Inhibitory effects of dietary soyasaponin on 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice.

Soyasaponins (SSs) abundant in soybean have anti-inflammatory activities; however, their therapeutic effects on allergic contact dermatitis (ACD) remain unknown. To assess the effects of SS-enriched diets on ACD, we used a mouse model of contact hypersensitivity (CHS). Mice were fed low-dose or high-dose SS-containing diets for 3 weeks prior to CHS induction with 2,4-dinitrofluorobenzene (DNFB). The low-dose SS diet attenuated DNFB-induced ear swelling and tissue oedema, and reduced the number of infiltrating Gr-1-positive myeloid cells. Low-dose, but not high-dose, SSs decreased chemokine (C-X-C motif) ligand 2 (CXCL2) and triggering receptor expressed on myeloid cells (TREM)-1 production in ear tissues, compared to a control. Taxonomic 16S rRNA analysis revealed significant alterations in faecal microbiota caused by CHS, which were reversed by low-dose SSs. The low-dose SS and non-CHS groups clustered together, while the high-dose SS group split between CHS and non-CHS clusters. Our results demonstrated that low-dose SSs alleviated CHS symptoms by attenuating inflammation and improving the intestinal microbiota composition, suggesting that dietary SSs may have beneficial effects on ACD.

Allergenic potential of rice-pollen proteins: expression, immuno-cross reactivity and IgE-binding.

Pollen proteins from several grass species have been identified and characterized as causative allergens in grass pollinosis. In contrast, allergenic potential of pollen proteins from rice, which belongs to the same Poaceae family, has not well been investigated, despite that a few clinical cases have been reported on rice-pollen allergy. In this study, to characterize expression and allergenic potential of pollen proteins from rice (Oryza sativa, ssp. japonica), rice putative proteins for ß-expansin (EXP), a Ca(2+)-binding protein (CBP)/polcalcin, extensin (EXT), profilin (PRF) and polygalacturonase (PGA) retrieved from a rice complete cDNA database were prepared as recombinant proteins, and the antibodies to these recombinant proteins were obtained. Immuno-blotting and immuno-histological analyses showed that rice putative EXP, EXT and PGA were expressed abundantly in anther tissue and pollen granules and immuno-cross reactive with pollen proteins from timothy grass. ELISA and immuno-dot blotting analyses using serum specimens from allergic patients showed that majority of the specimens was positive in the IgE-binding to EXP and EXT, but weakly to PGA and almost negative to PRF. EXP and EXT were suggested to be potentially allergenic in the rice-pollen allergy as well as the grass pollinosis.

Repression of mammary adipogenesis by genistein limits mammosphere formation of human MCF-7 cells.

Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and in rodent breast cancer models. Despite the suggested linkage between obesity and breast cancer, the local influence of bioactive dietary components on mammary adiposity for antitumor effects remains unknown. Herein, we report that post-weaning dietary exposure to soy protein isolate and its bioactive isoflavone genistein (GEN) lowered mammary adiposity and increased mammary tumor suppressor PTEN and E-cadherin expression in female mice, relative to control casein diet. To ascertain GEN's role in mammary adipose deposition that may affect underlying epithelial cell phenotype, we evaluated GEN's effects on SV40-immortalized mouse mammary stromal fibroblast-like (MSF) cells during differentiation into adipocytes. MSF cells cultured in a differentiation medium with 40 nM GEN showed reductions in mature adipocyte numbers, triglyceride accumulation, and Pparγ (Pparg) and fatty acid synthase transcript levels. GEN inhibition of adipose differentiation was accompanied by increased estrogen receptor ß (Erß (Esr2)) gene expression and was modestly recapitulated by ERß-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN). Reduction of Erß expression by siRNA targeting increased Pparγ transcript levels and stromal fibroblast differentiation into mature adipocytes; the latter was reversed by GEN but not by DPN. Conditioned medium from GEN-treated adipocytes diminished anchorage-independent mammosphere formation of human MCF-7 breast cancer cells. Our results suggest a mechanistic pathway to support direct regulation of mammary adiposity by GEN for breast cancer prevention.

IgG2 dominancy and carbohydrate recognition specificity of C3H/He mouse antibodies directed to cross-reactive carbohydrate determinants (CCDs) bearing beta-(1,2)-xylose and alpha-(1,3)-fucose.

Few common carbohydrate epitopes consisting of terminal beta-(1,2)-xylose and/or alpha-(1,3)-fucose residues are shared by a variety of glycoproteins from plants, insects and parasitic worms, termed cross-reactive carbohydrate determinant (CCD), and frequently recognized by IgE antibodies of patients with food and/or respiratory allergy, though clinical relevancy of such CCD-specific IgE is still controversial. Attention has also been focused on CCDs from the undesired post-translational modification of recombinant therapeutic proteins produced by transgenic plants and insects. In the present study, to clarify immunogenic potentials of CCD-bearing glycoproteins, the antibody response to a model plant glycoprotein, horseradish peroxidase (HRP) was investigated in a mouse model. C3H/He mice were immunized with HRP plus Al(OH)(3) or Freund's adjuvant, and IgG and IgE responses to CCDs in addition to HRP were analyzed by ELISA using some distinct glycoproteins with known N-glycan structures. IgE response to HRP was induced remarkably, whereas that to CCD was weaker and delayed. Moreover, apparent ratio of the CCD-specific antibodies to HRP-specific ones tended to be higher in IgG2a and IgG2b isotypes than IgG1, IgG3 and IgE. In contrast to rabbit antibodies, the CCD-specific antibodies from the mice gave poor reactivity with bromelain and honeybee phospholipase A2, suggesting the critical role of both beta-(1,2)-xylose and alpha-(1,3)-mannose in the CCD-recognition by the mouse antibodies. Moreover, the mouse antibodies showed weaker cross-reactivity to pollen- and insect-derived glycoproteins than the rabbit ones. Thus, in this mouse model, not only IgE but also IgG2 antibody responses to CCDs were induced by immunizing with a CCD-bearing glycoprotein, suggesting that CCDs affected not only Th2-type but also Th1-type antibody response at least in C3H/He mice.

Binding of Norovirus virus-like particles (VLPs) to human intestinal Caco-2 cells and the suppressive effect of pasteurized bovine colostrum on this VLP binding.

Noroviruses (NoVs), which cannot be grown in cell culture, are a major infectious agent of gastroenteritis. An in vitro assay system was established for the evaluation of NoV binding to enterocytes using virus-like particles (VLPs) produced in a baculovirus system expressing a NoV VP1 capsid protein. After confirmation of the purity by MS analysis, VLPs were incubated with human intestinal Caco-2 cells. NoV VLPs were detected clearly by confocal laser microscopy only on a certain population of Caco-2 cells, and were semi-quantified by immunoblotting of cell lysates. Then the suppressive effect of pasteurized bovine colostrum was analyzed on the VLP binding to Caco-2 cells by immunoblotting. The colostrum reduced VLP binding in a dose-dependent manner, at about 50% suppression with 12.5 microg of the colostral proteins. Furthermore, the colostrum contained IgG antibodies reacting to VLPs, suggesting that cross-reactive antibodies in the bovine colostrums block human NoV binding to intestinal cells.

Biotransformation of soy isoflavone-glycosides in laying hens: intestinal absorption and preferential accumulation into egg yolk of equol, a more estrogenic metabolite of daidzein.

Dietary soy-isoflavones have recently been noted as phytoestrogens with potentially beneficial effects on human health, and they are biologically transformed in the intestinal tract into aglycones and further into several specific metabolites. Here we report that in laying hens daidzin, a soy isoflavone-glycoside, in the diet was transformed into equol, absorbed, transported in circulating peripheral blood, and preferentially accumulated into egg yolk in its conjugated form. Laying hens were fed experimental diets containing two levels of soy isoflavone-glycosides (177 or 528 mg per 100 g diet) for 21 or 42 days, and blood and eggs were collected at 1- to 9-day intervals. HPLC analyses revealed that most of the isoflavones (daidzein, glycitein, and genistein) and a metabolite, equol, were present in blood and egg yolk in conjugated form. The concentration of equol-conjugates in blood plasma and egg yolk was higher than any of the other three isoflavone-conjugates analyzed and, especially in egg yolk, the equol-conjugates comprised no less than 60% of the total isoflavone-conjugates. The isoflavones, including equol, distributed mostly (95%) in the high-density fraction of blood serum, and more (65%) in the granule fraction of egg yolk. These results raise the possibility that feeding domestic animals soy-based fodder produces animal-based foods rich in a more active form of phytoestrogens.

A newly identified zona pellucida glycoprotein, ZPD, and dimeric ZP1 of chicken egg envelope are involved in sperm activation on sperm-egg interaction.

Fertilization begins with interaction between the sperm and the egg. The surface of the vertebrate oocyte is covered with the egg envelope, which is composed of ZP (zona pellucida) glycoproteins. We have identified two glycoproteins, ZP1/gp97 and ZPC/gp42, as the major components of the chicken egg envelope. In the present study, another 42 kDa protein, designated ZPD, has been found as a new major component of the chicken egg envelope. ZPD was specifically released from the egg envelope by ultrasonication treatment without urea. ZPD cDNA was cloned using a chicken granulosa cell cDNA pool. The deduced amino acid sequence showed that preproprotein of ZPD is composed of 418 amino acid residues with four potential N-glycosylation sites and includes a ZP domain, common in vertebrate ZP glycoproteins, and a transmembrane domain. ZPD belongs phylogenetically to a distinct group from known ZP glycoprotein subfamilies, ZPA, ZPB, and ZPC. In two-dimensional gel electrophoresis ZPD proteins were identified to be several isoforms with different pI values between 5 and 7. ZP1, ZPC and the newly identified ZPD were confirmed to be the major components of chicken egg envelope by MS of proteolytic digests of whole egg envelope. The in vitro incubation of chicken sperm with calcium ionophore A23187 induced sperm activation, resulting in the fragmentation and release of a 41 kDa PNA (peanut agglutinin)-positive glycoprotein and the decrease or loss of sperm PNA-stainability. The incubation with ZPD and dimeric ZP1, but not ZPC and monomeric ZP1, also induced the decrease or loss of sperm PNA-stainability, suggesting the in vitro sperm activation by these ZP components. Collectively, ZPD might bind loosely to egg envelope matrix and play a key role in the sperm activation on avian sperm-egg interaction.

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation.

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.

Accumulation of maize response regulator proteins in mesophyll cells after cytokinin treatment.

The maize response regulator genes ZmRR1 and ZmRR2 respond to cytokinin, and the translated products seem to be involved in nitrogen signal transduction mediated by cytokinin through the His-Asp phosphorelay. To elucidate the physiological function of the proteins, we examined the temporal and spatial distribution in maize leaves by immunochemical analysis and use of transgenic plants. ZmRR1 and ZmRR2 polypeptides could be distinctively detected by western blotting. The polypeptides accumulated in leaves within 5 h of the supply of nitrate to nitrogen-depleted maize, and the accumulation was transient. The extent of induction was larger in the leaf tip, which is rich in photosynthetically matured cells, than elsewhere. In leaves, the polypeptides accumulated mostly in mesophyll cells. Histochemical analyses of transgenic maize harboring a ZmRR1 promoter-beta-glucuronidase fusion gene also showed most of the expression to be in these cells. These results suggest that ZmRR1 and ZmRR2 are induced in mesophyll cells and function in nitrogen signal transduction mediated by cytokinin.