bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest
Authors: Capasso Holly , Palermo Carmela , Wan Shanhong , Rao Hui , John Ulrik P. , O'Connell Matthew J. , Walworth Nancy C.
Published: November 2002

In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.
Drosophilasyntaxin 16 is a Q-SNARE implicated in Golgi dynamics
Authors: Xu Hao , Boulianne Gabrielle L. , Trimble William S.
Published: November 2002

SNARE isoforms appear to regulate specific intracellular membrane trafficking steps. To identify new SNARE proteins in Drosophila melanogaster we used a yeast two-hybrid screen to search for proteins that interact with SNAP. Here we report the identification of the Drosophila homologue of syntaxin 16. dsyntaxin 16 binds SNAP in a concentration-dependent fashion and genetically interacts with NSF2. Like its mammalian homologue, dsyntaxin 16 is ubiquitously expressed and appears to be localized to the Golgi apparatus. In addition, membranes containing dsyntaxin 16 become aggregated upon Brefeldin A treatment and are dispersed during meiosis. Inhibition of dsyntaxin 16 function by overexpression of truncated forms in cultured Schneider cells indicates that dsyntaxin 16 may selectively regulate Golgi dynamics.
Identification of a novel sorting determinant for the regulated pathway in the secretory protein chromogranin A
Authors: Taupenot Laurent , Harper Kimberly L. , Mahapatra Nitish R. , Parmer Robert J. , Mahata Sushil K. , O'Connor Daniel T.
Published: November 2002

Chromogranin A (CgA) is the index member of the chromogranin/secretogranin(or `granin') family of regulated secretory proteins that are ubiquitously distributed in amine- and peptide-containing secretory granules of endocrine,neuroendocrine and neuronal cells. Because of their abundance and such widespread occurrence, granins have often been used as prototype proteins to elucidate mechanisms of protein targeting into dense-core secretory granules. In this study, we used a series of full-length, point mutant or truncated CgA-green fluorescent protein (GFP) chimeras to explore routing of CgA in neuroendocrine PC12 cells. Using sucrose gradient fractionation and 3D deconvolution microscopy to determine the subcellular localization of the GFP chimeras, as well as secretagogue-stimulated release, the present study establishes that a CgA-GFP fusion protein expressed in neuroendocrine PC12 cells is trafficked to the dense core secretory granule and thereby sorted to the regulated pathway for exocytosis. We show that information necessary for such trafficking is contained within the N-terminal but not the C-terminal region of CgA. We find that CgA's conserved N-terminal hydrophobic Cys17-Cys38 loop structure may not be sufficient for sorting of CgA into dense-core secretory granules, nor is its stabilization by a disulfide bond necessary for such sorting. Moreover, our data reveal for the first time that the CgA77-115 domain of the mature protein may be necessary (though perhaps not sufficient) for trafficking CgA into the regulated pathway of secretion.
β-Catenin is not required for proliferation and differentiation of epidermal mouse keratinocytes
Authors: Posthaus Horst , Williamson Lina , Baumann Dominique , Kemler Rolf , Caldelari Reto , Suter Maja M. , Schwarz Heinz , Müller Eliane
Published: November 2002

Despite the pivotal role of β-catenin in a variety of biological processes, conditional β-catenin gene ablation in the skin of transgenic mice failed to affect interfollicular epidermal morphogenesis. We elucidated the molecular mechanisms underlying this phenomenon. Long-term cultures of homozygous, heterozygous and β-catenin-null mutant keratinocytes were established to demonstrate that epidermal keratinocyte proliferation, cell cycle progression and cyclin D1 expression occur independently of β-catenin and correlate with repression of transcription from Tcf/Lef-responsive promoters. Moreover, during differentiation,β-catenin-null cells assemble normal intercellular adhesion junctions owing to the substitution of β-catenin with plakoglobin, whereas the expression of the other adhesion components remains unaffected. Taken together, our results demonstrate that epidermal proliferation and adhesion are independent of β-catenin.
E-cadherin-mediated interactions of thymic epithelial cells with CD103+ thymocytes lead to enhanced thymocyte cell proliferation
Authors: Kutleša Snježana , Wessels Johannes T. , Speiser Angelika , Steiert Inge , Müller Claudia A. , Klein Gerd
Published: November 2002

Cadherins are a family of cell adhesion molecules that mainly mediate homotypic homophilic interactions, but for E-cadherin, heterophilic interactions with the integrin αE(CD103)β7have also been reported. In the human thymus, where thymocytes develop in close contact with thymic stromal cells, E-cadherin expression was detected on thymic epithelial cells. By immunofluorescence staining, the strongest expression of E-cadherin was observed on medullary thymic epithelial cells. These cells also express cytosolic catenins, which are necessary to form functional cadherin-catenin complexes. Regardless of their developmental stage, human thymocytes do not express E-cadherin, indicating that homophilic interactions cannot occur. Flow cytometric analysis revealed that the E-cadherin ligand CD103 is expressed on subpopulations of the early CD4- CD8- double-negative and of the more mature CD8+ single-positive thymocytes. Using an in vitro cell adhesion assay, double-negative and CD8+ single-positive thymocytes adhered strongly to isolated thymic epithelial cells. These adhesive interactions could be inhibited by antibodies against E-cadherin or CD103. CD8+thymocytes showed a proliferative response when incubated with thymic epithelial cells. This mitogenic effect was inhibited by antibodies against CD103, which strongly indicates a direct involvement of the adhesive ligand pair CD103—E-cadherin in human thymocyte cell proliferation.
Nuclear localisation of cytosolic phospholipase A2-α in the EA.hy.926 human endothelial cell line is proliferation dependent and modulated by phosphorylation
Authors: Grewal Seema , Morrison Ewan E. , Ponnambalam Sreenivasan , Walker John H.
Published: November 2002

Cytosolic phospholipase A2-α (cPLA2-α) is a calcium-sensitive enzyme involved in receptor-mediated eicosanoid production. In resting cells, cPLA2-α is present in the cytosol and nucleus and translocates to membranes via its calcium-dependent lipid-binding (CaLB) domain following stimulation. cPLA2-α is also regulated by phosphorylation on several residues, which results in enhanced arachidonic acid release. Little is known about the factors controlling the nuclear localisation of cPLA2-α. Here the nuclear localisation of cPLA2-α in the EA.hy.926 human endothelial cell line was investigated. Nuclear localisation was dependent on proliferation, with subconfluent cells containing higher levels of nuclear cPLA2-α than contact-inhibited confluent or serum-starved cells. The broad-range protein kinase inhibitor staurosporine caused a decrease in the nuclear level of cPLA2-α, whereas the protein phosphatase inhibitor okadaic acid increased the level of nuclear cPLA2-α. Using inhibitors for specific mitogen-activated protein (MAP) kinases, both p42/44MAPK and p38MAPK were shown to be important in modulating nuclear localisation. Finally, inhibition of nuclear import and export using Agaricus bisporus lectin and leptomycin B, respectively, demonstrated that cPLA2-αcontains functional nuclear localisation and export signals. Thus we have identified a novel mode of regulation of cPLA2-α. This,together with the increasing body of evidence supporting the role of nuclear lipid second messengers in gene expression and proliferation, may have important implications for controlling the growth of endothelial cells in angiogenesis and tumour progression.
Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge
Authors: DesMarais Vera , Ichetovkin Ilia , Condeelis John , Hitchcock-DeGregori Sarah E.
Published: November 2002

Rapid polymerization of a network of short, branched actin filaments takes place at the leading edge of migrating cells, a compartment enriched in activators of actin polymerization such as the Arp2/3 complex and cofilin. Actin filaments elsewhere in the cell are long and unbranched. Results reported here show that the presence or absence of tropomyosin in these different actin-containing regions helps establish functionally distinct actin-containing compartments in the cell.

Tropomyosin, an inhibitor of the Arp2/3 complex and cofilin function, was localized in relation to actin filaments, the Arp2/3 complex, and free barbed ends of actin filaments in MTLn3 cells, which rapidly extend flat lamellipodia following EGF stimulation. All tropomyosin isoforms examined using indirect immunofluorescence were relatively absent from the dynamic leading edge compartment, but did colocalize with actin structures deeper in the lamellipodium and in stress fibers. An in vitro light microscopy assay revealed that tropomyosin protects actin filaments from cofilin severing. The results suggest that tropomyosin-free actin filaments under the membrane can participate in rapid, dynamic processes that depend on interactions between the activities of the Arp2/3 complex and ADF/cofilin that tropomyosin inhibits elsewhere in the cell.
Caspase-dependent initiation of apoptosis and necrosis by the Fas receptor in lymphoid cells: onset of necrosis is associated with delayed ceramide increase
Authors: Hetz Claudio A. , Hunn Martin , Rojas Patricio , Torres Vicente , Leyton Lisette , Quest Andrew F. G.
Published: November 2002

Engagement of the Fas receptor promotes apoptosis by activation of caspases. In addition, alterations in plasma membrane lipid orientation and intracellular ceramide levels are often observed. In A20 B-lymphoma cells,FasL-induced cell death and phosphatidylserine (PS) externalization were completely prevented by the generic caspase inhibitor z-VAD-fmk. By contrast,the caspase-3 inhibitor Ac-DEVD-cho only partially restored cell viability and had no effect on surface exposure of PS. Flow cytometric analysis after FasL treatment identified two populations of dead cells. In one, death was dependent on caspase-3 and paralleled by DNA fragmentation and cell shrinkage. In the second, death occurred in the absence of caspase-3 activity and apoptotic features but was also blocked by zVAD-fmk. By morphological criteria these were identified as apoptotic and necrotic cells, respectively. Using fluorescent substrates, caspase-3 activity was detected only in the apoptotic cell population, whereas caspase-8 activity was detected in both. Both forms of caspase-8-dependent cell death were also detected downstream of Fas in Jurkat T-cells, where Fas-dependent PS externalization and delayed ceramide production, which is similar to results shown here in A20 cells, have been reported. However, for Raji B-cells, lacking lipid scrambling and ceramide production in response to Fas activation, only apoptosis was detected. Short-chain C2- or C6-ceramides, but not the respective inactive dihydro compounds or treatment with bacterial sphingomyelinase, induced predominantly necrotic rather than apoptotic cell death in A20 B-, Raji B- and Jurkat T-cells. Thus, delayed elevation of ceramide is proposed to promote necrosis in those Fas-stimulated cells where caspase-8 activation was insufficient to trigger caspase-3-dependent apoptosis.
The `Spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families
Authors: Röper Katja , Gregory Stephen L. , Brown Nicholas H.
Published: November 2002

Recent studies have characterised a family of giant cytoskeletal crosslinkers encoded by the short stop gene in Drosophila and the dystonin/BPAG1 and MACF1 genes in mammals. We refer to the products of these genes as spectraplakins to highlight the fact that they share features with both the spectrin and plakin superfamilies. These genes produce a variety of large proteins, up to almost 9000 residues long, which can potentially extend 0.4 μm across a cell. Spectraplakins can interact with all three elements of the cytoskeleton: actin, microtubules and intermediate filaments. The analysis of mutant phenotypes in BPAG1 in mouse and short stop in Drosophila demonstrates that spectraplakins have diverse roles. These include linking the plasma membrane and the cytoskeleton, linking together different elements of the cytoskeleton and organising membrane domains.
Hedgehogs tryst with the cell cycle
Authors: Roy Sudipto , Ingham Philip W.
Published: November 2002

Hedgehog proteins play an essential role during pattern formation in animal development and, increasingly, much of our appreciation of their modes of action is emanating from studies of their signalling mechanisms at the cellular level. Recent work has provided insights into how Hedgehog controls the cell cycle in a variety of circumstances. The data suggest that this influence may be direct and operates through interaction of the signalling pathway with cell cycle regulators at multiple points within the cell cycle. These new findings have profound implications in the context of clinical conditions — especially cancers — that arise from de-regulated cell proliferation in response to aberrant Hedgehog signalling activity.
Assembly of the PINCH-ILK-CH-ILKBP complex precedes and is essential for localization of each component to cell-matrix adhesion sites
Authors: Zhang Yongjun , Chen Ka , Tu Yizeng , Velyvis Algirdas , Yang Yanwu , Qin Jun , Wu Chuanyue
Published: November 2002

PINCH, integrin-linked kinase (ILK) and calponin homology-containing ILK-binding protein (CH-ILKBP) form a ternary complex that plays crucial roles at cell-extracellular matrix adhesion sites. To understand the mechanism underlying the complex formation and recruitment to cell-adhesion sites we have undertaken a combined structural, mutational and cell biological analysis. Three-dimensional structure-based point mutations identified specific PINCH and ILK sites that mediate the complex formation. Analyses of the binding defective point mutants revealed that the assembly of the PINCH-ILK-CH-ILKBP complex is essential for their localization to cell-extracellular matrix adhesion sites. The formation of the PINCH-ILK-CH-ILKBP complex precedes integrin-mediated cell adhesion and spreading. Furthermore, inhibition of protein kinase C, but not that of actin polymerization, inhibited the PINCH-ILK-CH-ILKBP complex formation, suggesting that the PINCH-ILK-CH-ILKBP complex likely serves as a downstream effector of protein kinase C in the cellular control of focal adhesion assembly. Finally,we provide evidence that the formation of the PINCH-ILK-CH-ILKBP complex,while necessary, is not sufficient for ILK localization to cell-extracellular matrix adhesion sites. These results provide new insights into the molecular mechanism underlying the assembly and regulation of cell-matrix adhesion structures.
Molecular structure of cytoplasmic dynein 2 and its distribution in neuronal and ciliated cells
Authors: Mikami Atsushi , Tynan Sharon H. , Hama Taro , Luby-Phelps Katherine , Saito Tetsuichiro , Crandall James E. , Besharse Joseph C. , Vallee Richard B.
Published: November 2002

Cytoplasmic dynein is involved in a wide variety of cellular functions. In addition to the initially characterized form (MAP 1C/dynein 1), a second form of cytoplasmic dynein (dynein 2) has been identified and implicated in intraflagellar transport (IFT) in lower eukaryotes and in Golgi organization in vertebrates. In the current study, the primary structure of the full-length dynein 2 heavy chain (HC) was determined from cDNA sequence. The dynein 1 and dynein 2 sequences were similar within the motor region, and around the light intermediate chain (LIC)-binding site within the N-terminal stem region. The dynein 2 HC co-immunoprecipitated with LIC3, a homologue of dynein 1 LICs. Dynein 2 mRNA was abundant in the ependymal layer of the neural tube and in the olfactory epithelium. Antibodies to dynein 2 HC, LIC3 and a component of IFT particles strongly stained the ependymal layer lining the lateral ventricles. Both dynein 2 HC and LIC3 staining was also observed associated with connecting cilia in the retina and within primary cilia of non-neuronal cultured cells. These data support a specific role for dynein 2 in the generation and maintenance of cilia.
SARA, a FYVE domain protein, affects Rab5-mediated endocytosis
Authors: Hu Yang , Chuang Jen-Zen , Xu Kai , McGraw Timothy G. , Sung Ching-Hwa
Published: November 2002

Rab5, a member of the small GTPase family of proteins, is primarily localized on early endosomes and has been proposed to participate in the regulation of early endosome trafficking. It has been reported that phosphatidylinositol 3-kinases and FYVE domain proteins, such as EEA1, can be recruited onto early endosomes and act as Rab5 effectors. SARA (Smad anchor for receptor activation), also a FYVE domain protein, was initially isolated as a participant in signal transduction from the transforming growth factorβ receptor. Overexpressed SARA has been found on EEA1-positive early endosomes. In this report, we show that endogenous SARA is present on early endosomes and overexpression of SARA causes endosomal enlargement. Functionally, SARA overexpression significantly delays the recycling of transferrin. The transferrin receptor distributed on the cell surfaces was also greatly reduced in cells overexpressing SARA. However, the internalization rate of transferrin is not affected by SARA overexpression. The morphological and functional alterations caused by SARA overexpression resemble those caused by overexpression of Rab5:GTP mutant Rab5Q79L. Finally,all SARA-mediated phenotypic changes can be counteracted by overexpression Rab5:GDP mutant Rab5S34N. These results collectively suggested that SARA plays an important functional role downstream of Rab5-regulated endosomal trafficking.
An IP3-activated Ca2+ channel regulates fungal tip growth
Authors: Silverman-Gavrila Lorelei B. , Lew Roger R.
Published: November 2002

Hyphal extension in fungi requires a tip-high Ca2+ gradient,which is generated and maintained internally by inositol (1,4,5)-trisphosphate(IP3)-induced Ca2+ release from tip-localized vesicles and subapical Ca2+ sequestration. Using the planar bilayer method we demonstrated the presence of two types of IP3-activated Ca2+ channels in Neurospora crassa membranes with different conductances: one low (13 picosiemens), the other high (77 picosiemens). On sucrose density gradients the low conductance channel co-localized with endoplasmic reticulum and plasma membrane, and the high conductance channel co-localized with vacuolar membranes. We correlated the effect of inhibitors on channel activity with their effect on hyphal growth and Ca2+ gradients. The inhibitor of IP3-induced Ca2+ release, 2-aminoethoxidiphenylborate (2-APB), inhibits both channels, while heparin, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate,hydrochloride (TMB-8) and dantrolene inhibit only the large conductance channel. Because 2-APB inhibits hyphal growth and dissipates the tip-high cytosolic [Ca2+] gradient, whereas heparin microinjection, TMB-8 and dantrolene treatments do not affect growth, we suggest that the small conductance channel generates the obligatory tip-high Ca2+ gradient during hyphal growth. Since IP3 production must be catalyzed by tip-localized phospholipase C, we show that a number of phospholipase C inhibitors [neomycin,1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) (but not the inactive pyrrolidine U-73343),3-nitrocoumarin] inhibit hyphal growth and affect, similarly to 2-APB, the location of vesicular Ca2+ imaged by chlortetracycline staining.
Sbh1p, a subunit of the Sec61 translocon, interacts with the chaperone calnexin in the yeast Yarrowia lipolytica
Authors: Boisramé Anita , Chasles Marion , Babour Anna , Beckerich Jean-Marie , Gaillardin Claude
Published: November 2002

The core component of the translocation apparatus, Sec61p or α, was previously cloned in Yarrowia lipolytica. Using anti-Sec61p antibodies, we showed that most of the translocation sites are devoted to co-translational translocation in this yeast, which is similar to the situation in mammalian cells but in contrast to the situation in Saccharomyces cerevisiae, where post-translational translocation is predominant. In order to characterize further the minimal translocation apparatus in Y. lipolytica, the β Sec61 complex subunit, Sbh1p,was cloned by functional complementation of a Δsbh1,Δ sbh2 S. cerevisiae mutant. The secretion of the reporter protein is not impaired in the Y. lipolytica sbh1 inactivated strain. We screened the Y. lipolytica two-hybrid library to look for partners of this translocon component. The ER-membrane chaperone protein, calnexin, was identified as an interacting protein. By a co-immunoprecipitation approach, we confirmed this association in Yarrowia and then showed that the S. cerevisiae Sbh2p protein was a functional homologue of YlSbh1p. The interaction of Sbh1p with calnexin was shown to occur between the lumenal domain of both proteins. These results suggest that theβ subunit of the Sec61 translocon may relay folding of nascent proteins to their translocation.
The Ena/VASP enigma
Authors: Krause Matthias , Bear James E. , Loureiro Joseph J. , Gertler Frank B.
Published: November 2002

Ena/VASP proteins are actin-binding proteins that localize to actin stress fibres, the tips of filopodia and the lamellipodial leading edge. In the past few years, a number of seemingly conflicting studies have confused the Ena/VASP field, pointing to roles for these proteins in both promotion and inhibition of actin-dependent processes. Recent discoveries resolve these contradictions and suggest a novel mechanism of Ena/VASP function, in which the proteins function as `anti-capping' proteins that antagonize capping proteins at the barbed end of actin filaments.
Identification of an evolutionarily conserved superfamily of DOCK180-related proteins with guanine nucleotide exchange activity
Authors: Côté Jean-François , Vuori Kristiina
Published: November 2002

Mammalian DOCK180 protein and its orthologues Myoblast City (MBC) and CED-5 in Drosophila and Caenorhabditis elegans, respectively,function as critical regulators of the small GTPase Rac during several fundamentally important biological processes, such as cell motility and phagocytosis. The mechanism by which DOCK180 and its orthologues regulate Rac has remained elusive. We report here the identification of a domain within DOCK180 named DHR-2 (Dock Homology Region-2)that specifically binds to nucleotide-free Rac and activates Rac in vitro. Our studies further demonstrate that the DHR-2 domain is both necessary and sufficient for DOCK180-mediated Rac activation in vivo. Importantly, we have identified several novel homologues of DOCK180 that possess this domain and found that many of them directly bind to and exchange GDP for GTP both in vitro and in vivo on either Rac or another Rho-family member, Cdc42. Our studies therefore identify a novel protein domain that interacts with and activates GTPases and suggest the presence of an evolutionarily conserved DOCK180-related superfamily of exchange factors.
Large‐eddy simulation of the diurnal cycle of shallow cumulus convection over land
Authors: Brown A. R. , Cederwall R. T. , Chlond A. , Duynkerke P. G. , Golaz J.‐C. , Khairoutdinov M. , Lewellen D. C. , Lock A. P. , MacVean M. K. , Moeng C.‐H. , Neggers R. A. J. , Siebesma A. P. , Stevens B.
Published: November 2002

Large‐eddy simulations of the development of shallow cumulus convection over land are presented. Many characteristics of the cumulus layer previously found in simulations of quasi‐steady convection over the sea are found to be reproduced in this more strongly forced, unsteady case. Furthermore, the results are shown to be encouragingly robust, with similar results obtained with eight independent models, and also across a range of numerical resolutions. The datasets produced are already being used in the development and evaluation of parametrizations used in numerical weather‐prediction and climate models. © Royal Meteorological Society, 2002. A. R. Brown's, A. P. Lock's and M. K. MacVean's contributions are Crown copyright.
Equatorial components of global atmospheric angular momentum: Covariance functions
Authors: Egger Joseph , Hoinka Klaus‐Peter
Published: November 2002

Contributions to a dynamic climatology of the equatorial componentsMxandMyof global angular momentum are presented on the basis of ECMWF re‐analysis data. The covariance functions ofMx,Myand the related torques are evaluated using the observations. It is shown that the torques due to the equatorial bulge, and the torques due to the gradient of the geopotential, balance. Prognostic equations forMxandMyare derived on the basis of this result as are the equations for the covariance functions. All terms in these equations are discussed on the basis of the data. The autocovariance functions of both equatorial components decay rapidly for lags less than 5 days, and rather slowly for larger lags. The cross‐covariance function ofMxandMyimplies westward motion of the angular‐momentum vector. Atmospheric tides leave a clear imprint on the wind terms.

Mountain and friction torques have virtually no influence on the antisymmetric change of the autocovariance functions of the angular momenta with lag. This change is almost completely induced by the wind terms which represent the combined impact of the earth's rotation and its shape. The torques are, however, important in the symmetric part of the autocovariance equations where they balance the contributions by the wind terms. Moreover, the cross‐covariance equation forMxandMyindicates that both torques retard the westward motion of the angular‐momentum vector. Surprisingly, the correlation of friction torques and the related wind terms is small and positive while that with the mass terms is negative and large. The contributions by the wind terms in the covariance equations for mountain torques and angular momentum cancel almost exactly the orographic terms. The orographic terms are also of key importance in the covariance equations forMx,Myand the wind terms. The data quality is sufficient for the analysis of the auto‐ and cross‐covariance equations ofMxandMy, but not for some of the cross‐covariance equations ofMx,Myand the torques. Copyright © 2002 Royal Meteorological Society
Upper‐ and lower‐troposphere coupling processes involved in the FASTEX IOP16 frontal cyclone
Authors: Bouniol D. , Lemaître Y. , Protat A.
Published: November 2002

This paper investigates processes involved in the development of the secondary low sampled on 17 February 1997 during the field phase of the Fronts and Atlantic Storm‐Track EXperiment (FASTEX). This low is a fast‐ moving frontal wave that emerged from the trailing cold front of a parent low situated over Greenland.

The dynamics of this cyclone and its environment are described using the European Centre for Medium‐ Range Weather Forecasts analysis corresponding to Intensive Observing Period 16 of FASTEX. The ageostrophic circulation is retrieved and analysed in the upper and in the lower levels. In the upper levels it is found that the curvature of the upper‐level jet plays a crucial role in suppressing or amplifying the vertical motions. In the lower levels, in the region where the cloud head emerges, the vertical motion is scrutinized in the light of forcings which are acting. It is found that the vertical motions are the response to a geostrophic forcing reinforced by diabatic processes. The conjunction of vertical motions in the lower levels associated with vertical motions in the upper levels leads, in the developing period, to a coupling process that seems to be (following theoretical calculations) at the origin of the subsequent development of the cyclone.

The instability processes involved in the development of the studied secondary cyclone are also investigated. The atmosphere appears neutral for upright convection while the region of the coupling corresponds to a region which is potentially unstable with respect to slantwise convection. The tendency of the atmosphere is to relax this instability. The vertical motions of the coupling processes are then reinforced by the relaxation of slantwise instability. Copyright © 2002 Royal Meteorological Society
The effect of El Niño on intraseasonal Kelvin waves
Authors: Benestad Rasmus E. , Sutton Rowan T. , Anderson David L. T.
Published: November 2002

Intraseasonal Kelvin waves represent an important aspect of tropical ocean dynamics. There has been considerable speculation about whether intraseasonal Kelvin waves generated by intraseasonal wind events can trigger El Niño Southern Oscillation (ENSO) events, and therefore play a potentially important role in the prediction of El Niño. This study considers the different but related issue of how ENSO conditions influence intraseasonal Kelvin waves. Understanding this issue is an important part of clarifying the total role of scale interactions in the ENSO cycle.

Numerical experiments carried out with an ocean general circulation model forced with European Centre for Medium‐Range Weather Forecasts re‐analysis surface fluxes show that both the west‐to‐east energy transmission and speed of propagation of intraseasonal Kelvin waves are strongly influenced by the phase of ENSO. The waves are much more strongly damped during La Niña conditions than during El Niño, primarily as a result of increased viscous dissipation associated with enhanced vertical shear. We suggest that this mechanism represents a significant source of nonlinearity between El Niño and La Niña states. It may influence the onset, the lifetime and the magnitude of warm or cold anomalies. Copyright © 2002 Royal Meteorological Society
High‐resolution limited‐area ensemble predictions based on low‐resolution targeted singular vectors
Authors: Frogner Inger‐Lise , Iversen Trond
Published: November 2002

The operational limited‐area model, HIRLAM, at the Norwegian Meteorological Institute is used at 0.25° latitude/longitude resolution for ensemble weather prediction over Northern Europe and adjacent parts of the North Atlantic Ocean; this system is called LAMEPS. Initial and lateral boundary perturbations are taken from coarse‐resolution European Centre for Medium‐Range Weather Forecasts global ensemble members based on targeted singular vectors (TEPS). Five winter and five summer cases in 1997 consisting of 20 ensemble members plus one control forecast are integrated. Two sets of ensembles are generated, one for which both initial and lateral boundary conditions are perturbed, and another with only the initial fields perturbed. The LAMEPS results are compared to those of TEPS using the following measures: r.m.s. ensemble spread of 500 hPa geopotential height; r.m.s. ensemble spread of mean‐sea‐level pressure; Brier Skill Scores (BSS); Relative Operating Characteristic (ROC) curves; and cost/loss analyses. For forecasts longer than 12 hours, all measures show that perturbing the boundary fields is crucial for the performance of LAMEPS. For the winter cases TEPS has slightly larger ensemble spread than LAMEPS, but this is reversed for the summer cases. Results from BSS, ROC and cost/loss analyses show that LAMEPS performed considerably better than TEPS for precipitation, a result that is promising for forecasting extreme precipitation amounts. We believe this result to be linked to the high predictability of mesoscale flows controlled by complex topography. For two‐metre temperature, however, TEPS frequently performed better than LAMEPS. Copyright © 2002 Royal Meteorological Society
Radiative upper‐boundary conditions for a non‐hydrostatic atmosphere
Authors: Purser R. James , Kar Sajal K.
Published: November 2002

A non‐hydrostatic compressible model supports vertically propagating acoustic modes in addition to the modes of meteorological significance, such as the quasi‐geostrophically balanced, and gravity modes. The acoustic modes are stimulated inadvertently, either by initial conditions incompatibly balanced for the model discretization, or by physical processes injecting abrupt impulses of heat or motion into the model during the integration. A standard method for removing unwanted acoustic energy is through the inclusion in the model of three‐dimensional divergence damping. However, an alternative approach is to employ an upper‐boundary condition designed to radiate acoustic waves, at least partially, as they impinge on the model top. This note explores this latter option through the use of an upper‐boundary condition that incorporates a time filter to facilitate the selective absorption of both gravity and acoustic waves at the top. Copyright © 2002 Royal Meteorological Society
Relationship between thermodynamical parameters of activated complexes and initial reactants in the reaction of peroxides with amines
Authors: Turovskiy A. A. , Turovskiy N. A. , Kopylets V. I. , Zagladko E. A. , Zaikov G. A.
Published: November 2002

The linear dependence between heats of formation and entropies of initial reactants on the one hand and the corresponding thermodynamical parameters of activated complexes on the other hand was obtained for the reaction of peroxides with amines. This relationship allows one to estimate such parameters for other similar reactions of amines with peroxides. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 641–642, 2003
Morphology and thermal properties of PPS/ABS blend systems
Authors: Nam Jae‐Do , Kim Junmyung , Lee Sungjae , Lee Youngkwan , Park Chansuk
Published: November 2002

In this study the PPS/ABS blend system was investigated in order to collectively identify the relationship among blend morphology, chemical compatibilization, and thermal property. The ABS resin was chemically modified by the incorporation of maleic anhydride through reactive extrusion for enhanced compatibilization, and ABS/PPS and the modified ABS/PPS blends were prepared by a twin‐screw extruder. The effect of chemical modification of ABS on the morphological, mechanical, and thermal properties of the resulting blend was examined. A strong chemical interaction between PPS and MABS was observed by optical microscopy, scanning electron microscopy, and FTIR. The PPS/MABS blend showed a single glass‐transition temperature in dynamic mechanical analysis, demonstrating pseudo‐homogeneous phase morphology induced by chemical compatibilization. The PPS/MABS blend also exhibited an enhanced thermal stability and heat distortion temperature compared with the PPS/ABS blend. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 661–665, 2003
Relationship between thickness of polymer films and their oxidation on copper substrate
Authors: Lin D.G. , Vorobieva E.V.
Published: November 2002

This research was undertaken to understand how the thickness of polyethylene films oxidized on a copper substrate influences the accumulation of carbonyl groups (measured by an IR‐spectroscopy technique) and of metal from the substrate (determined by polarography analysis). It was found that the whole polymer became inhibited by the time the copper stopped transferring into the specimen. Plots of copper concentration versus film thickness have two thickness sections: section I is found between 0 and 70 μm and section II between 80 and 170 μm. Between these two sections the metal concentration varies drastically. This situation can be explained by two schemes by which PE changes to inhibited condition. According to Scheme I (for section I, short oxidation time) this change has only one step: the inhibited layer gradually becomes thicker beginning from the interface and moving toward the outer surface. The second scheme (for section II) shows that the polymer becomes inhibited in two steps. It is typical of thicker films. In this case the oxidation process shifts and localizes in the outer surface because of longer treatment. As a result, transfer of metal and formation of an inhibited layer are interrupted for some time. The metal accumulation in the film only resumes when low‐molecular‐weight products of thermooxidative degradation—formed in the specimen outer surface—enter the region of adhesional contact. A so‐called second transfer stage for metal is realized during which the whole polymer becomes inhibited. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 671–675, 2003
Hydrogels from glycidyl derivatives of poly(vinyl alcohol)
Authors: Ruiz J. , Mantecón A. , Cádiz V.
Published: November 2002

Poly(vinyl alcohol) (PVA) was modified with phthalic anhydride to obtain half esters with carboxylic acid groups, which made the reaction with epichlorohydrin easier. The oxirane ring underwent a further crosslinking that led to crosslinked polymers with polar groups capable of interacting strongly with water and therefore with properties of hydrogels. The curing kinetics of the crosslinking were studied by differential scanning calorimetry, and the dependence of the activation energy on conversion degree was studied by isoconversional kinetic analysis. Water absorption was determined gravimetrically as a function of time at room temperature. The swelling behavior of these hydrogels was related to the degree of crosslinking. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 693–698, 2003
Chemical modification of molten polyethylene by thermal decomposition of peroxyesters
Authors: Navarre Sébastien , Saule Myriam , Maillard Bernard
Published: November 2002

The functionalization of polyethylene was realized by the thermal decomposition of peroxyesters in molten polymer in the absence of any other additive. Ester and acid functions were introduced onto the polymer operating with various peroxyesters. This grafting resulted from the combination of a polymer radical, generated in the abstraction of a hydrogen from polyethylene by an alkoxy radical, with an acyloxy or carbon‐centered radical arising from the perester. A complete methodology was set up to identify and to titrate the functions present onto the polyethylene and to determine the extent of crosslinked polymer. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 699–707, 2003
Frequent activation of AKT2 kinase in human pancreatic carcinomas
Authors: Altomare Deborah A. , Tanno Satoshi , De Rienzo Assunta , Klein‐Szanto Andres J. , Tanno Sachie , Skele Kristine L. , Hoffman John P. , Testa Joseph R.
Published: November 2002

Activation of AKT/protein kinase B promotes a variety of biological activities important in tumorigenesis, such as cell survival and cell cycle progression. We previously demonstrated amplification and overexpression of the AKT2 gene in a subset of human pancreatic carcinomas. In this investigation, we assessed AKT2 catalytic activity in 50 frozen pancreatic tissues (37 carcinomas, four benign tumors, and nine normal pancreata) by in vitro kinase assay. Twelve of 37 (32%) pancreatic carcinomas showed markedly elevated levels of AKT2 activity compared to normal pancreata and benign pancreatic tumors. To delineate mechanisms contributing to AKT2 activation in malignant pancreatic tumors, we examined the status of upstream components of the phosphatidylinositol 3‐kinase (PI3K)/AKT pathway. Western blot analysis revealed loss of PTEN protein expression in two of the 12 pancreatic carcinomas with activated AKT2. In vitro PI3K assays demonstrated high levels of PI3K activity in seven carcinoma specimens that showed AKT2 activation. Immunohistochemical staining confirmed high levels of phosphorylated (active) AKT in malignant pancreatic tumors compared to normal pancreata. Overall, these data suggest that upstream perturbations of the PI3K/AKT pathway contribute to frequent activation of AKT2 in pancreatic cancer, which may contribute to the pathogenesis of this highly aggressive form of human malignancy. J. Cell. Biochem. 87: 470–476, 2002. © 2002 Wiley‐Liss, Inc.
Structural, antigenic and evolutionary analyses of immunoglobulins and T cell receptors
Authors: Marchalonis John J. , Jensen Ingvill , Schluter Samuel F.
Published: November 2002

We have had the pleasure of collaborating with Allen Edmundson for the past 15 years on the structure, binding properties and evolution of immunoglobulins and T cell receptors. Among the most significant contributions of our joint efforts were: (1) the predictive use of structural features of immunoglobulin domains to model the three‐dimensional structures of the immunoglobulin domains of human T‐cell receptor α and β chains as well as shark light chains and VH domains; (2) the finding that normal humans and other vertebrates express autoantibodies against combining site epitopes of their own T cell receptors; (3) the mapping of the peptide autoepitopes recognized in health, autoimmunity and retroviral infection; and (4) the determination that epitope recognition promiscuity is a characteristic property of the combining sites of IgM immunoglobulins ranging from those of sharks to those of humans. We briefly review the salient findings and status of these studies and indicate the future directions that we will pursue in their continuation. Copyright © 2002 John Wiley & Sons, Ltd.
Inhibition of HIV protease by monoclonal antibodies
Authors: Rezacova P. , Brynda J. , Fabry M. , Horejsi M. , Stouracova R. , Lescar J. , Chitarra V. , Riottot M. M. , Sedlacek J. , Bentley G. A.
Published: November 2002

The protease of HIV plays a critical role in the maturation of the infectious particles of the virus. The enzyme has therefore been extensively studied with the objective of developing therapeutics that inhibit viral proliferation. We have produced monoclonal antibodies specific for the HIV‐1 protease, and selected those that inhibit enzyme function for use as probes to study the enzyme's activity and as an eventual aid for the development of potential inhibitors targeted to regions other than the active site. We have characterized two such mAbs, F11.2.32 and 1696, which have inhibition constants in the low nanomolar range and which recognize epitopes from different regions of the protease. The crystal structures of the two antibodies, both in the free state as well as complexes with peptide fragments corresponding to their respective epitopes, have been solved. The structural analyses, taken together with other functional data on the antibodies, suggest mechanisms of protease inhibition by these antibodies. Copyright © 2002 John Wiley & Sons, Ltd.
Combinatorial methods for discovery of peptide ligands which bind to antibody‐like molecules
Authors: Tribbick Gordon , Rodda Stuart
Published: November 2002

In the 1980s, new methods of parallel peptide synthesis were used to make large libraries of peptides, which were then screened for binding to Bence–Jones dimers. Subsequent X‐ray crystallography of the Bence–Jones proteins, which had been infiltrated with peptide ligand, was used to determine the structural correlate of the binding data. The mode of binding was found to be not predictable and insight was gained into the forces determining how the so‐called mimotopes interacted with binding sites. Copyright © 2002 John Wiley & Sons, Ltd.
Bifluorophoric molecules as fluorescent beacons for antibody–antigen binding
Authors: Wei Ai‐Ping , Herron James N.
Published: November 2002

The quenching of fluorescence (up to 98%) by anti‐fluorescein antibodies is well documented in the literature. Here we report a system where, instead of quenching, bifluorophoric molecules are designed to increase in fluorescence upon binding by an anti‐fluorescein antibody. Bifluorophoric molecules are made of fluorescein (F) linked to tetramethylrhodamine (T) via varying numbers of methylene units, denoted as F‐(CH2)
n‐T. These F‐(CH2)
n‐T conjugates are almost nonfluorescent when free in solution due to intramolecular dimerization and stacking. Upon binding to an anti‐fluorescein antibody, however, up to 110‐fold increase in fluorescence was observed from the rhodamine moiety. This increase is believed to result from intramolecular dimer dissociation that dequenches the rhodamine fluorescence. Fluorescein fluorescence, on the other hand, remains quenched due to binding and intramolecular resonance energy transfer. Moreover, the excitation wavelength was at the absorption maxima of fluorescein, giving a Stoke's shift of about 90 nm. This system couples directly molecular recognition with a concurrent increase in fluorescence emission, obviating wash and incubation steps required by most assays. It is an important molecular reporter system for developing homogeneous assays. Copyright © 2002 John Wiley & Sons, Ltd.
Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD
Authors: Asvadi Parisa , Fletcher Anne , Raison Robert L.
Published: November 2002

In an attempt to generate recombinant anti‐D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti‐D antibody secreted by the lymphoblastoid cell line BTSN4. A single‐chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG‐CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity‐isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain‐treated D positive erythrocytes in the absence of a cross‐linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non‐covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non‐covalently associated multimeric forms of the antibody fragment. Copyright © 2002 John Wiley & Sons, Ltd.