Toxic peptides in Frazer's fraction interact with the actin cytoskeleton and affect the targeting and function of intestinal proteins

Celiac disease (CD) is a multisystemic autoimmune inflammation of the intestinal tract induced by wheat gluten and related cereals in HLA-DQ2/8 positive individuals. An essential role in the pathogenesis of CD is played by a fraction of the peptic-tryptic digest of gluten, Frazer's Fraction (FF). Here, we investigate the effects of FF on the integrity of intestinal cells with particular emphasis on brush border membrane (BBM) components, their subsequent trafficking and endocytosis.Caco-2 cells were incubated with FF at different concentrations. Thereafter, several protein and lipid components of treated and untreated cells were analysed at the molecular, functional and cellular levels. The control employed tryptic-peptic digests of ovalbumin.Our results show that FF directly interacts with actin in an alternating manner eliciting substantial alterations in its integrity and extent in the BBM. These alterations lead to an impaired trafficking of SI to the apical membrane and reduction in its enzymatic function. ApN and DPPIV follow a transcytotic pathway and are only partly affected by FF. By contrast, the trafficking of LPH remains unaffected concomitant with its actin-independent trafficking pattern. Finally, the endocytic pathway is substantially blocked in FF-treated cells leading to an accumulation of cholesterol, and sphingolipids in the BBM.We conclude that FF deteriorates the actin cytoskeleton in Caco-2 leading to reduced protein sorting and hampered endocytic events with subsequent alterations in the protein and lipid composition of the BBM. The reduced levels of the disaccharidase SI in the BBM suggest a potential pathomechanism of carbohydrate malabsorption in CD.     

不同光照强度下半夏化学成分含量的比较研究

利用遮荫网对卵叶型半夏（Pinellia ternata（Thunb.）Breit.）进行遮荫,秋季倒苗后,测定其化学成分含量,以研究光强对半夏化学成分含量的影响。结果显示：全光照下半夏的生物碱、鸟苷和蛋白质含量较高,55%遮荫处理半夏的块茎增重幅度较大,80%遮荫处理的还原糖和可溶性糖含量较高。不同光照强度下,半夏化学成分含量有显著的差异,全光照处理有利于半夏生物碱、鸟苷和蛋白质的积累,80%遮荫处理有利于其还原糖和可溶性糖的积累,55%遮荫处理有利于半夏的生长,但其化学成分含量低于全光照处理。     

Multiple recognition systems adopting four different glycotopes at the same domain for the Agaricus bisporus agglutinin-glycan interactions

For the GalNAcα1→ specific Agaricus bisporus agglutinin (ABA) from an edible mushroom, the mechanism of polyvalent Galβ1→3/4GlcNAcβ1→ complex in ABA-carbohydrate recognition has not been well defined since Gal and GlcNAc are weak ligands. By enzyme-linked lectinosorbent and inhibition assays, we show that the polyvalent Galβ1→3/4GlcNAcβ1→ in natural glycans also play vital roles in binding and we propose that four different intensities of glycotopes (Galβ1-3GalNAcα1-, GalNAcα1-Ser/Thr and Galβ1-3/4GlcNAcβ1-) construct three recognition systems at the same domain. This peculiar concept provides the most comprehensive mechanism for the attachment of ABA to target glycans and malignant cells at the molecular level.     

Squalene-based oil-in-water emulsion adjuvants perturb metabolism of neutral lipids and enhance lipid droplet formation

Oil-in-water emulsions are used as vaccine adjuvants, but the mechanism of action remains unknown. In this paper we used phagocytes (monocytes, macrophages, dendritic cells) and non-phagocytic cells (fibroblasts, skeletal muscle cells) to study internalization of emulsions in vitro, and to characterize the influence of emulsion uptake on cellular metabolism of neutral lipids. We found that all tested cell types endocytose the emulsion droplets, and that the uptake leads to an acute accumulation of neutral lipids in the form of cytoplasmic lipid droplets. The accumulated lipids comprise not only the delivered squalene, but also cholesteryl esters, triacylglycerols, fatty acids, and diacylglycerols. Lipid metabolism and innate immunity are closely linked, and accumulation of lipids in non-adipose tissues is known to induce inflammatory conditions. We propose that one aspect of o/w emulsion adjuvanticity could depend on their ability to rapidly change lipid metabolism of the target cells.     

Entomotoxic action of Sambucus nigra agglutinin i in Acyrthosiphon pisum aphids and Spodoptera exigua caterpillars through caspase‐3‐like‐dependent apoptosis

In this project, the toxicity and mechanism of action of the ricin‐B‐related lectin SNA‐I from elderberry (Sambucus nigra) in the pea aphid (Acyrthosiphon pisum) and the beet armyworm (Spodoptera exigua), two important pest insects in agriculture, were studied. SNA‐I is a chimeric lectin belonging to the class of ribosome‐inactivating proteins and consists of an A‐chain with N‐glycosidase activity and a carbohydrate‐binding B‐chain. Incorporation of 2 mg/ml of SNA‐I in the diet of neonates and adults of A. pisum caused 40–46% mortality within 2 days, while in third instars of S. exigua, the larval biomass was significantly reduced by 12% after feeding for 3 days on a diet containing 5 mg/g of SNA‐I. Interestingly, extracts of the (mid)gut of treated A. pisum and S. exigua demonstrated DNA fragmentation and this was accompanied with an increase in caspase‐3‐like activity. The involvement of cell death or apoptosis in the entomotoxicity of SNA‐I through induction of caspase‐3‐like activity was also confirmed by addition of the permeable caspase‐3 inhibitor III in the diet, leading to a rescue of the treated aphid neonates. Finally, similar to the chimeric lectin SNA‐I, the hololectin SNA‐II, consisting of two carbohydrate‐binding B‐chains caused high mortality to neonate A. pisum aphids with an LC₅₀ of 1.59 mg/ml, suggesting that the entomotoxic action of the lectins under study mainly relies on their carbohydrate‐binding activity. © 2010 Wiley Periodicals, Inc.     

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with ³⁵S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.Abbreviations ELISA enzyme-linked immunosorbent assay - GlcNAc N-acetylglucosamine - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat germ agglutinin     

Immunocytochemistry in plants with colloidal gold conjugates

Summary The chitin-binding lectin wheat germ agglutinin (WGA) is found at the periphery of wheat embryos, and a similar lectin is present at the root tips of older plants (Mishkind et al. 1982). Although a ferritin-conjugated secondary antibody is adequate for localizing WGA in embryos, native electron-opaque particles make the electron microscope identification of added label equivocal in other wheat tissues. As reported here, however, unambiguous ultrastructural localization of WGA-like lectin in adult wheat roots can be obtained with rabbit anti-WGA followed by colloidal gold-labeled goat anti-rabbit (GAR) IgG. Colloidal gold (CG) was prepared by the reduction of gold chloride with citrate, ascorbate or phosphorous. GAR IgG, prepared from serum by antigen affinity chromatograhy, was adsorbed to the gold particles to produce a stabilized suspension of GAR-CG. Localization was performed on 8–12 M frozen sections of tissue fixed in 4% paraformaldehyde, 0.3% glutaraldehyde, and 0.75% acrolein in phosphate-buffered saline containing 1M sucrose. Localization with GAR-CG was first compared to that ascertained in embryos using other probes and was then extended to the roots of adult plants. An advantage of the GARCG method is that it permits the visualization of antigen at both the light and electron microscope levels in the same section. At the light level, the anti-WGA-GAR-CG complex appears as a red stain that is localized in specific tissues of embryos and in the caps and outer layers of adult roots. Sections in which lectin was detected at the light microscope level were embedded in plastic and sectioned for subcellular examination. Electron dense gold particles indicative of WGA are found at the periphery of protein bodies in wheat embryos and in vacuoles of the roots of adult plants. Sections incubated with control IgG lack reaction product.     

Crystallization and preliminary X-ray diffraction studies of soybean agglutinin

Soybean agglutinin crystallizes in the monoclinic space group C2 with unit cell dimensions $\text{a = 118.6}\text{A}\text{?}\text{, b = 88.9}\text{A}\text{?}\text{, c = 165.9}\text{A}\text{?}\text{, β = 103.0 °}$ and one tetramer of 120,000 M_r per asymmetric unit. The crystals are suitable for high-resolution work.     

1,25-Dihydroxyvitamin D3 inhibits selectively the mitogenic stimulation of mouse medullary thymocytes

Mouse thymocytes were separated into cortical and medullary subpopulations by differential agglutination with peanut agglutinin. A high-affinity receptor for 1,25-dihydroxyvitamin D3 is present in medullary immunocompetent mouse thymocytes and is absent from cortical immature cells. 1,25-dihydroxyvitamin D3, at physiological concentrations, inhibits the mitogenic response of the medullary cells to phytohemagglutinin and interleukin-2, but has no effect on the cortical subpopulation. Other less active metabolites of vitamin D had little or no effect on medullary cell stimulation.     

The inhibitory effect of some lectins on the differentiation of Friend erythroleukemia cells

Growth and differentiation of Friend cells can be inhibited by treatment with concanavalin A and wheat-germ agglutinin. This inhibition is specific for cells that are in the early stages of the differentiation process (24–48 h after the addition of dimethyl sulfoxide) and is reversible upon treatment with the sugars competitive for these lectins. These results suggest a regulatory role for some plasma membrane-bound glycoproteins early in the differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide.