Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：公益社団法人 日本農芸化学会  

DOI： 10.1271/kagakutoseibutsu.57.205

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/cbf.3350

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/jb/mvy034

Web of Science

PubMed

Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/gtc.12521

Web of Science

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Electrophoresis Society  

<p>Milk contains two kinds of secretory membrane microvesicles, milk fat globule and exsosome. Using SDS-PAGE in combination with density gradient ultracentrifugation, a phosphatidyl serine (PS)-binding membrane protein, MFG-E8, of these membrane vesicles in mouse milk was investigated. Milk fat globules and exosomes in fresh milk from lactating mice were recovered mainly from the lower density (~1.0) fractions and higher density (1.07~1.13) fractions, respectively, and MFG-E8 distributed to the milk fat globule fractions. Such MFG-E8 distribution was changed after forced weaning (pups withdrawal), i.e., MFG-E8 was present in not only the milk fat globule fractions but also a wide density-range (1.0~1.1) of the fractions including the exosome fractions and soluble protein fractions. Fluorescence microscopy analysis of the milk fat globules using anti-MFG-E8 antibody and annexin V as a PS probe showed that both of MFG-E8 and PS on the surface of milk fat globules largely differed even in fresh milk, and increased both in vivo with time after weaning and in vitro during the milk incubation. Mammary epithelial cells are known as non-professional phagocytes, which play a role in clearance of neighboring apoptotic cells on the epithelial cell turnover in lactating mammary glands. Mammary epithelial HC-11 cells preferentially phagocytosed old milk-fat globules rich in the surface MFG-E8 and, moreover, such phagocytosis was inhibited by anti-MFG-E8 antibody. We hypothesize that mammary epithelial cells may remove aged milk fat globules labeled heavily with MFG-E8 from breast milk and maintain freshness of milk lipids as an important nutrient for nursing infants.</p>

DOI： 10.2198/electroph.61.115

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/09168451.2015.1116927

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M114.598821

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/dev.121863

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s11010-014-2128-x

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publishing type：Research paper (bulletin of university, research institution)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1271/bbb.121011

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1093/jb/mvs154

Language：English   Publishing type：Research paper (scientific journal)  

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 milk fat globule EGF factor VIII (MFG-E8) and annexin V-accessible phosphatidylserine on the surface of MFGs. The MFG content in milk markedly decreased to about 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 phosphatidylserine 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.

DOI： 10.1093/jb/mvs116

Language：English   Publishing type：Research paper (scientific journal)  

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.

DOI： 10.1271/bbb.120412

Language：English   Publishing type：Research paper (scientific journal)  

... These results suggest that degraded but Dectin - 1 - reactive β- glucan is discharged from macrophage cells ... ROS -production inhibitors reduced soluble β- glucan discharge from the ... Soluble β- glucan produced by in vitro ROS - driven degradation activated macrophages . ...

DOI： 10.1016/j.bbrc.2012.04.009

Language：English   Publishing type：Research paper (scientific journal)  

Microtubules form flexible fibers, which are utilized in cell proliferation and differentiation. Although the flexibility of microtubules was shown to be regulated by various microtubule-associated proteins, this regulation is still far from complete understanding. Here, we report a new potential regulator of microtubules in mammals. Gcap14 colocalizes with microtubules in mammalian cells transfected with Gcap14 expression vector. Association of Gcap14 with microtubules was confirmed by biochemical subcellular fractionation. Recombinant Gcap14 protein cosedimented with pure microtubules, indicating a direct binding between the two. Furthermore, recombinant Gcap14 was shown to have the ability of inducing microtubule bundling in vitro.

DOI： 10.1016/j.febslet.2012.04.008

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/jcs.087700

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.devcel.2011.02.001

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/dev.052605

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.cub.2006.06.032

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1042/BJ20051459

Language：English   Publishing type：Research paper (scientific journal)  

MFG-E8 (milk fat globule-EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains, a mucin-like domain and two-repeated discoidin-like domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. The immunofluorescent staining of intracellular and cell-surface proteins and biochemical analyses of cell-surface-biotinylated and secreted proteins demonstrated that both of the two C-domains were required for the membrane association. During the course of these studies for domain functions, MFG-E8, but not C-domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size-exclusion chromatography and ultracentrifugation analyses, the complexes were characterized to have a high-molecular mass, low density and higher sedimentation velocity and to be detergent-sensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epithelial cell line, COMMA-1D, was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore, the expression of MFG-E8 increased the number of these membrane vesicle secreted into the culture medium. These results suggest a possible role of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).

Language：English   Publishing type：Research paper (scientific journal)  

... Biochem., 64 (4), 785-792, 2000 M An ELISA - Based Assay for Detergent - solubilized Cellular {\ss

DOI： 10.1271/bbb.64.785

Language：English   Publishing type：Research paper (scientific journal)  

Expression of mRNA encoding MFG-E8, a milk fat-associated glycoprotein was investigated in mouse mammary gland. Two forms of mRNA, long and short variants, were shown to be expressed in the mammary tissue by RT-PCR analysis. Sequence analyses of these two variants and an isolated MFG-E8 gene segment indicated that the long and short mRNA variants resulted from an alternative splicing of a single pre-mRNA through in-flame inclusion and skipping of one exon, which encodes a proline/threonine (Pro/Thr)-rich domain. The long variant was expressed predominantly in mammary gland and the expression level was remarkably increased at late gestation and kept high during lactation. On the contrary, the short variant was detected ubiquitously in various tissues and its expression in the mammary gland was rather decreased in a lactation dependent manner. Expression of the long variant was also detected in a mouse mammary epithelial cell line, COMMA-1D, and enhanced by incubation with lactogenic hormones. Glycosylation inhibition analyses using tunicamycin and alpha-benzyl-GalNAc were conducted with COS7 cells transfected with plasmids expressing each mRNA variant, demonstrating that a fully glycosylated product of the long mRNA variant was not only N-glycosylated but also multiply O-glycosylated, whereas a product of the short one had only N-glycan(s). These results suggest that the alternative splicing plays a critical role for the mammary-specific and lactation-dependent expression of the MFG-E8 isoform and that the multiply O-glycosylated Pro/Thr-rich domain of this isoform is functionally important for formation of milk fat globules in mammary epithelial cells.

DOI： 10.1006/bbrc.1998.0107