bluuebunny/crossref_metadata_2025 huggingface dataset:
Regional arrangements for mode 4 in the services trade: lessons from the ASEAN experience
Authors: BHATNAGAR PRADIP , MANNING CHRIS
Published: September 2005

The paper deals with regional policies towards temporary labour migration (or the movement of natural persons) with specific reference to the liberalization of trade in services. The paper deals with policies towards labour migration, and progress in related Mode 4 negotiations within the GATS framework, among the Association of Southeast Asian Nations, or ASEAN. It finds that unilateral, national policies rather than regional or multilateral commitments dominate in policies towards temporary foreign workers. Despite advances on administrative issues, little progress has been made towards achieving ‘GATS-Plus’ outcomes, partly because of the lack of a strong political commitment among member countries to liberalizing the entry of foreign workers and partly because of real or imagined shortcomings in the ‘architecture’ for Mode 4 negotiations. Weak political will has led to relatively shallow commitments with a focus on skilled workers over unskilled migrants. With regard to architectural issues, we draw attention to the weaknesses of the ‘positive list’ approach in services trade negotiations, specifically with regard to the case of labour migration. However, the interpretation of the relationship between different modes of supply has also tended to inhibit regional negotiations for Mode 4. In particular, we point to the neglect of the potential importance of independent suppliers of services. It is concluded that the ASEAN countries need to place more emphasis on regional priorities and problems in seeking more open worker flows within the region.
Rien ne Va Plus? Distinguishing domestic regulation from market access in GATT and GATS
Authors: PAUWELYN JOOST
Published: September 2005

Depending on how one classifies market intervention, trade liberalization disciplines can be lenient or strict. Perhaps the most important distinction in this respect is that between government intervention labeled as a ‘market access restriction’ and that defined as ‘domestic regulation’. Both the GATT and the GATS declare market access restrictions (such as import quotas or limitations on the number of service suppliers) to be, in principle, prohibited. In contrast, domestic regulations (such as internal taxes, health standards, and safety requirements) are treated with much more deference. They are, in essence, only prohibited when discriminatory or more trade restrictive than necessary. Notwithstanding these major legal consequences, the distinction between market access and domestic regulation remains unclear. Based on a recent WTO dispute condemning the United States for banning online gambling, this article is an attempt to clarify the distinction. Starting from broad similarities, it finds crucial differences in this respect between GATT and GATS. For both, however, the paper's basic point is that a domestic regulation should not be regarded as a market access restriction simply because it has the effect of banning certain imports. To do otherwise risks seriously undermining the regulatory autonomy of WTO Members beyond anything imagined by the drafters of the WTO treaty.
United States – Anti-Dumping Act of 1916 (Original Complaint by the European Communities)– Recourse to arbitration by the United States under 22.6 of the DSU, WT/DS136/ARB, 24 February 2004
Authors: HOWSE ROBERT , STAIGER ROBERT W.
Published: September 2005

This arbitration on the level of countermeasures, pursuant to Dispute Settlement Understanding (DSU) 22.6, originates from findings against the United States with respect to a provision of the 1916 Anti-Dumping Act, which permits a private cause of action for treble damages against an alleged dumping importer or producer where the dumping meets certain criteria, notably including a predatory intention. An unusual feature of this case is that, apparently, the provision in question had not yet been used against the EC, the complainant. In any case, the provision in question was found to be in violation of the WTO Anti-Dumping Agreement, both because it would impose measures on dumping other than and in addition to anti-dumping duties, and because in so doing the procedural requirements of the Anti-Dumping Agreement would not be followed.
Synthesis of iron fertilization experiments: From the Iron Age in the Age of Enlightenment
Authors: de Baar Hein J. W. , Boyd Philip W. , Coale Kenneth H. , Landry Michael R. , Tsuda Atsushi , Assmy Philipp , Bakker Dorothee C. E. , Bozec Yann , Barber Richard T. , Brzezinski Mark A. , Buesseler Ken O. , Boyé Marie , Croot Peter L. , Gervais Frank , Gorbunov Maxim Y. , Harrison Paul J. , Hiscock William T. , Laan Patrick , Lancelot Christiane , Law Cliff S. , Levasseur Maurice , Marchetti Adrian , Millero Frank J. , Nishioka Jun , Nojiri Yukihiro , van Oijen Tim , Riebesell Ulf , Rijkenberg Micha J. A. , Saito Hiroaki , Takeda Shigenobu , Timmermans Klaas R. , Veldhuis Marcel J. W. , Waite Anya M. , Wong Chi‐Shing
Published: September 2005

Comparison of eight iron experiments shows that maximum Chl a, the maximum DIC removal, and the overall DIC/Fe efficiency all scale inversely with depth of the wind mixed layer (WML) defining the light environment. Moreover, lateral patch dilution, sea surface irradiance, temperature, and grazing play additional roles. The Southern Ocean experiments were most influenced by very deep WMLs. In contrast, light conditions were most favorable during SEEDS and SERIES as well as during IronEx‐2. The two extreme experiments, EisenEx and SEEDS, can be linked via EisenEx bottle incubations with shallower simulated WML depth. Large diatoms always benefit the most from Fe addition, where a remarkably small group of thriving diatom species is dominated by universal response of Pseudo‐nitzschia spp. Significant response of these moderate (10–30 μm), medium (30–60 μm), and large (>60 μm) diatoms is consistent with growth physiology determined for single species in natural seawater. The minimum level of “dissolved” Fe (filtrate < 0.2 μm) maintained during an experiment determines the dominant diatom size class. However, this is further complicated by continuous transfer of original truly dissolved reduced Fe(II) into the colloidal pool, which may constitute some 75% of the “dissolved” pool. Depth integration of carbon inventory changes partly compensates the adverse effects of a deep WML due to its greater integration depths, decreasing the differences in responses between the eight experiments. About half of depth‐integrated overall primary productivity is reflected in a decrease of DIC. The overall C/Fe efficiency of DIC uptake is DIC/Fe ∼ 5600 for all eight experiments. The increase of particulate organic carbon is about a quarter of the primary production, suggesting food web losses for the other three quarters. Replenishment of DIC by air/sea exchange tends to be a minor few percent of primary CO2 fixation but will continue well after observations have stopped. Export of carbon into deeper waters is difficult to assess and is until now firmly proven and quite modest in only two experiments.
On the sheltering effect of islands in ocean wave models
Authors: Ponce de León S. , Guedes Soares C.
Published: September 2005

The effect of resolving relatively small islands in a third‐generation wave model covering the whole North Atlantic is shown to have noticeable effects at long distances from the islands in addition to the local effects that have been reported earlier. To resolve the islands, a very fine grid is adopted locally, which is then nested in the overall model. The examples presented show the shadowing effect of Azores Islands for three specific wave conditions: when they come from the southwest, from the west, and from the northwest. The sheltering effect of the islands was demonstrated by a reduction of the significant wave height in the region on the leeward side of Azores Archipelago when comparing the wave model results with the island resolved and unresolved.
Relationship between ionic composition in PM10 and the synoptic‐scale and mesoscale weather conditions in a south China coastal city: A 4‐year study
Authors: Wai Ka‐Ming , Tanner Peter A.
Published: September 2005

Four‐year (1998–2001) data sets of chemical species in particulate matter of 10 μm in diameter or less (PM10) from a monitoring site in a south China coastal city, Hong Kong, comprising major crustal, marine, and anthropogenic species have been analyzed with respect to synoptic‐scale and mesoscale meteorological patterns. Their relationships are discussed using surface pressure maps, back trajectory analysis, chemical transport modeling results, and other relevant meteorological data. The mean concentrations of K+, non‐sea‐salt sulfate (nss‐SO42−), and NO3− in aerosols differed from that at a background site by no more than 15%, exemplifying the contributions of major sources from outside Hong Kong. Continental air masses associated with the East Asian winter monsoon always contain high contents of mineral dust and anthropogenic species such as nss‐SO42− when they pass over the desert/loess areas and urban cities/heavy industrial areas of mainland China before reaching Hong Kong. Clear decreasing temporal trends of the concentrations of the anthropogenic species nss‐SO42− and NO3− are evident during the sampling period. Emissions from biofuel combustion are mixed with urban/industrial plumes before reaching Hong Kong. Wet scavenging significantly depletes the concentrations of chemical species. Marine air masses associated with the summer monsoon or high‐pressure systems near Japan always contain large amounts of sea‐salt species, but there are noticeably different concentrations of crustal and anthropogenic species between these two weather patterns. The species K+, as a tracer for biomass burning, approaches background levels for summer monsoon air masses originating from Southeast Asia. The proximity and the arrival of the tropical cyclone reflect two extremes (very high/low) species levels. Stagnant air is not the only factor to govern the elevated species levels in aerosols, but the nature of air masses affecting Hong Kong before sampling should also be taken into consideration.
Three‐dimensional ionospheric tomography via band‐limited constrained iterative cross‐entropy minimization
Authors: Cornely Pierre‐Richard J. , Kuklinski Walter S.
Published: September 2005

The problem of reconstructing ionospheric electron density from ground‐based receiver to satellite total electron content (TEC) measurements is formulated as an underdetermined discrete linear inverse problem. If receivers and satellite orbit are coplanar, then a single two‐dimensional (2‐D) imaging plane can be used as a geometrical model. In most cases, receiver locations are determined by convenience and availability of sites, and thus a 3‐D imaging volume is required in order to capture the part of the electron density solution that is associated with gaps between receiver stations within a chain. It is well understood that spurious features, or “wings,” associated with these gaps can be produced in tomographic image reconstructions where ray path coverage from individual stations is lost or minimal. In this paper, a 3‐D reconstruction system that takes advantage of a multiple‐chain data acquisition geometry and provides a solution consistent with available TEC data everywhere within a receiver chain is presented. The reconstruction system exploits the fact that gaps, created by longitudinally unaligned receivers within a chain, can be captured as additional planes of constant longitudes within the 3‐D imaging volume. With parameterized ionospheric model (PIM)–generated data as a nonnegative prior estimate of the electron density, the reconstruction algorithm uses constraints based on prior knowledge of the 3‐D spatial Fourier transform of the prior electron density as a smoothing mechanism in the tomographic reconstruction process. Consequent to the underlined Fourier transform formulation, a unique solution is produced at locations that contributed to the measured TEC data, and a solution is interpolated within the remainder of the imaging volume. The band‐limited BISMART algorithm has been evaluated using a multiple‐receiver chain TEC data acquisition system, under known ionospheric conditions. The algorithm satisfactorily reconstructs density solutions, consistent with small‐scale enhancements, irregularities, and troughs in the auroral ionosphere, from the available TEC data. The quality of the density reconstructions, coupled with the computational efficiency of this algorithm, indicates the potential utility of this technique for real‐time three‐ and four‐dimensional ionospheric tomography.
Green's function analysis of an ideal hard surface rectangular waveguide
Authors: Huang Wei , Yakovlev Alexander B. , Kishk Ahmed A. , Glisson Allen W. , Eshrah Islam A.
Published: September 2005

Green's function analysis of an ideal hard surface rectangular waveguide is proposed for characterization of the modal spectrum of the structure. A decomposition of the hard surface waveguide into perfect electric conductor and perfect magnetic conductor waveguides allows the representation of dyadic Green's function as a superposition of transverse magnetic (TM) and transverse electric (TE) waveguide modes, respectively. In addition, a term corresponding to a transverse electromagnetic (TEM) mode is included in the eigenmode expansion of the Green's dyadic. It is shown that the TEM mode solution can be obtained by solving vector Helmholtz's equation in the zero cutoff limit with the corresponding boundary conditions of electric field on the ideal hard surface. The electric field distribution due to an arbitrarily oriented electric dipole source is illustrated for a few representative TM, TE, and TEM modes propagating in the ideal hard surface rectangular waveguide. The proposed model is verified by analyzing a realistic hard surface square waveguide using the Ansoft High‐Frequency Structure Simulator (HFSS).
Beaming of light at broadside through a subwavelength hole: Leaky wave model and open stopband effect
Authors: Jackson D. R. , Oliner A. A. , Zhao T. , Williams J. T.
Published: September 2005

The optical transmission through a subwavelength hole in a metal film is usually very small, and the beam radiated from its exit aperture is very broad. However, the transmission may be increased by orders of magnitude, and the output beam sharply narrowed, when the tiny hole is surrounded by a properly designed periodic structure, which may take the form of an array of grooves or indentations on the metal surface. We have shown that these dramatic effects are due to the excitation of a leaky surface plasmon mode by the periodic structure on the metal film. Following this understanding, we introduce a simple but effective leaky wave antenna model, which we use to further explain and to quantify these dramatic effects. Particular attention is given to optimizing the structure to achieve maximum radiation at broadside, which offers a significant challenge in view of the open stopband in precisely the broadside direction.
Fidelity of δ18O as a proxy for sea surface temperature: Influence of variable coral growth rates on the coral Porites lutea from Hainan Island, China
Authors: Shimamura Michiyo , Oba Tadamichi , Xu Guoqiang , Lu Bingquan , Wang Luejiang , Murayama Masafumi , Toyoda Kazuhiro , Winter Amos
Published: September 2005

The extremely high temporal resolution (nearly daily interval) measurement was conducted on the coral (Porites lutea) oxygen isotope compositions (δ18Oc) to examine the detailed relationship between δ18Oc and sea surface temperature (SST) and seawater oxygen isotope composition (δ18Ow) from Hainan Island, China. Although SST and sea surface salinity (SSS) or δ18Ow varied in a wide range at the studied site, the dynamic range of δ18Oc recorded in coral was much smaller than expected from SST and SSS (δ18Ow) changes. The extremely high (>30°C) and low (<22°C) SST and low (<27) SSS are not suitable for coral calcification, and coral could not record the information as their skeletal δ18O. Estimated coral extension rates (the overall rate is 15 mm/year) varied by a factor of about 18 within one year, from very slow in winter to very fast in spring. Light availability could affect the extension rate of coral skeleton through the activity of photosynthesis of symbiont algae. Such cessation and acceleration of calcification cause a serious distortion of the δ18O profile, which makes it difficult to make a correlation between δ18O and SST. Because high and low extension rates correspond to low‐ and high‐density bands, respectively, a detailed examination of the density structure may assist the correction for distortion of the δ18O profile.
Multispectral observations of auroral rays and curls
Authors: Ivchenko N. , Blixt E. M. , Lanchester B. S.
Published: September 2005

Two cases of discrete aurora are presented, in which auroral curls and auroral rays, respectively, were seen. The aurora was imaged by two spatially separated imagers with a long‐pass filter (mainly sensitive to N2 and N2+ emissions), and another imager with a narrow‐band 7325 Å filter (sensitive to forbidden O+ doublet). Also, spectra of the aurora were recorded. Using the multispectral imaging and spectra we find that the curls occurred in aurora caused by precipitation of energetic electrons with a lack of low‐energy population, while in the rays both high and low energy precipitation were present simultaneously. These findings are confirmed by the altitude determination from triangulation.
A stratospheric influence on the winter NAO and North Atlantic surface climate
Authors: Scaife Adam A. , Knight Jeff R. , Vallis Geoff K. , Folland Chris K.
Published: September 2005

The North Atlantic Oscillation (NAO) has a profound effect on winter climate variability around the Atlantic basin. Strengthening of the NAO in recent decades has altered surface climate in these regions at a rate far in excess of global mean warming. However, only weak NAO trends are reproduced in climate simulations of the 20th Century, even with prescribed climate forcings and historical sea‐surface conditions. Here we show that the unexplained strengthening of the NAO can be fully simulated in a climate model by imposing observed trends in the lower stratosphere. This implies that stratospheric variability needs to be reproduced in models to fully simulate surface climate variations in the North Atlantic sector. Despite having little effect on global mean warming, we show that downward coupling of observed stratospheric circulation changes to the surface can account for the majority of change in regional surface climate over Europe and North America between 1965 and 1995.
A nightside source of Saturn's kilometric radiation: Evidence for an inner magnetosphere energy driver
Authors: Farrell W. M. , Desch M. D. , Kaiser M. L. , Lecacheux A. , Kurth W. S. , Gurnett D. A. , Cecconi B. , Zarka P.
Published: September 2005

During Cassini's orbit insertion about Saturn, the spacecraft passed within 1.4 Rs of the planet passing from dayside into the nightside region. During this nightside passage, the onboard Radio and Plasma Wave (RPWS) instrument surprisingly detected Saturn kilometric radiation (SKR). Prior to this encounter, it was believed that SKR originated from a high‐latitude dayside source, and radio beams from such a source would not be viewable in this nearplanet night‐side location. Subsequent analysis presented here reveals that this SKR did indeed originate from the near‐midnight region on field lines near L ∼ 10–15. Such a radio source suggests the presence of an active region in the night‐side inner magnetosphere; this source possibly being near the outer edge of the icy‐moon created plasma torus surrounding the planet. The implication is that some of the SKR is driven by an internal energy source that may also account for recent UV aurora observations.
Cloud and aerosol measurements from GLAS: Overview and initial results
Authors: Spinhirne James D. , Palm Stephen P. , Hart William D. , Hlavka Dennis L. , Welton Ellsworth J.
Published: September 2005

Global space borne lidar profiling of atmospheric clouds and aerosol began in 2003 following the launch of the Geoscience Laser Altimeter System (GLAS) on the Ice, Cloud and land Elevation Satellite. GLAS obtains nadir profiles through the atmosphere in two wavelength channels, day and night, at a fundamental resolution of 76.8 m vertical and 172 m along track. The 532 nm channel uses photon‐counting detectors and resolves profiles of observed backscatter cross sections to 10−7 1/m‐sr. The 1064 nm channel employs analog detection adequate to 10−6 1/m‐sr and with greater dynamic range. By 2005 approximately seven months of global data are available. Processing algorithms produce data products for the corrected lidar signal, cloud and aerosol layer boundaries and optical thickness and extinction and backscatter cross sections. Operational sensitivity is shown by the frequency distribution for cloud optical thickness peaking at approximately 0.02.
Attribution of global surface warming without dynamical models
Authors: Stone Dáithí A. , Allen M. R.
Published: September 2005

Detection and attribution studies of observed surface temperature changes have served to consolidate our understanding of the climate system and its past and future behaviour. Most recent studies analysing up‐to‐date observations have relied on general circulation models (GCMs) to provide estimates of the responses to various external forcings. Here we revisit a methodology which instead estimates the responses using a simple model tuned directly to the observed record, paralleling a technique currently used with GCM output. The effects of greenhouse gases, tropospheric sulphate aerosols, and volcanic aerosols are all detected in the observed record, while the effects of solar irradiance are unclear. These results provide further observational constraints on past and future warming estimates consistent with those from recent studies with GCMs, supporting the notion that current estimates are robust against the modelling system used.
A large organic aerosol source in the free troposphere missing from current models
Authors: Heald Colette L. , Jacob Daniel J. , Park Rokjin J. , Russell Lynn M. , Huebert Barry J. , Seinfeld John H. , Liao Hong , Weber Rodney J.
Published: September 2005

Aircraft measurements of organic carbon (OC) aerosol by two independent methods over the NW Pacific during the ACE‐Asia campaign reveal unexpectedly high concentrations in the free troposphere (FT). Concentrations average 4 μg sm−3 in the 2–6.5 km column with little vertical gradient. These values are 10–100 times higher than computed with a global chemical transport model (CTM) including a standard 2‐product simulation of secondary organic aerosol (SOA) formation based on empirical fits to smog chamber data. The same CTM reproduces the observed vertical profiles of sulfate and elemental carbon aerosols, which indicate sharp decreases from the boundary layer to the FT due to wet scavenging. Our results suggest a large, sustained source of SOA in the FT from oxidation of long‐lived volatile organic compounds. We find that this SOA is the dominant component of aerosol mass in the FT, with implications for intercontinental pollution transport and radiative forcing of climate.
Permeability anisotropy induced by the shear displacement of rough fracture walls
Authors: Auradou H. , Drazer G. , Hulin J. P. , Koplik J.
Published: September 2005

The permeability anisotropy that results from a shear displacement between the complementary self‐affine walls of a rough fracture is investigated. Experiments in which a dyed fluid radially injected into a transparent fracture displaces a transparent one are presented. A clear anisotropy is observed in the presence of shear displacements and allows us to estimate the ratio of the permeabilities for flows parallel and perpendicular to . A simple model which accounts for the development of channels perpendicular to qualitatively explains these results and predicts a permeability decreasing (increasing) linearly with the variance of the aperture field for flow parallel (perpendicular) to the shear displacement. These predictions are then compared to the results of numerical simulations performed using a lattice Boltzmann technique and to the anisotropies measured in displacement experiments.
Development of a protein microarray library and a system for rheumatoid‐arthritis disease marker screening
Authors: Lebrun Stewart J. , Petchpud Wasinee , Tolentino Melissa
Published: September 2005

Early detection and intervention can delay the onset of symptoms of RA (rheumatoid arthritis), but current diagnostic tests suffer from low sensitivity and specificity, making such early disease detection difficult. Analysis of body fluids for the identification of multiple molecular markers and the subsequent determination of modifications in their associated protein structures emerges as a possibility for early detection. The structural modifications of these markers are thought to play a pivotal role in defining the pathological state of diseased joints. Therefore a proteome library and system for RA disease screening has been developed and investigated in the present study. The large (39 S) mammalian ribosomal subunit has been identified as a potential RA marker. This protein is known as L35, and antibodies to this protein have been found to be expressed in patients suffering from RA.
Optimal conditions for the papain digestion of polyclonal ovine IgG for the production of biotherapeutic Fab fragments
Authors: Cresswell Chrissie , Newcombe Anthony R. , Davies Susannah , Macpherson Ian , Nelson Paul , O'Donovan Kieran , Francis Richard
Published: September 2005

In the present paper, we describe a rapid method for the determination of optimum conditions for papain digestion of polyclonal ovine IgG (purified by Na2SO4 precipitation) for the production of biotherapeutic Fabs (antigen‐binding fragments). To determine the optimum conditions for digestion, a factorial approach to the design of experiments was undertaken. The resulting experimental data were used to construct the mathematical models using Design Expert 6.06® (Stat‐Ease, Minneapolis, MN, U.S.A.) to predict the optimum conditions for a robust IgG digestion step. Optimum conditions were evaluated experimentally, and the applicability of the conditions for large‐scale manufacture of biotherapeutic Fab fragments was assessed. The results and methods described in the present paper suggest that, provided the time and temperature are maintained at the high settings evaluated (24 h, 40 °C), the modelled data predict IgG digestion close to 100% for all the papain concentrations used. Provided papain is used at >2.5% (w/w), either time and/or temperature may be reduced. The results and methods described in the present paper may also be applicable to the generation of therapeutic Fab fragments from other immunoglobulins, including monoclonal antibodies purified from mammalian cell culture.
Decreased expression of AQP2 and AQP4 water channels and Na, K‐ATPase in kidney collecting duct in AQP3 null mice
Authors: Wan Kim Soo , Gresz Veronika , Rojek Aleksandra , Wang Weidong , Verkman A.S. , Frøkiær Jørgen , Nielsen Søren
Published: September 2005

Background information. Phenotype analysis has demonstrated that AQP3 (aquaporin 3) null mice are polyuric and manifest a urinary concentration defect. In the present study, we report that deletion of AQP3 is also associated with an increased urinary sodium excretion. To investigate further the mechanism of the decreased urinary concentration and significant natriuresis, we examined the segmental and subcellular localization of collecting duct AQPs [AQP2, p‐AQP2 (phosphorylated AQP2), AQP3 and AQP4], ENaC (epithelial sodium channel) subunits and Na, K‐ATPase by immunoperoxidase and immunofluorescence microscopy in AQP3 null (−/−), heterozygous (+/−) mice, wild‐type and unrelated strain of normal mice.

Results. The present study confirms that AQP3 null mice exhibit severe polyuria and polydipsia and demonstrated that they exhibit increased urinary sodium excretion. In AQP3 null mice, there is a marked down‐regulation of AQP2 and p‐AQP2 both in CNT (connecting tubule) and CCD (cortical collecting duct). Moreover, AQP4 is virtually absent from CNT and CCD in AQP3 null mice. Basolateral AQP2 was virtually absent from AQP3 null mice and normal mice in contrast with rat. Thus the above results demonstrate that no basolateral AQPs are expressed in CNT and CCD of AQP3 null mice. However, in the medullary‐collecting ducts, there is no difference in the expression levels and subcellular localization of AQP2, p‐AQP2 and AQP4 between AQP3 +/− and AQP3 null mice. Moreover, a striking decrease in the immunolabelling of the α1 subunit of Na, K‐ATPase was observed in CCD in AQP3 null mice, whereas a medullary‐collecting duct exhibited normal labelling. Immunolabelling of all the ENaC subunits in the collecting duct was comparable between the two groups.

Conclusions. The results improve the possibility that the severe urinary concentrating defect in AQP3 null mice may in part be caused by the decreased expression of AQP2, p‐AQP2 and AQP4 in CNT and CCD, whereas the increased urinary sodium excretion may in part be accounted for by Na, K‐ATPase in CCD in AQP3 null mice.
Training and aging modulate the loss‐of‐balance phenotype observed in a new ENU‐induced allele of Otopetrin1
Authors: Besson Vanessa , Nalesso Valérie , Herpin Alexandre , Bizot Jean‐Charles , Messaddeq Nadia , Romand Raymond , Puech Anne , Blanquet Véronique , Hérault Yann
Published: September 2005

Background information. The sensing of head movement in mammals depends upon the vestibular endorgan of the inner ear, a complex structure made up of the semicircular canals and otoliths. Due to the similarity between the human and mouse vestibular apparatus, the analysis of mutant mouse is a valuable strategy aiming to identify genes involved in the control of balance and movement.

Results. In the course of a genome‐wide chemical‐mutagenesis programme, we isolated a recessive mutation, named ied (inner ear defect), which induced a severe loss‐of‐balance. A detailed phenotypic analysis of the mutant mice demonstrates that the balance impairment does not affect the motor activity and can be rescued, in part, by training, despite a complete agenesis of otoconia in the utricule and the saccule of the inner ear. Molecular characterization of the ied mutation revealed a transversion that affects the splicing of the second exon of the Otopetrin1 gene located on mouse chromosome 5. The consequence of such a mutation leads to a disruption of the transcription of the gene.

Conclusions. The identification of the ied knock‐down allele strengthens the role of the Otopetrin1 in the sensing of balance. Moreover, the rescue of the ied mutant phenotype in specific behavioural tasks confirmed that other sensory inputs or neural plasticity can compensate, to some extent, for the loss‐of‐balance. In the future, the ied mutant mice might be helpful to study the genetic control of the compensation strategies developed by organisms to counteract balance defects.
Activation of Mrc1, a mediator of the replication checkpoint, by telomere erosion
Authors: Grandin Nathalie , Bailly Aymeric , Charbonneau Michel
Published: September 2005

Background information. In budding yeast, the loss of either telomere sequences (in telomerase‐negative cells) or telomere capping (in mutants of two telomere end‐protection proteins, Cdc13 and Yku) lead, by distinct pathways, to telomeric senescence. After DNA damage, activation of Rad53, which together with Chk1 represents a protein kinase central to all checkpoint pathways, normally requires Rad9, a checkpoint adaptor.

Results. We report that in telomerase‐negative (tlc1Δ) cells, activation of Rad53, although diminished, could still take place in the absence of Rad9. In contrast, Rad9 was essential for Rad53 activation in cells that entered senescence in the presence of functional telomerase, namely in senescent cells bearing mutations in telomere end‐protection proteins (cdc13‐1 yku70Δ). In telomerase‐negative cells deleted for RAD9, Mrc1, another checkpoint adaptor previously implicated in the DNA replication checkpoint, mediated Rad53 activation. Rad9 and Rad53, as well as other DNA damage checkpoint proteins (Mec1, Mec3, Chk1 and Dun1), were required for complete DNA‐damage‐induced cell‐cycle arrest after loss of telomerase function. However, unexpectedly, given the formation of an active Rad53–Mrc1 complex in tlc1Δ rad9Δ cells, Mrc1 did not mediate the cell‐cycle arrest elicited by telomerase loss. Finally, we report that Rad9, Mrc1, Dun1 and Chk1 are activated by phosphorylation after telomerase inactivation.

Conclusions. These results indicate that loss of telomere capping and loss of telomere sequences, both of which provoke telomeric senescence, are perceived as two distinct types of damages. In contrast with the Rad53–Rad9‐mediated cell‐cycle arrest that functions in a similar way in both types of telomeric senescence, activation of Rad53–Mrc1 might represent a specific response to telomerase inactivation and/or telomere shortening, the functional significance of which has yet to be uncovered.
Peroxin 5–peroxin 14 association in the protozoan Leishmania donovani involves a novel protein–protein interaction motif
Authors: Madrid Kleber P. , Jardim Armando
Published: September 2005

Import of proteins with a PTS1 (peroxisomal targeting signal 1) into the Leishmania glycosomal organelle involves docking of a PTS1-laden LdPEX5 [Leishmania donovani PEX5 (peroxin 5)] receptor to LdPEX14 on the surface of the glycosomal membrane. In higher eukaryotes, the PEX5–PEX14 interaction is mediated by a conserved diaromatic WXXXY/F motif. Site-directed and deletion mutageneses of the three WXXXY/F repeats in LdPEX5 did not abolish the LdPEX5–LdPEX14 association. Analysis of the equilibrium dissociation constant (Kd) revealed that ldpex5-W53A (Trp53→Ala), ldpex5-W293A, ldpex5-W176,293A and ldpex5-W53,176,293A mutant receptors were capable of binding LdPEX14 with affinities comparable with wild-type LdPEX5. That the diaromatic motifs were not required for the LdPEX5–LdPEX14 interaction was further verified by deletion analysis that showed that ldpex5 deletion mutants or ldpex5 fragments lacking the WXXXY/F motifs retained LdPEX14 binding activity. Mapping studies of LdPEX5 indicated that the necessary elements required for LdPEX14 association were localized to a region between residues 290 and 323. Finally, mutational analysis of LdPEX14 confirmed that residues 23–63, which encompass the conserved signature sequence AX2FLX7SPX6FLKGKGL/V present in all PEX14 proteins, are essential for LdPEX5 binding.
Structural and functional studies of vertebrate metallothioneins: cross-talk between domains in the absence of physical contact
Authors: Capasso Clemente , Carginale Vincenzo , Crescenzi Orlando , Di Maro Daniela , Spadaccini Roberta , Temussi Piero Andrea , Parisi Elio
Published: September 2005

In previous studies, we showed that the chemical and dynamic properties of fish and mouse MTs (metallothioneins) present a number of distinctive differences linked to their primary structures, and that phylogenetic relationships of mammal and fish MTs correlate with their three-dimensional structures. The different behaviours of MTs may also be linked to the interaction between their two domains. In the present study, we have compared the physicochemical properties of the isolated recombinant domains constituting Notothenia coriiceps and mouse MTs, and compared them with those of the corresponding whole MTs. NMR spectra of the separated domains of N. coriiceps are almost superimposable on those of the parent MT, suggesting an apparent lack of interaction between the two domains in the protein. However, certain dynamic and physicochemical features of the isolated domains are unlike those of the whole protein. In particular, the temperature-induced changes in the chiroptical properties, thiol reactivity of the Zn-MT domains and the Zn2+/Cd2+ rate of exchange are different for the two domains and with respect to the whole protein. Taken together, these results provide a strong argument in favour of the interaction of the two domains in the MT molecule, in spite of the elusive evidence provided by the structural analyses.
Porcine, mouse and human galactose 3-O-sulphotransferase-2 enzymes have different substrate specificities; the porcine enzyme requires basic compounds for its catalytic activity
Authors: Seko Akira , Sumiya Jun-ichi , Yamashita Katsuko
Published: September 2005

Sulphation of galactose at the C-3 position is one of the major post-translational modifications of colorectal mucin. Thus we partially purified a Gal 3-O-sulphotransferase from porcine colonic mucosa (pGal3ST) and studied its enzymatic characteristics. The enzyme was purified 48500-fold by sequential chromatographies on hydroxyapatite, Con A (concanavalin A)–Sepharose, porcine colonic mucin–Sepharose, Cu2+-chelating Sepharose and AMP–agarose. Interestingly, the purified pGal3ST required submillimolar concentrations of spermine or basic lipids, such as D-sphingosine and N,N-dimethylsphingosine, for enzymatic activity. pGal3ST recognized Galβ1→3GalNAc (core 1) as an optimal substrate, and had weaker activity for Galβ1→3GlcNAc (type 1) and Galβ1→4GlcNAc (type 2). Substrate competition experiments proved that a single enzyme catalyses sulphation of all three oligosaccharides. Among the four human Gal3STs cloned to date, the substrate specificity of pGal3ST is most similar to that of human Gal3ST-2, which is also strongly expressed in colonic mucosa, although the kinetics of pGal3ST and human Gal3ST-2 were rather different. To determine whether pGal3ST is the orthologue of human Gal3ST-2, a cDNA encoding porcine Gal3ST-2 was isolated and the enzyme was expressed in COS-7 cells for analysis of substrate specificity. This revealed that porcine Gal3ST-2 has the same specificity as pGal3ST, indicating that pGal3ST is indeed the porcine equivalent of Gal3ST-2. The substrate specificity of mouse Gal3ST-2 was also different from those of human and porcine Gal3ST-2 enzymes. Mouse Gal3ST-2 preferred core 1 and type 2 glycans to type 1, and the Km values were much higher than those of human Gal3ST-2. These results suggest that porcine Gal3ST-2 requires basic compounds for catalytic activity and that human, mouse and porcine Gal3ST-2 orthologues have diverse substrate specificities.
Function of the cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A: targeting adhesion proteins collagen I and von Willebrand factor
Authors: Serrano Solange M. T. , Jia Li-Guo , Wang Deyu , Shannon John D. , Fox Jay W.
Published: September 2005

The cysteine-rich domain of the haemorrhagic metalloproteinase atrolysin A was shown to inhibit collagen-stimulated platelet aggregation and to interact with MG-63 osteosarcoma cells via integrin α2β1 to inhibit adhesion to collagen I. In addition, we demonstrate by solid-phase binding assays that atrolysin A binds to collagen I and to vWF (von Willebrand factor) via exosites in the cysteine-rich domain. Interestingly, the binding site of the cysteine-rich domain on collagen I is distinct from the cell adhesion site, since the incubation of collagen-I-coated plates with the cysteine-rich domain did not prevent the adhesion of MG-63 cells to collagen. Finally, we show by surface plasmon resonance (BIAcore™) analyses that the cysteine-rich domain can block vWF binding to collagen I as well as the binding of collagen I to vWF. Taken together, these results indicate that this domain may function as a cell-surface-receptor-binding site and/or a substrate recognition exosite and may thus play a role in the pathologies associated with atrolysin A.
Unravelling the structure of the pneumococcal autolytic lysozyme
Authors: Monterroso Begoña , López-Zumel Consuelo , García José L. , Sáiz José L. , García Pedro , Campillo Nuria E. , Menéndez Margarita
Published: September 2005

The LytC lysozyme of Streptococcus pneumoniae forms part of the autolytic system of this important pathogen. This enzyme is composed of a C-terminal CM (catalytic module), belonging to the GH25 family of glycosyl hydrolases, and an N-terminal CBM (choline-binding module), made of eleven homologous repeats, that specifically recognizes the choline residues that are present in pneumococcal teichoic and lipoteichoic acids. This arrangement inverts the general assembly pattern of the major pneumococcal autolysin, LytA, and the lytic enzymes encoded by pneumococcal bacteriophages that place the CBM (made of six repeats) at the C-terminus. In the present paper, a three-dimensional model of LytC built by homology modelling of each module and consistent with spectroscopic and hydrodynamic studies is shown. In addition, the putative catalytic-pair residues are identified. Despite the inversion in the modular arrangement, LytC and the bacteriophage-encoded Cpl-1 lysozyme most probably adopt a similar global fold. However, the distinct choline-binding ability and their substrate-binding surfaces may reflect a divergent evolution directed by the different roles played by them in the host (LytC) or in the bacteriophage (Cpl-1). The tight binding of LytC to the pneumococcal envelope, mediated by the acquisition of additional choline-binding repeats, could facilitate the regulation of the potentially suicidal activity of this autolysin. In contrast, a looser attachment of Cpl-1 to the cell wall and the establishment of more favourable interactions between its highly negatively charged catalytic surface and the positively charged chains of pneumococcal murein could enhance the lytic activity of the parasite-encoded enzyme and therefore liberation of the phage progeny.
Oxidation of heparan sulphate by hypochlorite: role of N-chloro derivatives and dichloramine-dependent fragmentation
Authors: Rees Martin D. , Pattison David I. , Davies Michael J.
Published: September 2005

Activated phagocytes release the haem enzyme MPO (myeloperoxidase) and produce superoxide radicals and H2O2 via an oxidative burst. MPO uses H2O2 and Cl− to form HOCl, the physiological mixture of hypochlorous acid and its anion present at pH 7.4. As MPO binds to glycosaminoglycans, oxidation of extracellular matrix and cell surfaces by HOCl may be localized to these materials. However, the reactions of HOCl with glycosaminoglycans are poorly characterized. The GlcNAc (N-acetylglucosamine), GlcNSO3 (glucosamine-N-sulphate) and GlcNH2 [(N-unsubstituted) glucosamine] residues of heparan sulphate are potential targets for HOCl. It is shown here that HOCl reacts with each of these residues to generate N-chloro derivatives, and the absolute rate constants for these reactions have been determined. Reaction at GlcNH2 residues yields chloramines and, subsequently, dichloramines with markedly slower rates, k2∼3.1×105 and 9 M−1·s−1 (at 37 °C) respectively. Reaction at GlcNSO3 and GlcNAc residues yields N-chlorosulphonamides and chloramides with k2∼0.05 and 0.01 M−1·s−1 (at 37 °C) respectively. The corresponding monosaccharides display a similar pattern of reactivity. Decay of the polymer-derived chloramines, N-chlorosulphonamides and chloramides is slow at 37 °C and does not result in major structural changes. In contrast, dichloramine decay is rapid at 37 °C and results in fragmentation of the polymer backbone. Computational modelling of the reaction of HOCl with heparan sulphate proteoglycans (glypican-1 and perlecan) predicts that the GlcNH2 residues of heparan sulphate are major sites of attack. These results suggest that HOCl may be an important mediator of damage to glycosaminoglycans and proteoglycans at inflammatory foci.
The WNK1 and WNK4 protein kinases that are mutated in Gordon's hypertension syndrome phosphorylate and activate SPAK and OSR1 protein kinases
Authors: Vitari Alberto C. , Deak Maria , Morrice Nick A. , Alessi Dario R.
Published: September 2005

Mutations in the human genes encoding WNK1 [with no K (lysine) protein kinase-1] and the related protein kinase WNK4 are the cause of Gordon's hypertension syndrome. Little is known about the molecular mechanism by which WNK isoforms regulate cellular processes. We immunoprecipitated WNK1 from extracts of rat testis and found that it was specifically associated with a protein kinase of the STE20 family termed ‘STE20/SPS1-related proline/alanine-rich kinase’ (SPAK). We demonstrated that WNK1 and WNK4 both interacted with SPAK as well as a closely related kinase, termed ‘oxidative stress response kinase-1’ (OSR1). Wildtype (wt) but not catalytically inactive WNK1 and WNK4 phosphorylated SPAK and OSR1 to a much greater extent than with other substrates utilized previously, such as myelin basic protein and claudin-4. Phosphorylation by WNK1 or WNK4 markedly increased SPAK and OSR1 activity. Phosphopeptide mapping studies demonstrated that WNK1 phosphorylated kinase-inactive SPAK and OSR1 at an equivalent residue located within the T-loop of the catalytic domain (Thr233 in SPAK, Thr185 in OSR1) and a serine residue located within a C-terminal non-catalytic region (Ser373 in SPAK, Ser325 in OSR1). Mutation of Thr185 to alanine prevented the activation of OSR1 by WNK1, whereas mutation of Thr185 to glutamic acid (to mimic phosphorylation) increased the basal activity of OSR1 over 20-fold and prevented further activation by WNK1. Mutation of Ser325 in OSR1 to alanine or glutamic acid did not affect the basal activity of OSR1 or its ability to be activated by WNK1. These findings suggest that WNK isoforms operate as protein kinases that activate SPAK and OSR1 by phosphorylating the T-loops of these enzymes, resulting in their activation. Our analysis also describes the first facile assay that can be employed to quantitatively assess WNK1 and WNK4 activity.
Postprandial acylated ghrelin status following fat and protein manipulation of meals in healthy young women
Authors: Al Awar Rima , Obeid Omar , Hwalla Nahla , Azar Sami
Published: September 2005

The aim of the present study was to investigate the postprandial effect of diet composition on circulating acylated ghrelin levels in healthy women. A randomized cross-over study of three experimental treatments was performed. A total of 11 healthy young women of normal body weight completed the study. All 11 subjects consumed three iso-energetic meals of different macronutrient composition, a balanced meal (50% carbohydrates, 30% fat and 20% protein), a high-fat meal (45% carbohydrates, 45% fat and 10% protein) and a high-protein meal (45% carbohydrates, 20% fat and 35% protein), for breakfast on separate days. The test meals were administered 1 month apart. Blood samples were withdrawn immediately before and at 15, 30, 60, 120 and 180 min after the test meal for measurement of plasma acylated ghrelin, as well as serum glucose, insulin and triacylglycerol (triglyceride) levels. Acylated ghrelin fell significantly after ingestion of both the balanced and high-protein meals. Ghrelin persisted at significantly lower levels than baseline for a longer duration following the high-protein meal (P<0.05 at 15, 30, 60 and 120 min) compared with the balanced meal (P<0.05 at 30 and 60 min). Moreover, acylated ghrelin levels correlated negatively with the postprandial insulin levels. In conclusion, postprandial changes in acylated plasma ghrelin depend on the macronutrient composition of the meal and are possibly influenced by insulin.