Developing nation imperatives for a new law of the sea: UNCLOS I and III as stages in the international policy process

Abstract

This paper seeks to compare the two major law of the sea conferences of the post‐World War II period in terms of imperatives for moving toward a more equitable international system. Transitions in the international system which have taken place in the years between the conferences are analyzed, as well as differences in conference participation and procedures. The paper then focuses upon the necessity for policy to be formulated in such a way that imbalances in opportunities for utilization of the seas are redressed. Special attention is given the status of the landlocked and geographically disadvantaged states. The 1977 Negotiating Text is commented on from this perspective. Proposals for development of ocean law/policy in UNCLOS III more congruent with achievement of global equity are then set forth.     

Single and dual task gait training in people with Parkinson's Disease: A protocol for a randomised controlled trial

Background  

Difficulty performing more than one task at a time (dual tasking) is a common and disabling problem experienced by people with Parkinson disease (PD). If asked to perform another task when walking, people with PD often take shorter steps or walk more slowly. Currently there is uncertainty about whether clinicians should teach people with PD to avoid dual tasking or whether they should encourage them to practice dual tasking with the hope that practice will lead to enhanced performance. This study will address this issue by comparing single to dual task gait training.     

New pollen-specific receptor kinases identified in tomato,maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes

We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

Atypical memory phenotype T cells with low homeostatic potential and impaired TCR signaling and regulatory T cell function in Foxn1Delta/Delta mutant mice

Foxn1Delta/Delta mutants have a block in thymic epithelial cell differentiation at an intermediate progenitor stage, resulting in reduced thymocyte cellularity and blocks at the double-negative and double-positive stages. Whereas naive single-positive thymocytes were reduced >500-fold in the adult Foxn1Delta/Delta thymus, peripheral T cell numbers were reduced only 10-fold. The current data shows that Foxn1Delta/Delta peripheral T cells had increased expression of activation markers and the ability to produce IL-2 and IFN-gamma. These cells acquired this profile immediately after leaving the thymus as early as the newborn stage and maintained high steady-state proliferation in vivo but decreased proliferation in response to TCR stimulation in vitro. Single-positive thymocytes and naive T cells also had constitutively low alphabetaTCR and IL7R expression. These cells also displayed reduced ability to undergo homeostatic proliferation and increased rates of apoptosis. Although the frequency of Foxp3+CD4+CD25+ T cells was normal in Foxn1Delta/Delta mutant mice, these cells failed to have suppressor function, resulting in reduced regulatory T cell activity. Recent data from our laboratory suggest that T cells in the Foxn1Delta/Delta thymus develop from atypical progenitor cells via a noncanonical pathway. Our results suggest that the phenotype of peripheral T cells in Foxn1Delta/Delta mutant mice is the result of atypical progenitor cells developing in an abnormal thymic microenvironment with a deficient TCR and IL7 signaling system.     

Assembly and dynamics of Gp59-Gp32-single-stranded DNA (ssDNA), a DNA helicase loading complex required for recombination-dependent replication in bacteriophage T4

The Gp59 protein of bacteriophage T4 plays critical roles in recombination-dependent DNA replication and repair by correctly loading the replicative helicase, Gp41, onto recombination intermediates. Previous work demonstrated that Gp59 is required to load helicase onto single-stranded DNA that is saturated with Gp32, the T4 single-stranded DNA (ssDNA)-binding protein. Gp59 and Gp32 bind simultaneously to ssDNA, forming a Gp59-Gp32-ssDNA complex that is a key intermediate in helicase loading. Here we characterize the assembly and dynamics of this helicase loading complex (HLC) through changes in the fluorescent states of Gp32F, a fluorescein-Gp32 conjugate. Results show that HLC formation requires a minimum Gp32-ssDNA cluster size and that Gp59 co-localizes with Gp32-ssDNA clusters in the presence of excess free ssDNA. These and other results indicate that Gp59 targets helicase assembly onto Gp32-ssDNA clusters that form on the displaced strand of D-loops, which suggests a mechanism for the rapid initiation of recombination-dependent DNA replication. Helicase loading at the HLC requires ATP binding (not hydrolysis) by Gp41 and results in local remodeling of Gp32 within the HLC. Subsequent ATPase-driven translocation of Gp41 progressively disrupts Gp32-ssDNA interactions. Evidence suggests that Gp59 from the HLC is recycled to promote multiple rounds of helicase assembly on Gp32-ssDNA, a capability that could be important for the restart of stalled replication forks.     

Organ slices for the evaluation of human drug toxicity

Human organ slices, an in vitro model representing the multicellular and functional features of in vivo tissue, is a promising model for characterizing mechanisms of drug-induced organ injury and for identifying biomarkers of organ injury. Target organ injury is a significant clinical issue. In vitro models, which compare human and animal tissue to improve the extrapolation of animal in vivo studies for predicting human outcome, will contribute to improving drug candidate selection and to defining species susceptibilities in drug discovery and development programs. A critical aspect to the performance and outcome of human organ slice studies is the use of high quality tissue, and the use of culture conditions that support optimum organ slice survivability, in order to accurately reproduce mechanisms of organ injury in vitro. The attribute of organ slices possessing various cell types and interactions contributes to the overall biotransformation, inflammatory response and assessment of injury. Regional differences and changes in morphology can be readily evaluated by histology and special stains, similar to tissue obtained from in vivo studies. The liver is the major organ of which slice studies have been performed, however the utility of extra-hepatic derived slices, as well as co-cultures is increasing. Recent application of integrating gene expression, with human organ slice function and morphology demonstrate the increased potential of this model for defining the molecular and biochemical pathways leading to drug-induced tissue changes. By gaining a more detailed understanding of the mechanisms of drug-induced organ injury, and by correlating clinical measurements with drug-induced effects in the in vitro models, the vision of human in vitro models to identify more sensitive and discriminating markers of organ damage is attainable.     

The Membrane-binding Motif of the Chloroplast Signal Recognition Particle Receptor (cpFtsY) Regulates GTPase Activity

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) function in thylakoid biogenesis to target integral membrane proteins to thylakoids. Unlike cytosolic SRP receptors in eukaryotes, cpFtsY partitions between thylakoid membranes and the soluble stroma. Based on sequence alignments, a membrane-binding motif identified in Escherichia coli FtsY appears to be conserved in cpFtsY, yet whether the proposed motif is responsible for the membrane-binding function of cpFtsY has yet to be shown experimentally. Our studies show that a small N-terminal region in cpFtsY stabilizes a membrane interaction critical to cpFtsY function in cpSRP-dependent protein targeting. This membrane-binding motif is both necessary and sufficient to direct cpFtsY and fused passenger proteins to thylakoids. Our results demonstrate that the cpFtsY membrane-binding motif may be functionally replaced by the corresponding region from E. coli, confirming that the membrane-binding motif is conserved among organellar and prokaryotic homologs. Furthermore, the capacity of cpFtsY for lipid binding correlates with liposome-induced GTP hydrolysis stimulation. Mutations that debilitate the membrane-binding motif in cpFtsY result in higher rates of GTP hydrolysis, suggesting that negative regulation is provided by the intact membrane-binding region in the absence of a bilayer. Furthermore, NMR and CD structural studies of the N-terminal region and the analogous region in the E. coli SRP receptor revealed a conformational change in secondary structure that takes place upon lipid binding. These studies suggest that the cpFtsY membrane-binding motif plays a critical role in the intramolecular communication that regulates cpSRP receptor functions at the membrane.Proper compartmentalization of proteins relies on the ability of protein localization pathways to transport proteins efficiently from their sites of synthesis to their sites of function. The signal recognition particle (SRP)² and its receptor function in every kingdom of life to target proteins to the endoplasmic reticulum (eukaryotes), cytoplasmic membrane (prokaryotes), and thylakoid membrane (chloroplasts) (1). The targeting function of SRP relies on a conserved 54-kDa SRP subunit (SRP54; Ffh in Escherichia coli and cpSRP54 in chloroplasts) as well as a conserved SRP receptor (SRα; FtsY in E. coli and cpFtsY in chloroplasts). For cytosolic SRPs (SRP54 and Ffh), interactions with a substrate signal sequence and an SRP RNA moiety are prerequisite for interaction with the SRP receptor (SRα and FtsY) (2). GTP binding and hydrolysis by both SRP54 and SRα coordinate substrate release from SRP to the translocon and release of SRP from SRα. In chloroplasts, cpFtsY functions along with a unique SRP (cpSRP) to post-translationally target nuclear encoded proteins to thylakoid membranes (3). Light-harvesting chlorophyll a/b-binding proteins (LHCPs) imported into the chloroplast stroma are bound by cpSRP to form a soluble targeting complex, which directs the LHCP substrate to the thylakoid membrane translocon Alb3 (Albino3) in a GTP- and cpFtsY-dependent manner (14, 36). Although many general steps of SRP protein targeting seem largely conserved across evolutionary boundaries, the nature and dynamics of the receptor appear to have diverged.In eukaryotic systems, SRα is peripherally bound to the membrane through association with the integral membrane subunit SRβ. In contrast, no chloroplast or bacterial homolog of SRβ has been identified. cpFtsY and E. coli FtsY (EcFtsY) are found partitioned between the membrane and the stroma or cytosol, respectively. The membrane-binding capacity of EcFtsY serves to stimulate GTPase activity and appears critical in that only membrane-associated EcFtsY supports the release of nascent chains from SRP to the translocon (4, 5). However, the partitioning activity is not strictly required because EcFtsY tethered to the membrane is functional in vivo (37). Given the conserved nature of partitioning among bacterial and chloroplast SRP receptors, partitioning may play an, as of yet, unidentified role in protein targeting by SRP. Nevertheless, differences in lipid composition between bacterial and thylakoid membranes make it interesting to speculate that there are mechanistic differences in membrane partitioning.Like many prokaryotic FtsY homologs (e.g. Thermus aquaticus), cpFtsY lacks the N-terminal acidic domain (A domain) implicated in EcFtsY membrane binding (6). Although the highly conserved FtsY GTPase domain (NG domain) of EcFtsY (EcFtsY_NG) fails to support protein targeting, the addition of the last A domain residue, Phe-196 of a conserved double-Phe motif (EcFtsY_NG+1), restores protein targeting in vivo (7). In vitro studies also show that EcFtsY_NG+1 retains the capacity to bind membranes and support integration of SRP-dependent substrates, although at significantly reduced levels compared with full-length EcFtsY (8). A resolved structure of EcFtsY_NG+1 suggests that the amphipathic nature of the region containing Phe-196 plays a critical role in membrane association (9). Furthermore, it has been demonstrated that liposomes stimulate GTP hydrolysis rates of SRP with EcFtsY_NG+1, but not with EcFtsY_NG, supporting the idea that the A domain in its entirety is not strictly required.For cpFtsY, the necessity and functional role(s) of partitioning between a thylakoid-bound and a soluble phase, as well as the role of N-terminal residues in these functions, remain unknown. In addition, both the conformational state of membrane-bound cpFtsY and EcFtsY and the mechanism responsible for controlling membrane partitioning and altered GTPase activity remain unclear. Because of the gain of function exhibited by EcFtsY_NG+1 and the conserved nature of the surrounding motif (9), it seems likely that this conserved region is necessary to support membrane binding and corresponding functions not only in EcFtsY but also in FtsY homologs.To examine the functional role of the N-terminal region of cpFtsY, we have utilized deletion and point mutants in assays that reconstitute cpFtsY activities, including the cpSRP-dependent integration of LHCP. Together, our data indicate that the conserved lipid-binding motif identified in bacterial FtsY homologs is present in cpFtsY and is both necessary and sufficient for thylakoid binding and critical for LHCP targeting.