Transcription termination at intrinsic terminators: the role of the RNA hairpin.

Intrinsic termination of transcription in Escherichia coli involves the formation of an RNA hairpin in the nascent RNA. This hairpin plays a central role in the release of the transcript and polymerase at intrinsic termination sites on the DNA template. We have created variants of the lambda tR2 terminator hairpin and examined the relationship between the structure and stability of this hairpin and the template positions and efficiencies of termination. The results were used to test the simple nucleic acid destabilization model of Yager and von Hippel and showed that this model must be modified to provide a distinct role for the rU-rich sequence in the nascent RNA, since a perfect palindromic sequence that is sufficiently long to form an RNA hairpin that could destabilize the entire putative 12-bp RNA-DNA hybrid does not trigger termination at the expected positions. Rather, our results show that both a stable terminator hairpin and the run of 6-8 rU residues that immediately follows are required for effective intrinsic termination and that termination occurs at specific and invariant template positions relative to these two components. Possible structural or kinetic modifications of the simple model are proposed in the light of these findings and of recent results implicating "inchworming" and possible conformational heterogeneity of transcription complexes in intrinsic termination. Thus, these findings argue that the structure and dimensions of the hairpin are important determinants of the termination-elongation decision and suggest that a complete mechanism is likely to involve specific interactions of the polymerase, the RNA terminator hairpin, and, perhaps, the dT-rich template sequence that codes for the run of rU residues at the 3' end of the nascent transcript.

Cotranslational folding and calnexin binding during glycoprotein synthesis.

To analyze cotranslational folding of influenza hemagglutinin in the endoplasmic reticulum of live cells, we used short pulses of radiolabeling followed by immunoprecipitation and analysis with a two-dimensional SDS/polyacrylamide gel system which was nonreducing in the first dimension and reducing in the second. It separated nascent glycopolypeptides of different length and oxidation state. Evidence was obtained for cotranslational disulfide formation, generation of conformational epitopes, N-linked glycosylation, and oligosaccharide-dependent binding of calnexin, a membrane-bound chaperone that binds to incompletely folded glycoproteins via partially glucose-trimmed oligosaccharides. When glycosylation or oligosaccharide trimming was inhibited, the folding pathway was perturbed, suggesting a role for N-linked oligosaccharides and calnexin during translation of hemagglutinin.

Low concentrations of diacylglycerol promote the binding of apolipophorin III to a phospholipid bilayer: a surface plasmon resonance spectroscopy study.

The binding of the exchangeable apolipoprotein apolipophorin III (apoLp-III) to an egg phosphatidylcholine bilayer as a function of the concentration of diacylglycerol (DG) in the bilayer was studied by surface plasmon resonance spectroscopy. At a DG concentration of 2 mol % in the bilayer, the binding of apoLp-III reached saturation. Under saturating conditions, apoLp-III forms a closely packed monolayer approximately 55 A thick, in which each molecule of protein occupies approximately 500 A2 at the membrane surface. These dimensions are consistent with the molecular size of the apoLp-III molecule determined by x-ray crystallography, if apoLp-III binds to the bilayer with the long axis of the apoLp-III normal to the membrane surface. In the absence of protein, the overall structure of the lipid bilayer was not significantly changed up to 2.5 mol% DG. However, at 4 and 6 mol % DG, the presence of nonbilayer structures was observed. The addition of apoLp-III to a membrane containing 6 mol % DG promoted the formation of large lipid-protein complexes. These data support a two-step sequential binding mechanism for binding of apoLp-III to a lipid surface. The first step is a recognition process, consisting of the adsorption of apoLp-III to a nascent hydrophobic defect in the phospholipid bilayer caused by the presence of DG. This recognition process might depend on the presence of a hydrophobic sensor located at one of the ends of the long axis of the apoLp-III molecule but would be consolidated through H-bond and electrostatic interactions. Once primary binding is achieved, subsequent enlargement of the hydrophobic defect in the lipid surface would trigger the unfolding of the apolipoprotein and binding via the amphipathic alpha-helices. This two-step sequential binding mechanism could be a general mechanism for all exchangeable apolipoproteins. A possible physiological role of the ability of apoLp-III to bind to lipid structures in two orientations is also proposed.

In vitro cleavage and joining at the viral origin of replication by the replication initiator protein of tomato yellow leaf curl virus.

Replication of the single-stranded DNA genome of geminiviruses occurs via a double-stranded intermediate that is subsequently used as a template for rolling-circle replication of the viral strand. Only one of the proteins encoded by the virus, here referred to as replication initiator protein (Rep protein), is indispensable for replication. We show that the Rep protein of tomato yellow leaf curl virus initiates viral-strand DNA synthesis by introducing a nick in the plus strand within the nonanucleotide 1TAATATT decreases 8AC, identical among all geminiviruses. After cleavage, the Rep protein remains bound to the 5' end of the cleaved strand. In addition, we show that the Rep protein has a joining activity, suggesting that it acts as a terminase, thus resolving the nascent viral single strand into genome-sized units.

Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast.

The CDC47 gene was isolated by complementation of a cdc47 temperature-sensitive mutant in Saccharomyces cerevisiae and was shown to encode a predicted polypeptide, Cdc47, of 845 aa. Cdc47 belongs to the Cdc46/Mcm family of proteins, previously shown to be essential for initiation of DNA replication. Using indirect immunofluorescence microscopy and subcellular fractionation techniques, we show that Cdc47 undergoes cell cycle-regulated changes in its subcellular localization. At mitosis, Cdc47 enters the nucleus, where it remains until soon after the initiation of DNA replication, when it is rapidly exported back into the cytoplasm. Cdc47 protein levels do not vary with the cell cycle, but expression of CDC47 and nascent synthesis of Cdc47 occur late in the cell cycle, coinciding with mitosis. Together, these results show that Cdc47 is not only imported into the nucleus at the end of mitosis but is also exported back into the cytoplasm at the beginning of S phase. The observation that Cdc47 is exported from the nucleus at the beginning of S phase has important implications for how initiation of DNA replication is controlled.

Coaliciones promotoras y transparencia en México: el caso de las políticas de derecho de acceso a la información en la administración pública federal 2002-2014

Mexico is now one of the countries with better policies on transparency and access to public information, according to various indicators and academics. Just fifteen years ago, Mexico was a country that lacked legal instruments thereon, whereby the institutions were deeply opaque and citizens could not exercise this right of access to public information. The development of the right of access to public information, in both law and public policy, a milestone in the history of Mexico. It has been, therefore gestation, as its formulation and implementation. In Mexico there have existed diverse social movements that have promoted democratization and the defense of human rights. In the framework of these movements the fight registers for the right of access to the public information that one presents as a successful model of civic action and government intervention, without for it, not to know the challenges that his deepening has still and take root both in the company and in the political class in general. How was it achieved to construct a new institutional of transparency that was functional? How was it possible that the above mentioned change was achieved? These are questions that interests formulated to the political science and to the public administration for the analysis of the change and improvement of institutions. The study of the political change is relevant since the public policies precisely try to solve a problem, to transform a reality but not always the change is achieved, is not even realized of successful form. In a nascent democratic regime, it turns out important to know what factors can collaborate in the conformation of a public successful sustainable politics in the time. Even more, on having treated itself about a substantive politics that it gives content and viability itself to the democracy in a marked country historically and culturally for the opaqueness and the corruption...

Safeguarding the EDTIB: the Case for Supervising non-EU FDI in the Defence Sector. Security Policy Brief No. 41, December 2012

It is time for the EU member states to start collectively supervising non-EU FDI in Europe’s defence industries and infrastructures. This should be a prudent element of the nascent EDTIB and a way to maintain European security by encouraging greater coordination between the national supervisory frameworks.

Nietzsche, la pulsion, l’histoire

Cette étude vise à comprendre le rôle de l’histoire dans la philosophie de Nietzsche et à faire ressortir son lien étroit avec l’articulation du corps vivant comme fil conducteur philosophique. Notre objectif principal est de montrer comment cette philosophie aphoristique a su maintenir les préoccupations historiographiques de jeunesse en permutant leur sens à l’aune de la pulsionnalité interprétative du corps. Prenant notre départ des considérations critiques écrites lors de son professorat à Bâle, nous démontrons que le sens historique se manifeste alors comme une sensibilité historique déterminée par une saisie intuitive, mais existentiellement difficile, du passé. Nous procédons ensuite à décrire comment le renouveau philosophique de sa période intermédiaire peut être vu comme une réduction pathologique de l’histoire de la métaphysique qui emprunte ses éléments critiques au scepticisme de Michel de Montaigne, à l’évolutionnisme naissant et au développement du néo-kantisme. Cette réduction, qui ramène l’expression des valeurs morales et métaphysiques au corps vécu (Leib). Par sa déconstruction de la subjectivité au profit d’une réalité pulsionnelle primordiale, mais irréductible, nous démontrons ensuite comment Nietzsche a su réinterpréter l’hérédité biologique comme une mémoire physiologique incorporée dont l’expression première est la reconduction de la notion d’espèce à celle de type. Enfin, par un retour à la sensibilité historique et notre analyse du phénomène historique en tant que tel, nous proposons de comprendre l’articulation ultime de la philosophie nietzschéenne comme une philosophie historique qui ne cherche pas à comprendre ou à expliquer le devenir, mais en opérer la synthèse par le truchement de « l’instant décisif ».

Improving the role of equity crowdfunding in Europe's capital markets. Bruegel Policy Contribution Issue 2014/09, August 2014

Crowdfunding is a growing phenomenon that encompasses several different models of financing for business or other ventures. Despite the hype, equity crowdfunding is still the smallest part of the crowdfunding market. Because of its legal framework, Europe has been at the forefront of equity crowdfunding market development. Equity crowdfunding is more complex than other forms of crowdfunding and requires proper checks and balances if it is to provide a viable channel for financial intermediation in the seed and early-stage market in Europe. It is important to explore this new channel of funding for young and innovative firms given the critical role these start-ups can play job creation and economic growth in Europe. We assess the potential role of equity crowdfunding in the overall seed and early-stage financing market and highlight the potential risks of equity crowdfunding. We describe the current state of play in this nascent industry, considering both the innovations introduced by market operators and existing regulation. Currently in Europe there is a patchwork of national legal frameworks related to equity crowdfunding and this should be addressed in a harmonised way.

How two become one: HJURP dimerization drives CENP-A assembly

CENP‐A containing nucleosomes epigenetically specify centromere position on chromosomes. Deposition of CENP‐A into chromatin is mediated by HJURP, a specific CENP‐A chaperone. Paradoxically, HJURP binding sterically prevents dimerization of CENP‐A, which is critical to form functional centromeric nucleosomes. A recent publication in The EMBO Journal (Zasadzińska et al, 2013) demonstrates that HJURP itself dimerizes through a C‐terminal repeat region, which is essential for centromeric assembly of nascent CENP‐A.

Human Capital and New Venture Performance: The Moderating Role of Social Relations

With whom should entrepreneurs create their firms in order to enhance nascent venture performance? Conventional wisdom suggests that the stronger human capital and social relations in nascent venture teams are, the better the nascent venture’s performance. We draw from social embeddedness literature, however, and argue that the positive effect of team members’ human capital on three different dimensions of nascent venture performance is weaker when team members exhibit strong social relations. Our analysis of 488 nascent venture teams in the PSED II dataset confirms our predictions, showing that nascent ventures of teams with strong human capital but weaker social relations exhibit the best performance. The study thus offers valuable contributions particularly to literature on entrepreneurial teams the determinants of new venture performance.

Nietzsche, la pulsion, l’histoire

Cette étude vise à comprendre le rôle de l’histoire dans la philosophie de Nietzsche et à faire ressortir son lien étroit avec l’articulation du corps vivant comme fil conducteur philosophique. Notre objectif principal est de montrer comment cette philosophie aphoristique a su maintenir les préoccupations historiographiques de jeunesse en permutant leur sens à l’aune de la pulsionnalité interprétative du corps. Prenant notre départ des considérations critiques écrites lors de son professorat à Bâle, nous démontrons que le sens historique se manifeste alors comme une sensibilité historique déterminée par une saisie intuitive, mais existentiellement difficile, du passé. Nous procédons ensuite à décrire comment le renouveau philosophique de sa période intermédiaire peut être vu comme une réduction pathologique de l’histoire de la métaphysique qui emprunte ses éléments critiques au scepticisme de Michel de Montaigne, à l’évolutionnisme naissant et au développement du néo-kantisme. Cette réduction, qui ramène l’expression des valeurs morales et métaphysiques au corps vécu (Leib). Par sa déconstruction de la subjectivité au profit d’une réalité pulsionnelle primordiale, mais irréductible, nous démontrons ensuite comment Nietzsche a su réinterpréter l’hérédité biologique comme une mémoire physiologique incorporée dont l’expression première est la reconduction de la notion d’espèce à celle de type. Enfin, par un retour à la sensibilité historique et notre analyse du phénomène historique en tant que tel, nous proposons de comprendre l’articulation ultime de la philosophie nietzschéenne comme une philosophie historique qui ne cherche pas à comprendre ou à expliquer le devenir, mais en opérer la synthèse par le truchement de « l’instant décisif ».

Channel Migration and Avulsion Hazards at Sunshine Point Campground, Mount Rainier National Park, Washington

In November 2006, the flood of record on the upper Nisqually River destroyed part of Sunshine Point Campground in Mount Rainier National Park, Washington. The Nisqually River migrated north and reoccupied five acres of its floodplain; Tahoma Creek partially avulsed into the west floodplain, topping banks of an undersized channel and flooding the campground. I assessed hazards to infrastructure at the old campground location, where the Park proposes to rebuild the remaining campground roads and sites. This assessment focuses on two major hazards: northward Nisqually River migration, which may reincorporate the floodplain into the river destroying infrastructure; and Tahoma Creek avulsions, which may flood the campgroud and deposit sediment burying campground infrastructure. I quantify northward migration by: estimating migration rates and changes to channel width; evaluating river occupation of the pre- and post-2006 campground; and estimating scour depths at revetments protecting the campground. I digitized the Nisqually River channels and channel centerlines from maps and images between 1955 and 2013 into a GIS, which I used to estimate migration rate and river width changes. Centerline migration rates average 9 ft/yr along the length of the Nisqually River study reach; at Sunshine Point lateral migration rates average 11 ft/yr. Maximum migration along the study reach was 19 ft/yr between 2006 and 2009. Greater than average migration rates and channel widths correspond to river confluences and include the Tahoma Creek confluence at Sunshine Point. To determine historical channel locations and the frequency that the river occupied different parts of its floodplain, I digitized the river from maps and images between 1903 and 2013. The Nisqually River flows through Sunshine Point Campground in eight out of 15 historical images. I assess scour at revetments protecting infrastructure from the Nisqually River during a 100-year recurrence interval flood using measured cross-sections. During a 100-year flood, the Nisqually River may scour up to 10 feet below the bed elevation. These scour depths can destabilize critical revetments leaving loose unconsolidated riverbanks exposed to Nisqually River flows. To determine the causes, locations, and frequency of flood hazards from Tahoma Creek avulsions, I field map avulsion channels and compare the results with imagery and channel width changes between 1955 and 2013. Mapped avulsion channels occur with swaths of dead vegetation or nascent vegetation; both dead and recent vegetation are visibly distinct from surrounding vegetation in aerial images. Times of changes to these vegetation anomalies correspond to increases in Tahoma Creek channel width. Avulsions have occurred at least three times in the study period: pre-1955, between 1979 and 1984, and in 2006. The 1984 and 2006 avulsions both occur after increases in Tahoma Creek reach averaged width. The NPS is considering two options to rebuild Sunshine Point Campground, both at the same location. The hazards posed by the Nisqually River and Tahoma Creek at Sunshine Point will affect both construction options equally. Migration hazards to the campground may be reduced by limiting the proposed campground infrastructure to an elevated ridge that has not been occupied by the Nisqually River since 1903. The hazards of damage from migration may be reduced by revetments, which were effective in preventing northward Nisqually River migration in 1959 and 1965. Tahoma Creek avulsions are related increased of Tahoma Creek reach averaged widths, which are near a 58- year maximum, and occurred during a 10-year flood in 1984. The campground may be as susceptible to flooding from avulsions during as little as a 10-year flood. A large avulsion may occur with the next significant Tahoma Creek width increase. Glacial retreat has been shown to increase debris flow activity and increase sediment delivery to Mount Rainier rivers. Increased sediment discharge has been correlated with aggradation, which will further encourage Tahoma Creek avulsions.

Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NSO) cells. Four homogeneous NSO cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab clatasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (C) 2004 Wiley Periodicals, Inc.