Myc represses transcription of the growth arrest gene gas1

Cell proliferation is regulated by the induction of growth promoting genes and the suppression of growth inhibitory genes. Malignant growth can result from the altered balance of expression of these genes in favor of cell proliferation. Induction of the transcription factor, c-Myc, promotes cell proliferation and transformation by activating growth promoting genes, including the ODC and cdc25A genes. We show that c-Myc transcriptionally represses the expression of a growth arrest gene, gas1. A conserved Myc structure, Myc box 2, is required for repression of gas1, and for Myc induction of proliferation and transformation, but not for activation of ODC. Activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen was sufficient to repress gas1 gene transcription. These findings suggest that transcriptional repression of growth arrest genes, including gas1, is one step in promotion of cell growth by Myc.

Genetic instability of cancer cells is proportional to their degree of aneuploidy

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnauer et al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997) Nature (London) 386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Fibronectin Regulates Assembly of Actin Filaments and Focal Contacts in Cultured Cells via the Heparin-binding Site in Repeat III13

Fibroblasts, when plated on the extracellular matrix protein fibronectin (FN), rapidly spread and form an organized actin cytoskeleton. This process is known to involve both the central α5β1 integrin-binding and the C-terminal heparin-binding regions of FN. We found that within the heparin-binding region, the information necessary for inducing organization of stress fibers and focal contacts was located in a 29–amino acid segment of FN type III module 13 (III13). We did not find a cytoskeleton-organizing role for repeat III14, which had previously been implicated in this process. Within III13, the same five basic amino acids known to be most important for heparin binding were also necessary for actin organization. A substrate of III13 alone was only weakly adhesive but strongly induced formation of filopodia and lamellipodia. Stress fiber formation required a combination of III13 and III7–11 (which contains the integrin α5β1 recognition site), either as a single fusion protein or as separate polypeptides, and the relative amounts of the two binding sites appeared to determine whether stress fibers or filopodia and lamellipodia were the predominant actin structures formed. We propose that a balance of signals from III13 and from integrins regulates the type of actin structures assembled by the cell.

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

Characterization of Cell–Matrix Adhesion Requirements for the Formation of Fascin Microspikes

Cell adhesion to thrombospondin-1 (TSP-1) correlates with assembly of cell–substratum contact structures that contain fascin microspikes. In this analysis, cell-matrix requirements for assembly of fascin microspikes were examined in detail. In six cell lines, cell spreading on a TSP-1 substratum correlated with expression of fascin protein and formation of fascin microspikes. Microspikes were not formed by H9c2 cells adherent on fibronectin, vitronectin, collagen IV, or platelet factor 4. However, both fascin microspikes and focal contacts were assembled by cells adherent on laminin-1. Using mixed substrata containing different proportions of TSP-1, and fibronectin, fascin microspike formation by H9c2 and C2C12 cells was found to be reduced on substrata containing 25% fibronectin and abolished on substrata containing 75% fibronectin. Adhesion to intermediate mixtures of TSP-1 and fibronectin resulted in coassembly of fascin microspikes and focal contacts, colocalization of fascin with actin stress fiber bundles and altered distributions of β1 integrins, cortical α-actinin, and tropomyosin. In cells adherent on 50% TSP-1:50% fibronectin, GRGDSP peptide treatment decreased focal contact assembly and altered cytoskeletal organization but did not inhibit microspike assembly. Treatment with chondroitin sulfate A or p-nitrophenol β-d-xylopyranoside decreased microspike formation and modified cytoskeletal organization but did not inhibit focal contact formation. In polarized migratory and postmitotic C2C12 cells, fascin microspikes and ruffles were localized at leading edges and TSP matrix deposition was also concentrated in this region. Depletion of matrix TSP by heparin treatment correlated with decreased microspike formation and cell motility. Thus, the balance of adhesive receptors ligated at the cell surface during initial cell–matrix attachment serves to regulate the type of substratum adhesion contact assembled and subsequent cytoskeletal organization. A role for fascin microspikes in cell motile behavior is indicated.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492–742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein

The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chemical reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide derivative of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Analysis of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue extracts appears to be less stable than T. The existence of T in tissues under normal physiological conditions has important implications for its function both in zinc metabolism and the redox balance of the cell.

Potential responses of soil organic carbon to global environmental change

Recent improvements in our understanding of the dynamics of soil carbon have shown that 20–40% of the approximately 1,500 Pg of C stored as organic matter in the upper meter of soils has turnover times of centuries or less. This fast-cycling organic matter is largely comprised of undecomposed plant material and hydrolyzable components associated with mineral surfaces. Turnover times of fast-cycling carbon vary with climate and vegetation, and range from <20 years at low latitudes to >60 years at high latitudes. The amount and turnover time of C in passive soil carbon pools (organic matter strongly stabilized on mineral surfaces with turnover times of millennia and longer) depend on factors like soil maturity and mineralogy, which, in turn, reflect long-term climate conditions. Transient sources or sinks in terrestrial carbon pools result from the time lag between photosynthetic uptake of CO2 by plants and the subsequent return of C to the atmosphere through plant, heterotrophic, and microbial respiration. Differential responses of primary production and respiration to climate change or ecosystem fertilization have the potential to cause significant interrannual to decadal imbalances in terrestrial C storage and release. Rates of carbon storage and release in recently disturbed ecosystems can be much larger than rates in more mature ecosystems. Changes in disturbance frequency and regime resulting from future climate change may be more important than equilibrium responses in determining the carbon balance of terrestrial ecosystems.

The effect of carbon nanofillers on the performance of electromechanical polyaniline-based composite actuators

Different types of crystalline carbon nanomaterials were used to reinforce polyaniline for use in electromechanical bilayer bending actuators. The objective is to analyze how the different graphitic structures of the nanocarbons affect and improve the in situ polymerized polyaniline composites and their subsequent actuator behavior. The nanocarbons investigated were multiwalled carbon nanotubes, nitrogen-doped carbon nanotubes, helical-ribbon carbon nanofibers and graphene oxide, each one presenting different shape and structural characteristics. Films of nanocarbon-PAni composite were tested in a liquid electrolyte cell system. Experimental design was used to select the type of nanocarbon filler and composite loadings, and yielded a good balance of electromechanical properties. Raman spectroscopy suggests good interaction between PAni and the nanocarbon fillers. Electron microscopy showed that graphene oxide dispersed the best, followed by multiwall carbon nanotubes, while nitrogen-doped nanotube composites showed dispersion problems and thus poor performance. Multiwall carbon nanotube composite actuators showed the best performance based on the combination of bending angle, bending velocity and maximum working cycles, while graphene oxide attained similarly good performance due to its best dispersion. This parallel testing of a broad set of nanocarbon fillers on PAni-composite actuators is unprecedented to the best of our knowledge and shows that the type and properties of the carbon nanomaterial are critical to the performance of electromechanical devices with other conditions remaining equal.

Aplicación de un modelo geográfico con información climática para el cálculo del balance hídrico de la comarca de la Marina Baja (Alicante)

Este trabajo analiza las nuevas tendencias en la creación y gestión de información geográfica, para la elaboración de modelos inductivos basados exclusivamente en bases de datos geográficas. Estos modelos permiten integrar grandes volúmenes de datos de características heterogéneas, lo que supone una gran complejidad técnica y metodológica. Se propone una metodología que permite conocer detalladamente la distribución de los recursos hídricos naturales en un territorio y derivar numerosas capas de información que puedan ser incorporadas a estos modelos «ávidos de datos» (data-hungry). La zona de estudio escogida para aplicar esta metodología es la comarca de la Marina Baja (Alicante), para la que se presenta un cálculo del balance hídrico espacial mediante el uso de herramientas estadísticas, geoestadísticas y Sistemas de Información Geográfica. Finalmente, todas las capas de información generadas (84) han sido validadas y se ha comprobado que su creación admite un cierto grado de automatización que permitirá incorporarlas en análisis de Minería de Datos más amplios.

Paid to Inhabitants of Cambridge For Supplies in the Hall, [1799 June]

One octavo-sized folded leaf containing a short handwritten list of payments made to Cambridge for supplies between 1796 and 1799.

A Legal and Economic Assessment of European Takeover Regulation. CEPS Paperbacks. December 2012

Takeovers are one-off events, altering control and strategy within an organisation. But the chances of becoming the target of a bid, even where remote, daily influence corporate decision-making. Takeover rules are therefore central to company law and the balance of power among managers, shareholders and stakeholders alike. This study analyses the corporate governance drivers underpinning takeover bid regulations and assesses the implementation of the EU Directive on takeover bids and compares it with the legal framework of nine other major jurisdictions, including the US. It finds that similar rules have different effects depending on company-level and country-level characteristics and considers the use of modular legislation and optional provisions to cater for them.

Proportionality needed in the subsidiarity debate in the EU – Appraisal of the British and Dutch initiatives. CEPS Essay No. 11, 8 April 2014

In a new CEPS Essay, Michael Emerson assesses the initiatives taken by the UK and Dutch governments to cut out excessive EU regulatory intrusion, namely in the form of the ongoing British Balance of Competences Review and the Dutch list of 54 items of EU regulation that they would like to see repealed or reformed. He concludes that while one can approve of a campaign for better EU regulation and for cutting out unnecessary micro-regulation, it would require impressive commitment by all member states and the EU institutions to follow the best features of the British and Dutch leads for this to have a real effect in the fight against populist euroscepticism. In his view, that battle will have to be won primarily with bigger weapons – some combination of better macroeconomic results, bigger foreign policy achievements and the emergence of a European-level political leadership to which the people can relate. In short, there has to be due proportionality in the diagnosis of the responsibility of inadequate subsidiarity for the EU’s ills.

Simulation of Land Use and Investment Behaviour under Different Policy Scenarios: Results of the extended farm/household model. Factor Markets Working Paper No. 27, July 2012

Factor markets are a central issue in analyses of farm development and of agricultural sector vitality. Among the different production factors, land is one of the most studied. Several studies seek to estimate the effect of government policy payments on land value or land rental prices. The studies mostly agree that government payments and other types of policy support are significant in explaining land prices and account for a large share of them. In October 2011, the European Commission published a new policy proposal for the common agricultural policy (CAP) up to 2020. The proposed regulation includes a shift from historical to regional payments. The objective of this paper is to provide an ex ante analysis of the impact of the new CAP policy instruments on the land market. In particular, the effect of the regionalisation of payments in Italy is examined. The analysis is based on the use of a mathematical programming model to simulate the changes in land demand for a farm in Emilia Romagna. The results highlight the relevance of the new policy mechanism in determining a change in land demand. Yet the effect is highly dependent on initial ownership of entitlements under the historical payment scheme.

Is the EMU ready for future shocks? An overview of available backstops. European Policy Brief No. 20, March 2014

In recent years much has been accomplished to make the EMU more resilient to banking crises, sovereign-debt crises or balance-of-payment crises. Several ‘backstops’ or financial safety nets were progressively put in place to absorb the shocks that could have otherwise broken the EMU as a system. These substantial advances reflected a gradual, trial-and-error approach rather than a grand design that would have completely overhauled the EMU architecture. While flexibility and realism have advantages, complacency is a clear risk. With no roadmap to follow, efforts to complete the architecture of the EMU may fade with time. Maintaining a sense of direction is crucial while potential vulnerabilities remain.