Search

Radial neuronal migration is a key neurodevelopmental event for proper cortical laminar organization. Newly born neurons migrate in a complex three-dimensional environment generated by the extracellular matrix (ECM), secreted factors, and neighboring cells. However, the molecular mechanisms by which the ECM controls radial migration remain largely unknown. We herein identified neurocan (NCAN), a risk factor for neuropsychiatric disorders, as a major chondroitin sulfate proteoglycan produced by developing cortical neurons. NCAN binds to both radial glial cell-derived tenascin-C (TNC) and hyaluronan (HA), a large linear polysaccharide, forming a ternary complex of NCAN, TNC, and HA. Developing cortical neurons make contact with the ternary complex during migration. The enzymatic or genetic disruption of the ternary complex impairs radial migration by suppressing the multipolar-to-bipolar transition. Furthermore, TNC in the ternary complex promotes neuronal morphological maturation. The present results provide evidence for the cooperative role of neuron- and radial glial cell-derived ECM molecules in cortical development.

Biological degradation of cellulose from dead plants in nature and plant biomass from agricultural and food-industry waste is important for sustainable carbon recirculation. This study aimed at searching diverse cellulose-degrading systems of wild filamentous fungi and obtaining fungal lines useful for cellooligosaccharide production from agro-industrial wastes. Fungal lines with cellulolytic activity were screened and isolated from stacked rice straw and soil in subtropical fields. Among 13 isolated lines, in liquid culture with a nutrition-limited cellulose-containing medium, four lines of Aspergillus spp. secreted 50–60 kDa proteins as markedly dominant components and gave clear activity bands of possible endo-β-1,4-glucanase in zymography. Mass spectroscopy (MS) analysis of the dominant components identified three endo-β-1,4-glucanases (GH5, GH7 and GH12) and two cellobiohydrolases (GH6 and GH7). Cellulose degradation by the secreted proteins was analysed by LC-MS-based measurement of derivatized reducing sugars. The enzymes from the four Aspergillus spp. produced cellobiose from crystalline cellulose and cellotriose at a low level compared with cellobiose. Moreover, though smaller than that from crystalline cellulose, the enzymes of two representative lines degraded powdered rice straw and produced cellobiose. These fungal lines and enzymes would be effective for production of cellooligosaccharides as cellulose degradation-intermediates with added value other than glucose.

Hunger and malnutrition, especially children, are still global issues today. Rice is a staple food for more than half of the world population and important nutritional source of not only carbohydrate but also protein. In recent aging societies, protein-energy malnutrition in elderly people emerges also as a social issue. Malnutrition in elderly people raises the risk of falling into age-related chronic diseases. Nutritional care can prevent elderly people from such age-related diseases. Rice and rice flour would be good foodstuff for preparation of diet suitable for and preferred by elderly people. Protein content of rice grains, like the other cereal grains, is less than 10% by weight, which is a little lower than meat and cheese, but higher than dairy milk and yoghurt. Nutritional quality of rice proteins is higher than the other cereal grains. Such relatively higher nutritional quality of rice proteins could be due to high copies of glutelin genes evolved from an ancestral gene common to soybean glycinin and resultant high content of legume-type seed storage proteins. Recently, rice flour became to be utilized for various processed food. The rice seed proteins as well as starch are accumulated in specific organelles termed protein bodies and amyloplast in the cells of endosperm and aleurone layer. By milling rice grains to flour particles consisting of protein and starch nanoparticles, processing characteristics of rice starch and proteins could be changed. To develop rice-based processed food for prevention of malnutrition, rice flour particles from various different rice sources could be blended for desired nutritional composition without spoiling the value of product food.

Mammary gland involution is a regressive process for the gland to return to its prepregnancy state after lactation and comprises an initial reversible and second remodeling stage. Although many genes and the multiple expression profiles of their mRNAs have been found in this process, the mechanisms controlling the profiles are largely unknown. In this study, we identified and analyzed transcription factor Sox4 in mammary gland involution. Elevated expression of Sox4 gene in the first stage (48 h after weaning) was observed at the mRNA and protein levels in the mouse mammary gland. Immunohistochemistry of the involuting gland indicated that Sox4 was located in the nuclei of epithelial cells. Nuclear Sox4 was also detected in the second stage, but unlikely to be involved in cell death, one of the characteristic events of involution. To clarify the functional roles of Sox4 in involution, we introduced a model, including a normal mammary epithelial cell line, for finding candidate target genes of this transcription factor and examined its effect on tenascin C mRNA expression., ファイル公開：2020/10/07

Background Perineuronal nets (PNNs) are insoluble aggregates of extracellular matrix molecules in the brain that consist of hyaluronan (HA) and chondroitin sulfate proteoglycans (CSPGs). PNNs promote the acquisition and storage of memories by stabilizing the formation of synapses in the adult brain. Although the deterioration of PNNs has been suggested to contribute to the age-dependent decline in brain function, the molecular mechanisms underlying age-related changes in PNNs remain unclear. Methods The amount and solubility of PNN components were investigated by sequential extraction followed by a disaccharide analysis and immunoblotting. We examined the interaction between HA and aggrecan, a major HA-binding CSPG, by combining mass spectrometry and pull-down assays. Results The solubility and amount of HA increased in the brain with age. Among several CSPGs, the solubility of aggrecan was selectively elevated during aging. In contrast to alternations in biochemical properties, the expression of PNN components at the transcript level was not markedly changed by aging. The increased solubility of aggrecan was not due to the loss of HA-binding properties. Our results indicated that the degradation of high-molecular-mass HA induced the release of the HA-aggrecan complex from PNNs in the aged brain. Conclusion The present study revealed a novel mechanism underlying the age-related deterioration of PNNs in the brain.

While transcription plays an obviously important role in gene expression, translation has recently been emerged as a key step that defines the composition and quality of the proteome in the cell of higher eukaryotes including mammals. Selective translation is supposed to be regulated by the structural heterogeneity of cytoplasmic ribosomes including differences in protein composition and chemical modifications. However, the current knowledge on the heterogeneity of mammalian ribosomes is limited. Here, we report mammalian Serbp1 as a ribosome‐associated protein. The translated products of Serbp1 gene, including the longest isoform, were found to be localized in the nucleolus as well as in the cytoplasm. Subcellular fractionation indicated that most of cytoplasmic Serbp1 molecules were precipitated by ultracentrifugation. Proteomic analysis identified Serbp1 in the cytoplasmic ribosomes of the rodent testis. Polysome profiling suggested that Serbp1, as a component of the small 40S subunit, was included in translating ribosomes (polysomes). Cosedimentation of Serbp1 with the 40S subunit was observed after dissociation of the ribosomal subunits. Serbp1 was also included in the ribosomes of human cancer cells, which may lead to a mechanistic understanding of an emerging link between Serbp1 and tumour progression., ファイル公開：2019/08/01

Milk fat globule-EGF factor 8 (MFG-E8) is a divalent-binding secretory protein possessing an Arg-Gly-Asp (RGD) motif and a phosphatidylserine (PS)-binding motif. This protein has been shown to be involved in mammary gland development and morphogenesis. Integrin-binding activity is necessary for these MFG-E8-dependent cell processes. Although the target cells and molecules of MFG-E8 in the cellular microenvironment are important to understand its physiological function, its localization is largely unclear. Here, we found that mouse MFG-E8 localized to the basal lamina of the mammary gland during involution. In a model system of mammary COMMA-1D cells, exogenously and endogenously expressed MFG-E8 was deposited in the extracellular matrix (ECM) with membranous particles dependently on the PS-binding motifs in the discoidin domains that were essential for association ability to extracellular vesicles (EVs). These data showed the basal MFG-E8 localization mechanism in which EVs served as a scaffold. Such an immobilized MFG-E8 associating with cell substrata but not soluble one in the culture media promoted integrin-dependent suppression of β-casein expression. These results suggest that MFG-E8 requires EVs to transduce cellular signals from the basolateral side of the adhesion cells by accumulating in ECM., ファイル公開日: 2018/10/01

Rice are consumed mainly as polished rice grains. In the threshing and polishing processes of paddy rice, a considerable amount of husk and bran are separated as by-products. Rice bran is utilized for oil production, whereas rice husk as well as straw is not fully utilized. Defatted rice bran is rich in proteins and non-digestible polysaccharides, while husk and straw consist mainly of plant cell wall components, including cellulose, hemicellulose and pectin. Such non-digestible polysaccharides function in gastrointestinal lumen as dietary fiber, though physiological functions and their application are limited. Non-digestible oligosaccharides have recently been interested as prebiotics from a viewpoint of health benefit via utilization by intestinal microbiota. A diversity of non-digestible polysaccharides in rice bran and husk are good and ecological sources for production of both prebiotic and potentially prebiotic oligosaccharides. In this review, we summarize non-digestible polysaccharides constituting cell wall of rice grains including husk and degradation of the polysaccharides into oligo- and monosaccharides by microbial glycoside hydrolases. Prebiotic potential of such oligosaccharides derived from rice non-digestible polysaccharides are also introduced. Finally, our recent attempt for effective production of cello-oligosaccharides by regulated enzymatic degradation is briefly described.

Mammalian fertilisation begins when sperm interacts with the egg zona pellucida (ZP), whose ZP1 subunit is important for fertility by covalently cross-linking ZP filaments into a three-dimensional matrix. Like ZP4, a structurally-related component absent in the mouse, ZP1 is predicted to contain an N-terminal ZP-N domain of unknown function. Here we report a characterisation of ZP1 proteins carrying mutations from infertile patients, which suggests that, in human, filament cross-linking by ZP1 is crucial to form a stable ZP. We map the function of ZP1 to its ZP-N1 domain and determine crystal structures of ZP-N1 homodimers from a chicken homolog of ZP1. These reveal that ZP filament cross-linking is highly plastic and can be modulated by ZP1 fucosylation and, potentially, zinc sparks. Moreover, we show that ZP4 ZP-N1 forms non-covalent homodimers in chicken but not in human. Together, these data identify human ZP1 cross-links as a promising target for non-hormonal contraception., Glycoprotein ZP1 is a component of the oocyte’s zona pellucida (ZP), and mutations in human ZP1 are linked to female infertility. Here, using structure-function analysis, the authors suggest that filament cross-linking by ZP1 is required to form a stable ZP in human, and infertility mutations interfere with cross-linking.

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28–58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.

Background Hyaluronan (HA) is a major component of the extracellular matrix (ECM) and is involved in many cellular functions. In the adult brain, HA forms macromolecular aggregates around synapses and plays important roles in neural plasticity. In contrast to the well-characterized function of HA in the adult brain, its roles in the developing brain remain largely unknown. Methods Biochemical and histochemical analyses were performed to analyze the amount, solubility, and localization of HA in the developing mouse brain. By combining in utero labeling, cell isolation, and in vitro cultures, we examined the expression of hyaluronan synthase (HAS) and morphological maturation of cortical neurons. Results The amount of HA increased during perinatal development and decreased in the adult. HA existed as a soluble form in the early stages; however, its solubility markedly decreased during postnatal development. HA localized in cell-sparse regions in the embryonic stages, but was broadly distributed during the postnatal development of the cerebral cortex. Developing cortical neurons expressed both Has2 and Has3, but not Has1, suggesting the autonomous production of HA by neurons themselves. HA formed a pericellular matrix around the cell bodies and neurites of developing cortical neurons, and the inhibition of HA synthesis reduced neurite outgrowth. Conclusion The formation of the pericellular HA matrix is essential for the proper morphological maturation of developing neurons. General significance This study provides new insights into the roles of hyaluronan in the brain. development.

Birds are oviparous vertebrates in terrestrial animals. Birds' eggs accumulate mass of egg yolk during the egg development and are accordingly much larger than the eggs of viviparous vertebrates. Despite such difference in size and contents, the birds' eggs are surrounded with the egg-coat morphologically and compositionally resembling the mammalian egg-coat, zona pellucida. On the other hand, there are some differences in part between the two egg-coats, though relationships of such structural differences to any biological roles specific for the extracellular matrix of birds' eggs are not fully understood. In birds, unlike mammals, ZP proteins constituting the egg-coat are highly conserved and therefore those of chicken are described as a representative of birds. The egg-coat ZP proteins, ZP1, ZP3, and ZPD as the majors, accumulate and form the matrix by self-assembly around the egg rapidly growing in the ovarian follicle, in which ZP1 is from liver and both ZP3 and ZPD are from follicular granulosa cells. Although details of the egg-coat-sperm interaction on fertilization remain to be investigated, the lytic degradation process of egg-coat matrix for the sperm penetration has become to be clarified gradually. ZP1 is the primary target of sperm acrosin, and the limited cleavage in the specific region leading to the loss of intermolecular cross-linkages is crucial for the lysis of egg-coat matrix. Possible roles of the ZP1 with the additional sequence characteristic to birds are discussed from a viewpoint of giving both robustness and elastomeric nature to the egg-coat matrix for the birds' eggs.

Casein (CN) is the major milk protein to nourish infants but, in certain population, it causes cow’s milk allergy, indicating the uptake of antigenic CN and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of CN was investigated. Confocal microscopy using component-specific antibodies showed that αs1-CN antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular CN signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein (EEA1) and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4°C. Liquid chromatography coupled with mass spectroscopy analysis of the protein fraction in the basal-side medium identified the αs1-CB fragment including the N-terminal region and the αs2-CN fragment containing the central part of polypeptide at 100–1,000 fmol per well levels. Moreover, β-CN C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that CNs are partially degraded by cellular proteases and/or peptidases and immunologically active CN fragments are transported to basal side of the cell monolayers., ファイル公開日: 2019/08/01

Birds are oviparous vertebrates in terrestrial animals. Birds' eggs accumulate mass of egg yolk during the egg development and are accordingly much larger than the eggs of viviparous vertebrates. Despite such difference in size and contents, the birds' eggs are surrounded with the egg-coat morphologically and compositionally resembling the mammalian egg-coat, zona pellucida. On the other hand, there are some differences in part between the two egg-coats, though relationships of such structural differences to any biological roles specific for the extracellular matrix of birds' eggs are not fully understood. In birds, unlike mammals, ZP proteins constituting the egg-coat are highly conserved and therefore those of chicken are described as a representative of birds. The egg-coat ZP proteins, ZP1, ZP3, and ZPD as the majors, accumulate and form the matrix by self-assembly around the egg rapidly growing in the ovarian follicle, in which ZP1 is from liver and both ZP3 and ZPD are from follicular granulosa cells. Although details of the egg-coat-sperm interaction on fertilization remain to be investigated, the lytic degradation process of egg-coat matrix for the sperm penetration has become to be clarified gradually. ZP1 is the primary target of sperm acrosin, and the limited cleavage in the specific region leading to the loss of intermolecular cross-linkages is crucial for the lysis of egg-coat matrix. Possible roles of the ZP1 with the additional sequence characteristic to birds are discussed from a viewpoint of giving both robustness and elastomeric nature to the egg-coat matrix for the birds' eggs.

Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the Km value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 μm. Despite the relatively low Km value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGT(R377Q) were not affected. Importantly, the interaction between UDP-GlcNAc and EOGT(R377Q) was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS.

In birds in the reproductive season, an egg is ovulated without cumulus cells from the largest follicle with the highest hierarchy in the ovary. The outermost part of the ovulated eggs is the perivitelline layer, a glycoprotein matrix consisting of a few ZP-glycoproteins. The fertilization starts from sperm penetration of the perivitelline layer predominantly in the germinal disc region, followed by uptake of the sperm into the egg, and goes through by the fusion of sperm male pronucleus with the female pronucleus in the egg. A series of these fertilization steps occurs in the infundibulum of the oviduct within a short period after ovulation. Some pioneering microstructural studies using electron microscopy and supporting biochemical data from later studies indicate that, in avian fertilization, sperm interacts with the perivitelline layer covering the germinal disc, locally degrade and dissolve the matrix of the perivitelline layer, and penetrate it through the hole made proteolytically at the sperm-binding site on the perivitelline layer. Several molecules and structures presumably involved in the sperm-perivitelline interaction have been characterized, especially sperm proteases and their targets in the egg perivitelline layer. On the other hand, no molecules involved in the sperm-egg membrane fusion for the male pronucleus uptake into the egg have yet been identified or characterized and, moreover, no orthologue but one have been annotated so far in the chicken genome for the mouse genes involved in the sperm-egg membrane fusion.

In birds in the reproductive season, an egg is ovulated without cumulus cells from the largest follicle with the highest hierarchy in the ovary. The outermost part of the ovulated eggs is the perivitelline layer, a glycoprotein matrix consisting of a few ZP-glycoproteins. The fertilization starts from sperm penetration of the perivitelline layer predominantly in the germinal disc region, followed by uptake of the sperm into the egg, and goes through by the fusion of sperm male pronucleus with the female pronucleus in the egg. A series of these fertilization steps occurs in the infundibulum of the oviduct within a short period after ovulation. Some pioneering microstructural studies using electron microscopy and supporting biochemical data from later studies indicate that, in avian fertilization, sperm interacts with the perivitelline layer covering the germinal disc, locally degrade and dissolve the matrix of the perivitelline layer, and penetrate it through the hole made proteolytically at the sperm-binding site on the perivitelline layer. Several molecules and structures presumably involved in the sperm–perivitelline interaction have been characterized, especially sperm proteases and their targets in the egg perivitelline layer. On the other hand, no molecules involved in the sperm–egg membrane fusion for the male pronucleus uptake into the egg have yet been identified or characterized and, moreover, no orthologue but one have been annotated so far in the chicken genome for the mouse genes involved in the sperm–egg membrane fusion.

Effect of deletion of acid resistant genes of E. coli on the high-pressure carbon dioxide (HPC) resistance was investigated. Genes coding amino acid decarboxylases, such as lysine, arginine, and glutamate decarboxylase, were found to contribute to HPC resistance. Protonophore-treated cells showed hypersensitivity to HPC, confirming that HPC induced cytoplasm acidification and exerted severe damage on cells by intrusion of gaseous carbon dioxide into cytoplasm.

Oral immunotherapy for food allergy has been the focus of a lot of attention recently. The patients have to eat allergenic food instead of eliminating it in this therapy and there is no established standard method yet. To promote clear understanding and improvement of oral immunotherapy, the present study using B10.A mice investigated the effect of multiple oral administration of a model antigen, egg-white lysozyme, on both the antibody response and the anaphylactic reaction induced by subsequent administration of lysozyme. Various doses of egg-white lysozyme (0-100 mg/mouse) were administered to mice intragastrically for 6 d; then additional lysozyme was administered via the intraperitoneal route in all groups. Lysozyme-specific antibody responses were promptly induced by the first oral administration and enhanced by intraperitoneal administration. An anaphylactic reaction was further induced in these sensitized mice by intragastric administration of lysozyme, and the symptoms of shock were compared in order to evaluate the effects of pretreatment. Interestingly, the decrease in rectal temperature which is one of the common anaphylactic symptoms in mice was suppressed in all of the oral pre-administration groups, and the effects were highest in the group that received 20 mg. Consequently, this study using B10.A mice has shown that sensitization can be induced by intragastric administration of lysozyme instead of oral tolerance; however, anaphylactic shock induced by subsequent intragastric administration of lysozyme is suppressed. This mouse model would be useful for assessing the method of oral immunotherapy.

Recent studies have revealed heterogeneity in the structure of eukaryotic cytoplasmic ribosomes, including a difference in protein composition. It has been proposed that this heterogeneity, or the specialized ribosome, contributes to tissue development and homeostasis through selective mRNA translation, although this remains largely unclear. Our previous proteomic survey of rodent ribosomes found the testis-specific ribosomal proteins L10-like and L39-like, which are paralogues of the X-linked ribosomal proteins L10 and L39, respectively. We have hypothesized that the rodent testis provides a good model for examining the possible functional importance of ribosome heterogeneity. In the present study, a new paralogue of X-linked ribosomal protein S4 has been identified in the mouse testis. The gene encoding this paralogue was autosomal, intronless and expressed predominantly in the testis. It appeared that this paralogue was included in polysomes as a component of the ribosome. Although these properties were similar to those of the ribosomal proteins L10-like and L39-like, this S4 paralogue and L10-like showed partially different expression patterns in spermatogenic cells. These findings are discussed in relation to the unique evolution of genes encoding a paralogue of ribosomal protein S4 in mammals and to the significance of testis-specific paralogues of ribosomal proteins in active ribosomes during spermatogenesis.

Advanced glycation end products (AGEs) are a heterogeneous and complex group of compounds that are formed when reducing sugars, such as dehydroascorbic acid, react in a nonenzymatic way with amino acids in proteins and other macromolecules. AGEs are prevalent in the diabetic vasculature and contribute to the development of atherosclerosis. The presence and accumulation of AGEs in many different cell types affect the extracellular and intracellular structure and function. In the present study, we studied the immune response to the dehydroascorbic acid-derived AGEs and provide multiple lines of evidence suggesting that the AGEs could be an endogenous source of innate epitopes recognized by natural IgM antibodies. Prominent IgM titers to the AGEs were detected in the sera of normal mice and were significantly accelerated by the immunization with the AGEs. Patients with systemic lupus erythematosus (SLE), a potentially fatal systemic autoimmune disease characterized by the increased production of autoantibodies, showed significantly higher serum levels of the IgM titer against the AGEs than healthy individuals. A progressive increase in the IgM response against the AGEs was also observed in the SLE-prone mice. Strikingly, a subset of monoclonal antibodies, showing a specificity toward the AGEs, prepared from normal mice immunized with the AGEs and from the SLE mice cross-reacted with the double-stranded DNA. Moreover, they also cross-reacted with several other modified proteins, including the acetylated proteins, suggesting that the multiple specificity of the antibodies might be ascribed, at least in part, to the increased electronegative potential of the proteins. These findings suggest that the protein modification by the endogenous carbonyl compounds, generating electronegative proteins, could be a source of multispecific natural antibodies.

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.

Bovine trypsin was modified with glucose through the Maillard reaction at 50°C and 65% RH for various periods (1 to 8 days). Tryptic activity against both benzoyl-L-arginine-p-nitroanilide and two protein substrates was enhanced with increases in the reaction period, and reached the maximum after a 4-day reaction. Although there were no big differences in pH dependency of trypsin activity between native and modified trypsins, the Km of the modified trypsin decreased to about half the native one. The modified trypsin retained its original activity almost completely after incubation in a buffer solution of pH 8.0 at 37°C for 72 h, while native trypsin was greatly inactivated. Furthermore, tryptic acitivity at high temperature, residueal activity after heating, and differential scanning calorimetric analysis showed that the modified trypsin was more heat-stable than native trypsin.

In lactating mammary glands, milk fat is secreted as fat globules surrounded by a cell plasma membrane containing characteristic membrane-associated proteins. Among these, butyrophilin has been shown to be specific and intrinsic to the fat globule membrane, whereas milk-fat globule EGF-factor VIII (MFG-E8) is uncertain. We characterized in the present study MFG-E8 in milk fat globules and in the culture medium of HC11 mammary epithelial cells. MFG-E8 was immunologically detected in the mammary tissues of both pregnant and lactating mice. Double-immunofluorescence staining for MFG-E8 and butyrophilin showed diversity in the MFG-E8-staining intensity among different fat globules in milk. HC11 cells secreted monomeric MFG-E8 with phosphatidylserine-binding activity, despite no fat globules being detected in the cells. This secretion was upregulated by not only prolactin but also by insulin or EGF. These results suggest that milk MFG-E8 was not equally present among fat globules and not necessarily intrinsic to the fat globules.

Milk fat globules (MFGs) secreted by lactating mammary gland are unique lipid surrounded by a phospholipid bi-layer. We report here post-weaning changes in MFG EGF factor VIII (MFG-E8) and annexin V-accessible phosphatidyl-l-serine on the surface of MFGs. The MFG content in milk markedly decreased to about one-half within 2 days after forced weaning, despite a slight increase in milk protein content. Immunofluorescence-staining of MFGs using anti-MFG-E8 and annexin V indicated that MFG-E8 was present on some, but not all, MFGs before weaning, whereas most of MFGs were MFG-E8-positive and annexin V-negative after weaning. Free MFG-E8 with binding activity to phosphatidyl-l-serine was present abundantly in the post-weaning milk, and indeed exhibited binding to MFGs in pre-weaning milk. MFGs were taken up by HC11 mouse mammary epithelial cells in vitro, and those from post-weaning milk were remarkable for such cellular uptake. Moreover, the uptake of MFGs by the cells was inhibited by an anti-MFG-E8 antibody. Taken together, these findings suggest that MFG-E8 plays a critical role in regulation of MFG dynamics after weaning or during the suckling interval through the control of MFG-epithelial cell interaction in lactating mammary glands.

At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin–proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody.In vitropenetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation.

The vertebrate egg coat corresponding to mammalian zona pellucida is a filamentous matrix composed of highly and heterogeneously glycosylated proteins designated ZP glycoproteins including ZP1 to 4, ZPD and ZPAX, and play important roles in species-specific egg-sperm interactions. Recent advance in structural biology of chicken ZP3 provided new insights into molecular mechanisms of the egg-coat function involving its carbohydrate moieties. In this study, chicken ZP3 was separated into four major and distinct isoforms with different pI in 2D-PAGE. To investigate the meanings of the ZP3 heterogeneity in egg-sperm interactions, we preliminary analyzed glycan diversity on the molecules by using lectin-staining assays. The four major ZP3 isoforms 4-7 (from acidic to basic) were recognized equally with PNA (Galβ1-3GalNAc), but the isoforms 5-7 were recognized dominantly with WGA ((β-GlcNAc)n, clustered Sia), PHA-E (bi- and triantennary N-glycan containing Galβ1-4GlcNAcβ1-2Manα1-6) and RCA I (terminal Galβ1-4GlcNAc), respectively. Despite such sugar chain diversity among the ZP3 isoforms, a partner in the egg coat, ZP1, showed specific binding to each isoform equally. Localization of ZP1 and ZP3 in the egg-coat matrix were also analyzed.

O-linked-β-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shown whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.

Rice seed proteins are known to be a causative antigen in some patients with food allergy, especially cereal allergy, with clinical symptoms such as eczema and dermatitis. The α-amylase/trypsin inhibitors (14-16 kDa), α-globulin (26 kDa) and β-glyoxalase I (33 kDa) are regarded as major potential allergens of rice (Oryza sativa L.) seed based on specific recognition by serum IgE from allergy patients. In order to suppress the production of these major allergens in rice grains, a mutant in the 'Koshihikari' background lacking the 26 kDa allergen (GbN-1) was used as a host for RNA silencing. A binary vector harboring two RNA interference (RNAi) gene cassettes for suppression of 14-16 kDa and 33 kDa allergens driven by the 13 kDa and 10 kDa prolamin endosperm-specific promoters, respectively, was introduced into the GbN-1 genome by Agrobacterium-mediated transformation. In the most promising transgenic line, the content of the three potential allergens was remarkably reduced to a very faint level without a change in seed phenotype. IgE binding of 15 patients' sera to the transgenic rice seed mostly deficient in the three major allergens was on average only about 10% that of the control wild-type rice, suggesting that these three accounted for the great majority of rice seed causative allergens recognized by patients' IgE and that the sequential allergen deletion/reduction strategy works in the development of hypo-allergenic rice lines.

SummaryZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 Å resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-glycan indicate how sperm binding could trigger downstream events via intramolecular signaling.

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.

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-M(r) glycoprotein fraction (F1) from cow's milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 degrees C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.

Involution of the mammary gland is a regressive phase that occurs after lactation, and requires reprogramming of gene expression for the tissue to return to a pre-pregnant state. Although the transcriptome of the mammary gland demonstrates complex changes at the mRNA level, the molecular mechanisms governing post-transcriptional control remain obscure. In the present study, we isolated cytoplasmic mRNA-protein complexes (mRNPs) from the mouse mammary gland at the early involution stage using discontinuous sucrose density ultracentrifugation. mRNPs including untranslated mRNAs were then purified with oligo(dT) immobilized on cellulose or paramagnetic beads. Proteins in the purified complexes were subjected to one/two-dimensional gel electrophoresis followed by mass spectrometry. This identified heterogeneous nuclear ribonucleoprotein A/B (Hnrpab), along with three other heterogeneous nuclear ribonucleoproteins. Hnrpab in the mRNPs reproducibly increased within 48 h after weaning and became one of the major components. When a vector expressing Hnrpab was transfected into two different cell lines, their growth was suppressed, demonstrating that this protein has cytostatic activity. These results suggest that early involution can be used as a model for understanding the mechanism of post-transcriptional control of gene expression, responsible for modulation of cell function. Copyright © 2010 John Wiley & Sons, Ltd.

By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems.

We previously reported that 3T3-L1 and rat primary adipocytes secreted microvesicles, known as adipocyte-derived microvesicles (ADMs). In the present study, we further characterized the 3T3-L1 ADMs and found that they exhibited angiogenic activity in vivo. Antibody arrays and gelatin zymography analyses revealed that several angiogenic and antiangiogenic proteins, including leptin, TNFalpha, acidic fibroblast growth factor (FGFa), interferon-gamma, and matrix metalloprotease (MMP)-2 and MMP-9, were present in the ADMs. Gene expression of most of these angiogenic factors was induced in the adipose tissue of diet-induced obese mice. Furthermore, leptin, TNFalpha, and MMP-2 were up-regulated at the protein level in the adipocyte fractions prepared from epididymal adipose tissues of high-fat-diet-induced obese mice. ADMs induced cell migration and tube formation of human umbilical vein endothelial cells, which were partially suppressed by neutralizing antibodies to leptin, TNFalpha, or FGFa but not to interferon-gamma. Supporting these data, a mixture of leptin, TNFalpha, and FGFa induced tube formation. ADMs also promoted cell invasion of human umbilical vein endothelial cells through Matrigel, which was suppressed by the addition of the MMP inhibitor 1,10'-phenanthroline and a neutralizing antibody to MMP-2 but not to MMP-9. These results suggest that ADMs are associated with multiple angiogenic factors and play a role in angiogenesis in adipose tissue.

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.

Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome.