The transition of mouse pluripotent stem cells from the naïve to the primed state requires Fas signaling through 3-O sulfated heparan sulfate structures recognized by the HS4C3 antibody

The characteristics of pluripotent embryonic stem cells of human and mouse are different. The properties of human embryonic stem cells (hESCs) are similar to those of mouse epiblast stem cells (mEpiSCs), which are in a later developmental pluripotency state, the so-called “primed state” compared to mouse embryonic stem cells (mESCs) which are in a naïve state. As a result of the properties of the primed state, hESCs proliferate slowly, cannot survive as single cells, and can only be transfected with genes at low efficiency. Generating hESCs in the naïve state is necessary to overcome these problems and allow their application in regenerative medicine. Therefore, clarifying the mechanism of the transition between the naïve and primed states in pluripotent stem cells is important for the establishment of stable methods of generating naïve state hESCs. However, the signaling pathways which contribute to the transition between the naïve and primed states are still unclear. In this study, we carried out induction from mESCs to mEpiSC-like cells (mEpiSCLCs), and observed an increase in the activation of Fas signaling during the induction. The expression of Fgf5, an epiblast marker, was diminished by inhibition of Fas signaling using the caspase-8 and -3 blocking peptides, IETD and DEVD, respectively. Furthermore, during the induction, we observed increased expression of 3-O sulfated heparan sulfate (HS) structures synthesized by HS 3-O-sulfotransferase (3OST), which are recognized by the HS4C3 antibody (HS4C3-binding epitope). Knockdown of 3OST-5 reduced Fas signaling and the potential for the transition to mEpiSCLCs. This indicates that the HS4C3-binding epitope is necessary for the transition to the primed state. We propose that Fas signaling through the HS4C3-binding epitope contributes to the transition from the naïve state to the primed state.     

Isolation and Purification of Fucoidin from Brown Seaweed Pelvetia wrightii

A highly purified fucoidin was isolated from Pelvetia wrightii by an improved method, which involves the removal of alginate with calcium chloride solution and purification with cetylpyridinium chloride (CPC).

To this end, the critical salt concentrations of the cetylpyridinium complex of alginic acid and fucoidin in salt solutions (KCl, NaCl, CaCl₂) were determined.

The fucoidin of this alga contained both fucose and galactose as its constituents, in a ratio of approximately 10:1, and it is considered to be a galactofucan sulfate.     

GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.     

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2–PTEN–AKT Pathway and Placental Cell Proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2–PTEN–AKT pathway and placental cell proliferation.     

Purification and characterization of glutathione S-transferase isozymes in dog lens.

1. Two isozymes of glutathione S-transferase (GST-dl1 and GST-dl2) were purified to homogeneity from dog lens. 2. The subunit size and the isoelectric point were determined to be 24,000 and > pI 9.5 for GST-dl1 and 22,000 and pI 8.1 for GST-dl2. 3. It was judged that GST-dl1 is a class alpha enzyme and GST-dl2 belongs to class pi on the basis of their immunological properties and N-terminal amino acid sequences. 4. The expression pattern of glutathione S-transferase isoenzymes in dog lens is different from that in pig, rat and bovine lenses.     

Cloning and functional characterization of chicken p160 coactivator family members

The factors SRC-1, TIF2 and ACTR were identified as interacting with nuclear receptors in a highly ligand-dependent manner. Because the molecular mass of each of these factors is approximately 160 kDa, they are collectively termed p160 coactivators. So far, p160 coactivators have been cloned from human, mouse and Xenopus. We report here the cloning of the chicken homologues of p160 coactivator members. As in human and mouse, chicken has three p160 coactivators. Each gene encodes an approximately 160 kDa protein which exhibits 70-80% amino acid sequence identity to human and mouse p160 coactivators. Chicken p160 coactivators also have the property of interacting with several liganded nuclear receptors. Moreover, we describe an imperfect LXXLL sequence, termed NR box 4, which is located downstream of NR box 3 and conserved between evolutionarily diverse species. The loss of NR box 4 results in a decrease of interaction with the nuclear receptor, which indicates that NR box 4 is required for efficient interaction.