Antisense-mediated depletion of p300 in human cells leads to premature G1 exit and up-regulation of c-MYC

The cAMP-response element-binding protein (CREB)-binding protein and p300 are two highly conserved transcriptional coactivators and histone acetyltransferases that integrate signals from diverse signal transduction pathways in the nucleus and also link chromatin remodeling with transcription. In this report, we have examined the role of p300 in the control of the G1 phase of the cell cycle in nontransformed immortalized human breast epithelial cells (MCF10A) and fibroblasts (MSU) by using adenovirus vectors expressing p300-specific antisense sequences. Quiescent MCF10A and MSU cells expressing p300-specific antisense sequences synthesized p300 at much reduced levels and exited G1 phase without serum stimulation. These cells also showed an increase in cyclin A and cyclin A- and E-associated kinase activities characteristic of S phase induction. Further analysis of the p300-depleted quiescent MCF10A cells revealed a 5-fold induction of c-MYC and a 2-fold induction of c-JUN. A direct target of c-MYC, CAD, which is required for DNA synthesis, was also found to be up-regulated, indicating that up-regulation of c-MYC functionally contributed to DNA synthesis. Furthermore, S phase induction in p300-depleted cells was reversed when antisense c-MYC was expressed in these cells, indicating that up-regulation of c-MYC may directly contribute to S phase induction. Adenovirus E1A also induced DNA synthesis and increased the levels of c-MYC and c-JUN in serum-starved MCF10A cells in a p300-dependent manner. Our results suggest an important role of p300 in cell cycle regulation at G1 and raise the possibility that p300 may negatively regulate early response genes, including c-MYC and c-JUN, thereby preventing DNA synthesis in quiescent cells.

Quantitative ER ↔ Golgi Transport Kinetics and Protein Separation upon Golgi Exit Revealed by Vesicular Integral Membrane Protein 36 Dynamics in Live CellsV⃞

To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 ± 0.44%/min for ER → Golgi, and 7.68 ± 1.94%/min for Golgi → ER transport, revealing a half-time of 113 ± 70 min for leaving the ER and 1.67 ± 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF4− treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components.

Surface antigen cross-linking triggers forced exit of a protozoan parasite from its host.

We used the common fish pathogen Ichthyophthirius multifiliis as a model for studying interactions between parasitic ciliates and their vertebrate hosts. Although highly pathogenic, Ichthyophthirius can elicit a strong protective immune response in fish after exposure to controlled infections. To investigate the mechanisms underlying host resistance, a series of passive immunization experiments were carried out using mouse monoclonal antibodies against a class of surface membrane proteins, known as immobilization antigens (or i-antigens), thought to play a role in the protective response. Such antibodies bind to cilia and immobilize I. multifiliis in vitro. Surprisingly, we found that passive antibody transfer in vivo caused rapid exit of parasites from the host. The effect was highly specific for a given I. multifiliis serotype. F(ab)2 subfragments had the same effect as intact antibody, whereas monovalent Fab fragments failed to protect. The activity of Fab could, nevertheless, be restored after subsequent i.p. injection of bivalent goat anti-mouse IgG. Parasites that exit the host had detectable antibody on their surface and appeared viable in all respects. These findings represent a novel instance among protists in which protective immunity (and evasion of the host response) result from an effect of antibody on parasite behavior.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.

Pressure radiation from a perforated duct exit region

Acknowledgements The authors are grateful to the following bodies that provided financial support for the project: (i) China Scholarship Council, (ii) National Natural Science Foundation of China (Grant no. U1334201) and (iii) UK Engineering and Physical Sciences Research Council (Grant no. EP/G069441/1).

Dual Regulation of the mitotic exit network (MEN) by PP2A-Cdc55 phosphatase.

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A(Cdc55) phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A(Cdc55) phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A(Cdc55) regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A(Cdc55) initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A(Cdc55) functions affect the regulation of various MEN components, contributing to mitotic exit.

Una técnica de análisis parcial sobre textos no restringidos (SUPP) aplicada a un sistema de extracción de información (EXIT)

En este trabajo presentamos la integración de una técnica de análisis parcial definida sobre textos no restringidos (SUPP) sobre un sistema de extracción de información (EXIT) en el dominio restringido de los textos notariales, en concreto, en las escrituras de compraventa de inmuebles. La principal contribución está centrada en la identificación de sintagmas nominales definidos, acrónimos y alias para su resolución posterior de correferentes.

The long haul: managing exit from financial assistance. Bruegel Policy Contribution Issue 2014/03, 20 February 2014

Countries can make a clean exit from financial assistance, or enter a new programme or a precautionary programme, depending on the sustainability of their public debt and their vulnerability to shocks. Ireland made a clean exit in December 2013, supported by significant budgetary and current-account adjustment and signs of economic recovery. But Irish debt sustainability is not guaranteed and prudence will be needed to avoid future difficulties. A clean exit for Portugal is not recommended when its programme ends in May 2014, because compared to Ireland it faces higher interest rates, has poorer growth prospects and has probably less ability to generate a consistently high primary surplus. A precautionary arrangement would be advisable. In case debt sustainability proves difficult to achieve later, some form of debt restructuring may prove necessary. It is unlikely that Greece will be able to exit its programme in December 2014. A third programme should be put in place to take Greece out of the market until 2030, accompanied by enhanced budgetary and structural reform commitments by Greece, a European boost to economic growth in the euro-area periphery and willingness on the part of lenders to reduce loan charges below their borrowing costs, should public debt levels prove unsustainable despite Greece meeting the loan conditions. Even assuming all goes well, the three countries will be subject to enhanced post-pro-gramme surveillance for decades. Managing such long-term relationships will be a key challenge.

New market conditions and "exit strategies" for the German arms industry. OSW Commentary No. 130, 12.03.2014

Due to changing internal and external conditions, the German arms industry is facing serious challenges as are its counterparts across Europe. The arms sales market in Germany is contracting – orders from the Bundeswehr are slowing down and the Federal Ministry of Defence is planning to change the way it cooperates with German arms producers. In addition, member states of NATO and the EU, major customers of German arms manufacturers, are reducing their defence spending, which will spell a fall in their orders for new armament and military equipment. In response to the new circumstances, the German arms industry is beginning to organise itself and increase its lobbying efforts in Berlin and, with the support of the federal government, it has been implementing specific measures in several areas. German companies are interested in securing new markets outside NATO and the EU and are also exploring opportunities for mergers and joint ventures with other German and foreign companies, and are seeking to create more conducive conditions for business on the EU and NATO markets.

The Role of Pluriactivity in Farm Exit and Labour Supply Decisions. Factor Markets Working Document No. 67, August 2013

EDITED VERSION TO BE PUBLISHED SOON Pluriactivity has been a topic of research in agriculture for the best part of a century. It is a term which has both broad and narrow definitions and hence is subject to multiple interpretations. This paper considers two forms of pluriactivity: within the farm gate pluriactivity, also commonly referred to as farm diversification, and beyond the farm-gate pluriactivity, also known as multiple job holding. Previous studies of pluriactivity have shown that it can inhibit the natural process of structural change in the farm sector, by allowing small and unprofitable farms to survive with the support of income from outside the sector. In this paper, two empirical models of pluriactivity are estimated using farm level data for Ireland. The first examines the impact of on-farm diversification on off-farm labour supply, while the second investigates the relationship between off-farm labour supply and farm exit which is specified in the context of retirement and non-succession. The result of the first model suggests that farms that engage in within the farm gate pluriactivity are less likely to engage in beyond the farm gate pluriactivity, in other words more diversified farmers are less likely to work off farm. The second model confirms previous findings in the literature that part-time farmers have a reduced probability of having a farm successor. While the model results are specific to the Irish case, they do provide some value insights into the impacts of pluriactivity on structural change in farming.

Fostering social cohesion: the missing link in the EU's exit strategy from the crisis. EPC Discussion Paper, 31 March 2015

In this Discussion Paper, Claire Dhéret argues that the EU should consider the level of social cohesion as a concrete policy priority in its long-term exit strategy from the crisis.

Exit from Brexit? Not likely but uncertainty prevails. EPC Commentary, 12 July 2016

Theresa May, who is set to become UK Prime Minister on Wednesday, has made it clear that “Brexit means Brexit”. Stating this so bluntly has become necessary as a number of voices have speculated that there may be a way back from the brink, claiming that the result of the referendum is not very decisive and that citizens have been misled by the lies of the Brexit campaign, not realising the magnitude of the decision. There certainly seems a wish in the Chancellery in Berlin and in some other capitals that Britain can be kept in, accompanied by a willingness to table compromises to smooth the process.

Failure or Voluntary Exit? Reassessing the Female Underperformance Hypothesis

We reevaluate the female underperformance hypothesis by challenging the assumption that female-owned ventures are more likely to fail. Instead of equating exit with failure, we draw on exit literature and feminist theories to argue that female entrepreneurs are actually more likely than males to exit voluntarily. We argue for further gender differences by using an even more fine-grained conceptualization of entrepreneurial exit (failure, exit for personal reasons, and exit for other professional/financial opportunities). Post-hoc analyses also point to withingender heterogeneity depending on family status. A sample probe of 219 Spanish entrepreneurs who had exited their business supports our overall reasoning.