Nuclear-Gene Mutations Suppress a Defect in the Expression of the Chloroplast-Encoded Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase1

The green alga Chlamydomonas reinhardtii mutant 76–5EN lacks photosynthesis because of a nuclear-gene mutation that specifically inhibits expression of the chloroplast gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39). Photosynthesis-competent revertants were selected from mutant 76–5EN to explore the possibility of increasing Rubisco expression. Genetic analysis of 10 revertants revealed that most arose from suppressor mutations in nuclear genes distinct from the original 76–5EN mutant gene. The revertant strains have regained various levels of Rubisco holoenzyme, but none of the suppressor mutations increased Rubisco expression above the wild-type level in either the presence or absence of the 76–5EN mutation. One suppressor mutation, S107–4B, caused a temperature-conditional, photosynthesis-deficient phenotype in the absence of the original 76–5EN mutation. The S107–4B strain was unable to grow photosynthetically at 35°C, but it expressed a substantial level of Rubisco holoenzyme. Whereas the 76–5EN gene encodes a nuclear factor that appears to be required for the transcription of the Rubisco large-subunit gene, the S107–4B nuclear gene may be required for the expression of other chloroplast genes.

Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists.

Full activation of T cells requires signaling through the T-cell antigen receptor (TCR) and additional surface molecules interacting with ligands on the antigen-presenting cell. TCR recognition of agonist ligands in the absence of accessory signals frequently results in the induction of a state of unresponsiveness termed anergy. However, even in the presence of costimulation, anergy can be induced by TCR partial agonists. The unique pattern of early receptor-induced tyrosine phosphorylation events induced by partial agonists has led to the hypothesis that altered TCR signaling is directly responsible for the development of anergy. Here we show that anergy induction is neither correlated with nor irreversibly determined by the pattern of early TCR-induced phosphorylation. Rather, it appears to result from the absence of downstream events related to interleukin 2 receptor occupancy and/or cell division. This implies that the anergic state can be manipulated independently of the precise pattern of early biochemical changes following TCR occupancy, a finding with implications for understanding the induction of self-tolerance and the use of partial agonist ligands in the treatment of autoimmune diseases.

Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.

Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.

Sterol regulatory element binding protein binds to a cis element in the promoter of the farnesyl diphosphate synthase gene.

Sterol-regulated transcription of the gene for rat farnesyl diphosphate (FPP) synthase (geranyl-diphosphate:isopentenyl-diphosphate geranyltranstransferase, EC 2.5.1.10) is dependent in part on the binding of the ubiquitous transcription factor NF-Y to a 6-bp element within the proximal promoter. Current studies identify a second element in this promoter that is also required for sterol-regulated transcription in vivo. Mutation of three nucleotides (CAC) within this element blocks the 8-fold induction of FPP synthase promoter-reporter genes that normally occurs when the transfected cells are incubated in medium deprived of sterols. Gel mobility-shift assays demonstrate that the transcriptionally active 68-kDa fragment of the sterol regulatory element (SRE-1)-binding protein assays (SREBP-1) binds to an oligonucleotide containing the wild-type sequence but not to an oligonucleotide in which the CAC has been mutated. DNase 1 protection pattern (footprint) analysis indicates that SREBP-1 binds to nucleotides that include the CAC. Both the in vivo and in vitro assays are affected by mutagenesis of nucleotides adjacent to the CAC. Coexpression of SREBP with a wild-type FPP synthase promoter-reporter gene in CV-1 cells results in very high levels of reporter activity that is sterol-independent. In contrast, the reporter activity remained low when the promoter contained a mutation in the CAC trinucleotide. We conclude that sterol-regulated transcription of FPP synthase is controlled in part by the interaction of SREBP with a binding site that we have termed SRE-3. Identification of this element may prove useful in the identification of other genes that are both regulated by SREBP and involved in lipid biosynthesis.

Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach.

Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type III secretion mechanism. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis and Yersinia enterocolitica translocate cytotoxin YopE across the host cell plasma membrane. Several lines of evidence suggest that tyrosine phosphatase YopH follows the same pathway. We analyzed internalization of YopE and YopH into murine PU5-1.8 macrophages by using recombinant Y. enterocolitica producing truncated YopE and YopH proteins fused to a calmodulin-dependent adenylate cyclase. The YopE-cyclase and YopH-cyclase hybrids were readily secreted by Y. enterocolitica. The N-terminal domain required for secretion was not longer than 15 residues of YopE and 17 residues of YopH. Internalization into eukaryotic cells, revealed by cAMP production, only required the N-terminal 50 amino acid residues of YopE and the N-terminal 71 amino acid residues of YopH. YopE and YopH are thus modular proteins composed of a secretion domain, a translocation domain, and an effector domain. Translocation of YopE and YopH across host cell's membranes was also dependent on the secretion of YopB and YopD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bacterial pathogens.

The Creation of a Legal Ordinance and Program Manual for the Purpose of Illicit Discharge Detection and Elimination

Illegal dumping and improper disposal of pollutants in urban areas can contribute significant pollutant loads to the municipal separate storm sewer system (MS4) and natural environments. Illicit discharges to the MS4 can pose a significant risk to human and environmental health. The Clean Water Act requires that municipalities implement a legal mechanism and plan to detect and eliminate illicit discharges to the MS4. The methodology for program creation included the analysis of other municipal illicit discharge programs, review of state and federal guidance publications, and the review of illicit discharge case-studies. This paper describes a systematic approach applied to the creation and implementation of a legal ordinance and program manual designed for the purpose of illicit discharge detection and elimination (IDDE).

Applying reactive models to column experiments to assess the hydrogeochemistry of seawater intrusion: optimising ACUAINTRUSION and selecting cation exchange coefficients with PHREEQC

Three sets of laboratory column experimental results concerning the hydrogeochemistry of seawater intrusion have been modelled using two codes: ACUAINTRUSION (Chemical Engineering Department, University of Alicante) and PHREEQC (U.S.G.S.). These reactive models utilise the hydrodynamic parameters determined using the ACUAINTRUSION TRANSPORT software and fit the chloride breakthrough curves perfectly. The ACUAINTRUSION code was improved, and the instabilities were studied relative to the discretisation. The relative square errors were obtained using different combinations of the spatial and temporal steps: the global error for the total experimental data and the partial error for each element. Good simulations for the three experiments were obtained using the ACUAINTRUSION software with slight variations in the selectivity coefficients for both sediments determined in batch experiments with fresh water. The cation exchange parameters included in ACUAINTRUSION are those reported by the Gapon convention with modified exponents for the Ca/Mg exchange. PHREEQC simulations performed using the Gains-Thomas convention were unsatisfactory, with the exchange coefficients from the database of PHREEQC (or range), but those determined with fresh water – natural sediment allowed only an approximation to be obtained. For the treated sediment, the adjusted exchange coefficients were determined to improve the simulation and are vastly different from those from the database of PHREEQC or batch experiment values; however, these values fall in an order similar to the others determined under dynamic conditions. Different cation concentrations were simulated using two different software packages; this disparity could be attributed to the defined selectivity coefficients that affect the gypsum equilibrium. Consequently, different calculated sulphate concentrations are obtained using each type of software; a smaller mismatch was predicted using ACUAINTRUSION. In general, the presented simulations by ACUAINTRUSION and PHREEQC produced similar results, making predictions consistent with the experimental data. However, the simulated results are not identical to the experimental data; sulphate (total S) is overpredicted by both models, most likely due to such factors as the kinetics of gypsum, the possible variations in the exchange coefficients due to salinity and the neglect of other processes.

Growth of Carbon Nanotubes on Surfaces: The Effects of Catalyst and Substrate

We report a study of synthesising air-stable, nearly monodispersed bimetallic colloids of Co/Pd and Fe/Mo of varying compositions as active catalysts for the growth of carbon nanotubes. Using these catalysts we have investigated the effects of catalyst and substrate on the carbon nanostructures formed in a plasma-enhanced chemical vapour deposition (PECVD) process. We will show how it is possible to assess the influence of both the catalyst and the support on the controlled growth of carbon nanotube and nanofiber arrays. The importance of the composition of the catalytic nuclei will be put into perspective with other results from the literature. Furthermore, the influence of other synthetic parameters such as the nature of the nanoparticle catalysts will also be analysed and discussed in detail.

Facilitation Among Saproxylic Insects Inhabiting Tree Hollows in a Mediterranean Forest: The Case of Cetonids (Coleoptera: Cetoniidae) and Syrphids (Diptera: Syrphidae)

Tree hollows offer an ideal niche for saproxylic insects in mature Mediterranean forests, where Diptera and Coleoptera are the richest groups. Co-occurrence is frequently observed among many species of both groups in these microhabitats, and some of these species have been considered to facilitate the presence of other species by acting as ecosystem engineers. One of the systems that is found in Mediterranean tree hollows is formed by cetonid (Coleoptera: Cetoniidae) and syrphid (Diptera: Syrphidae) larvae. Here, cetonid larvae feed on wood and litter and produce a substrate that is easier to decompose. To assess the possible role of these larvae as facilitating agents for the saproxylic guild, we studied whether the presence of saprophagous Syrphidae inside tree hollows is associated with the activity of cetonid larvae. Furthermore, in laboratory conditions, we tested whether cetonid larvae activity can improve the development and fitness of the saprophagous syrphid species. Our results show that “cetonid activity” was the variable that best explained the presence of saprophagous syrphid species in natural conditions. Myathropa florea (L., 1758) was one of the species most influenced by this activity. The laboratory experiment gave similar results, demonstrating that an enriched substrate with Cetonia aurataeformis Curti, 1913 larval feces improves syrphid larval growth rate and fitness of adults (measured as longer wing length) of M. florea.

Performance Assessment of a Polymer Electrolyte Membrane Electrochemical Reactor under Alkaline Conditions – A Case Study with the Electrooxidation of Alcohols

A novel polymer electrolyte membrane electrochemical reactor (PEMER) configuration has been employed for the direct electrooxidation of propargyl alcohol (PGA), a model primary alcohol, towards its carboxylic acid derivatives in alkaline medium. The PEMER configuration comprised of an anode and cathode based on nanoparticulate Ni and Pt electrocatalysts, respectively, supported on carbonaceous substrates. The electrooxidation of PGA was performed in 1.0 M NaOH, where a cathode based on a gas diffusion electrode was manufactured for the reduction of oxygen in alkaline conditions. The performance of a novel alkaline anion-exchange membrane based on Chitosan (CS) and Poly(vinyl) alcohol (PVA) in a 50:50 composition ratio doped with a 5 wt.% of poly (4-vinylpyridine) organic ionomer cross-linked, methyl chloride quaternary salt resin (4VP) was assessed as solid polymer electrolyte. The influence of 4VP anionic ionomer loading of 7, 12 and 20 wt.% incorporated into the electrocatalytic layers was examined by SEM and cyclic voltammetry (CV) upon the optimisation of the electroactive area, the mechanical stability and cohesion of the catalytic ink onto the carbonaceous substrate for both electrodes. The performance of the 4VP/CS:PVA membrane was compared with the commercial alkaline anion-exchange membrane FAA −a membrane generally used in direct alcohol alkaline fuel cells- in terms of polarisation plots in alkaline conditions. Furthermore, preparative electrolyses of the electrooxidation of PGA was performed under alkaline conditions of 1 M NaOH at constant current density of 20 mA cm−2 using a PEMER configuration to provide proof of the principle of the feasibility of the electrooxidation of other alcohols in alkaline media. PGA conversion to Z isomers of 3-(2-propynoxy)-2-propenoic acid (Z-PPA) was circa 0.77, with average current efficiency of 0.32. Alkaline stability of the membranes within the PEMER configuration was finally evaluated after the electrooxidation of PGA.

The Influence of Geological Process on Interpreting Ediacaran Fossils in the Catalina Formation, Bonavista Peninsula, Newfoundland

Understanding the nature of the earliest complex fossils has presented many challenges over the past century since Billings first described Ediacaran fossils from Newfoundland in 1872. Previous studies have documented abundant Ediacaran fossils in the Bonavista Peninsula of Newfoundland. This thesis focuses on the H14 surface north of Catalina, which contains a nearly monospecific assemblage that includes hundreds of specimens of the rangeomorph, Fractofusus andersoni. Three factors need to be considered when trying to interpret these organisms. The first of these three factors is structural deformation. The area has undergone deformation during the formation of the Appalachian orogenic belt. This has distorted both fossil shape and orientation, requiring retrodeformation to restore the shapes and relationships of fossils to their original form. Two additional taphonomic factors influencing fossil visibility are: partly or completely ash covered fossils and the removal of fossil impressions from the bedding plane by modern weathering. These processes hinder acceptance of some previously published interpretations.

What scenarios for the Euro-Mediterranean in 2030 in the wake of the Arab spring? MEDPRO Policy Paper No. 2, October 2011 (Updated 25 February 2013)

In the wake of the Arab Spring, the Southern Mediterranean region has reached a turning point in its history, presenting many opportunities and challenges for the EU. In this MEDPRO Policy Paper, Rym Ayadi and Carlo Sessa explore various possible scenarios that could play out in EU-Mediterranean relations over the next two decades but find, lamentably, that the EU has set itself on a ‘business as usual’ course, leaving the region open to further polarisation and the involvement of other external players.

The relief of Vienna in vain. Assessing the Implications of Turkish EU-membership for Council of Ministers’ Decision-making. IES WORKING PAPER 1/2011

This research forecasts the implications of Turkish membership for decision-making effectiveness and dynamics within the Council of Ministers of the European Union (EU). Effectiveness is determined in this research by 'passage probability': the chance that a random proposal as put forth by the European Commission (EC) is accepted by the Council of Ministers. Dynamics are determined by means of the Shapley-Shubik Index (SSI), which plots power values of individual member states by forecasting a number of possible EU enlargement scenarios. This study falsifies earlier research by Baldwin and Widgrén.1 It finds that the implications of Turkish EU-membership for EU decision-making efficiency are ambiguous and depend on the number of other candidate states entering the EU alongside Turkey, as well as the timeslot - 2014 or 2020 - at which the accession would take place. Moreover, this study asserts that Turkish EU-accession would result in unequal- but generally negative - power changes among other EU member states, although member states with similar demographic weight will experience comparable changes. Finally, it appears that the larger a EU member state is, the more power it loses if Turkey would join the EU.

Social Citizenship and Institution Building: EU-Enlargement and the Restructuring of Welfare States in East Central Europe. CES Germany & Europe Working Paper, No. 01.2, April 2001

With the EU-enlargement process well underway, this paper focuses on social citizenship as a conceptual frame for analyzing the restructuring of social institutions in applicant countries in East Central Europe. So far, comparative welfare state analysis has concentrated mainly on the developed economies of the OECD-countries; there is little systematic analytical work on the transitions in post-communist Europe. Theoretically, this paper builds on comparative welfare state analysis as well as on new institutionalism. The initial hypothesis is built on the assumption that emerging patterns of social support and social security diverge from the typology described in the comparative welfare state literature inasmuch as the transformation of postcommunist societies is distinctly different from the building of welfare states in Europe. The paper argues that institutionbuilding is shaped by and embedded in the process of European integration and part of governance in the EU. Anticipating full membership in the European Union, the applicant countries have to adapt to the rules and regulations of the EU, including the "social acquis." Therefore, framing becomes an important feature of institutional changes. The paper seeks to identify distinct patterns and problems of the institutionalization of social citizenship.

The revision of the Posted Workers Directive: towards a sufficient policy adjustment? EPC Discussion Paper, 20 April 2016

Labour mobility within the European Union continues to be a limited phenomenon. This concerns both long-term intra-EU mobility and more temporary forms of mobility such as posting of workers, i.e. workers posted to another member state in the framework of cross-border service provision. Yet, despite the limited nature of posting, this topic is far from being absent from the public and political debates. Several factors contribute to this. Firstly, a surge in the number of posted workers has been noticed over the recent years and increased attention has therefore been paid to this issue. Quite a few economic sectors, including construction, manufacturing, and social work, are very concerned by this trend. Secondly, several types of abuses have been recorded such as letter-box companies, bogus self-employment and exploitation of the posted workers' vulnerable situation. Thirdly, questions have been raised as to whether the balance struck by the EU legislator in 1996 (when adopting the Posted Workers Directive) between the freedom to provide crossborder services and the workers' social rights is still valid today. These elements highlight the need for a policy adjustment in order to preserve the legitimacy of the citizens' and workers' freedom to move and, to a certain extent, of the social dimension of the European project. In this context, the European Commission published a proposal to revise the 1996 Directive in order to strike a better balance between economic and social rights. But is this proposal sufficient to ensure a level playing field between economic actors and equal treatment between workers? How will this proposal affect the implementation of other EU initiatives aiming to tackle fraud and abuse? What else is needed to address the tensions between the Single Market principles and the EU's social objectives? This discussion paper, published in the context of the Dutch Presidency and the ongoing negotiations of a revised Directive on posted workers, focuses on these questions while proposing some concrete solutions for a fairer policy framework.