bluuebunny/crossref_metadata_2025 huggingface dataset:
Greenland ice sheet surface mass balance 1991–2000: Application of Polar MM5 mesoscale model and in situ data
Authors: Box Jason E. , Bromwich David H. , Bai Le‐Sheng
Published: August 2004

The Polar Pennsylvania State University–National Center for Atmospheric Research Fifth‐Generation Mesoscale Model (Polar MM5) regional climate model was run over the North Atlantic region for 1991–2000. We analyze 24‐km output over the Greenland ice sheet to evaluate spatial and temporal variability of the surface mass balance and its subcomponents. The model output is compared with 3 years of automatic weather station (AWS) data from 17 sites to identify biases. Using the in situ data, we derive simple corrections for biases in melt energy and in water vapor fluxes from the surface and from blowing snow. The simulated accumulation rate is in agreement with AWS and snow pit observations. Estimates for runoff and the surface mass balance distribution over the ice sheet are produced using modeled melt volume and a meltwater retention scheme. From the decade investigated, the magnitude of interannual variability in surface mass balance components is tentatively established. The largest variability is concentrated along the ice sheet margin, where both accumulation and ablation rates are largest. The simulated interannual fluctuations suggest a large absolute variability, ±187 km3 yr−1 for total ice sheet surface mass balance. Variability in simulated equilibrium line altitude is suggestive of a dominance of thermal variability in the south with increasing importance of accumulation variability with increasing latitude. Empirical functions for the sensitivity of surface mass balance to temperature and precipitation anomalies are presented. The precise locations and regions of maximum and minimum surface energy and mass fluxes are suggested. Using an estimate for iceberg discharge and bottom melting, the total ice sheet mass balance is estimated be −78 km3 yr−1, producing 2.2 mm of eustatic sea level rise over the 1991–2000 decade and contributing 15% to the observed (1.5 mm yr−1) global sea level rise. The more negative mass balance is attributed to including blowing‐snow sublimation loss and to regional warming in the 1990s.
Diffractive vector and scalar integrals for bistatic radio holographic remote sensing
Authors: Pavelyev A. G. , Liou Y. A. , Wickert J.
Published: August 2004

We introduce new vector diffractive integrals, which can be used for the radio holographic remote sensing of the atmosphere and terrestrial surfaces. These integrals are exact relationships connecting the electromagnetic fields known at some interface or curve in space with radio fields on the terrestrial surface or inside the atmosphere. They allow one to restore the radio image of the atmosphere or Earth surface in the investigated regions using a radio hologram registered in space by a small instrument installed on the low Earth orbit satellite. The high‐precision radio signals of the Global Positioning System (GPS) navigational satellites can be used as a source of the radio emission for radio holograms. We indicated a connection between the vector diffractive integrals and scalar diffractive integral, which is now applied for the GPS occultation investigation of Earth's atmosphere under an assumption of the spherical symmetry. For the atmosphere itself the accuracy of the scalar theory corresponds to the accuracy of the GPS occultation measurements. The most significant factor that affects the polarization is the reflection from the surface. The use of vector theory can thus be useful for the investigation of Earth's atmosphere by detecting the reflected rays. We show that the reference signal needed for restoration of the radio field from the registered radio hologram is coinciding with the Green function of the scalar wave equation corresponding to a three‐dimensional inhomogeneous medium. This substantiates the radio holographic–focused synthetic aperture principle (RFSA) in its application to the atmosphere and surface research. We validated the high vertical resolution of the RFSA method by obtaining radio image of the atmosphere and Earth's surface. Zverev's diffractive integral is used to compare the canonical transform (CT), back propagation (BP), and RFSA methods. For comparison, a general inverse operator (GIO) is introduced. The CT and BP transforms can be obtained by application of the GIO transform to Zverev's diffractive integral. The CT method can resolve physical rays in multipath situations under an assumption of the global spherical symmetry of the atmosphere and ionosphere. The RFSA method can account for the multipath in the case when the global spherical symmetry is absent by using the appropriate model of the refractivity and has a promise to be effective for operational data analysis.
Limits of applicability of the advection‐dispersion model in aquifers containing connected high‐conductivity channels
Authors: Liu Gaisheng , Zheng Chunmiao , Gorelick Steven M.
Published: August 2004

The macrodispersion model from stochastic transport theory is demonstrated to be of limited utility when applied to heterogeneous aquifer systems containing narrow connected pathways. This is so even when contrasts in hydraulic conductivity (K) are small and variance in ln K is less than 0.10. We evaluated how well an advection‐dispersion model (ADM) could be used to represent solute plumes transported through mildly heterogeneous three‐dimensional (3‐D) systems characterized by a well‐connected dendritic network of 10 cm wide high‐K channels. Each high‐K channel network was generated using an invasion percolation algorithm and consisted of ∼10% by volume high‐K regions. Contrasts in K between the channels and matrix were varied systematically from 2:1 to 30:1, corresponding to ln K values ranging from 0.04 to 1.05. Simulations involved numerical models with 3‐D decimeter discretization, and each model contained 2–4 million active cells. Transport through each channel network considered only the processes of advection and molecular diffusion. In every case, the temporal change in the second spatial moment of concentrations was linear, with R2 values ranging from 0.97 to 0.99. The third spatial moment, or alternatively, the skewness coefficient values, indicated significant tailing downstream of the plume center. For each case, a corresponding ADM was used to simulate transport through the system. The corresponding ADM employed the effective mean hydraulic conductivity that reproduced the total discharge through the channel network system under an identical ambient gradient. Dispersivity values used in the ADM were obtained from the temporal change in the second spatial moments of concentrations for the plumes in the channel network systems and ranged from 0.014 m to 0.85 m. The results indicate that as the conductivity contrast between the channels and matrix increased, the simulated plumes in the channel network system became more and more asymmetric, with little solute dispersed upstream of the plume center and extensive downstream spreading of low concentrations. Distinctly different spreading was found upstream versus downstream of the plume center. The ADM failed to capture this asymmetry. Comparison of each plume in the channel network system with the corresponding plume produced using the corresponding ADM showed a maximum correlation of only 0.64 and a minimum fractional error of 0.29 for cases in which the log K variance was ∼0.20 (ln K variance was ∼1.0). At early times the correlations were as low as 0.40. The greatest correlation occurred at late times and for cases in which a wide source was considered.
Topographic variations in chaos on Europa: Implications for diapiric formation
Authors: Schenk Paul M. , Pappalardo Robert T.
Published: August 2004

Disrupted terrain, or chaos, on Europa, might have formed through melting of a floating ice shell from a subsurface ocean [Carr et al., 1998; Greenberg et al., 1999], or breakup by diapirs rising from the warm lower portion of the ice shell [Head and Pappalardo, 1999; Collins et al., 2000]. Each model makes specific and testable predictions for topographic expression within chaos and relative to surrounding terrains on local and regional scales. High‐resolution stereo‐controlled photoclinometric topography indicates that chaos topography, including the archetypal Conamara Chaos region, is uneven and commonly higher than surrounding plains by up to 250 m. Elevated and undulating topography is more consistent with diapiric uplift of deep material in a relatively thick ice shell, rather than melt‐through and refreezing of regionally or globally thin ice by a subsurface ocean. Vertical and horizontal scales of topographic doming in Conamara Chaos are consistent with a total ice shell thickness >15 km. Contact between Europa's ocean and surface may most likely be indirectly via diapirism or convection.
Orbital‐scale anti‐phase variation of sea surface temperature in mid‐latitude North Pacific margins during the last 145,000 years
Authors: Yamamoto Masanobu , Oba Tadamichi , Shimamune Junko , Ueshima Toshinori
Published: August 2004

The late Quaternary records of alkenone sea surface temperature (SST) in the Japan and California margins showed orbital‐scale anti‐phase SST variations between the two margins. This east‐west seesaw‐like change agreed well with the long‐term El Niño‐Southern Oscillation (ENSO) behavior predicted by the Zebiak‐Cane ENSO model [Clement et al., 1999] as regards both the timing and frequency during 0–60 ka and 120–145 ka, and is attributed to the precession‐controlled change in tropical ENSO behavior. This anti‐phase SST change was not clearly demonstrated during 60–120 ka. This finding suggests that the influence of tropical climatic dynamics on the mid‐latitude North Pacific varied in response to glacial‐interglacial cycles.
Neutron diffraction study of the contribution of grain contacts to nonlinear stress‐strain behavior
Authors: Darling T. W. , TenCate J. A. , Brown D. W. , Clausen B. , Vogel S. C.
Published: August 2004

Repeatable, hysteretic loops in quasi‐static loading measurements on rocks are well known; the fundamental processes responsible for them are not. The grain contact region is usually treated as the site of these processes, but there is little supporting experimental evidence. We have performed simultaneous neutron diffraction and quasi‐static loading experiments on a selection of rocks to experimentally isolate the response of these contact regions. Neutron diffraction measures strain in the lattice planes of the bulk of the grain material, so differences between this strain and the macroscopic response yield information about grain contact behavior. We find the lattice responds linearly to stress in all cases, oblivious to the macroscopic unrecoverable strains, curvature, and hysteresis, localizing these effects to the contacts. Neutron diffraction shows that the more granular rocks appear to distribute stresses so that the same strain appears in all the grains, independent of crystallographic orientation.
Measurements of aerosol optical depths and black carbon over Bay of Bengal during post‐monsoon season
Authors: Sumanth E. , Mallikarjuna K. , Stephen Joshi , Moole Mahesh , Vinoj V. , Satheesh S. K. , Moorthy K. Krishna
Published: August 2004

Extensive and collocated measurements of aerosol optical depths (AODs), mass concentration of composite aerosols and aerosol black carbon (BC) were made over the Bay of Bengal (onboard R/V Sagar Kanya during its cruise #197) region during post‐Indian summer monsoon period. These data along with MODIS (onboard TERRA satellite) data shows that aerosol visible AODs were in the range of 0.2 to 0.7 (with mean value of 0.43 ± 0.12) and Angstrom wavelength exponent of ∼0.93 ± 0.21 which in conjunction with a single scattering albedo (ω) of ∼0.94 indicates the presence of weakly absorbing aerosols. The BC mass fraction was ∼2.9 ± 1.1% during post‐monsoon season compared to 5.8 ± 0.6% (with ω of ∼0.88) during pre‐monsoon season. The presence of aerosols over Bay of Bengal during post‐monsoon season decreases the short wave radiation reaching the surface by as much as 25 W m−2 and increases top of the atmosphere reflected radiation by 14 W m−2.
Octasaccharide is the minimal length unit required for efficient binding of cyclophilin B to heparin and cell surface heparan sulphate
Authors: VANPOUILLE Christophe , DENYS Agnès , CARPENTIER Mathieu , PAKULA Rachel , MAZURIER Joël , ALLAIN Fabrice
Published: August 2004

Cyclophilin B (CyPB) is a heparin-binding protein first identified as a receptor for cyclosporin A. In previous studies, we reported that CyPB triggers chemotaxis and integrin-mediated adhesion of T-lymphocytes by way of interaction with two types of binding sites. The first site corresponds to a signalling receptor; the second site has been identified as heparan sulphate (HS) and appears crucial to induce cell adhesion. Characterization of the HS-binding unit is critical to understand the requirement of HS in pro-adhesive activity of CyPB. By using a strategy based on gel mobility shift assays with fluorophore-labelled oligosaccharides, we demonstrated that the minimal heparin unit required for efficient binding of CyPB is an octasaccharide. The mutants CyPBKKK− [where KKK− refers to the substitutions K3A(Lys3→Ala)/K4A/K5A] and CyPBΔYFD (where Tyr14-Phe-Asp16 has been deleted) failed to interact with octasaccharides, confirming that the Y14FD16 and K3KK5 clusters are required for CyPB binding. Molecular modelling revealed that both clusters are spatially arranged so that they may act synergistically to form a binding site for the octasaccharide. We then demonstrated that heparin-derived octasaccharides and higher degree of polymerization oligosaccharides inhibited the interaction between CyPB and fluorophore-labelled HS chains purified from T-lymphocytes, and strongly reduced the HS-dependent pro-adhesive activity of CyPB. However, oligosaccharides or heparin were unable to restore adhesion of heparinase-treated T-lymphocytes, indicating that HS has to be present on the cell membrane to support the pro-adhesive activity of CyPB. Altogether, these results demonstrate that the octasaccharide is likely to be the minimal length unit required for efficient binding of CyPB to cell surface HS and consequent HS-dependent cell responses.
Structure and cholesterol domain dynamics of an enriched caveolae/raft isolate
Authors: GALLEGOS Adalberto M. , McINTOSH Avery L. , ATSHAVES Barbara P. , SCHROEDER Friedhelm
Published: August 2004

Despite the importance of cholesterol in the formation and function of caveolar microdomains in plasma membranes, almost nothing is known regarding the structural properties, cholesterol dynamics or intracellular factors affecting caveolar cholesterol dynamics. A non-detergent method was employed to isolate caveolae/raft domains from purified plasma membranes of murine fibroblasts. A series of fluorescent lipid probe molecules or a fluorescent cholesterol analogue, dehydroergosterol, were then incorporated into the caveolae/raft domains to show that: (i) fluorescence polarization of the multiple probe molecules {diphenylhexatriene analogues, DiI18 (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), parinaric acids and NBD-stearic acid {12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-octadecanoic acid} indicated that acyl chains in caveolae/raft domains were significantly less ‘fluid’ (i.e. more rigid) and the transbilayer ‘fluidity gradient’ was 4.4-fold greater than in plasma membranes; (ii) although sterol was more ordered in caveolae/raft domains than plasma membranes, spontaneous sterol transfer from caveolae/raft domains was faster (initial rate, 32%; half-time, t1/2, 57%) than from the plasma membrane; (iii) although kinetic analysis showed similar proportions of exchangeable and non-exchangeable sterol pools in caveolae/raft domains and plasma membranes, addition of SCP-2 (sterol carrier protein-2) 1.3-fold more selectively increased sterol transfer from caveolae/raft domains by decreasing the t1/2 (50%) and increasing the initial rate (5-fold); (iv) SCP-2 was also 2-fold more selective in decreasing the amount of non-exchangeable sterol in caveolae/raft domains compared with plasma membranes, such that nearly 80% of caveolar/raft sterol became exchangeable. In summary, although caveolae/raft lipids were less fluid than those of plasma membranes, sterol domains in caveolae/rafts were more spontaneously exchangeable and more affected by SCP-2 than those of the bulk plasma membranes. Thus caveolae/raft domains isolated without the use of detergents display unique structure, cholesterol domain kinetics and responsiveness to SCP-2 as compared with the parent plasma membrane.
Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria-associated membrane) and/or mitochondria?
Authors: BIONDA Clara , PORTOUKALIAN Jacques , SCHMITT Daniel , RODRIGUEZ-LAFRASSE Claire , ARDAIL Dominique
Published: August 2004

Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB1 (fumonisin B1) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydrosphingosine N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate, generating ceramide 1-phosphate, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.
Purification of Drosophila DNA polymerase ζ by REV1 protein-affinity chromatography
Authors: TAKEUCHI Ryo , OSHIGE Masahiko , UCHIDA Makiyo , ISHIKAWA Gen , TAKATA Kei-ichi , SHIMANOUCHI Kaori , KANAI Yoshihiro , RUIKE Tatsushi , MORIOKA Hiroshi , SAKAGUCHI Kengo
Published: August 2004

Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol ζ (DNA polymerase ζ) from Drosophila melanogaster (Dmpol ζ). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol ζ was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2′,3′-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)10:1 as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol ζ showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol ζ were not detected. Drosophila REV7 interacted with Dmpol ζ in vitro, but did not influence the DNA synthesis activity of Dmpol ζ. The present study is the first report about characterization of purified pol ζ from multicellular organisms, and the second concerning the characterization of yeast pol ζ.
Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase with an unusual lectin domain from the platyhelminth parasite Echinococcus granulosus
Authors: FREIRE Teresa , FERNÁNDEZ Cecilia , CHALAR Cora , MAIZELS Rick M. , ALZARI Pedro , OSINAGA Eduardo , ROBELLO Carlos
Published: August 2004

As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 °C, in the range 6.5–7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.
Enhancement of correct protein folding in vivo by a non-lytic baculovirus
Authors: HO Yu , LO Huei-Ru , LEE Tzu-Ching , WU Carol P. Y. , CHAO Yu-Chan
Published: August 2004

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.
Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells
Authors: WALIA Baljit , CASTANEDA Florencia E. , WANG Lixin , KOLACHALA Vasantha L. , BAJAJ Rahul , ROMAN Jesse , MERLIN Didier , GEWIRTZ Andrew T. , SITARAMAN Shanthi V.
Published: August 2004

Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role in mucosal immune responses during inflammation.
Identification of the growth hormone-releasing peptide binding site in CD36: a photoaffinity cross-linking study
Authors: DEMERS Annie , McNICOLL Normand , FEBBRAIO Maria , SERVANT Marc , MARLEAU Sylvie , SILVERSTEIN Roy , ONG Huy
Published: August 2004

The GHRPs (growth hormone-releasing peptides) are a class of small synthetic peptides known to stimulate GH release through binding of a G-protein-coupled receptor (designated GHS-R). We have found that hexarelin, a hexapeptide member of the GHRPs, binds to another protein identified as CD36, a scavenger receptor that is expressed in various tissues, including monocytes/macrophages and the endothelial microvasculature. CD36 is involved in the endocytosis of oxLDL (oxidized low-density lipoprotein) by macrophages, and in the modulation of angiogenesis elicited by thrombospondin-1 through binding to endothelial cells. To define the binding domain for hexarelin on CD36, covalent photolabelling of CD36 followed by enzymic and chemical degradation of the photoligand–receptor complex was performed. A 8 kDa photolabelled fragment corresponding to the CD36-(Asn132–Glu177) sequence has been identified as the hexarelin-binding site. Chemical cleavage of this fragment with CNBr resulted in the release of the free ligand, suggesting that Met169 is the contact point for the ligand within the receptor binding pocket. We conclude that the binding domain for hexarelin on CD36 overlaps with that for oxLDL, which corresponds to residues Gln155–Lys183 of CD36. Hence hexarelin might interfere with the CD36-mediated uptake of modified lipoproteins by macrophages. This may contribute, at least in part, to the anti-atherosclerotic effect of GHRPs in apolipoprotein E-deficient mice.
Interaction of the tyrosine phosphatase SHP-2 with Gab2 regulates Rho-dependent activation of the c-fosserum response element by interleukin-2
Authors: ARNAUD Mary , MZALI Rym , GESBERT Franck , CROUIN Catherine , GUENZI Christine , VERMOT-DESROCHES Claudine , WIJDENES John , COURTOIS Geneviève , BERNARD Olivier , BERTOGLIO Jacques
Published: August 2004

Gab2 (Grb2-associated binder-2), a member of the IRS (insulin receptor substrate)/Gab family of adapter proteins, undergoes tyrosine phosphorylation in response to cytokine or growth factor stimulation and serves as a docking platform for many signal transduction effectors, including the tyrosine phosphatase SHP-2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase]. Here, we report that, following IL-2 (interleukin-2) stimulation of human T lymphocytes, SHP-2 binds tyrosine residues 614 and 643 of human Gab2 through its N- and C-terminal SH2 domains respectively. However, the sole mutation of Tyr-614 into phenylalanine is sufficient to prevent Gab2 from recruiting SHP-2. Expression of the Gab2 Tyr-614→Phe (Y614F) mutant, defective in SHP-2 association, prevents ERK (extracellular-signal-regulated kinase) activation and expression of a luciferase reporter plasmid driven by the c-fos SRE (serum response element), indicating that interaction of SHP-2 with Gab2 is required for ERK activation in response to IL-2. Further investigation of IL-2-dependent induction of SRE showed that expression of a constitutively active mutant of the RhoA GTPase synergizes with IL-2 for SRE-driven transcription, whereas a dominant-negative mutant reduces the IL-2 response. Thus, in response to IL-2, full induction of the SRE requires ERK-dependent as well as Rho-dependent signals that target the Ets-box and the CArG-box respectively. We also report that the synergy between Gab2/SHP-2 and RhoA for IL-2-dependent CArG-box-driven transcription depends upon MEK (mitogen-activated protein kinase/ERK kinase) activation, and is likely to involve regulation of the serum response factor co-activator MAL. Our studies thus provide new insights into the role of Gab2 and SHP-2 in IL-2 signal transduction.
Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast
Authors: MORGAN Clive P. , INSALL Robert , HAYNES Lee , COCKCROFT Shamshad
Published: August 2004

The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253–257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the full-length 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.
Intracellular ceramide synthesis and protein kinase Cζ activation play an essential role in palmitate-induced insulin resistance in rat L6 skeletal muscle cells
Authors: POWELL Darren J. , TURBAN Sophie , GRAY Alexander , HAJDUCH Eric , HUNDAL Harinder S.
Published: August 2004

Non-esterified fatty acids (NEFAs) have been implicated in the pathogenesis of skeletal muscle insulin resistance that may develop, in part, as a consequence of a direct inhibitory effect on early insulin signalling events. Here we report work investigating the mechanism by which palmitate (a saturated free fatty acid) inhibits insulin action in rat L6 myotubes. Palmitate suppressed the insulin-induced plasma membrane recruitment and phosphorylation of protein kinase B (PKB) and this was associated with a loss in insulin-stimulated glucose transport. The inhibition in PKB was not due to a loss in insulin receptor substrate (IRS)1 tyrosine phosphorylation, IRS-1/p85 (phosphoinositide 3-kinase) association or suppression in phosphatidyl 3,4,5 triphosphate synthesis, but was attributable to an elevated intracellular synthesis of ceramide (6-fold) from palmitate and a concomitant activation of protein kinase PKCζ (5-fold). Inhibitors of serine palmitoyl transferase suppressed the intracellular synthesis of ceramide from palmitate, prevented PKCζ activation, and antagonized the inhibition in PKB recruitment/phosphorylation and the loss in insulin-stimulated glucose transport elicited by the NEFA. Inhibiting the palmitate-induced activation of PKCζ with Ro 31.8220, also prevented the loss in the insulin-dependent phosphorylation of PKB caused by palmitate. These findings indicate that intracellular ceramide synthesis and PKCζ activation are important aspects of the mechanism by which palmitate desensitizes L6 muscle cells to insulin.
δ-Catenin/NPRAP (neural plakophilin-related armadillo repeat protein) interacts with and activates sphingosine kinase 1
Authors: FUJITA Toshitada , OKADA Taro , HAYASHI Shun , JAHANGEER Saleem , MIWA Noriko , NAKAMURA Shun-ichi
Published: August 2004

Sphingosine kinase (SPHK) is a key enzyme catalysing the formation of sphingosine 1-phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events acting through intracellular, as well as extracellular, mechanisms. However, the molecular mechanism of intracellular actions of SPP remains unclear. Here, we have identified δ-catenin/NPRAP (neural plakophilin-related armadillo repeat protein) as a potential binding partner for SPHK1 by yeast two-hybrid screening. From co-immunoprecipitation analyses, the C-terminal portion of δ-catenin/NPRAP containing the seventh to tenth armadillo repeats was found to be required for interaction with SPHK1. Endogenous δ-catenin/NPRAP was co-localized with endogenous SPHK1 and transfected δ-catenin/NPRAP was co-localized with transfected SPHK1 in dissociated rat hippocampal neurons. MDCK (Madin–Darby canine kidney) cells stably expressing δ-catenin/NPRAP contained elevated levels of intracellular SPP. In a purified system δ-catenin/NPRAP stimulated SPHK1 in a dose-dependent manner. Furthermore, δ-catenin/NPRAP-induced increased cell motility in MDCK cells was completely inhibited by dimethylsphingosine, a specific inhibitor of SPHK1. These results strongly suggest that at least some of δ-catenin/NPRAP functions, including increased cell motility, are mediated by an SPHK–SPP signalling pathway.
Tumour necrosis factor α decreases glucose-6-phosphatase gene expression by activation of nuclear factor κB
Authors: GREMPLER Rolf , KIENITZ Anne , WERNER Torsten , MEYER Marion , BARTHEL Andreas , AILETT Fabienne , SUTHERLAND Calum , WALTHER Reinhard , SCHMOLL Dieter
Published: August 2004

The key insulin-regulated gluconeogenic enzyme G6Pase (glucose-6-phosphatase) has an important function in the control of hepatic glucose production. Here we examined the inhibition of G6Pase gene transcription by TNF (tumour necrosis factor) in H4IIE hepatoma cells. TNF decreased dexamethasone/dibtuyryl cAMP-induced G6Pase mRNA levels. TNFα, but not insulin, led to rapid activation of NFκB (nuclear factor κB). The adenoviral overexpression of a dominant negative mutant of IκBα (inhibitor of NFκB α) prevented the suppression of G6Pase expression by TNFα, but did not affect that by insulin. The regulation of G6Pase by TNF was not mediated by activation of the phosphoinositide 3-kinase/protein kinase B pathway, extracellular-signal-regulated protein kinase or p38 mitogen-activated protein kinase. Reporter gene assays demonstrated a concentration-dependent down-regulation of G6Pase promoter activity by the transient overexpression of NFκB. Although two binding sites for NFκB were identified within the G6Pase promoter, neither of these sites, nor the insulin response unit or binding sites for Sp proteins, was necessary for the regulation of G6Pase promoter activity by TNFα. In conclusion, the data indicate that the activation of NFκB is sufficient to suppress G6Pase gene expression, and is required for the regulation by TNFα, but not by insulin. We propose that NFκB does not act by binding directly to the G6Pase promoter.
Thermal and conformational stability of Ssh10b protein from archaeon Sulfolobus shibattae
Authors: XU Su , QIN Sanbo , PAN Xian-Ming
Published: August 2004

The secondary structure of the DNA binding protein Ssh10b is largely unaffected by change in temperature between 25 °C and 85 °C, indicating that the protein is highly thermostable. Here, we report the temperature-dependent equilibrium denaturation of Ssh10b in the presence of guanidine hydrochloride (GdnHCl). It was found that the transition midpoint values of the temperature (Tm), and changes of enthalpy (ΔHm) and entropy (ΔSm) of Ssh10b unfolding were linearly decreasing with increasing GdnHCl concentration. The true values of the thermodynamic parameters, Tm=402 K, ΔHm=590±40 kJ·mol−1 and ΔSm=1.4±0.15 kJ·T−1·mol−1, were obtained by linear extrapolation to 0 M GdnHCl. The value of the heat capacity change of Ssh10b unfolding, ΔCp=3.8±0.2 kJ·T−1·mol−1 (approx. 19 J T−1·mol residue−1), was obtained from the measured thermodynamic parameters. This is significantly smaller than that of the average value for mesophilic proteins (50 J·K−1·mol residue−1) or the value calculated from the Ssh10b structural data (64 J T−1·mol residue−1). A consequence of the small ΔCp is that the ΔG of Ssh10b is larger than that of mesophilic proteins, while the values of ΔH and T*ΔS are smaller. The small ΔCp of Ssh10b appears to result mainly from the presence of compactness in the denatured state.
A proteome analysis of the arsenite response in cultured lung cells: evidence for in vitro oxidative stress-induced apoptosis
Authors: LAU Andy T. Y. , HE Qing-Yu , CHIU Jen-Fu
Published: August 2004

Arsenite is well documented as a chemotherapeutic agent capable of inducing cell death. However, the cellular response at the molecular level has not been studied extensively. In the present study, we provide for the first time a proteomic analysis of rat LECs (lung epithelial cells) treated with arsenite, with the aim of identifying defence proteins, probably expressed to protect the cells during the course of arsenic-induced apoptosis. Comparative proteome analysis was conducted on LECs and LECs treated with 40 μM arsenite to identify global changes in their protein expression profiles. Over 1000 protein spots were separated by two-dimensional electrophoresis and visualized by silver staining. Seven proteins changed expression levels significantly and were identified by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry and database searching. The proteins up-regulated were mostly HSPs (heat-shock proteins) and antioxidative stress proteins, including HSP70, aldose reductase, haem oxygenase-1, HSP27, ferritin light chain and αB-crystallin. The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase was down-regulated. Pretreatment with the thiol antioxidants glutathione or N-acetylcysteine before arsenite insult effectively abrogated the induction of these defence proteins and sustained cell viability, whereas antioxidants were protective only at earlier time points if they were added to cells after arsenite. Taken together, our results demonstrate that high levels of arsenite cause oxidative stress-induced apoptosis.
Isolation and functional expression of human COQ2, a gene encoding a polyprenyl transferase involved in the synthesis of CoQ
Authors: FORSGREN Margareta , ATTERSAND Anneli , LAKE Staffan , GRÜNLER Jacob , SWIEZEWSKA Ewa , DALLNER Gustav , CLIMENT Isabel
Published: August 2004

The COQ2 gene in Saccharomyces cerevisiae encodes a Coq2 (p-hydroxybenzoate:polyprenyl transferase), which is required in the biosynthetic pathway of CoQ (ubiquinone). This enzyme catalyses the prenylation of p-hydroxybenzoate with an all-trans polyprenyl group. We have isolated cDNA which we believe encodes the human homologue of COQ2 from a human muscle and liver cDNA library. The clone contained an open reading frame of length 1263 bp, which encodes a polypeptide that has sequence homology with the Coq2 homologues in yeast, bacteria and mammals. The human COQ2 gene, when expressed in yeast Coq2 null mutant cells, rescued the growth of this yeast strain in the absence of a non-fermentable carbon source and restored CoQ biosynthesis. However, the rate of CoQ biosynthesis in the rescued cells was lower when compared with that in cells rescued with the yeast COQ2 gene. CoQ formed when cells were incubated with labelled decaprenyl pyrophosphate and nonaprenyl pyrophosphate, showing that the human enzyme is active and that it participates in the biosynthesis of CoQ.
Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding
Authors: ALQAWI Omar , BATES Susan , GEORGES Elias
Published: August 2004

ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp1000) selected with doxorubicin and verapamil. Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/AdrVp1000 cells. This mutation was shown to modulate the transport of Rh123 (rhodamine 123). In the present study, we have used a previously characterized photoreactive drug analogue of Rh123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg482→Thr mutation on Rh123 binding and transport by ABCG2. Our results show that both wild-type (ABCG2R482) and mutant (ABCG2T482) ABCG2 bound directly to IAARh123. Surprisingly, however, wild-type ABCG2R482, which does not transport Rh123, was more intensely photolabelled than mutant ABCG2T482. In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2. For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2. Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.
Mapping of an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine mono- and bi-layer binding site in erythroid β-spectrin
Authors: HRYNIEWICZ-JANKOWSKA Anita , BOK Ewa , DUBIELECKA Patrycja , CHORZALSKA Anna , DIAKOWSKI Witold , JEZIERSKI Adam , LISOWSKI Marek , SIKORSKI Aleksander F.
Published: August 2004

It has been shown previously that binding of vesicles and monolayers containing PE (phosphatidylethanolamine) by either erythroid or non-erythroid spectrin proved sensitive to inhibition by purified erythrocyte ankyrin. We tested the lipid-binding affinities of the purified ankyrin-binding domain of β-spectrin and of its truncated mutants in four ways, by analysing: (1) penetration of ‘loose’ PE/PC (phosphatidylcholine) monolayers; (2) binding to liposomes in suspension; (3) competition with spectrin for liposomes; and (4) binding of a PE/PC monolayer in a surface plasmon resonance system. The results obtained indicated that the full-length ankyrin-binding domain bound PE/PC mono- and bi-layers with moderate affinity, penetrated monolayers and competed with spectrin for liposomes. Moreover, its truncated mutants that retained the N-terminal part, in contrast with those lacking eight or 38 N-terminal residues (which bound lipid mono- and bi-layers with lower affinity), bound PE/PC mono- and bi-layers with an affinity and capacity comparable with those of the full-length ankyrin-binding domain, and this activity was inhibited by purified erythrocyte ankyrin. The full-length domain, in contrast with the mutant lacking 38 N-terminal residues, induced a small increase in the fluidity of PE/PC membranes when probed with 5′-doxyl stearate, similar to the effect of purified spectrin. Therefore we conclude that the binding site for PE-rich lipids, which is sensitive to ankyrin inhibition, is located in a 38-residue N-terminal fragment of the β-spectrin ankyrin-binding domain, and that the first eight residues play a key role in this activity.
Cloning, expression in Escherichia coli and characterization of the recombinant Neu5Acα2,6Galβ1,4GlcNAc-specific high-affinity lectin and its mutants from the mushroom Polyporus squamosus
Authors: TATENO Hiroaki , WINTER Harry C. , GOLDSTEIN Irwin J.
Published: August 2004

Lectin from the mushroom Polyporus squamosus (PSL) has a unique carbohydrate-binding specificity for sialylated glycoconjugates containing Neu5Acα2,6Galβ1,4Glc/GlcNAc trisaccharide sequences of asparagine-linked glycoproteins. In the present study, we elucidate the molecular basis for its binding specificity as well as establish a consistent source of this useful lectin using a bacterial expression system. cDNA cloning revealed that PSL contains a ricin B chain-like (QXW)3 domain at its N-terminus that is composed of three homologous subdomains (α, β and γ). A recombinant lectin was expressed in Escherichia coli as a fully active, soluble form. It agglutinated rabbit erythrocytes and showed the highest affinity for Neu5Acα2,6Galβ1,4GlcNAc, but not for the sialyl α2,3-linked isomer. We also investigated the structure–function relationship of PSL. A monomeric C-terminal deletion mutant lacking 40% of the lectin's molecular mass retained sugar-binding activity, indicating that the carbohydrate-binding sites are situated in the N-terminal portion of the lectin, whereas the C-terminal portion probably functions in oligomerization and structural stabilization. Mutant constructs that have single amino acid substitutions in the putative sugar-binding sites, based on sequence alignment with the ricin B-chain, indicate that the β and γ subdomains are most probably sugar-binding sites. The recombinantly expressed lectin will be a valuable reagent for the detection of the Neu5Acα2,6Galβ1,4GlcNAc sequence of asparagine-linked glycans.