein expression levels were lowered by 17.8% and 43.5% (p < 0.05) respectively. However, in the GalNAc pre-treated groups, occludin and claudin-3 mRNAs were reduced by 1.6% (p > 0.05) and 2.7% (p < 0.01), respectively, while the corresponding proteins were reduced by 4.3% and 7.2% (p < 0.05). In conclusion, GalNAc may prevent the effect of SBA on membrane permeability and tight junction proteins on IPEC-J2s.

The fungal chimerolectin MOA inhibits protein and DNA synthesis in NIH/3T3 cells and may induce BAX-mediated apoptosis.

The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense.

HGA-2, a novel galactoside-binding lectin from the sea cucumber Holothuria grisea binds to bacterial cells.

A novel lectin, HGA-2, was isolated from the sea cucumber Holothuria grisea. The protein was isolated by a single chromatographic step using a column of Guar Gum as affinity. HGA-2 showed an apparent molecular mass of 17 kDa and 34 kDa under reducing and nonreducing conditions, respectively. The hemagglutinating activity was specific for rabbit erythrocytes, showing no activity for human blood A, B and O. Its hemagglutinating activity was inhibited by carbohydrates containing galactose, with higher affinity for GalNAc and glycoprotein porcine stomach mucin (PSM). HGA-2 was stable at pH 6-10, significantly declining at pH 5 and a temperature of 40°C, with its activity being abolished at 100 °C. The HGA-2 protein was found to be Ca(2+)-dependent; it was highly toxic against Artemia nauplii and able to recognize and agglutinate cells of Escherichia coli. Amino acid sequences of tryptic peptides of HGA-2 strongly suggest that HGA-2 is a member of the C-type lectin family.

[Correlation of Pinellia ternata agglutinin and Pinellia ternata raphides' toxicity].

OBJECTIVE: To study the correlation of Pinellia ternata agglutinin (PTA) and toxicity of P. ternata raphides and to find out the toxic mechanism of P. ternata. METHOD: PTA has obvious effect of pro-inflammation. The model of rats peritonitis was used to study the dose-toxicity and time-toxicity relationship of the effect by detecting the releases of inflammatory mediators PGE2 in the exudates. The model of Draize rabbit eye test was applied to determine the correlation of PTA and toxicity of raphides by pathological examination. RESULT: PTA enhanced the content of PGE2 and protein in rats peritoneal cavities concentration dependently. With PTA concentration increased, PTA enhanced the inflammation induced by raphides to rabbit eyes, but PTA alone had no toxicity response. CONCLUSION: PTA had obvious effect of pro-inflammation. The toxic mechanism of P. ternata was PTA induced inflammation only when the raphides pierce into the organization.

Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with insect tissues and cells.

This project studied in detail the insecticidal activity of a fungal lectin from the sclerotes of Sclerotinia sclerotiorum, referred to as S. sclerotiorum agglutinin or SSA. Feeding assays with the pea aphid (Acyrthosiphon pisum) on an artificial diet containing different concentrations of SSA demonstrated a high mortality caused by this fungal lectin with a median insect toxicity value (LC50) of 66 (49-88) µg/ml. In an attempt to unravel the mode of action of SSA the binding and interaction of the lectin with insect tissues and cells were investigated. Histofluorescence studies on sections from aphids fed on an artificial liquid diet containing FITC-labeled SSA, indicated the insect midgut with its brush border zone as the primary target for SSA. In addition, exposure of insect midgut CF-203 cells to 25 µg/ml SSA resulted in a total loss of cell viability, the median cell toxicity value (EC50) being 4.0 (2.4-6.7) µg/ml. Interestingly, cell death was accompanied with DNA fragmentation, but the effect was caspase-3 independent. Analyses using fluorescence confocal microscopy demonstrated that FITC-labeled SSA was not internalized in the insect midgut cells, but bound to the cell surface. Prior incubation of the cells with saponin to achieve a higher cell membrane permeation resulted in an increased internalization of SSA in the insect midgut cells, but no increase in cell toxicity. Furthermore, since the toxicity of SSA for CF-203 cells was significantly reduced when SSA was incubated with GalNAc and asialomucin prior to treatment of the cells, the data of this project provide strong evidence that SSA binds with specific carbohydrate moieties on the cell membrane proteins to start a signaling transduction cascade leading to death of the midgut epithelial cells, which in turn results in insect mortality. The potential use of SSA in insect control is discussed.

Immunomodulatory effects of Viscum album agglutinin-I on natural immunity.

In 24 h cultured human peripheral blood mononuclear cells, treated with various (1 microgram/ml to 1 ng/ml) concentrations of Viscum album agglutinin-I, quantitative assessment of DNA breaks labelled with terminal deoxynucleotidyl transferase revealed a dose-dependent Viscum album agglutinin-I-induced apoptosis above a lectin concentration of 10 ng/ml. After 24 h incubation of peripheral blood mononuclear cells with non-cytotoxic concentrations of Viscum album agglutinin-I (10 and 1 ng/ml), messenger (m)RNA expression and secretion of a panel of cytokines were evaluated by reverse polymerase chain reaction and by enzyme-linked immunosorbent assay (ELISA), respectively. The lectin induced expression of interleukin-1 alpha, interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, interferon-gamma, granulocyte-monocyte colony stimulating factor and interleukin-10 genes, but no expression of interleukin-2 or interferon-gamma production could be detected. In addition, cellular components of the natural immune system (such as monocytes and granulocytes) bound Viscum album agglutinin-I molecules to a higher degree than lymphocytes. To establish the modulatory potency of Viscum album agglutinin-I on the natural immunity of human subjects, four randomized, double-blind crossover trials were performed on healthy volunteers. In contrast to the significant lectin-induced increases in number and activity of natural killer cells observed in animal models, in the first and second trial human healthy individuals showed no significant differences between their natural killer responses following an injection of lectin-enriched preparation or saline. Due to considerable intrinsic fluctuation of these parameters, a third and fourth double-blind trial with freshly isolated Viscum album agglutinin-I was performed using a more rapidly detectable parameter, the priming of granulocytes. Here, significant lectin-induced increases were found.

[Effect of various heat treatments in the hemagglutinin content and in the protein quality of beans (Phaseolus vulgaris)]. / Efecto de diversos tratamientos térmicos en el contenido de hemaglutininas y en la calidad protéica del frijol (Phaseolus vulgaris).

The effect of pre-soaking raw seed beans upon detoxification and the biological quality of its protein were evaluated. In whole raw seed beans (Phaseolus vulgaris) var. "tórtola", the net protein utilization (NPU), true digestibility and hemagglutinin titer were determined after 60', 90' and 120' of heat treatment, with and without 14 hours of pre-soaking. It is concluded that soaking prior to cooking is not necessary to eliminate the toxicity of dry beans, but that it does contribute to the softening of seeds and reduction of cooking time. The hemagglutinin levels of six commercial bean flours were evaluated, concluding that almost all of them presented toxic levels. The effect of the cooking methods upon the toxicity of bean flours was studied. Two raw bean flours, var. "tórtola" and "burro" at 10% and 20%, were cooked employing different boiling times (5, 10, 15 and 30'). The two raw samples contained high hemagglutinin levels which were inactivated at 10% with 10' cooking. The presence of toxic levels was detected at 20% after 15' cooking and these were eliminated at 30' of cooking.