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p57Kip2 Stabilizes the MyoD Protein by Inhibiting Cyclin E-Cdk2 Kinase Activity in Growing Myoblasts
Reynaud, Emmanuel G.
Pelpel, Karine
Guillier, Martine
Leibovitch, Marie Pierre
Leibovitch, Serge A.
Molecular and Cellular Biology
We show that expression of p57Kip2, a potent tight-binding inhibitor of several G1 cyclin–cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation. We examined the effect of p57Kip2 on the activity of the transcription factor MyoD. In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors. In addition, p57Kip2, p21Cip1, and p27Kip1 but not p16Ink4a induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57Kip2. Forced expression of p57Kip2 correlated with hypophosphorylation of MyoD in C2C12 myoblasts. A dominant-negative Cdk2 mutant arrested cells at the G1 phase transition and induced hypophosphorylation of MyoD. Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57Kip2. In addition, the NH2 domain of p57Kip2 necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts. Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57Kip2 could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.
Related Articles
Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells ▿ †
Croucher, David R.
Rickwood, Danny
Tactacan, Carole M.
Musgrove, Elizabeth A.
Daly, Roger J.
Molecular and Cellular Biology
The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
Related Articles
A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition
Fang, Yong
Wang, Yihong
Wang, Yulei
Meng, Yan
Zhu, Junlan
Jin, Honglei
Li, Jingxia
Zhang, Dongyun
Yu, Yonghui
Wu, Xue-Ru
Huang, Chuanshu
Biochemical Journal
2014;463(Pt 3):383-392.
p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis.
An inverse relationship between p27Kip1 and EGFR expression in parental T24 human bladder cancer cells and various human cancer tissues was found. Depletion of p27Kip1 in cells markedly elevated EGFR expression through transcriptional repression of Egfr by p27Kip1 via the JNK/c-Jun cascade.
bladder cancer; c-Jun N-terminal kinase (JNK)/c-Jun pathway; epidermal growth factor receptor (EGFR); p27Kip1; signal transduction pathway; AP-1, activator protein 1; BME, basal medium Eagle; CDK, cyclin-dependent kinase; DMEM, Dulbecco's modified Eagle's medium; EGFR, epidermal growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HSF-1, heat-shock factor 1; Hsp, heat-shock protein; IHC, immunohistochemistry; JNK, c-Jun N-terminal kinase; MEF, mouse embryonic fibroblast; RT, reverse transcription; SP1, specificity protein 1
Related Articles
Phosphorylation of p27Kip1 by JAK2 directly links cytokine receptor signaling to cell cycle control
Jäkel, H
Weinl, C
Hengst, L
Janus kinase 2 (JAK2) couples ligand activation of cell surface cytokine receptors to the regulation of cellular functions including cell cycle progression, differentiation and apoptosis. It thereby coordinates biological programs such as development and hematopoiesis. Unscheduled activation of JAK2 by point mutations or chromosomal translocations can induce hyperproliferation and hematological malignancies. Typical signal transduction by the JAK2 tyrosine kinase comprises phosphorylation of STAT transcription factors. In this study, we describe the identification of the cyclin-dependent kinase (CDK) inhibitor p27Kip1 as a novel JAK2 substrate. JAK2 can directly bind and phosphorylate p27Kip1. Both, the JAK2 FERM domain and its kinase domain bind to p27Kip1. JAK2 phosphorylates tyrosine residue 88 (Y88) of p27Kip1. We previously reported that Y88 phosphorylation of p27Kip1 by oncogenic tyrosine kinases impairs p27Kip1-mediated CDK inhibition, and initiates its ubiquitin-dependent proteasomal degradation. Consistently, we now find that active oncogenic JAK2V617F reduces p27Kip1 stability and protein levels in patient-derived cell lines harboring the mutant JAK2V617F allele. Moreover, tyrosine phosphorylation of p27Kip1 is impaired and p27Kip1 expression is restored upon JAK2V617F inactivation by small hairpin RNA-mediated knockdown or by the pyridone-containing tetracycle JAK inhibitor-I, indicating that direct phosphorylation of p27Kip1 can contribute to hyperproliferation of JAK2V617F-transformed cells. Activation of endogenous JAK2 by interleukin-3 (IL-3) induces Y88 phosphorylation of p27Kip1, thus unveiling a novel link between cytokine signaling and cell cycle control in non-transformed cells. Oncogenic tyrosine kinases could use this novel pathway to promote hyperproliferation in tumor cells.
cell cycle control; CDK inhibitors; p27Kip1; tyrosine kinases; JAK2; JAK2V617F
Related Articles
Partial hepatectomy in rats results in immediate down-regulation of p27Kip1 in residual liver tissue by transcriptional and post-translational processes
Rohlfing, Anne-Katrin
Trescher, Karoline
Hähnel, Juliane
Müller, Christian
Hildebrandt, Jan-Peter
Frontiers in Physiology
Purpose: The cyclin-dependent kinase (Cdk) inhibitor p27Kip1 may be involved in regulating re-entry of residual hepatocytes into the cell cycle upon loss of liver tissue by partial hepatectomy (PH). As yet, changes in Kip1 expression during the initial period following PH are not well-characterized. We investigated immediate changes in Kip1 mRNA and protein levels as well as changes in Kip1 phosphorylation in liver tissue within the relevant time window between surgery and the onset of DNA synthesis at 10–12 h.
Methods: We used real-time PCR, quantitative Western blotting, and immune histochemistry on tissue samples of adult rats obtained during or between 2 and 10 h after surgical removal of two thirds of the liver to analyze Kip1 mRNA or protein levels, respectively, or to quantify nuclear expression of Kip1.
Results: Kip1 mRNA was down-regulated within 4 h after PH by 60% and remained unchanged thereafter up to 10 h. With a lag phase of 2–3 h, Kip1-protein was down-regulated to a level of 40% of the control. The level of Thr187-phosphorylated Kip1 started to increase at 4 h and reached a maximum level at 8–10 h after PH. Kip1 immunoreactivity was observed in 30% of the hepatocytes before PH. Within 6–8 h after PH, more than half of the hepatocytes lost nuclear Kip1 signals. Kip1-specific micro-RNAs (miRNA221, miRNA222) were not changed upon PH.
Conclusions: A portion of hepatocytes in adult rats constitutively express Kip1 and down-regulate Kip1 immediately upon PH. This response involves transcriptional processes (loss of Kip1 mRNA) as well as accelerated degradation of existing protein (increase in pThr187-phosphorylation mediating polyubiquitinylation and proteasomal degradation of Kip1). Kip1 down-regulation occurs precisely within the intervall between surgery and onset of DNA synthesis which supports the hypothesis that it mediates activation of G0/0S-phase Cdk/cyclin-complexes and re-entry of hepatocytes into the cell cycle.
cell cycle regulator; cyclin-dependent kinase inhibitor; Kip1; compensatory growth; liver regeneration; rat hepatocytes; cell proliferation
Related Articles
The QKI-6 and QKI-7 RNA Binding Proteins Block Proliferation and Promote Schwann Cell Myelination
Larocque, Daniel
Fragoso, Gabriela
Huang, Jinghan
Mushynski, Walter E.
Loignon, Martin
Richard, Stéphane
Almazan, Guillermina
PLoS ONE
The quaking viable (qkv) mice have uncompacted myelin in their central and peripheral nervous system (CNS, PNS). The qk gene encodes 3 major alternatively spliced isoforms that contain unique sequence at their C-terminus dictating their cellular localization. QKI-5 is a nuclear isoform, whereas QKI-6 and QKI-7 are cytoplasmic isoforms. The qkv mice harbor an enhancer/promoter deletion that prevents the expression of isoforms QKI-6 and QKI-7 in myelinating cells resulting in a dysmyelination phenotype. It was shown that QKI regulates the differentiation of oligodendrocytes, the myelinating cells of the CNS, however, little is known about the role of the QKI proteins, or RNA binding proteins in PNS myelination.
Methodology/Principal Findings
To define the role of the QKI proteins in PNS myelination, we ectopically expressed QKI-6 and QKI-7 in primary rat Schwann cell/neuron from dorsal root ganglia cocultures. We show that the QKI isoforms blocked proliferation and promoted Schwann cell differentiation and myelination. In addition, these events were coordinated with elevated proteins levels of p27KIP1 and myelin basic protein (MBP), markers of Schwann cell differentiation. QKI-6 and QKI-7 expressing co-cultures contained myelinated fibers that had directionality and contained significantly thicker myelin, as assessed by electron microscopy. Moreover, QKI-deficient Schwann cells had reduced levels of MBP, p27KIP1 and Krox-20 mRNAs, as assessed by quantitative RT-PCR.
Our findings suggest that the QKI-6 and QKI-7 RNA binding proteins are positive regulators of PNS myelination and show that the QKI RNA binding proteins play a key role in Schwann cell differentiation and myelination.
Related Articles
Homeodomain Transcription Factor Phox2a, via Cyclic AMP-Mediated Activation, Induces p27Kip1 Transcription, Coordinating Neural Progenitor Cell Cycle Exit and Differentiation▿ †
Paris, Maryline
Wang, Wen-Horng
Shin, Min-Hwa
Franklin, David S.
Andrisani, Ourania M.
Molecular and Cellular Biology
Mechanisms coordinating neural progenitor cell cycle exit and differentiation are incompletely understood. The cyclin-dependent kinase inhibitor p27Kip1 is transcriptionally induced, switching specific neural progenitors from proliferation to differentiation. However, neuronal differentiation-specific transcription factors mediating p27Kip1 transcription have not been identified. We demonstrate the homeodomain transcription factor Phox2a, required for central nervous system (CNS)- and neural crest (NC)-derived noradrenergic neuron differentiation, coordinates cell cycle exit and differentiation by inducing p27Kip1 transcription. Phox2a transcription and activation in the CNS-derived CAD cell line and primary NC cells is mediated by combined cyclic AMP (cAMP) and bone morphogenetic protein 2 (BMP2) signaling. In the CAD cellular model, cAMP and BMP2 signaling initially induces proliferation of the undifferentiated precursors, followed by p27Kip1 transcription, G1 arrest, and neuronal differentiation. Small interfering RNA silencing of either Phox2a or p27Kip1 suppresses p27Kip1 transcription and neuronal differentiation, suggesting a causal link between p27Kip1 expression and differentiation. Conversely, ectopic Phox2a expression via the Tet-off expression system promotes accelerated CAD cell neuronal differentiation and p27Kip1 transcription only in the presence of cAMP signaling. Importantly, endogenous or ectopically expressed Phox2a activated by cAMP signaling binds homeodomain cis-acting elements of the p27Kip1 promoter in vivo and mediates p27Kip1-luciferase expression in CAD and NC cells. We conclude that developmental cues of cAMP signaling causally link Phox2a activation with p27Kip1 transcription, thereby coordinating neural progenitor cell cycle exit and differentiation.
Related Articles
Vitamin E δ-Tocotrienol Induces p27Kip1-Dependent Cell-Cycle Arrest in Pancreatic Cancer Cells via an E2F-1-Dependent Mechanism
Hodul, Pamela J.
Dong, Yanbin
Husain, Kazim
Pimiento, Jose M.
Chen, Jiandong
Zhang, Anying
Francois, Rony
Pledger, Warren J.
Coppola, Domenico
Sebti, Said M.
Chen, Dung-Tsa
Malafa, Mokenge P.
PLoS ONE
Vitamin E δ-tocotrienol has been shown to have antitumor activity, but the precise molecular mechanism by which it inhibits the proliferation of cancer cells remains unclear. Here, we demonstrated that δ-tocotrienol exerted significant cell growth inhibition pancreatic ductal cancer (PDCA) cells without affecting normal human pancreatic ductal epithelial cell growth. We also showed that δ-tocotrienol-induced growth inhibition occurred concomitantly with G1 cell-cycle arrest and increased p27Kip1 nuclear accumulation. This finding is significant considering that loss of nuclear p27Kip1 expression is a well-established adverse prognostic factor in PDCA. Furthermore, δ-tocotrienol inactivated RAF-MEK-ERK signaling, a pathway known to suppress p27Kip1 expression. To determine whether p27Kip1 induction is required for δ-tocotrienol inhibition of PDCA cell proliferation, we stably silenced the CDKN1B gene, encoding p27Kip1, in MIAPaCa-2 PDCA cells and demonstrated that p27Kip1 silencing suppressed cell-cycle arrest induced by δ-tocotrienol. Furthermore, δ-tocotrienol induced p27Kip1 mRNA expression but not its protein degradation. p27Kip1 gene promoter activity was induced by δ-tocotrienol through the promoter's E2F-1 binding site, and this activity was attenuated by E2F-1 depletion using E2F-1 small interfering RNA. Finally, decreased proliferation, mediated by Ki67 and p27Kip1 expression by δ-tocotrienol, was confirmed in vivo in a nude mouse xenograft pancreatic cancer model. Our findings reveal a new mechanism, dependent on p27Kip1 induction, by which δ-tocotrienol can inhibit proliferation in PDCA cells, providing a new rationale for p27Kip1 as a biomarker for δ-tocotrienol efficacy in pancreatic cancer prevention and therapy.
Related Articles
Gfer inhibits Jab1-mediated degradation of p27kip1 to restrict proliferation of hematopoietic stem cells
Teng, Ellen C.
Todd, Lance R.
Ribar, Thomas J.
Lento, William
Dimascio, Leah
Means, Anthony R.
Sankar, Uma
Molecular Biology of the Cell
Genes that promote hematopoietic stem cell (HSC) quiescence play important roles in the maintenance of their function. Here we show a novel role for the evolutionarily conserved flavin adenine dinucleotide (FAD)-dependent sulfhydryl oxidase, growth factor erv1-like (Gfer) in the restriction of HSC proliferation through its inhibition of Jab1-mediated turnover of p27kip1.
Growth factor erv1-like (Gfer) is an evolutionarily conserved sulfhydryl oxidase that is enriched in embryonic and adult stem cells and plays an essential prosurvival role in pluripotent embryonic stem cells. Here we show that knockdown (KD) of Gfer in hematopoietic stem cells (HSCs) compromises their in vivo engraftment potential and triggers a hyper-proliferative response that leads to their exhaustion. KD of Gfer in HSCs does not elicit a significant alteration of mitochondrial morphology or loss of cell viability. However, these cells possess significantly reduced levels of the cyclin-dependent kinase inhibitor p27kip1. In contrast, overexpression of Gfer in HSCs results in significantly elevated total and nuclear p27kip1. KD of Gfer results in enhanced binding of p27kip1 to its inhibitor, the COP9 signalosome subunit jun activation-domain binding protein 1 (Jab1), leading to its down-regulation. Conversely, overexpression of Gfer results in its enhanced binding to Jab1 and inhibition of the Jab1-p27kip1 interaction. Furthermore, normalization of p27kip1 in Gfer-KD HSCs rescues their in vitro proliferation deficits. Taken together, our data demonstrate the presence of a novel Gfer-Jab1-p27kip1 pathway in HSCs that functions to restrict abnormal proliferation.
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Repression of p27kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells.
Agrawal, D
Hauser, P
McPherson, F
Dong, F
Garcia, A
Pledger, W J
Molecular and Cellular Biology
We have investigated the regulation of p27kip1, a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G0) to a proliferative (G1) state. The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1, as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1. Analysis of metabolically labelled cells revealed that cyclin D2, cyclin D3, and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor (PDGF) treatment. Furthermore, the decline in p27kip1 and reduced association with cyclin D3, initiated by the addition of PDGF but not plasma-derived factors, suggested that these changes are involved in competence, the first step in the exit from G0. Synthesis of p27kip1 as determined by incorporation of [35S]methionine was repressed upon mitogenic stimulation, and PDGF was sufficient to elicit this repression within 2 to 3 h. Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation. Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1. Northern (RNA) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA, suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism.
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Hes1 Directly Controls Cell Proliferation through the Transcriptional Repression of p27Kip1
Murata, Kaoru
Hattori, Masakazu
Hirai, Norihito
Shinozuka, Yoriko
Hirata, Hiromi
Kageyama, Ryoichiro
Sakai, Toshiyuki
Minato, Nagahiro
Molecular and Cellular Biology
A transcriptional regulator, Hes1, plays crucial roles in the control of differentiation and proliferation of neuronal, endocrine, and T-lymphocyte progenitors during development. Mechanisms for the regulation of cell proliferation by Hes1, however, remain to be verified. In embryonic carcinoma cells, endogenous Hes1 expression was repressed by retinoic acid in concord with enhanced p27Kip1 expression and cell cycle arrest. Conversely, conditional expression of a moderate but not maximal level of Hes1 in HeLa cells by a tetracycline-inducible system resulted in reduced p27Kip1 expression, which was attributed to decreased basal transcript rather than enhanced proteasomal degradation, with concomitant increases in the growth rate and saturation density. Hes1 induction repressed the promoter activity of a 5′ flanking basal enhancer region of p27Kip1 gene in a manner dependent on Hes1 expression levels, and this was mediated by its binding to class C sites in the promoter region. Finally, hypoplastic fetal thymi, as well as livers and brains of Hes1-deficient mice, showed significantly increased p27Kip1 transcripts compared with those of control littermates. These results have suggested that Hes1 directly contributes to the promotion of progenitor cell proliferation through transcriptional repression of a cyclin-dependent kinase inhibitor, p27Kip1.
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The Cyclin-Dependent Kinase Inhibitor p57Kip2 Regulates Cell Cycle Exit, Differentiation, and Migration of Embryonic Cerebral Cortical Precursors
Tury, Anna
Mairet-Coello, Georges
DiCicco-Bloom, Emanuel
Cerebral Cortex (New York, NY)
Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57Kip2 and p27Kip1, control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57Kip2 remain poorly defined. Using in vivo and culture approaches, we show p57Kip2 overexpression at E14.5–15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57Kip2-deficient precursors. Studies at later ages indicate p57Kip2 overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57Kip2 overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57Kip2 was twice as effective as p27Kip1 in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57Kip2 alone inhibits in vivo migration. Furthermore, p57Kip2 effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27Kip1 studies report cell cycle-independent functions. These observations suggest p57Kip2 coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27Kip1.
gliogenesis; in utero electroporation; neurite outgrowth; neurogenesis; transfection
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Forkhead Transcription Factor FKHR-L1 Modulates Cytokine-Dependent Transcriptional Regulation of p27KIP1
Dijkers, Pascale F.
Medema, Rene H.
Pals, Cornelieke
Banerji, Lolita
Thomas, N. Shaun B.
Lam, Eric W.-F.
Burgering, Boudewijn M. T.
Raaijmakers, Jan A. M.
Lammers, Jan-Willem J.
Koenderman, Leo
Coffer, Paul J.
Molecular and Cellular Biology
Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27KIP1 through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27KIP1 promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27KIP1 appears to be critical in the regulation of cell survival since mere ectopic expression of p27KIP1 was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27KIP1 null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27KIP1 transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.
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E Proteins and Id2 Converge on p57Kip2 To Regulate Cell Cycle in Neural Cells
Rothschild, Gerson
Zhao, Xudong
Iavarone, Antonio
Lasorella, Anna
Molecular and Cellular Biology
A precise balance between proliferation and differentiation must be maintained during neural development to obtain the correct proportion of differentiated cell types in the adult nervous system. The basic helix-loop-helix (bHLH) transcription factors known as E proteins and their natural inhibitors, the Id proteins, control the timing of differentiation and terminal exit from the cell cycle. Here we show that progression into S phase of human neuroblastoma cells is prevented by E proteins and promoted by Id2. Cyclin-dependent kinase inhibitors (CKI) have been identified as key effectors of cell cycle arrest in differentiating cells. However, p57Kip2 is the only CKI that is absolutely required for normal development. Through the use of global gene expression analysis in neuroblastoma cells engineered to acutely express the E protein E47 and Id2, we find that p57Kip2 is a target of E47. Consistent with the role of Id proteins, Id2 prevents activation of p57Kip2 expression, and the retinoblastoma tumor suppressor protein, a known Id2 inhibitor, counters this activity. The strong E47-mediated inhibition of entry into S phase is entirely reversed in cells in which expression of p57Kip2 is silenced by RNA interference. During brain development, expression of p57Kip2 is opposite that of Id2. Our findings identify p57Kip2 as a functionally relevant target recruited by bHLH transcription factors to induce cell cycle arrest in developing neuroblasts and suggest that deregulated expression of Id proteins may be an epigenetic mechanism to silence expression of this CKI in neural tumors.
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Polypyrimidine Tract-Binding Protein Enhances the Internal Ribosomal Entry Site-Dependent Translation of p27Kip1 mRNA and Modulates Transition from G1 to S Phase
Cho, Sungchan
Kim, Jong Heon
Back, Sung Hoon
Jang, Sung Key
Molecular and Cellular Biology
The p27Kip1 protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27Kip1 is directed by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of p27Kip1 mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27Kip1 mRNA through an interaction with the IRES element. We found that addition of PTB to an in vitro translation system and overexpression of PTB in 293T cells augmented the IRES activity of p27Kip1 mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27Kip1 mRNA. Moreover, the G1 phase in the cell cycle (which is maintained in part by p27Kip1) was shortened in cells depleted of PTB by siRNA knockdown. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation in HL60 cells was used to examine PTB-induced modulation of p27Kip1 protein synthesis during differentiation. The IRES activity of p27Kip1 mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27Kip1 mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27Kip1 mRNA.
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Differential Regulation of P27Kip1 Expression by Mitogenic and Hypertrophic Factors
Servant, Marc J.
Coulombe, Philippe
Turgeon, Benjamin
Meloche, Sylvain
The Journal of Cell Biology
Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G1 cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G1 phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27Kip1 expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27Kip1 protein. The time course of p27Kip1 decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27Kip1 synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-β1 by aortic SMC. These results identify p27Kip1 as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.
growth factors; cell cycle; CDK inhibitors; gene expression; smooth muscle cells
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ErbB2 Potentiates Breast Tumor Proliferation through Modulation of p27Kip1-Cdk2 Complex Formation: Receptor Overexpression Does Not Determine Growth Dependency
Lane, Heidi A.
Beuvink, Iwan
Motoyama, Andrea B.
Daly, John M.
Neve, Richard M.
Hynes, Nancy E.
Molecular and Cellular Biology
Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G1 arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27Kip1, and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27Kip1 onto Cdk2 complexes, an event preceding increased p27Kip1 expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27Kip1 sequestration) and the loss of p27Kip1 from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27Kip1 redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27Kip1 protein levels remained constant. Antisense-mediated inhibition of p27Kip1 expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27Kip1 accumulation, p27Kip1 redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G1 block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27Kip1 sequestration proteins, thus deregulating the G1/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.
Related Articles
Expression of constitutively active 4EBP-1 enhances p27Kip1 expression and inhibits proliferation of MCF7 breast cancer cells
Jiang, Hong
Coleman, Jennifer
Miskimins, Robin
Miskimins, W Keith
Cancer Cell International
Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells.
A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1.
Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.
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Review of cellular and molecular pathways linking thrombosis and innate immune system during sepsis
Konecny, Filip A.
Journal of Research in Medical Sciences : The Official Journal of Isfahan University of Medical Sciences
Cellular and molecular pathways link thrombosis and innate immune system during sepsis. Extrinsic pathway activation of protease thrombin through FVIIa and tissue factor (TF) in sepsis help activate its endothelial cell (EC) membrane Protease Activated Receptor 1 (PAR-1). Thrombin adjusts the EC cycle through activation of G proteins (G12/13), and later through Rho GEFs (guanine nucleotide exchange factors), and provides a path for Rho GTPases mediated cytoskeletal responses involved in shape change and permeability of the EC membrane leading to an increase of leakage of plasma proteins.
At the same time, thrombin stimulates spontaneous mitogenesis by inducing activation of the cell cycle from G0-G1 to S by down-regulation of p27Kip1, a negative regulator of the cell cycle, in association with the up-regulation of S-phase kinase associated protein 2 (Skp2). After transport in cytoplasm, p27 Kip1 binds to RhoA thus prevent activation of RhoA by GEFs, thus inhibit GDP-GTP exchange mediated by GEFs. In cytoplasm, releasing factor (RF) p27-RF-Rho is able to free RhoA. P27 RF-Rho binds p27kip1 and prevents p27kip1 from binding to RhoA. Exposed RhoA is later able to increase the expression of the F-box protein Skp2, after its Akt triggered 14-3-3-β-dependent cytoplasm relocation. Skp2 increases cytoplasm ubiquitination-dependent degradation of p27Kip1. Additionally, after septic induction of canonical NF-kB pathway in EC through TLR4/IRAK4/TRAF/IkB, an IKKα dimer phosphorylates the p52 precursor NF-kB2/p100, leading to p100 processing and translocation of RelB/p52 to the nucleus. By controlling the NF-kB-RelB complex, IKKα signaling regulates the transcription of the Skp2 and correspondingly p27Kip1.
Inherited Immune System; Thrombin; PAR; TLR; Cell Cycle
Related Articles
A novel role for the cyclin-dependent kinase inhibitor p27Kip1 in angiotensin II–stimulated vascular smooth muscle cell hypertrophy
Braun-Dullaeus, Ruediger C.
Mann, Michael J.
Ziegler, Almut
von der Leyen, Heiko E.
Dzau, Victor J.
Journal of Clinical Investigation
Angiotensin II (Ang II) has been shown to stimulate either hypertrophy or hyperplasia. We postulated that the differential response of vascular smooth muscle cells (VSMCs) to Ang II is mediated by the cyclin-dependent kinase (Cdk) inhibitor p27Kip1, which is abundant in quiescent cells and drops after serum stimulation. Ang II treatment (100 nM) of quiescent VSMCs led to upregulation of the cell-cycle regulatory proteins cyclin D1, Cdk2, proliferating cell nuclear antigen, and Cdk1. p27Kip1 levels, however, remained high, and the activation of the G1-phase Cdk2 was inhibited as the cells underwent hypertrophy. Overexpression of p27Kip1 cDNA inhibited serum-stimulated [3H]thymidine incorporation compared with control-transfected cells. This cell-cycle inhibition was associated with cellular hypertrophy, as reflected by an increase in the [3H]leucine/[3H]thymidine incorporation ratio and by an increase in forward-angle light scatter during flow cytometry at 48 hours after transfection. The role of p27Kip1 in modulating the hypertrophic response of VSMCs to Ang II was further tested by antisense oligodeoxynucleotide (ODN) inhibition of p27Kip1 expression. Ang II stimulated an increase in [3H]thymidine incorporation and the percentage of S-phase cells in antisense ODN–transfected cells but not in control ODN–transfected cells. We conclude that p27Kip1 plays a role in mediating VSMC hypertrophy. Ang II stimulation of quiescent cells in which p27Kip1 levels are high results in hypertrophy but promotes hyperplasia when levels of p27Kip1 are low, as in the presence of other growth factors.
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Oligodendroglial Maturation Is Dependent on Intracellular Protein Shuttling
Göttle, Peter
Sabo, Jennifer K.
Heinen, André
Venables, Gene
Torres, Klintsy
Tzekova, Nevena
Parras, Carlos M.
Kremer, David
Hartung, Hans-Peter
Cate, Holly S.
Küry, Patrick
The Journal of Neuroscience
Multiple sclerosis is an autoimmune disease of the CNS resulting in degeneration of myelin sheaths and loss of oligodendrocytes, which means that protection and electrical insulation of axons and rapid signal propagation are impaired, leading to axonal damage and permanent disabilities. Partial replacement of lost oligodendrocytes and remyelination can occur as a result of activation and recruitment of resident oligodendroglial precursor cells. However, the overall remyelination capacity remains inefficient because precursor cells often fail to generate new oligodendrocytes. Increasing evidence points to the existence of several molecular inhibitors that act on these cells and interfere with their cellular maturation. The p57kip2 gene encodes one such potent inhibitor of oligodendroglial differentiation and this study sheds light on the underlying mode of action. We found that subcellular distribution of the p57kip2 protein changed during differentiation of rat, mouse, and human oligodendroglial cells both in vivo and in vitro. Nuclear export of p57kip2 was correlated with promoted myelin expression, higher morphological phenotypes, and enhanced myelination in vitro. In contrast, nuclear accumulation of p57kip2 resulted in blocked oligodendroglial differentiation. Experimental evidence suggests that the inhibitory role of p57kip2 depends on specific interactions with binding proteins such as LIMK-1, CDK2, Mash1, and Hes5 either by controlling their site of action or their activity. Because functional restoration in demyelinating diseases critically depends on the successful generation of oligodendroglial cells, a therapeutic need that is currently unmet, the regulatory mechanism described here might be of particular interest for identifying suitable drug targets and devising novel therapeutic approaches.
inhibitor; mode of action; myelin repair; nuclear export; p57kip2; regeneration
Related Articles
p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.
Shiyanov, P
Hayes, S
Chen, N
Pestov, D G
Lau, L F
Raychaudhuri, P
Molecular Biology of the Cell
p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.
Related Articles
Epidermal growth factor upregulates Skp2/Cks1 and p27kip1 in human extrahepatic cholangiocarcinoma cells
Kim, Ja-yeon
Kim, Hong Joo
Park, Jung Ho
Park, Dong Il
Cho, Yong Kyun
Sohn, Chong Il
Jeon, Woo Kyu
Kim, Byung Ik
Kim, Dong Hoon
Chae, Seoung Wan
Sohn, Jin Hee
World Journal of Gastroenterology : WJG
AIM: To evaluate the expression status of S-phase kinase-associated protein 2 (Skp2)/cyclin-dependent kinases regulatory subunit 1 (Cks1) and p27kip1, and assess the prognostic significance of Skp2/Cks1 expression with p27kip1 in patients with extrahepatic cholangiocarcinoma.
METHODS: Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December 1994 to March 2008 were enrolled. Immunohistochemical staining for Skp2, Cks1, p27kip1, and Ki67, along with other relevant molecular biologic experiments, were performed.
RESULTS: By Cox regression analyses, advanced age (> 65 years), advanced AJCC tumor stage, poorly differentiated histology, and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma. Exogenous epidermal growth factor (EGF, especially 0.1-10 ng/mL) significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1 in SNU-1196, SNU-1079, and SNU-245 cells. The protein levels of Skp2/Cks1 (from nuclear lysates) and p27kip1 (from cytosolic lysate) were also significantly increased in these cells. There were significant reductions in the protein levels of Skp2/Cks1 and p27kip1 (from nuclear lysate) after the treatment of LY294002. By chromatin immunoprecipitation assay, we found that E2F1 transcription factor directly binds to the promoter site of Skp2.
CONCLUSION: Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma. EGF upregulates the mRNA and protein levels of Skp2/Cks1 and p27kip1 via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.
S-phase kinase-associated protein 2; Cyclin-dependent kinases regulatory subunit 1; P27kip1; Cholangiocarcinoma; E2F1; PI3K/Akt
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A 39-kD DNA-binding protein from mouse brain stimulates transcription of myelin basic protein gene in oligodendrocytic cells
The Journal of Cell Biology
The MB1 regulatory sequence of the myelin basic protein (MBP) gene spanning between nucleotides -14 to -50 with respect to the transcription start site is critical for cell type-specific transcription of the MBP gene, which encodes the major protein component of myelin sheath in cells derived from the central nervous system (CNS). This regulatory sequence has the ability to interact with a developmentally controlled DNA-binding protein from mouse brain that stimulates transcription of MBP promoter in an in vitro system (Haas, S., J. Gordon, and K. Khalili. 1993. Mol. Cell. Biol. 13:3103-3112). Here, we report the purification of a 39-kD protein from mouse brain tissue at the peak of myelination and MBP production that binds to the MB1 regulatory motif. Following partial amino acid sequence analysis, we have identified a complementary DNA encoding a 39-kD DNA-binding protein called pur alpha. Expression of pur alpha cDNA in the prokaryotic and eukaryotic cells resulted in the synthesis of a protein with characteristics similar to the purified brain-derived 39-kD protein in band shift competition assays. Cotransfection of the recombinant pur alpha expressor plasmid with MBP promoter construct indicated that Pur alpha stimulates transcription of the MBP promoter in oligodendrocytic cells, and that the nucleotide sequence required for binding of the 39-kD Pur alpha to DNA within the MB1 region is crucial for this activity. Moreover, transient expression of Pur alpha caused elevation in the level of endogenous MBP RNA in oligodendrocytic cells. Thus, Pur alpha, a sequence-specific DNA-binding protein upon binding to MB1 regulatory region may play a significant role in determining the cell type-specific expression of MBP in brain.
Related Articles
Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1
Kadowaki, Maiko
Mizuno, Shiro
Demura, Yoshiki
Ameshima, Shingo
Miyamori, Isamu
Ishizaki, Takeshi
Respiratory Research
Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.
We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.
Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.
Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.
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2000 Chrysler Concorde Q&A
2000 Chrysler Concorde Question: My car will not idle fast enough. Is this an easy fix?
It is idling at or below 5000 rpms and will die when sitting still. Drives great going down the road. Just had new H2O pump put on and timing belt. - Visitor
Answer 1
5000 RPM is way to high, an engine should idle at 500 to 700RPMs. A lot of big work was just recently done on your car, I suspect some vacuum hose or electrical connector was inadvertently knocked off causing your idle problem. The idle speed is electronically controlled on your car by the computer in your car but even it does not have the scope to raise the idle if there is a vacuum leak. Do a visual check in the engine compartment yourself to see if a vacuum hose is off or see if you hear a hissing sound at engine idle when you lift the hood. Is there a Check Engine Light on? Many automotive repair shops have a machine that generates an inert non flammable low pressure smoke that looks similar to cigar smoke. The intake manifold is sealed off, and smoke is put into the engines intake manifold. The smoke then fills the engines crankcase and if there are any vacuum leaks from gaskets, hoses, or housing smoke emits from the source of the leak. Some mechanics will use propane or aerosol carburetor cleaner, with the engine running they will spray the cleaner or propane around the intake manifold and vacuum hoses. A leak will cause a "lean mixture" condition, when the flammable fuel (carburetor cleaner or propane) comes in contact with the source of the vacuum leak the engine idle speed will raise as the engine has received "fuel". this method of finding a vacuum leak works but is not recommended as it can easily cause a fire. Even just having a professional grade scan tool attached to your car will retrieve live sensor data that may indicate where the problem really is, for example if the fuel trim data shows adding fuel by 15% or so there is likely to be a leak, if it does not show "adding fuel" there is probably not a vacuum leak but its hard to give advice without being able to do first hand basic tests. - patrick mannion
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subset: commoncrawl


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Alera SedinaAG Series Hutch With Sliding Doors - Espresso
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A contemporary classic with clean lines, elevated top and modern silver metalwork. Thermally fused wood grain laminate is scratch-, dent- and water-resistant. Compartmentalized storage with adjustable shelves and two sliding doors.
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realmagick.com The shrine of knowledge.
Viluppuram Kalrayan Hills
A selection of articles related to viluppuram kalrayan hills.
Original articles from our library related to the Viluppuram Kalrayan Hills. See Table of Contents for further available material (downloadable resources) on Viluppuram Kalrayan Hills.
- Lughnasadh
- Lughnasadh (pronounced Loo-nas-ah), August 1st, is the first harvest festival in the Celtic and Neo-Pagan Year. Like all Celtic Fire Festivals, it begins on the eve of the actual day. Although it later became known as Christian Lammas, it still survives in...
Holidays >> Lammas
- King James Bible: Luke, Chapter 23
- Chapter 23 23:1 And the whole multitude of them arose, and led him unto Pilate. 23:2 And they began to accuse him, saying, We found this fellow perverting the nation, and forbidding to give tribute to Caesar, saying that he himself is Christ a King. 23:3 And...
New Testament >> Luke
- Indian History Rewritten
- The Indian civilisation, considered to be the product of clash and subsequent inter mixture of the white-skinned civilised invaders and the dark savages native to India, has been rightly questioned in recent years, and the mischievously fabricated history of...
History & Anthropology >> Indic / Vedic
- The Celtic Vedic Connection, Part I
- Of all the great ancient cultures perhaps no two share more parallels than those of the Celtic and Vedic peoples. A deep rooted affinity runs between them, what is present in one is mirrored in the other. Myths, Gods, Goddesses, even fairy tales bear a...
Religions >> Druidism
- Elemental Associations
- The earliest of Greek natural philosophers, Thales, in considering which was the most basic element in the cosmos, from which the cosmos was made, supposed that the cosmos was made of Water. Later, Heraclitus decided that the most basic element in the cosmos...
Magick >> The Elements
- King James Bible: Numbers, Chapter 23
- Chapter 23 23:1 And Balaam said unto Balak, Build me here seven altars, and prepare me here seven oxen and seven rams. 23:2 And Balak did as Balaam had spoken; and Balak and Balaam offered on every altar a bullock and a ram. 23:3 And Balaam said unto Balak,...
Old Testament >> Numbers
Viluppuram Kalrayan Hills is described in multiple online sources, as addition to our editors' articles, see section below for printable documents, Viluppuram Kalrayan Hills books and related discussion.
Suggested Pdf Resources
- 1 Villupuram. Kalrayan Hills. 2 Thiruvannamalai.
- www.tn.gov.in
- TRIBAL DEVELOPMENT PLAN January 25, 2005
- Feb 28, 2005 Vellimalai of Kalrayan Hills in Villupuram District with an outlay of Rs. 2.95 Crores .
- www-wds.worldbank.org
- www.iamwarm.gov.in
- Ethnomedicinal studies of the woody species of Kalrayan
- Long) and in the Vellimali forest of Kalrayan hills. (Fig.1) (600-900m elevation) located in Villupuram district of Tamil Nadu (11°38´ and 12°01 N Lat.
- nopr.niscair.res.in
- The Impact of NREGA on Rural-Urban Migration: Field survey of
- The Kalrayan Hills Story According to the Government. 4. Findings .
- ccs.in
Suggested Web Resources
- District Tourist Places - Viluppuram District
- Viluppuram is the district headquarters of the Viluppuram District, which was Nestled on three hills, and enclosed by a huge rampart 60 feet thick, stands the .. Kanchipuram and settled in kalrayan hills.
- www.viluppuram.tn.nic.in
- Kalrayan Hills - Wikipedia, the free encyclopedia
- The Kalrayan Hills (Tamil : கல்வராயன் மலை) are a major range of The range serves as a boundary between the Salem and Villupuram districts.
- en.wikipedia.org
- The Hindu : Destination Cool
- www.hindu.com
- 1 Villupuram. Kalrayan Hills. 2 Thiruvannamalai.
- www.tn.gov.in
- Kalrayan Hills, Tamil Nadu
- Jan 11, 2010 Kalrayan Hills, Tamil Nadu - Informative & researched article on Kalrayan Hills, It separates the district of Salem and Villupuram districts.
- www.indianetzone.com
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As an Allstate Agent in Menlo Park, I know many local families. My knowledge and understanding of the people in this community help me provide customers with an outstanding level of service. I look forward to helping families like yours protect the things that are important - your family, home, car, boat, and more.
At Allstate, we believe that the solution to stress-free and hassle-free life insurance enrollment is simple: it involves taking the time to discuss your needs and budget. In many cases, a term life insurance policy may be what you're looking for. It offers flexible layers of coverage and customizable term lengths. Contact agent Harold Lorber at (650)462-8733 to learn more about Allstate's term life insurance options.
Harold Lorber
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Menlo Park, CA, 94025
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just 1 little mm... Noté 4 sur 5 étoiles
worx good so far, but the option "prevent goBack/goForward" doesn't work with Tab History Menu 2.1.1 (https://addons.mozilla.org/en-US/firefox/addon/tab-history-menu/).
activated "prevent goBack/goForward" but still CAN go back or forwards by just clicking tab and then select.
5 Stars if this could be fixed.


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Bad karma: we can't find that page!
You asked for http://www.pantherrules.com/pantherrules/panthers/nfl-draft/meet-the-rookies-amini-silatolu.html, but despite our computers looking very hard, we could not find it. What happened ?
- the link you clicked to arrive here has a typo in it
- or somehow we removed that page, or gave it another name
- or, quite unlikely for sure, maybe you typed it yourself and there was a little mistake ?
It's not the end of everything though : you may be interested in the following pages on our site:
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text: I recently had a lower venous exam to check on the progress of a baker's cyst in my left popliteal area. During the scan, we noticed several enlarged lymph nodes in the common femoral junction. I have had no fever, pain or any indication of anything systemic going on. No insect bites or any kind of sores are evident on the skin. Are enlarged lymph nodes always attributed to a systemic infection?


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Find out more about 8tracks-com.
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VIEWS: 0 PAGES: 1 POSTED ON: 9/13/2012 Public Domain
NOTICE OF REGULAR MEETING OF THE Crandall Economic Development Corporation For the City of Crandall Notice is hereby given that a Regular Meeting of the CEDC will be held on the 21st day of August, 2012, at 6:30 PM in the Creekview Golf Club meeting room located at 1602 E. Highway 175 Crandall, Texas at which time the following subjects will be discussed, to-wit: 1. Call to Order: Regular Meeting 2. Invocation 3. Pledge of Allegiance 4. Citizens – At this time citizens shall be allowed to speak (3 minutes each) on any matter other than personnel matters on a first-come, first-served basis provided they are listed on the sign-in sheet. No Board action is to be taken until such matter is placed on the Agenda and posted in accordance with law. 5. Approval of Minutes 6. Director's Report: Meeting with Mayor Cody Frazier. SWOT Analysis 7. Treasurer's Report 8. Discuss and take action: CEDC budget for 2012-2013 9. Discuss and take action: Secretary report and recommendations on attendance. 9. Adjourn As authorized by Section 551.071 of the Texas Government Code, this meeting may be convened into closed Executive Session for the purpose of seeking confidential legal advice from the City Attorney on any agenda item listed herein. I, the undersigned authority, do hereby certify that the above Notice of Meeting of the CEDC is a true and correct copy of said Notice and that I posted a true and correct copy of said Notice on the bulletin board, at the City Hall of Crandall, Texas, a place convenient and readily accessible to the general public at all times, and said Notice was posted on August 16, 2012 and remained continuously for at least 72 hours preceding the scheduled time of said meeting. Persons with disabilities who plan to attend this meeting and who may need auxiliary aids or services such as interpreters for persons who are deaf or hearing impaired, readers, large print or Braille, are requested to contact Bobbie Jo Taylor at 972.427.3771 three (3) days prior to the meeting. DATED this the 16thth day of August, 2012. By: Bobbie Jo Taylor, Assistant to the City Secretary CARRYING OF A CONCEALED HANDGUN IS PROHIBITED AND VIOLATORS WILL BE ARRESTED FOR CRIMINAL TRESPASS. Texas Penal Code 30.05(a)
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Printable Area
Photo by HeatherFeather
Photo by HeatherFeather
"I found a variation of this recipe on another website, but the original did not contain nearly as many spices or flavorings. I also prefer to use my Cinnamon Bun Icing on top of these. If you do not have nonfat dry milk powder, you may use 3/4 cup low-fat or nonfat milk, warmed to lukewarm in place of the water and powdered milk. These are lightly spiced, and if you use the cloves, they will have a darker color. Not very sweet, the icing provides most of the sweetness."
Serving Size: 1 (76 g)
Servings Per Recipe: 12


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Home > Arts & Events >
A show at any of Gagosian's influential galleriesfive in New York, two in London, one in L.A., one in Rome, two in Paris, one in Geneva, one in Athens, and one in Hong Kong is the flashing neon announcement that an artist has made it. (When he poached John Currin, Gagosian reportedly added another digit to Currin's prices.) In his elegant Madison Ave. space, he's shown the works of Cy Twombly, and a superb exhibit of Picasso's sculptures. His cathedral-like warehouse space in Chelsea opened with a massive steel installation by Richard Serra, followed by a stern Anselm Kiefer show of steel bookcases filled with lead books and sunflowers and grit-studded canvases. His 2000 Damien Hirst exhibit is legendary as one of the most extravagantly produced shows in recent history, with a three-story-high anatomical model and an ob-gyn examination chair sunken in a fish tank. And the museum-quality show of De Kooning paintings was a stunner, proving Gagosian one of the very few galleries capable of competing with museums.
Fish in the Dark at Cort Theatre
Larry David makes his Broadway debut as playwright and star performer in this comedy about a death in a family. More »
"Yoko Ono: One Woman Show, 1960-1971" at The Museum of Modern Art


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Audio Samples
Album Summary
Notes & Reviews:
American Record Guide, November/December 2014
In another non-Italian performance (Freiburg) Bollon conducts with passion, orchestra and chorus easily sustain the pace. Filianoti is almost ideal as Federico, a beautiful sound with genuine Italian passion. He manages his robust voice well, scaling it back for the more intimate passages. The sweet retarded L'Innocente of Lee is quite touching. Orozco and Landolfi are the robust Metigio and a dignified Baldassare.
During the Italian verismo Francesco Cilea was the master of subtle portraits of the human soul. Leopoldo Marenco's libretto was based on Alphonse Daudet's drama L'Arlesienne. For its concert performance of L'Arlesiana, the Freiburg Philharmonic Orchestra engaged a singer on his way to international renown: Giuseppe Filianoti, who not only has scored a brilliant success in this role in Carnegie Hall but also on the stages of the New York Metropolitan Opera and the Milan Scala.Notes & Reviews:
Recording information: Rolf Böhme Saal, Konzerthaus Freiburg (07/12/2012-07/17/2012).
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Works DetailsFrancesco Cilèa (Composer) (1866 - 1950) : L'Arlesiana, opera
- Conductor: Fabrice Bollon
- Ensemble: Opernchor und Kinderchor des Theaters Freiburg
- Notes: Rolf Böhme Saal, Konzerthaus Freiburg (07/12/2012-07/17/2012)
- Running Time: 4 min. 40 sec.
- Period Time: Post Romantic
- Form: Opera/Operetta
- Written: 1897


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Outdoor Equipment
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- Secondary Container, 1 gal.Secondary Container,1 gal.
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Big Bad's BadgesFoursquareTen Hundred
Over 1000 check-ins!? Dayum. You're a powerful beast of exploration and discovery. You're what makes Foursquare awesome. We wish we could give you 1,000 hugs.
Big Bad unlocked the Ten Hundred badgeAugust 26, 2012
Download Foursquare for your smart phone and start exploring the world around you!


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Friday, July 3 and Saturday, July 4 for Independence Day.
The collection is an adaptation of a volume of images and information collected by library staff to form the Biographical Sketches of the Mayors of the City of Milwaukee.
Want to have your very own personal Business Librarian, at least for 45 minutes? Sign up for a Book A Business Librarian session during the first week of May!
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From NewsBank, find indexing of and full articles in the Milwaukee Journal Sentinel (from 1990-present) and other Wisconsin, national and world newspapers.
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Used Volvo V50
Expert Reviews
Kelley Blue Book ® - 2009 Volvo V50 OverviewVehicle Overview from Kelley Blue Book
Overview: EDITORS' NOTES The 2009 Volvo V50 is a compact premium wagon derived from the same chassis as the...
2005 Volvo V50 First DriveYouthful Exuberance
QUEBEC CITY, Canada - Fahrenheit and Celsius converge when the temperature reaches 40 below zero, the forecasted wind chill on the day of our first test drive of the 2005 Volvo...
2005 Volvo V50 PreviewOn target to do it all
The Volvo V40 never proved compelling to Americans seeking a modicum of luxury and utility for just under $30,000, selling in low numbers to Volvo fans and safety-conscious...
More Reviews See all reviews for the Volvo V50
Browse thousands of Used Volvo V50 autos online from the comfort of your own home. Once on the Autobytel.com website, select the car that best fits your needs. A certain Volvo V50 for sale can have many listings spanning the last few generations. See Pre Owned Volvo V50 Prices or read expert reviews.
If you are considering the purchase of a pre owned automobile, this is a great place to start. Autobytel.com has multiple vehicles in our online database from a number of car dealers across the country. View Used Volvo V50 Prices on every vehicle. Get a short synopsis of each Volvo V50 for sale. Pick a vehicle and get the lowdown on standard equipment, price, location of the dealer, and mileage.
Select a Used Volvo V50 to find specific facts about each model. At the vehicle page, there is much more information. View information about the dealer where the automobile is located. Contact the sales department through the Autobytel.com website if you want more information on a Used Volvo V50. See a nice variety of photos including the interior and exterior shots. Get the VIN number and find out what city the vehicle is located in. The Autobytel.com site has a tool to find average prices on any Pre Owned Volvo V50. This is useful as it allows you to spot a used V50 that is a bargain. Lastly, check the features equipped on each individual Volvo V50 for sale.
Start your Pre Owned Volvo V50 shopping experience by choosing a make or type. Cut right to the chase with the quick search function on the home page if you know the exact automobile you are interested in. Find pre owned vehicles for sale in your region by entering a local zip code or by choosing the nearest large city. Choosing a metro is convenient because there are generally more used car listings. It may be tough to locate a Volvo V50 for sale if you reside in an area with a small local population. Using a large metro as your search base will definitely broaden the number of Used Volvo V50 vehicles for sale.
After you land on the page with the used automobiles; you can select the information you are interested in. Display the Used Volvo V50 automobiles by a couple of different ways including: vehicle mileage, cost, and range. When there are numerous listings, the data can be separated even further. See Pre Owned Volvo V50 Prices broken down in increments. Choose car you want displayed that have a certain amount of mileage. Find Used Volvo V50 vehicles according to model year.


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Connect With Us800-299-9470
How Will My Product(s) Be Delivered?When purchasing a large appliance from AppliancesConnection, a representative will contact you 48 hours prior to arriving to your location to schedule a delivery date and a 4 hour time window. Upon delivery, your product will be unloaded and ready for you to open and inspect. If you have chosen White Glove service, the item will be placed in your room of choice and the debris will be removed.
Learn more about our shipping policy.
Free Scheduled DeliveryDelivery will be scheduled when item reaches your local distribution center. Signature will be required.
The Mackinaw collection includes a traditional dining set to meet any needs. With options from casual to formal, these charming dining pieces will blend beautifully with your home decor. Soft lines, elegant curves, turned details, and intricate pressed accents make these pieces special. The pieces are all crafted of oak, with a warm medium finish that will brighten your room. Tables with convenient leaves make theses dining sets great for both everyday use, or entertaining large groups. Mix and match these pieces for the perfect set to fit your home and meet your needs. The Mackinaw collection is a great option if you are looking for Traditional furniture.
Coaster is one of the innovators of Ready to Assemble furniture. It has grown to be a name customers can trust when it comes to quality furniture for the right price. This classic nostalgia table features a light oak veneer top. The empire base is sturdy and traditional, while offering plenty of support for dinner parties or family get-togethers. The 18 inch leaf extension makes this table convenient for everyday use and big enough for holiday dinners. The traditional look of this table is sure to please, and will be a staple in your home for years to come.
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Aurora AU1050XE Paper Shredder
The Aurora AU1050XE was the least expensive of all the 10-sheet shredders in our test and it churned through sheets, credit cards, staples, and paperclips at an impressive speed. Otherwise this model was merely mediocre, only making it through a couple hundred sheets or so before automatically shutting off (to avoid overheating) and jamming fairly easily when we inserted a couple more sheets than the recommended amount. We found it finicky to feed too, with papers get stuck in the input between two pieces of plastic. You'll have to struggle with the Aurora's heavy shred mechanism every time you need to empty the bin.
Type: 10-Sheet Cross-Cut
Manufacturer's Warranty: 1 year on product, 5 years on cutter
Reviewed: July 2012
Price When Reviewed: $54.99
- Fast shred speed
- Helpful manual
- Responsive customer service
- Noisy operation
- Difficult to input paper and empty basket
What do you think?
Krups 4-Slice Belgian Waffle Maker #F654-75
Although the Krups wasn't able to produce crisp waffles from a mix, it made nicely crisp waffles from scratch and was the most consistent...
Hamilton Beach Mess Free Belgian Style Waffle Maker #26041
Hamilton Beach's Mess Free Belgian Style Waffle Maker makes one round waffle that can be split into eight individual waffle sticks.
Proctor Silex Large Belgian Waffle Maker #26016A
This attractive Belgian Waffle Maker bakes four decently sized square waffles, so you can have breakfast for two ready in just one go.
Waring Pro Belgian Waffle Maker #WMK250SQ
The rotating Belgian Waffle Maker from Waring Pro churns out breakfast in as little as two minutes' time, making it one of the fastest...
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Tim McGraw Announces Dates for Two Lanes of Freedom 2013 Tour
Truck yeah! Country music superstar Tim McGraw will release a new album titled 'Two Lanes of Freedom' on Feb. 5, and in support, he'll hit the road on a 30-date tour.
Truck yeah! Country music superstar Tim McGraw will release a new album titled 'Two Lanes of Freedom' on Feb. 5, and in support, he'll hit the road on a 30-date tour.
Country superstars Tim McGraw and Faith Hill will join Dixie Chicks singer Natalie Maines, Emmylou Harris and an all-star cast of music's brightest for a salute to Bruce Springsteen at the upcoming MusiCares gala on Feb. 8 in Los Angeles. The official charity of the Recording Academy has named the blue-collar rocker their Person of the Year.
Apparently, two albums featuring Tim McGraw songs are scheduled to be released in the next 30 days. The singer's former label home, Curb Records, just announced that 'Tim McGraw and Friends' will be available on January 22.
We all remember Tim McGraw as part of a VERY UNLIKELY pair with Usher singing their song, 'Over and Over Again'. Welp, this time it's a little closer than not, but Tim is still caught 'Rapping' it up in Colt Ford's new song.
Tim McGraw is one of those Country Super Stars that you can't help but love to death! He's so charitable and gracious and all the things that makes his fans proud to call themselves his fans. So take a look behind the scene of the music video being made in the new music video !
Have you every wondered what goes on behind the scenes of a country music video shoot? Thanks to Tim McGraw, now you can find out. The country legend will use a live video stream to give fans an insider look at his 'One of Those Nights' video shoot.
Tim McGraw might be surrounded by pickup trucks in the video for his single, 'Truck Yeah,' but the superstar says his regular ride is not quite so macho or glamorous.
Tim McGraw surprised 30 soldiers and their families during a Veteran's Day appreciation luncheon at Outback Steakhouse in Brentwood, Tenn. on Thursday. The singer gave out handshakes, words of encouragement and thank yous to the men and women for their service.
Chris Lucas of the LoCash Cowboys is sporting some new ink this week after a trip to the tattoo parlor. The hit country songwriter went under the needle in order to celebrate the success of the gold-selling Tim McGraw single 'Truck Yeah,' which he helped write.
The night was filled with top-notch performances and even a Willie Nelson tribute, to boot! Get all the results from Country's BIGGEST night right here!
Tim McGraw is basking in the joy of scoring yet another gold-selling single with his latest hit, 'Truck Yeah.' The song has exceeded 500,000 digital downloads sold. The amped-up tune is the first release from McGraw's highly-anticipated debut album on Big Machine Records, the one he has quietly been working on over the past several months.
A panel of three judges from Tennessee's Court of Appeals ruled in favor of country music superstar Tim McGraw on Tuesday (Sept. 25), handing him a victory in the latest round of his ongoing lawsuit with his former label, Curb Records.


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Mann Center for the Performing Arts Young People's Concert Series 2015 "Magnificent Magical Mondays"
- Music
- 5201 Parkside Avenue, Philadelphia, PA, 19131
Philadelphia Lantern Slide Salon
Wagner Free Institute of Science
Thursday, October 10, 2013 from 6:00 PM to 8:00 PM (EDT)
The Wagner Free Institute of Science is proud to announce its first-ever lantern slide salon highlighting the historic lantern slide collections of some of the region's most significant cultural and educational institutions: The Athenaeum of Philadelphia, Independence Seaport Museum, Morris Arboretum, Swarthmore College Peace Collection, Temple University Special Collections and Research Center, the University of Pennsylvania Museum of Archaeology and Anthropology, and the Wagner Free Institute of Science.
Predating the motion picture, lantern slides were used for entertainment and to illustrate educational programs and courses. This salon will bring together some unique and rarely-seen images that depict a vast range of subjects: from the local—early engineering feats and turn-of-the century views of Philadelphia's neighborhoods—to the international—early 20th Century Japanese life, 1930s peace demonstrations in Berlin, the Jewish immigrant experience and more.
The salon will be held in the Wagner's unchanged Victorian lecture hall using an original lantern slide projector. Join us for a rare treat as we revive an old technology in order to see globally significant lantern slide collections drawn from the libraries and archives of Philadelphia's most distinguished institutions.
This program is part of the Philadelphia Archives Month
and part of the Global Philadelphia™ Exposition.
Benjamin Franklin Bridge under construction, lantern slide collection of the Wagner Free Institute of science.
Advertisement for the "Stereopticon & Magic Lantern," 1876. Wagner Free Institute of Science Library and Archives.
Join millions of people on Eventbrite.
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


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I am proud of America and what it stands for. I may not have liked every policy or inaction, but that's what happens sometimes when anyone can run for office. This also what makes this form of government great.
But how can we truly be one people if we catagorize ourselves? I am an American. The millions of our ancestors that emigrated to this country only wanted to be Americans as well.


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Wright Shines In Debut As Orioles Blank Angels 3-0
Mike Wright took a three-hitter into the eighth inning of his major league debut, and the Baltimore Orioles ended the Los Angeles Angels' five-game winning streak with a 3-0 victory Sunday.
Angels Beat Orioles 6-1
Matt Shoemaker scattered three hits and allowed one run over seven innings, leading Los Angeles to a season-high five-game winning streak.
Weaver Solid as Angels Beat Orioles 3-1
Left-hander Wei-Yin Chen (1-2) had an effective outing for Baltimore overshadowed by Weaver. He allowed two runs and seven hits and tied a season high with seven strikeouts with a walk over seven innings. It was the fifth time this season that Chen allowed two runs or fewer and didn't earn a victory.
Jeffry Todd Of MLBtradeRumors.com Joins Scott And Jeremy To Discuss Possible Trade Rumors
Although the Orioles do have somewhat of a stronghold on first place, the AL central is much closer than Baltimore's division.
"It could still go either way," said Todd. "The Royals have a game and half on them, and Detroit has the better roster and they could still pull away with the division. But their rotation is shallow now. They might lose a couple due to that and it could be the difference."
Dave Szymborski MLB Analyst For ESPN Joins Scott And Jeremy
"Davis has had a very up and down career," said Szymborski. "He's hitting .200 or less of late, and it's not really sustainable."
Though many in Baltimore are a bit panicked after Machado's injury, Szymborski points out that every team has injuries, and many also have far worse scenarios as a road to the playoffs.
Pujols' RBI In 13th Lifts Angels Over Orioles 1-0
Albert Pujols hit an RBI single in the 13th inning, and the Los Angeles Angels beat the Baltimore Orioles 1-0 Thursday night to avert a three-game series sweep.
Gausman Sharp As Orioles Beat Angels 4-3
For much of the first half of the season, the Baltimore Orioles were selective and cautious with their use of rookie Kevin Gausman.
Orioles Beat Angels 7-6 In 12 Innings
The Baltimore Orioles have a knack for winning in extra innings, and it has nothing to do with luck.
Female Demonstration Pilot Named To USNA's Blue Angels Squadron
A female Navy Academy graduate has been named to the Blue Angels Squadron.
Angels Beat O's On Bases-Loaded Walk In 8th
Kole Calhoun drew a bases-loaded walk from reliever Brian Matusz in the eighth inning after a tying RBI double by Erick Aybar, and the Los Angeles Angels pulled out a 3-2 victory over the Baltimore Orioles on Wednesday night.
Schoop's 2-Run HR Helps O's Beat Angels 4-2
Miguel Gonzalez took a two-hit shutout into the eighth inning, Jonathan Schoop homered and J.J. Hardy hit two RBI doubles to lead the Baltimore Orioles to a 4-2 victory over the Los Angeles Angels on Tuesday night.
Jones Powers Orioles Over Shoemaker, Angels 4-2
Two misplaced pitches to Adam Jones cost Los Angeles Angels rookie Matt Shoemaker in an otherwise dominant outing against the Baltimore Orioles.


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VIEWS: 0 PAGES: 19 CATEGORY: Communications POSTED ON: 4/16/2012 Public Domain
BACKGROUND While increasingly unfettered mobility is a normal part of modern life, it is desirable for those in positions of authority to assert a level of control over the mobility of those for whom they are responsible. Many social groups require theimposition of such mobility control, requiring that a level of authority be established and maintained between group members, for example the authority of a parent over a son or daughter within a family, the authority of an employer over an employee in awork environment, and the authority of a teacher over a student at a school. In consideration of safety or the law, teachers or parents may wish that children for whom they are responsible avoid certain locations or adhere to certain pre-defined routeson their way to and from school or other destinations. Employers, in an effort to enhance worker productivity, may wish to enforce schedules defining where employees should be at particular times during the work day. The growing ubiquity of locatable mobile devices such as mobile telephones, cellular-enabled personal computers and GPS systems potentially provides those in positions of authority with at least the means to locate someone over whom theymaintain authority. Regardless, independent minds, whether children or adults, are apt to consider controls placed on their mobility to be especially onerous and therefore may endeavor to hinder attempts to assert control or otherwise express theirdissent. It would be desirable to provide an effective method for establishing and maintaining mobility control over a user of a locatable mobile device.SUMMARY The present invention provides a computer-implemented method of rewarding a user based on compliance with predetermined geographic limitations. The method includes receiving from a first user and storing an indication of one or more geographicareas. Mobile device location information of a user mobile device associated with a second user is received from the user mobile
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"They wanted to glean good ideas and figured their opponent the CIA was doing it, so they had to do it too," Grady said.
The government's community service website, serve.gov, also features a USDA "Let's glean!"
"The biggest mistake people make is to glean from their most recent CSI episode, which is that one size fits all," he added.
early 14c., from Old French glener (Modern French glaner) "to glean," from Late Latin glennare "make a collection," perhaps from Gaulish (cf. Old Irish do-glinn "he collects, gathers," Celt. glan "clean, pure"). Figurative sense was earlier in English than the literal one of "gather grain left by the reapers" (late 14c.). Related: Gleaned; gleaning.
The corners of fields were not to be reaped, and the sheaf accidentally left behind was not to be fetched away, according to the law of Moses (Lev. 19:9; 23:22; Deut. 24:21). They were to be left for the poor to glean. Similar laws were given regarding vineyards and oliveyards. (Comp. Ruth 2:2.)