Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells

The Tol2 transposable element of the Japanese medaka fish belongs to the hAT family of transposons including hobo of Drosophila, Ac of maize, and Tam3 of snapdragon. To date, Tol2 is the only natural transposon in vertebrates that has ever been shown to encode a fully functional transposase. It has not been known, however, whether Tol2 can transpose in vertebrates other than fish. We report here transposition of Tol2 in mouse embryonic stem (ES) cells. We constructed a transposon donor plasmid containing a nonautonomous Tol2 element with the neomycin resistance gene and a helper plasmid capable of expressing the transposase and introduced the donor plasmid with various amounts of the helper plasmid by electroporation into mouse ES cells. The number of G418-resistant ES colonies increased as the amount of helper plasmid was increased, in a dose-dependent manner, indicating that the transposase activity elevated the integration efficiency. These G418-resistant ES colonies were cloned and the structure of the junction of the integrated Tol2 element and the genomic DNA was analyzed by inverse PCR. In those clones, Tol2 was surrounded by mouse genomic sequences and an 8-bp direct repeat was created adjacent to both ends of Tol2, indicating that Tol2 was integrated in the genome through transposition. The Tol2 transposon system is thus active in mouse as well as in fish. We propose that it should be used as a genetic tool to develop novel gene transfer, transgenesis, and mutagenesis methods in mammals.     

Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration

The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.     

Analyses to clarify rich fractions in hepatic progenitor cells from human umbilical cord blood and cell fusion

Umbilical cord blood (UCB) is a source of hematopoietic stem cells and other stem cells, and human UCB cells have been reported to contain transplantable hepatic progenitor cells. However, the fractions of UCB cells in which hepatic progenitor cells are rich remain to be clarified. In the present study, first, the fractionated cells by CD34, CD38, and c-kit were transplanted via portal vein of NOD/SCID mice, and albumin mRNA expression was examined in livers at 1 and 3 months posttransplantation. At 1 and 3 months, albumin mRNA expression in CD34+UCB cells-transplanted livers was higher than that in CD34- cells-transplanted livers. Albumin mRNA expression in CD34+CD38+ cells-transplanted livers was higher than that in CD34+CD38- cells-transplanted [corrected] liver at 1 month. However, it was much higher [corrected] in CD34+CD38- cell-transplanted livers at 3 months. Similar expression of albumin mRNA was obtained between CD34+CD38+c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, and between CD34+CD38-c-kit+ cells- and CD34+CD38-c-kit- cells-transplanted livers, respectively. Second, fluorescence in situ hybridization and immunohistochemistry were performed to examine whether UCB cells really transdifferentiated into hepatocytes or they only fused with mouse hepatocytes. In mouse liver sections, of 1.2% cells which had human chromosomes, 0.9% cells were due to cell fusion, whereas 0.3% cells were transdifferentiated into human hepatocytes. These results suggest that CD34+UCB cells are rich fractions in hepatic progenitor cells, and that transdifferentiation from UCB cells into hepatocytes as well as cell fusion simultaneously occur in this situation.     

Proteasome-dependent decrease in Akt by growth factors in vascular smooth muscle cells

Akt is activated by growth factors to regulate various aspects of vascular smooth muscle cell function. Platelet-derived growth factor (PDGF) and insulin-like growth factor-1 activated Akt in vascular smooth muscle cells with a rapid reduction of total Akt protein that lasted for several hours. The downregulation of Akt required phosphatidylinositol 3-kinase activity, but not intrinsic Akt activity. The downregulation of Akt was abrogated by MG-132, a proteasome inhibitor, but not by inhibitors of calpain or cathepsins. Akt was found in ubiquitin immune complex after PDGF treatment. Proteasome-dependent degradation of Akt may provide a counter-regulatory mechanism against overactivation of Akt.     

Participation of proteasomes in Xenopus embryogenesis

We examined the effects of various protease substrates on Xenopus laevis embryogenesis. Thirty-three peptidyl-MCA substrates were added to the culture medium in which Xenopus embryos were developing. Five of the 33 substrates were found to inhibit embryogenesis at the early gastrula stage or much earlier ones. These results suggest that proteases that hydrolyze these substrates are involved in embryonic development. We found that the developmental stage of embryos is crucial for these substrates to inhibit their development. We purified a protease that hydrolyzes Pyr-Arg-Thr-Lys-Arg-MCA, a substrate that inhibits embryogenesis, from Xenopus embryos. This protease turned out to be a component of proteasomes. We found that 4 of the 5 substrates that inhibit embryogenesis are among the proteasome substrates. Thus, we concluded that proteasomes play a crucial role in the development of Xenopus embryos. Possibly, various catalytic subunits in proteasomes function independently, in stage-specific manners.