Abstract. Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin . A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.541) fragment lacking NHZ-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was -325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as C ELLULAR adhesion to specific extracellular matrix components is required for a variety of biological events including cell proliferation, embryonic development, wound healing, and disease pathogenesis . The adhesive glycoprotein fibronectin has been an especially important model system for deriving insights into the mechanisms of cell adhesion and migration (8,9,23,36 an immunogen and their epitopes were characterized . Two separate mAbs, one binding close to the RGD site and the other to a site -15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by >90% . In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT1080 cells, functions which were also dependent on function of the a5ß1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the a5ßß fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.cell surface receptors. These binding activities are contained in distinct, protease-resistant domains. The best characterized cell-binding domain of fibronectin occupies its central region . This central cell-binding domain of fibronectin mediates cell adhesion with a wide variety of cell types (1, 45, 46) through a (Gly)-Arg-Gly-Asp-(Ser) sequence . For example, synthetic peptides containing this sequence block fibronectin...

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Sperm‐egg fusion is believed to be mediated via specific molecular interactions. Integrin α6β1 is a strong candidate for a sperm receptor on the egg plasma membrane. However, the ability of the egg integrin α6β1 to interact with molecules on intact sperm has not yet been proven. In this report, possible involvement of integrin α6β1 in sperm‐egg interactions was examined by biochemical and immunocytochemical analyses. To identify egg molecules that specifically interact with sperm, we first incubated sperm with biotin‐labeled egg surface proteins. Under this condition, solubilized proteins from eggs inhibited sperm‐egg fusion. Western blot analysis under reducing conditions indicated that a major‐labeled band of 135 kDa bound to sperm. An immunodepletion experiment using the anti‐integrin α6 antibody GoH3 indicated that the 135 kDa egg surface molecule that bound to sperm was the integrin α6 subunit. To investigate the potential involvement of integrin α6β1 in sperm‐egg fusion, we next examined the localization of integrin α6 and β1 subunits before and after fertilization by confocal laser microscopy. At an early stage of sperm‐egg fusion, the integrin α6 and β1 subunits were accumulated at the sperm binding site. The frequency of cluster formation was closely related to that of sperm‐egg fusion, indicating that integrin receptors are accumulated by sperm destined for fusion. Taken together, these results strongly suggest that the integrin α6β1 is involved in sperm‐egg binding leading to fusion via direct association of the integrin α6 with sperm. Mol. Reprod. Dev. 56:412–423, 2000. © 2000 Wiley‐Liss, Inc.

BackgroundEnvironmental challenges during development affect the fetal epigenome, but the period(s) vulnerable to epigenetic dysregulation is(are) not clear. By employing a soy phytoestrogen, genistein, that is known to alter the epigenetic states of the Avy allele during embryogenesis, we have explored the sensitive period for epigenetic regulation. The post-implantation period, when de novo DNA methylation actively proceeds, is amenable to in vitro analysis using a mouse embryonic stem (ES) cell differentiation system.Methods and FindingsMouse ES cells were differentiated in the presence or absence of genistein, and DNA methylation patterns on day 10 were compared by microarray-based promoter methylation analysis coupled with a methylation-sensitive endonuclease (HpaII/McrBC)-dependent enrichment procedure. Moderate changes in methylation levels were observed in a subset of promoters following genistein treatment. Detailed investigation of the Ucp1 and Sytl1 promoters further revealed that genistein does not affect de novo methylation occurring between day 0 and day 4, but interferes with subsequent regulatory processes and leads to a decrease in methylation level for both promoters.ConclusionGenistein perturbed the methylation pattern of differentiated ES cells after de novo methylation. Our observations suggest that, for a subset of genes, regulation after de novo DNA methylation in the early embryo may be sensitive to genistein.