Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.

Solution structure of the Shc SH2 domain complexed with a tyrosine-phosphorylated peptide from the T-cell receptor.

She is a widely expressed adapter protein that plays an important role in signaling via a variety of cell surface receptors and has been implicated in coupling the stimulation of growth factor, cytokine, and antigen receptors to the Ras signaling pathway. She interacts with several tyrosine-phosphorylated receptors through its C-terminal SH2 domain, and one of the mechanisms of T-cell receptor-mediated Ras activation involves the interaction of the Shc SH2 domain with the tyrosine-phosphorylated zeta chain of the T-cell receptor. Here we describe a high-resolution NMR structure of the Shc SH2 domain complexed to a phosphopeptide (GHDGLpYQGLSTATK) corresponding to a portion of the zeta chain of the T-cell receptor. Although the overall architecture of the protein is similar to other SH2 domains, distinct structural differences were observed in the smaller beta-sheet, BG loop, (pY + 3) phosphopeptide-binding site, and relative position of the bound phosphopeptide.

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Utilization of ADR Concepts to Bridge the Gap Between the Extractive Industry and Environmental Interests: An Anticipatory Cooperative Effort (ACE)

The extractive industry, more than any other sector of the economy, often finds itself mired in conflicts with various environmental and community interests. As traditional legal avenues of resolution gave way to the collaborative ideas of alternative dispute resolution, the outcomes, especially the relational outcomes, were less than desirable. This capstone project proposes that an Anticipatory Cooperative Effort (ACE) can help to bridge the gap between industry and environmental interests by encouraging a pro-active and pre-emptive engagement. The point of the ACE concept is not that it defines a new set of principles so much as it repositions where established ADR principles are entertained.

Resolution of the committee concerning the way through Rogers's farm, 1762 February 27

One octavo-sized leaf containing a one-page handwritten draft of a resolution by a Harvard Corporation Committee appointed to "lay out an High Way thro' Rogers's Farm & determine about the Cost of the Sd way & the making the fences to enclose it." The resolution permits the town of Waltham to lay a highway on the farm's property as long as it is enclosed by a stone wall.

Tackling barriers to trade in the Single Market. ACES Cases No. 2014.2

This paper focuses on the challenges operating in the single market due to continued persistence of regulatory barriers to trade, despite being considered one of the most integrated and successful areas of market integration. We use a unique data set on infringements to the free movement of goods to assess the types of barriers that firms encounter, their impact and variation across states and sectors, and their resolution method - through Court decisions or the pre-litigation, administrative means available within the infringement proceedings mechanism to restore compliance. We also resort to the Solvit dataset provided to the authors by the Commission to analyse some features and the effectiveness of this informal mechanism in dealing with discriminatory domestic trade and regulatory practices. We examine four key questions: What are the most problematic policy areas in terms of barriers to trade that undermine the single market? What different dispute resolution mechanisms are utilized to address trade barriers and thus improve the functioning of the single market? Under what conditions are different enforcement mechanisms and strategies more likely to be used to resolve barriers for businesses operating in the single market? How important and effective are the more informal strategies in improving market access? In doing so, our goal is to link the research on trade barriers to that of implementation and compliance to assess the diverse strategies undertaken to reduce regulatory barriers to trade.

Tropical Atlantic SST during the mid-Pleistocene transition (MPT) of ODP Hole 175-1077B

We compare a new mid-Pleistocene sea surface temperature (SST) record from the eastern tropical Atlantic to changes in continental ice volume, orbital insolation, Atlantic deepwater ventilation, and Southern Ocean front positions to resolve forcing mechanisms of tropical Atlantic SST during the mid-Pleistocene transition (MPT). At the onset of the MPT, a strong tropical cooling occurred. The change from a obliquity- to a eccentricity-dominated cyclicity in the tropical SST took place at about 650 kyr BP. In orbital cycles, tropical SST changes significantly preceded continental ice-volume changes but were in phase with movements of Southern Ocean fronts. After the onset of large-amplitude 100-kyr variations, additional late glacial warming in the eastern tropical Atlantic was caused by enhanced return flow of warm waters from the western Atlantic driven by strong trade winds. Pronounced 80-kyr variations in tropical SST occurred during the MPT, in phase with and likely directly forced by transitional continental ice-volume variations. During the MPT, a prominent anomalous long-term tropical warming occurred, likely generated by extremely northward displaced Southern Ocean fronts. While the overall pattern of global climate variability during the MPT was determined by changes in mean state and frequency of continental ice volume variations, tropical Atlantic SST variations were primarily driven by early changes in Subantarctic sea-ice extent and coupled Southern Ocean frontal positions.

Sea surface temperature reconstruction based on alkenones of sediment core M35003-4

Evidence for abrupt climate changes on millennial and shorter timescales is widespread in marine and terrestrial climate records (Dansgard et al., 1993, doi:10.1038/364218a0; Bond et al., 1993, doi:10.1038/365143a0; Charles et al., 1996, doi:10.1016/0012-821X(96)00083-0, Bard et al., 1997, doi:10.1038/385707a0). Rapid reorganization of ocean circulation is considered to exert some control over these changes (Broecker et al., 1985, doi:10.1038/315021a0), as are shifts in the concentrations of atmospheric greenhouse gases (Broecker, 1994, doi:10.1038/372421a0). The response of the climate system to these two influences is fundamentally different: slowing of thermohaline overturn in the North Atlantic Ocean is expected to decrease northward heat transport by the ocean and to induce warming of the tropical Atlantic (Crowley, 1992, doi:10.1029/92PA01058; Manabe and Stouffer, 1997, doi:10.1029/96PA03932), whereas atmospheric greenhouse forcing should cause roughly synchronous global temperature changes (Manabe et al., 1991, doi:10.1175/1520-0442(1991)004<0785:TROACO>2.0.CO;2). So these two mechanisms of climate change should be distinguishable by the timing of surface-water temperature variations relative to changes in deep-water circulation. Here we present a high-temporal-resolution record of sea surface temperatures from the western tropical North Atlantic Ocean which spans the past 29,000 years, derived from measurements of temperature-sensitive alkenone unsaturation in sedimentary organic matter. We find significant warming is documented for Heinrich event H1 (16,900-15,400 calendar years bp) and the Younger Dryas event (12,900-11,600 cal. yr bp), which were periods of intense cooling in the northern North Atlantic. Temperature changes in the tropical and high-latitude North Atlantic are out of phase, suggesting that the thermohaline circulation was the important trigger for these rapid climate changes.

Morphometric measurements throughout ontogeny of selected planktic foraminiferal test species

Development plays an important part in shaping adult morphology and morphological disparity, yet its influence on evolutionary processes is seldom explored because of a lack of preservation of ontogenetic stages in the fossil record. By preserving their entire ontogenetic history within their test, and with the advent of high-resolution imaging techniques, planktic foraminifera allow us to investigate the influence of developmental constraints on disparity. Using Synchrotron radiation X-ray tomographic microscopy (SRXTM), we reconstruct the ontogenetic progression of seven species across several of the major morphotypic groups of planktic foraminifera, including morphotypes of a species exhibiting high phenotypic plasticity and closely related pseudo-cryptic sister-taxa. We show differences in growth patterns between the globigerinid species, which appear more tightly regulated within the framework of isometry from the neanic stage, and the globorotaliid species, whose adult stages present allometric trends. Morphological change through ontogeny results in a change in surface area to volume ratios. Different metabolic processes therefore dominate at different stages of ontogeny, changing the vulnerability of the organism to environmental influences over growth, from factors affecting diffusion rates in the juvenile to those affecting energy supply in the adult. These findings identify some of the parameters within which evolutionary mechanisms have to act.

Why Do Some International Institutions Contain Strong Dispute Settlement Provisions: New Evidence from Preferential Trade Agreements

To understand why some international institutions have stronger dispute settlement mechanisms (DSMs) than others, we investigate the dispute settlement provisions of nearly 600 preferential trade agreements (PTAs), which possess several desirable case-selection features and are evoked more than is realized. We broaden the study of dispute settlement design beyond “legalization” and instead reorient theorizing around a multi-faceted conceptualization of the strength of DSMs. We posit that strong DSMs are first and foremost a rational response to features of agreements that require stronger dispute settlement, such as depth and large memberships. Multivariate empirical tests using a new data set on PTA design confirm these expectations and reveal that depth – the amount of policy change specified in an agreement – is the most powerful and consistent predictor of DSM strength, providing empirical support to a long-posited but controversial conjecture. Yet power also plays a sizeable role, since agreements among asymmetric members are more likely to have strong DSMs due to their mutual appeal, as are those involving the United States. Important regional differences also emerge, as PTAs across the Americas are designed with strong dispute settlement, as are Asian PTAs, which contradicts the conventional wisdom about Asian values and legalization. Our findings demonstrate that rationalism explains much of international institutional design, yet it can be enhanced by also incorporating power-based and regional explanations.

The Extensive (but Fragile) Authority of the WTO Appellate Body

The authority of an international court (IC) is not necessarily evolutionary and its development unidirectional. This article addresses the authority of the Appellate Body (AB) of the World Trade Organization (WTO) and shows how it rapidly and almost immediately became extensive, but has since exhibited signs of becoming more fragile. The article applies a typology of IC authority developed by Alter, Helfer and Madsen (2014) and explains the transformation from narrow authority (a dispute resolution venue under the GATT based on political negotiations) to extensive authority (a judicialized WTO dispute settlement system with a sophisticated case law) and presents empirical indicators of the rise of the AB’s authority. Such rapid development of extensive authority is arguably a unique case in international politics at the multilateral level. That authority nonetheless remains fragile, and shows signs that it could decline significantly for reasons we explain.

Mediation & conference programs in the federal courts of appeals : a sourcebook for judges and lawyers / Robert J. Niemic.

Shipping list no.: 97-0241-P.

Testimony delivered to the Commission on the Future of Worker-Management Relations, U.S. Department of Labor, Washington, D.C. /

"April 6, 1994."

Lessons from tomographic studies of the mammalian Golgi

Basic structure studies of the biosynthetic machinery of the cell by electron microscopy (EM) have underpinned much of our fundamental knowledge in the areas of molecular cell biology and membrane traffic. Driven by our collective desire to understand how changes in the complex and dynamic structure of this enigmatic organelle relate to its pivotal roles in the cell, the comparatively high-resolution glimpses of the Golgi and other compartments of the secretory pathway offered to us through EM have helped to inspire the development and application of some of our most informative, complimentary (molecular, biochemical and genetic) approaches. Even so, no one has yet even come close to relating the basic molecular mechanisms of transport, through and from the Golgi, to its ultrastructure, to everybody's satisfaction. Over the past decade, EM tomography has afforded new insights into structure -function relationships of the Golgi and provoked a re-evaluation of older paradigms. By providing a set of tools for structurally dissecting cells at high-resolution in three-dimensions (3D), EM tomography has emerged as a method for studying molecular cell biology in situ. As we move rapidly toward the establishment of molecular atlases of organelles through advances in proteomics and genomics, tomographic studies of the Golgi offer the tantalizing possibility that one day, we will be able to map the spatio-temporal coordinates of Golgi-related proteins and lipids accurately in the context of 4D cellular space. (c) 2005 Elsevier B.V. All rights reserved.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.