Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides

Protein TrwC is the conjugative relaxase responsible for DNA processing in plasmid R388 bacterial conjugation. TrwC has two catalytic tyrosines, Y18 and Y26, both able to carry out cleavage reactions using unmodified oligonucleotide substrates. Suicide substrates containing a 30-Sphosphorothiolate linkage at the cleavage site displaced TrwC reaction towards covalent adducts and thereby enabled intermediate steps in relaxase reactions to be investigated. Two distinct covalent TrwC–oligonucleotide complexes could be separated from noncovalently bound protein by SDS–PAGE. As observed by mass spectrometry, one complex contained a single, cleaved oligonucleotide bound to Y18, whereas the other contained two cleaved oligonucleotides, bound to Y18 and Y26. Analysis of the cleavage reaction using suicide substrates and Y18F or Y26F mutants showed that efficient Y26 cleavage only occurs after Y18 cleavage. Strand-transfer reactions carried out with the isolated Y18–DNA complex allowed the assignment of specific roles to each tyrosine. Thus, only Y18 was used for initiation. Y26 was specifically used in the second transesterification that leads to strand transfer, thus catalyzing the termination reaction that occurs in the recipient cell.

Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.

The vegetation and climate history of the last glacial cycle in a new pollen record from Lake Fimon (southern Alpine foreland, N-Italy)

The sediments of Like Fimon N Italy contain the first continuous archive of the Late Pleistocene environmental and climate history of the southern Alpine foreland We present here the detailed palynological record of the interval between Termination II and the List Glacial Maximum The age-depth model is obtained by radiocarbon dating in the uppermost part of the record Downward we con elated major forest expansion and contraction events to isotopic events in the Greenland Ice core records via a stepping-stone approach involving intermediate correlation to isotopic events dated by TIMS U/Th in Alpine and Apennine stalagmites and to pollen records from mime cores of the Iberian margin Modelled ages obtained by Bayesian analysis of deposition are thoroughly consistent with actual ages with maximum offset of +/- 1700 years Sharp expansion of broad-leaved temperate forest and of sudden water table rise mark the onset of the Last Interglacial after a treeless steppe phase at the end of penultimate glaciation This event is actually a two-step process which matches the two step rise observed in the isotopic record of the nearby Antro del Corchia stalagmite respectively dated to 132 5 +/- 2 5 and 129 +/- 1 5 ka At the interglacial decline mixed oak forests were replaced by oceanic mixed forests the latter persisting further for 7 ka till the end of the Eemian succession Warm-temperate woody species are still abundant at the Eemian end corroborating a steep gradient between central Europe and the Alpine divide at the inception of the last glacial After a stadial phase marked by moderate forest decline a new expansion of warm broad leaved forests interrupted by minor events and followed by mixed oceanic forests can be identified with the north-alpine Saint Germain I The spread of beech during the oceanic phase is a valuable circumalpine marker The subsequent stadial-interstadial succession lacking the telocratic oceanic phase is also consistent with the evidence at the north alpine foreland The Middle Wurmian (full glacial) is marked by persistence of mixed forests dominated by conifers but with significant lime and other broad leaved species A major Arboreal Pollen decrease is observed at modelled age of 38 7 +/- 0 5 ka (larch expansion and last occurrence of lime) which his been related to Heinrich Event 4 The evidence of afforestation persisting south of the Alps throughout most of MIS 3 contrasts with a boreal and continental landscape known for the northern alpine foreland pointing to a sharp rainfall boundary at the Alpine divide and to southern air circulation This is in agreement with the Alpine paleoglaciological record and is supported by the pressure and rainfall patterns designed by mesoscale paleoclimate simulations Strenghtening the continental high pressure during the full glacial triggered cyclogenesis in the middle latitude eastern Europe and orographic rainfall in the eastern Alps and the Balkanic mountains thus allowing forests development at current sea level altitudes (C) 2010 Elsevier Ltd All rights reserved

The development of regret

In two experiments, 4- to 9-year-olds played a game in which they
selected one of two boxes to win a prize. On regret trials the unchosen
box contained a better prize than the prize children actually
won, and on baseline trials the other box contained a prize of the
same value. Children rated their feelings about their prize before
and after seeing what they could have won if they had chosen
the other box and were asked to provide an explanation if their
feelings had changed. Patterns of responding suggested that regret
was experienced by 6 or 7 years of age; children of this age could
also explain why they felt worse in regret trials by referring to
the counterfactual situation in which the prize was better. No evidence
of regret was found in 4- and 5-year-olds. Additional findings
suggested that by 6 or 7 years, children’s emotions were
determined by a consideration of two different counterfactual
scenarios.

The Cold War and British Debates over the Boycott of the 1980 Moscow Olympics

This article examines the debate precipitated by the Thatcher government's (unsuccessful) attempt to secure a British boycott of the 1980 Moscow Olympics in response to the Soviet invasion of Afghanistan. Aware that it faced a struggle to win over the autonomous British Olympic Association, but with Thatcher in particular keen to support the United States, the government's case that the invasion required a specific response in the form of a boycott was steadily overshadowed as the public debate increasingly focused on arguments over human rights and détente and the use of state power.

Physical Models of Wind Instruments : A Generalized Excitation Coupled with a Modular Tube Simulation Platform

To develop real-time simulations of wind instruments, digital waveguides filters can be used as an efficient representation of the air column. Many aerophones are shaped as horns which can be approximated using conical sections. Therefore the derivation of conical waveguide filters is of special interest. When these filters are used in combination with a generalized reed excitation, several classes of wind instruments can be simulated. In this paper we present the methods for transforming a continuous description of conical tube segments to a discrete filter representation. The coupling of the reed model with the conical waveguide and a simplified model of the termination at the open end are described in the same way. It turns out that the complete lossless conical waveguide requires only one type of filter.Furthermore, we developed a digital reed excitation model, which is purely based on numerical integration methods, i.e., without the use of a look-up table.

Hidden costs of control: four repetitions and an extension

We report four repetitions of Falk and Kosfeld's (Am. Econ. Rev. 96(5):1611-1630, 2006) low and medium control treatments with 476 subjects. Each repetition employs a sample drawn from a standard subject pool of students and demographics vary across samples. We largely confirm the existence of hidden costs of control but, contrary to the original study, hidden costs of control are usually not substantial enough to significantly undermine the effectiveness of economic incentives. Our subjects were asked, at the end of the experimental session, to complete a questionnaire in which they had to state their work motivation in hypothetical scenarios. Our questionnaires are identical to the ones administered in Falk and Kosfeld's (Am. Econ. Rev. 96(5):1611-1630, 2006) questionnaire study. In contrast to the game play data, our questionnaire data are similar to those of the original questionnaire study. In an attempt to solve this puzzle, we report an extension with 228 subjects where performance-contingent earnings are absent i.e. both principals and agents are paid according to a flat participation fee. We observe that hidden costs significantly outweigh benefits of control under hypothetical incentives.

Politics, policy preferences and the evolution of Irish bureaucracy:A framework for analysis

Analysis of the Irish state's administrative system is an unaccountably neglected area of systematic academic inquiry. This is all the more difficult to account for in view of the dynamic relationship between government actors and the public bureaucracy in realizing political goals. This paper identifies some distinguishing institutional features and dominant trends in Irish politico-administrative governance, and suggests avenues for future inquiry. The paper begins with an examination of the literature on administrative system change, with a focus on the New Public Management literature. Following this, the Irish case is profiled, identifying the evolution of ministerial departments and of state agencies by successive Irish governments, including patterns of agency creation and termination over time. Particular attention is given to the period 1989-2010, which has been one of quite rapid and complex organizational change within the state's bureaucratic apparatus. © 2012 Political Studies Association of Ireland.

Survival without disability to age 5 years after neonatal caffeine therapy for apnea of prematurity

Very preterm infants are prone to apnea and have an increased risk of death or disability. Caffeine therapy for apnea of prematurity reduces the rates of cerebral palsy and cognitive delay at 18 months of age.