Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early Gi phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (GO). A Gi cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced Gl arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the Gl arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, Gl progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early Gl progression and growth in the absence of nutrients. INTRODUCTIONThe immunosuppressant rapamycin and the related compound FK506 exert their immunosuppressive effects by inhibiting intermediate steps in signal transduction that lead to T cell activation and proliferation (Heitman et al

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Sixty-two explants from peripheral blood, bone marrow and cerebral fluid of children with acute lymphoblastic leukemia (ALL) and leukemic transformed non-Hodgkin lymphoma (NHL) were cultivated for at least 8 weeks. Although lymphatic cells persisted up to 16 weeks in tissue culture, no proliferation was observed in 54 cultures. From the remaining cultures, eight permanently growing cell lines were obtained. Five of these were EBNA (Epstein-Barr virus-specific nuclear antigen)-positive. Three, however, were ENBA-negative and lacked Epstein-Barr virus genomes. Two cell lines (KM-3 and SH-2) expressed neither B nor T cell characteristics. One line (JM) expressed T cell characteristics and complement receptors. The growing lymphatic cells represented leukemic cells, since the pattern of cytochemical staining and that of membrane receptors of lymphoblasts from the same donor prior to cultivation were identical. All leukemic cell lines were derived from patients in relapse. In contrast, no proliferation of leukemic cells occurred in explains from patients revealing the first manifestation of the disease. These results suggest enhanced growth potential of lymphoblasts resisting antileukemic therapy.

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Also, two minor isomers were detected 6 3.07 (dt, J = 10, 2.5 Hz) and 6 3.31 (dm, J = I O Hz) in a 95.1:2.6:2.3 ratio. (la,2cr,3a)-N-Methyl-6-isopropylidene-3-propy~-4-cyclohexen~l,2dicarboximide: 'H NMR (CDC13, 200 MHz) 6 0.95 (t, J = 7.2 Hz, 3 H), 1.43-1.89 (m, 4 H), 1.91 (s, 3 H), 2.00 (s, 3 H), 2.29 (m, 1 H), 2.79 ,925,905, 840,790,735 cm-l; MS, m / e 364, 320, 308, 211. (la,2cr,3a)-N-Methyl-S-( tert -butyldimethylsiloxy)-6-isopropylidene-3-propyl-4-cyclohexene-l,2-carboximide: IH NMR (CDC13, 200 MHz) 1.30-1.86 (m. 4 H), 1.96 (s, 3 H), 2.13 (m, 1 H), 2.86 (s, 3 H), 3.06 (dd,

Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin ␣. The in vivo and in vitro data indicated that the prothymosin ␣-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin ␣ was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin ␣ by using prothymosin ␣ overproduction and mRNA interference approaches. Our data attribute to prothymosin ␣ the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin ␣ and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.

The subcellular distribution of Tor1p and Tor2p, two phosphatidylinositol kinase homologs and targets of the immunosuppressive drug rapamycin in Saccharomyces cerevisiae, was analyzed. We found that Tor protein is peripherally associated with membranes. Subcellular fractionation and immunofluorescence studies showed that Tor1p and Tor2p associate with the plasma membrane and a second fraction that is distinct from Golgi, vacuoles, mitochondria, and nucleus and may represent vesicular structures. Pulse-chase experiments showed that association of Tor protein with plasma membrane and the second compartment is fast, does not appear to involve components of endocytic, secretory, or Golgi to vacuole transport pathways, and is not affected by the immunosuppressive drug rapamycin. Deletion analysis reveals that two domains within Tor2p independently mediate localization to both compartments. These domains are composed of HEAT repeats that are thought to act as protein-protein interaction surfaces. Our studies therefore place Tor proteins at the site of action of their known downstream effectors and suggest that they may be part of a multiprotein complex.

Repair of the bicuspid aortic valve combined with root remodeling leads to excellent 10- and 15-year results. Cusp calcification and the need for partial cusp replacement are associated with valve failure.