Human Silent Information Regulator Type 1 (SIRT1) is an NAD+-dependent deacetylase protein which is an intermediary of cellular metabolism in gene silencing and aging. SIRT1 has been extensively investigated and shown to delay senescence; however, less is known about the regulation of SIRT1 during aging. In this study, we show that the peroxisome proliferator-activated receptor-γ (PPARγ), which is a ligand-regulated modular nuclear receptor that governs adipocyte differentiation and inhibits cellular proliferation, inhibits SIRT1 expression at the transcriptional level. Moreover, both PPARγ and SIRT1 can bind the SIRT1 promoter. PPARγ directly interacts with SIRT1 and inhibits SIRT1 activity, forming a negative feedback and self-regulation loop. In addition, our data show that acetylation of PPARγ increased with increasing cell passage number. We propose that PPARγ is subject to regulation by acetylation and deacetylation via p300 and SIRT1 in cellular senescence. These results demonstrate a mutual regulation between PPARγ and SIRT1 and identify a new posttranslational modification that affects cellular senescence.

Sir2, a NAD-dependent deacetylase, modulates lifespan in yeasts, worms and flies. The SIRT1, mammalian homologue of Sir2, regulates signaling for favoring survival in stress. But whether SIRT1 has the function to influence cell viability and senescence under non-stressed conditions in human diploid fibroblasts is far from unknown. Our data showed that enforced SIRT1 expression promoted cell proliferation and antagonized cellular senescence with the characteristic features of delayed Senescence-Associated β-galactosidase (SA-β-gal) staining, reduced Senescence-Associated Heterochromatic Foci (SAHF) formation and G1 phase arrest, increased cell growth rate and extended cellular lifespan in human fibroblasts, while dominant-negative SIRT1 allele (H363Y) did not significantly affect cell growth and senescence but displayed a bit decreased lifespan.. Western blot results showed that SIRT1 reduced the expression of p16INK4A and promoted phosphorylation of Rb. Our data also exposed that overexpression of SIRT1 was accompanied by enhanced activation of ERK and S6K1 signaling. These effects were mimicked in both WI38 cells and 2BS cells by concentration-dependent resveratrol, a SIRT1 activator. It was noted that treatment of SIRT1-.transfected cells with Rapamycin, a mTOR inhibitor, reduced the phosphorylation of S6K1 and the expression of Id1, implying that SIRT1-induced phosphorylation of S6K1 may be partly for the decreased expression of p16INK4A and promoted phosphorylation of Rb in 2BS. It was also observed that the expression of SIRT1 and phosphorylation of ERK and S6K1 was declined in senescent 2BS. These findings suggested that SIRT1-promoted cell proliferation and antagonized cellular senescence in human diploid fibroblasts may be, in part, via the activation of ERK/ S6K1 signaling.

The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/ interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.The ras genes encode signal-transducing guanine nucleotide-binding proteins that are involved in mitogenic response and differentiation in eucaryotes (10,11,20,27). To carry out these functions, Ras proteins make physical contact with a variety of proteins that are classified as either regulators of Ras activity or signalling effectors. The regulators of Ras include the GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors. These proteins are able to directly stimulate the intrinsic properties of Ras that control its guanine nucleotide-bound state and hence its signalling activity (2, 6). In some cases, these proteins may also serve in an effector capacity to propagate signals from Ras. In the yeast Saccharomyces cerevisiae, adenylyl cyclase has been shown to act as the primary effector of Ras (29,30). In mammalian cells, Raf1 was identified as a common downstream component of Ras signalling pathways. Mammalian Raf1 and yeast adenylyl cyclase were shown to physically interact with Ras in both two-hybrid and direct binding experiments (13,18,19,31,33,34,36). Recently, direct interaction of phosphatidylinositol 3-kinase with Ras has also been demonstrated (23). These interactions display the predicted hallmarks of Ras-effector binding; they are enhanced by GTP-bound Ras and they are blocked by Ras effector domain mutations (26,35). Interestingly, although these Ras effectors (yeast adenylyl cyclase, Raf1, and phosphatidylinositol 3-kinase) have the same Ras interaction characteristics, they do not have any significant sequence similarity.We have previously described the isolation of human cDNAs that, when expressed in S. cerevisiae, are able to suppress an activated RAS2 allele (RAS2 V19 ). Yeast cells carrying this mutation have elevated cyclic AMP (cAMP) levels as a result of the overstimulation of adenylyl cyclase by Ras. These cells are exquisi...

Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the ''effector domain'' of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL-SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.RAS is a membrane-associated small G protein that is indirectly coupled to both receptor and nonreceptor tyrosine kinases. RAS activation is regulated at the level of guanine nucleotide binding: the GTP-and GDP-bound states of RAS are distinct in their structures (1, 2) and in their capacity to dispatch downstream signals through effector proteins. The interactions between RAS and its effectors require both the GTP-dependent structural confirmation of RAS and the presence of particular RAS residues including a short sequence referred to as the ''effector domain.'' Some effector pathways can lead to cell transformation and tumorigenesis when constitutively activated by a mutant RAS that is unregulated and predominantly in the active form.The first identified and best characterized effector of mammalian RAS is RAF1. Interaction with RAS leads to events that stimulate the protein kinase activity of RAF1 (3-5). Other potential RAS effectors have now emerged (reviewed in ref. 6). These include PI3 kinase (7), RAL-GDS (8-10), and RIN1 (11). Although these proteins do not share obvious sequence commonality, they each bind RAS in a manner that is conditional upon RAS activation (GTP binding) and an intact effector domain. In the case of RAF1, expression of the RAS-binding domain (RBD) alone has been shown to block normal RAS signaling (12, 13), presumably due to a nonproductive interaction with RAS that blocks access to wild-type RAF1 and other effector molecules.

Abstract-This paper investigates a genetic algorithm based hyperheuristic (hyper-GA) for scheduling geographically distributed training staff and courses. The aim of the hyper-GA is to evolve a good-quality heuristic for each given instance of the problem and use this to find a solution by applying a suitable ordering from a set of low-level heuristics. Since the user only supplies a number of low-level problem-specific heuristics and an evaluation function, the hyperheuristic can easily be reimplemented for a different type of problem, and we would expect it to be robust across a wide range of problem instances. We show that the problem can be solved successfully by a hyper-GA, presenting results for four versions of the hyper-GA as well as a range of simpler heuristics and applying them to five test data set

RIN1 was originally identified by its ability to physically bind to and interfere with activated Ras in yeast. Paradoxically, RIN1 potentiates the oncogenic activity of the BCR-ABL tyrosine kinase in hematopoietic cells and dramatically accelerates BCR-ABL-induced leukemias in mice. RIN1 rescues BCR-ABL mutants for transformation in a manner distinguishable from the cell cycle regulators c-Myc and cyclin D1 and the Ras connector Shc. These biological effects require tyrosine phosphorylation of RIN1 and binding of RIN1 to the Abl-SH2 and SH3 domains. RIN1 is tyrosine phosphorylated and is associated with BCR-ABL in human and murine leukemic cells. RIN1 exemplifies a new class of effector molecules dependent on the concerted action of the SH3, SH2, and catalytic domains of a cytoplasmic tyrosine kinase.

Sinusoidal modulation of radial speed around a circular path of tangentially oriented Gabor patches results in the percept of modulation of the radius. These patterns have been called motion radial frequency (RF) patterns. Sensitivity to these patterns has been attributed to global summation of local speed by mechanisms, analogous to those proposed to explain sensitivity to spatial RF patterns, which are sensitive to particular radial frequencies of speed modulation. We demonstrate that: Adaptation to spatial RF patterns results in a phase specific after effect in motion RF patterns and vice versa; the rate of change of perceived displacement of a Gabor patch with a moving grating with increasing speed of that grating, is independent of whether the patch is or is not incorporated into a circular path; in the absence of local motion direction cues, global integration across 1, 2 and 3 cycles of motion and spatial RF modulation conforms to a power function with the same index; and finally that the magnitude of the motion position illusion is sufficient and necessary to account for sensitivity to motion RF patterns. We therefore propose that motion RF patterns are analyzed by the same mechanisms responsible for sensitivity to spatial RF patterns using perceived rather than actual local positions of the Gabor patches.

Mechanisms governing the transcription of p16INK4a , one of the master regulators of cellular senescence, have been extensively studied. However, little is known about chromatin dynamics taking place at its promoter and distal enhancer. Here, we report that Forkhead box A1 protein (FOXA1) is significantly upregulated in both replicative and oncogene-induced senescence, and in turn activates transcription of p16INK4a through multiple mechanisms. In addition to acting as a classic sequence-specific transcriptional activator, FOXA1 binding leads to a decrease in nucleosome density at the p16INK4a promoter in senescent fibroblasts. Moreover, FOXA1, itself a direct target of Polycomb-mediated repression, antagonizes Polycomb function at the p16 INK4a locus. Finally, a systematic survey of putative FOXA1 binding sites in the p16INK4a genomic region revealed an B150 kb distal element that could loop back to the promoter and potentiate p16INK4a expression. Overall, our findings establish several mechanisms by which FOXA1 controls p16 INK4a expression during cellular senescence.