A stem cell-derived gene (Sddr) negatively regulates differentiation of embryonic stem cells.

Embryonic stem (ES) cells, derived from the inner cell mass of blastocysts, are pluripotent and continue to self-renew. To better understand the molecular mechanisms underlying self-renewal, we have been searching for a gene(s) which is specifically expressed in self-renewing ES cells. Here we report the isolation and characterization of a novel gene, Sddr (stem cell-derived differentiation regulator). Sddr was highly expressed in undifferentiated ES cells, and its expression was downregulated upon differentiation. In addition to ES cells, Sddr expression was observed strongly in ovary, and weakly in lung. Immunostaining and cellular fractionation analyses suggested that Sddr is a cytoplasmic protein associated with the cytoskeleton. Sddr-null ES cells showed no remarkable abnormalities in their undifferentiated state. In contrast, in differentiating Sddr-null cells, induction of several differentiation-associated markers was enhanced, and downregulation of self-renewal marker genes was accelerated, as compared with wild-type cells. These results suggest that although it is dispensable for ES cell self-renewal, Sddr is a negative regulator of ES cell differentiation.

Generation of thymine radicals from 6-alkoxy-5-bromo-5,6-dihydrothymine derivatives by radiolytic reduction and photolytic dehalogenation.

The reduction of 6-alkoxy-5-bromo-5,6-dihydrothymine derivatives ( 1a, b) by hydrated electrons (e aq (-)) generated in the radiolysis of deoxygenated aqueous solution was investigated. As the major products, 1-(6'-alkoxy-5',6'-dihydrothymin-5'-yl)thymines ( 6a, b), 5-(hydroxymethyl)uracil ( 8), 6-alkoxy-5,6-dihydrothymines ( 9a, b), and thymine ( 10) were produced in sufficient yields. This product distribution is indicative of the generation of 6-alkoxy-5,6-dihydrothymin-5-yl radicals ( 2a, b) as primary intermediates that undergo elimination of alkoxide ions (RO (-)) into thymine radical cations ( 3) followed by deprotonation at the N1 to form N-centered thymine radicals ( 4). The transient absorption spectra of the 5-yl radicals 2a- c were observed by means of nanosecond laser flash photolysis of 1a, b and 5-bromo-6-ethoxy-5,6-dihydrothymidine ( 1c) in deoxygenated aqueous solution, in which homolytic C5-Br bond dissociation occurred. In contrast to the reaction characteristics in aqueous solutions, the dimeric products were not obtained in acetonitrile, probably because in-cage hydrogen abstraction from the C5 methyl group by bromine atom leads to formation of methide type intermediates 20.

Beta-catenin up-regulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells.

It is well known that mouse embryonic stem (ES) cells can be maintained by the presence of leukemia inhibitory factor (LIF). Recent studies have revealed that Wnt also exhibits activity similar to LIF. The molecular mechanism behind the maintenance of ES cells by these factors, however, is not fully understood. In this study, we found that LIF enhances level of nuclear beta-catenin, a component of the Wnt signaling pathway. Expression of an activated mutant of beta-catenin led to the long-term proliferation of ES cells, even in the absence of LIF. Furthermore, it was found that beta-catenin up-regulates Nanog in an Oct-3/4-dependent manner and that beta-catenin physically associates with Oct-3/4. These results suggest that up-regulating Nanog through interaction with Oct-3/4 involves beta-catenin in the LIF- and Wnt-mediated maintenance of ES cell self-renewal.