A plastid enzyme arrested in the step of precursor translocation in vivo.

The key enzyme of chlorophyll biosynthesis in higher plants, NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR, EC 1.3.1.33), accumulates in its precursor form (pPORA) in barley. pPORA is bound to the chloroplasts and is able to interact with the enzyme's substrate, Pchlide, at both the cytosolic as well as the stromal side of the plastid envelope. The interaction with intraplastidic Pchlide, formed in ATP-containing chloroplasts upon feeding with -aminolevulinic acid, drives vectorial translocation of pPORA across the plastid envelope membranes. In contrast, exogenously applied Pchlide causes the release of the envelope-bound precursor protein to the cytosol. Both processes compete with each other if intra- and extraplastidic Pchlide are applied simultaneously. A cytosolic heat shock cognate protein of Mr 70,000 present in wheat germ and barley leaf protein extracts appears to prevent the release of the pPORA to the cytosol in vivo, however.

Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis.

We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme. The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids. The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa). A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence. In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane. A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA. Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs. The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.

STAT3 activation is a critical step in gp130-mediated terminal differentiation and growth arrest of a myeloid cell line.

Myeloid leukemia M1 cells can be induced for growth arrest and terminal differentiation into macrophages in response to interleukin 6 (IL-6) or leukemia inhibitory factor (LIF). Recently, a large number of cytokines and growth factors have been shown to activate the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In the case of IL-6 and LIF, which share a signal transducing receptor gp130, STAT3 is specifically tyrosine-phosphorylated and activated by stimulation with each cytokine in various cell types. To know the role of JAK-STAT pathway in M1 differentiation, we have constructed dominant negative forms of STAT3 and established M1 cell lines that constitutively express them. These M1 cells that overexpressed dominant negative forms showed no induction of differentiation-associated markers including Fc gamma receptors, ferritin light chain, and lysozyme after treatment with IL-6. Expression of either c-myb or c-myc was not downregulated. Furthermore, IL-6- and LIF-mediated growth arrest and apoptosis were completely blocked. Thus these findings demonstrate that STAT3 activation is the critical step in a cascade of events that leads to terminal differentiation of M1 cells.

Barriers to protein folding: formation of buried polar interactions is a slow step in acquisition of structure.

In the MYL mutant of the Arc repressor dimer, sets of partially buried salt-bridge and hydrogen-bond interactions mediated by Arg-31, Glu-36, and Arg-40 in each subunit are replaced by hydrophobic interactions between Met-31, Tyr-36, and Leu-40. The MYL refolding/dimerization reaction differs from that of wild type in being 10- to 1250-fold faster, having an earlier transition state, and depending upon viscosity but not ionic strength. Formation of the wild-type salt bridges in a hydrophobic environment clearly imposes a kinetic barrier to folding, which can be lowered by high salt concentrations. The changes in the position of the transition state and viscosity dependence can be explained if denatured monomers interact to form a partially folded dimeric intermediate, which then continues folding to form the native dimer. The second step is postulated to be rate limiting for wild type. Replacing the salt bridge with hydrophobic interactions lowers this barrier for MYL. This makes the first kinetic barrier rate limiting for MYL refolding and creates a downhill free-energy landscape in which most molecules which reach the intermediate state continue to form native dimers.

Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast.

Temperature-sensitive alleles in four genes (slu7-1, prp16-2, prp17-1, and prp18-1) are known to confer a specific block to the second chemical step of pre-mRNA splicing in vivo in the yeast Saccharomyces cerevisiae. Previous studies showed that Prp16p and Prp18p are required solely for the second step in vitro. The RNA-dependent ATPase, Prp16p, functions at a stage in splicing when ATP is required, whereas Prp18p functions at an ATP-independent stage. Here we use immunodepletion to show that the roles of Slu7p and Prp17p are also confined to the second step of splicing. We find that extracts depleted of Prp17p require both Prp17p and ATP for slicing complementation, whereas extracts depleted of Slu7p require only the addition of Slu7p. These different ATP requirements suggest that Prp16p and Prp17p function before Prp18p and Slu7p. Although SLU7 encodes an essential gene product, we find that a null allele of prp17 is temperature-sensitive for growth and has a partial splicing defect in vitro. Finally, high-copy suppression experiments indicate functional interactions between PRP16 and PRP17, PRP16 and SLU7, and SLU7 and PRP18. Taken together, the results suggest that these four factors may function within a multi-component complex that has both an ATP-dependent and an ATP-independent role in the second step of pre-mRNA splicing.

Size-Dependent and Step-Modulated Supramolecular Electrochemical Properties of Catechol-Derived Adlayers at Pt(hkl) Surfaces

The electrochemical reactivity of catechol-derived adlayers is reported at platinum (Pt) single-crystal electrodes. Pt(111) and stepped vicinal surfaces are used as model surfaces possessing well-ordered nanometer-sized Pt(111) terraces ranging from 0.4 to 12 nm. The electrochemical experiments were designed to probe how the control of monatomic step-density and of atomic-level step structure can be used to modulate molecule–molecule interactions during self-assembly of aromatic-derived organic monolayers at metallic single-crystal electrode surfaces. A hard sphere model of surfaces and a simplified band formation model are used as a theoretical framework for interpretation of experimental results. The experimental results reveal (i) that supramolecular electrochemical effects may be confined, propagated, or modulated by the choice of atomic level crystallographic features (i.e.monatomic steps), deliberately introduced at metallic substrate surfaces, suggesting (ii) that substrate-defect engineering may be used to tune the macroscopic electronic properties of aromatic molecular adlayers and of smaller molecular aggregates.

Beyond the H2/CO2 upper bound: one-step crystallization and separation of nano-sized ZIF-11 by centrifugation and its application in mixed matrix membranes

The synthesis of nano-sized ZIF-11 with an average size of 36 ± 6 nm is reported. This material has been named nano-zeolitic imidazolate framework-11 (nZIF-11). It has the same chemical composition and thermal stability and analogous H2 and CO2 adsorption properties to the conventional microcrystalline ZIF-11 (i.e. 1.9 ± 0.9 μm). nZIF-11 has been obtained following the centrifugation route, typically used for solid separation, as a fast new technique (pioneering for MOFs) for obtaining nanomaterials where the temperature, time and rotation speed can easily be controlled. Compared to the traditional synthesis consisting of stirring + separation, the reaction time was lowered from several hours to a few minutes when using this centrifugation synthesis technique. Employing the same reaction time (2, 5 or 10 min), micro-sized ZIF-11 was obtained using the traditional synthesis while nano-scale ZIF-11 was achieved only by using centrifugation synthesis. The small particle size obtained for nZIF-11 allowed the use of the wet MOF sample as a colloidal suspension stable in chloroform. This helped to prepare mixed matrix membranes (MMMs) by direct addition of the membrane polymer (polyimide Matrimid®) to the colloidal suspension, avoiding particle agglomeration resulting from drying. The MMMs were tested for H2/CO2 separation, improving the pure polymer membrane performance, with permeation values of 95.9 Barrer of H2 and a H2/CO2 separation selectivity of 4.4 at 35 °C. When measured at 200 °C, these values increased to 535 Barrer and 9.1.

The Single Supervisory Mechanism: A Sound First Step in Europe’s Banking Union? Egmont European Affairs Paper, March 2013

The financial and economic crisis has hit Europe in its core. While the crisis may not have originated in the European Union, it has laid bare structural weaknesses in the EU’s policy framework. Both public finances and the banking sector have been heavily affected. For a long time, the EU failed to take into account sufficiently the perverse link that existed between the two. Negative evolutions in one field of the crisis often dragged along the other in its downward spiral. In June 2012, in the early hours of a yet another EU Summit, the leaders of the eurozone finally decided to address the link between the banking and sovereign debt crises. Faced with soaring public borrowing costs in Spain and Italy, they decided to allow for the direct European recapitalisation of banks when the Member State itself would no longer be in a position to do so. In exchange, supervision of the banking sector would be lifted to the European level by means of a Single Supervisory Mechanism. The Single Supervisory Mechanism, or SSM in the EU jargon, is a first step in the broader revision of policies towards banks in Europe. The eventual goal is the creation of a Banking Union, which is to carry out effective surveillance and – if needed – crisis management of the banking sector. The SSM is to rely on national supervisors and the ECB, with the ECB having final authority on the matter. The involvement of the latter made it clear that the SSM would be centred on the eurozone – while it is to remain open to other Member States willing to join. Due to the ongoing problems and the link between the creation of the SSM and the recapitalisation of banks, the SSM became one of the key legislative priorities of the EU. In December 2012, Member States reached an agreement on the design of the SSM. After discussions with the European Parliament (which were still ongoing at the time of writing), the process towards making the SSM operational can be initiated. The goal is to have the SSM fully up and running in the first half of 2014. The decisions that were taken in June 2012 are likely to have had a bigger impact than the eurozone’s Heads of State and Government could have realised at the time for two important reasons. On the one hand, creating the SSM necessitates a full Banking Union and therefore shared risk. On the other hand, the decisions improved the ECB’s perception of the willingness of governments to take far-reaching measures. This undoubtedly played a significant role in the creation of the Outright Monetary Transactions programme by the ECB, which has led to a substantial easing of the crisis in the short-term. 1 These short-term gains should now be matched with a stable long-term framework for bank supervision and crisis management. The agreement on the SSM should be the first step in the direction of this goal. This paper provides an analysis of the SSM and its role in the creation of a Banking Union. The paper starts with a reminder of why the EU decided to put in place the SSM (§1) and the state of play of the ongoing negotiations on the SSM (§2). Subsequently, the supervisory responsibilities of the SSM are detailed, including its scope and the division of labour between the national supervisors and the ECB (§3). The internal functioning of the SSM (§4) and its relation to the other supervisors are discussed afterwards (§5). As mentioned earlier, the SSM is part of a wider move towards a Banking Union. Therefore, this paper sheds light on the other building blocks of this ambitious project (§6). The transition towards the Banking Union is important and will prove to be a bumpy ride. Before formulating a number of conclusions, this Working Paper therefore provides an overview of the planned road ahead (§7).