Although interactions between cell surface proteins and extracellular ligands are key to initiating embryonic stem cell differentiation to specific cell lineages, the plasma membrane protein components of these cells are largely unknown. We describe here a group of proteins expressed on the surface of the undifferentiated mouse embryonic stem cell line D3. These proteins were identified using a combination of cell surface labeling with biotin, subcellular fractionation of plasma membranes, and mass spectrometry-based protein identification technology. From 965 unique peptides carrying biotin labels, we assigned 324 proteins including 235 proteins that have putative signal sequences and/or transmembrane segments. Receptors, transporters, and cell adhesion molecules were the major classes of proteins identified. Besides known cell surface markers of embryonic stem cells, such as alkaline phosphatase, the analysis identified 59 clusters of differentiation-related molecules and more than 80 components of multiple cell signaling pathways that are characteristic of a number of different cell lineages. We identified receptors for leukemia-inhibitory factor, interleukin 6, and bone morphogenetic protein, which play critical roles in the maintenance of undifferentiated mouse embryonic stem cells. We also identified receptors for growth factors/cytokines, such as fibroblast growth factor, platelet-derived growth factor, ephrin, Hedgehog, and Wnt, which transduce signals for cell differentiation and embryonic development. Finally we iden- Embryonic stem (ES)1 cells are a unique type of cultured cells defined by two functional properties, self-renewal and pluripotency. In cultured mouse ES cells, the soluble cytokine leukemia-inhibitory factor (LIF) can support the undifferentiated state and promote self-renewal, whereas the formation of embryoid bodies followed by the addition of growth factors induces differentiation of the cells to specific fates (1-4). Interactions between cell surface proteins and soluble factors or insoluble ligands play important roles in regulating ES cell functions. However, the molecular mechanisms involved in these cellular processes remain unclear because we lack a thorough understanding of the properties and functions of ES cell surface proteins. The study of ES cell surface proteins is also attractive because some of these proteins can be used as non-destructive markers to characterize and/or isolate specific cell types. Thus, a large scale identification of ES cell surface proteins is key to understanding the regulation of ES cell function and to developing new research tools.Recent advances in MS-based proteomics have enabled us to identify a large number of proteins from a variety of membrane preparations (5-7). However, it is difficult to isolate From the ‡Division

A protein subset expressed in the mouse embryonic stem (ES) cell line, E14-1, was characterized by mass spectrometry-based protein identification technology and data analysis. In total, 1790 proteins including 365 potential nuclear and 260 membrane proteins were identified from tryptic digests of total cell lysates. The subset contained a variety of proteins in terms of physicochemical characteristics, subcellular localization, and biological function as defined by Gene Ontology annotation groups. In addition to many housekeeping proteins found in common with other cell types, the subset contained a group of regulatory proteins that may determine unique ES cell functions. We identified 39 transcription factors including Oct-3/4, Sox-2, and undifferentiated embryonic cell transcription factor I, which are characteristic of ES cells, 88 plasma membrane proteins including cell surface markers such as CD9 and CD81, 44 potential proteinaceous ligands for cell surface receptors including growth factors, cytokines, and hormones, and 100 cell signaling molecules. The subset also contained the products of 60 ES-specific and 41 stemness genes defined previously by the DNA microarray analysis of Ramalho-Santos et al. (Ramalho-Santos et al., Science 2002, 298, 597-600), as well as a number of components characteristic of differentiated cell types such as hematopoietic and neural cells. We also identified potential post-translational modifications in a number of ES cell proteins including five Lys acetylation sites and a single phosphorylation site. To our knowledge, this study provides the largest proteomic dataset characterized to date for a single mammalian cell species, and serves as a basic catalogue of a major proteomic subset that is expressed in mouse ES cells.

An extensive X‐ray study of CeO2–Nd2O3 solid solutions was performed, and the densities of solid solutions containing various concentrations of NdO1.5 were measured using several techniques. Solid solutions containing 0–80 mol% NdO1.5 were synthesized by coprecipitation from Ce(NO3)3 and Nd(NO3)3 aqueous solutions, and the coprecipitated samples were sintered at 1400°C. A fluorite structure was observed for CeO2–NdO1.5 solid solutions with 0–40 mol% NdO1.5, which changed to a rare earth C‐type structure at 45–75 mol% NdO1.5. The change in the lattice parameters of CeO2–NdO1.5 solid solutions, when plotted with respect to the NdO1.5 concentration, showed that the lattice parameters followed Vegard's law in both the fluorite and rare earth C‐type regions. The maximum solubility limit for NdO1.5 in CeO2 solid solution was approximately 75 mol%. The relationship between the density and the Nd concentration indicated that the defect structure followed the anion vacancy model over the entire range (0–70 mol% NdO1.5) of solid solution.

Fluorescence quenching by guanine allows DNA hybridization to be monitored and any point mutations in oligonucleotides to be detected. However, fluorescence quenching is often affected by untargeted guanine located in a protruding end (single-strand DNA) of the probe-target DNA duplex resulting in an unsatisfactory sensitivity. In the present study, we used enzymatic digestion of the protruding end of a probetarget DNA duplex to avoid interference by untargeted guanine on fluorescence quenching for detection of a nucleobase mutation. Enzymatic digestion of the protruding end of the DNA duplex fully prevented interference by untargeted guanine, and produced a marked difference in the quenching ratios (36% for wild-type, and 0% for mutant).