T cell differentiation and repertoire selection depend critically on several distinct thymic epithelial cell types, whose lineage relationships are unclear. We have investigated these relationships via functional analysis of the epithelial populations within the thymic primordium. Here, we show that mAbs MTS20 and MTS24 identify a population of cells that, when purified and grafted ectopically, can differentiate into all known thymic epithelial cell types, attract lymphoid progenitors, and support CD4(+) and CD8(+) T cell development in nude mice. In contrast, other epithelial populations in the thymic primordium can fulfill none of these functions. These data establish that the MTS20(+)24(+) population is sufficient to generate a functional thymus in vivo and thus argue strongly that all thymic epithelial cell types derive from a common progenitor cell.

Aging is a universal but poorly understood biological process. Free radicals accumulate with age and have been proposed to be a major cause of aging. We measured genome-wide changes in transcript levels as a function of age in Drosophila melanogaster and compared these changes with those caused by paraquat, a free-radical generator. A number of genes exhibited changes in transcript levels with both age and paraquat treatment. We also found genes whose transcript levels changed with age but not with paraquat treatment. This study suggests that free radicals play an important role in regulating transcript levels in aging but that they are not the only factors. This genome-wide survey also identifies candidates for molecular markers of aging.A ging is a biological process universal to eukaryotic organisms, and its underlying mechanisms are under intensive study. Genetic analyses of yeast, nematode, fly, and mouse have uncovered a number of genes, whether mutated or misexpressed, that would increase the lifespans of these organisms (1). These genes include superoxide dismutase, a free-radical scavenger; methuselah, a potential G protein-coupled receptor, in Drosophila melanogaster; and p66 shc , an oxidative stress-response gene, in mice (2-5). Lifespan-related genes in Caenorhabditis elegans include clk-1, a gene involved in ubiquinone biosynthesis, and a group of genes involved in an insulin receptor-like signaling pathway: daf-2, age-1, and daf-16 (6, 7). Many mutations that increase lifespan also confer resistance to oxidative stress (1). This finding supports the free-radical hypothesis of aging, which suggests that reactive oxygen species that accumulate with increasing age cause oxidative damage to macromolecules (including nucleic acids, proteins, and lipids) and are causally linked to aging and death (8, 9). Free radicals have been found to regulate the expression of a number of genes that include antioxidant defense genes involved in repairing oxidative damage, as well as genes involved in inducing apoptosis (10, 11). The aging process is also accompanied by changes in the expression patterns of a number of genes (12)(13)(14). How the regulation of gene expression in aging correlates with that in response to oxidative stress, however, is understood poorly. Materials and MethodsConstruction of Microarray. We constructed a microarray containing 7,829 expressed sequence tags (ESTs). This set includes 221 ESTs provided by our lab and 7,608 ESTs generously supplied by K. White (Stanford University, Stanford, CA) and K. Burtis (University of California, Davis). The ESTs were amplified (as described by White et al., ref. 15), and the DNA was mechanically spotted onto polylysine-coated slides (as described by DeRisi et al., ref. 16).Calculation of Survival Rate and Preparation of Fly Tissues. Male flies (w 1118 ) raised in standard cornmeal agar medium, were collected within 24 h after eclosion (17). Approximately 200 flies were maintained in constant darkness in each food bottle at 26-27°C and 60-70% humidity an...

We have generated embryonic stem (ES) cells and transgenic mice carrying a tau-tagged green fluorescent protein (GFP) transgene under the control of a powerful promoter active in all cell types including those of the central nervous system. GFP requires no substrate and can be detected in fixed or living cells so is an attractive genetic marker. Tau-tagged GFP labels subcellular structures, including axons and the mitotic machinery, by binding the GFP to microtubules. This allows cell morphology to be visualized in exquisite detail. We test the application of cells derived from these mice in several types of cell-mixing experiments and demonstrate that the morphology of tau-GFP-expressing cells can be readily visualized after they have integrated into unlabeled host cells or tissues. We anticipate that these ES cells and transgenic mice will prove a novel and powerful tool for a wide variety of applications including the development of neural transplantation technologies in animal models and fundamental research into axon pathfinding mechanisms. A major advantage of the tau-GFP label is that it can be detected in living cells and labeled cells and their processes can be identified and subjected to a variety of manipulations such as electrophysiological cell recording.

Verotoxins (VTs) are important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), a group of bacteria associated with severe disease sequelae in humans. The potent cytotoxic activity of VTs is important in pathogenicity, resulting in the death of cells expressing receptor Gb3 (globotriaosylceramide). EHEC, particularly serotype O157:H7, frequently colonize reservoir hosts (such as cattle) in the absence of disease, however, the basis to avirulence in this host has been unclear. The objective of this study was assessment of interaction between VT and intestinal epithelium, which represents the major interface between the host and enteric organisms. Bovine intestinal epithelial cells expressed Gb3 in vitro in primary cell cultures, localizing specifically to proliferating crypt cells in corroboration with in situ immunohistological observations on intestinal mucosa. Expression of receptor by these cells contrasts with the absence of Gb3 on human intestinal epithelium in vivo. Despite receptor expression, VT exhibited no cytotoxic activity against bovine epithelial cells. Sub-cellular localization of VT indicated that this toxin was excluded from endoplasmic reticulum but localized to lysosomes, corresponding with abrogation of cytotoxicity. VT intracellular trafficking was unaffected by treatment of primary cell cultures with methyl-beta-cyclodextrin, indicating that Gb3 in these cells is not associated with lipid rafts but is randomly distributed in the membrane. The combination of Gb3 isoform, membrane distribution and VT trafficking correlate with observations of other receptor-positive cells that resist verocytotoxicity. These studies demonstrate that intestinal epithelium is an important determinant in VT interaction with major implications for the differential consequences of EHEC infection in reservoir hosts and humans.

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