bluuebunny/crossref_metadata_2025 huggingface dataset:
E-Cadherin Suppresses Cellular Transformation by Inhibiting β-Catenin Signaling in an Adhesion-Independent Manner
Authors: Gottardi Cara J. , Wong Ellen , Gumbiner Barry M.
Published: July 2002

E-cadherin is a tumor suppressor protein with a well-established role in cell–cell adhesion. Adhesion could contribute to tumor suppression either by physically joining cells or by facilitating other juxtacrine signaling events. Alternatively, E-cadherin tumor suppressor activity could result from binding and antagonizing the nuclear signaling function of β-catenin, a known proto-oncogene. To distinguish between an adhesion- versus a β-catenin signaling–dependent mechanism, chimeric cadherin constructs were expressed in the SW480 colorectal tumor cell line. Expression of wild-type E-cadherin significantly inhibits the growth of this cell line. Growth inhibitory activity is retained by all constructs that have the β-catenin binding region of the cytoplasmic domain but not by E-cadherin constructs that exhibit adhesive activity, but lack the β-catenin binding region. This growth suppression correlates with a reduction in β-catenin/T cell factor (TCF) reporter gene activity. Importantly, direct inhibition of β-catenin/TCF signaling inhibits the growth of SW480 cells, and the growth inhibitory activity of E-cadherin is rescued by constitutively activated forms of TCF. Thus, the growth suppressor activity of E-cadherin is adhesion independent and results from an inhibition of the β-catenin/TCF signaling pathway, suggesting that loss of E-cadherin expression can contribute to upregulation of this pathway in human cancers. E-cadherin–mediated growth suppression was not accompanied by overall depletion of β-catenin from the cytosol and nucleus. This appears to be due to the existence of a large pool of cytosolic β-catenin in SW480 cells that is refractory to both cadherin binding and TCF binding. Thus, a small pool of β-catenin that can bind TCF (i.e., the transcriptionally active pool) can be selectively depleted by E-cadherin expression. The existence of functionally distinct pools of cytosolic β-catenin suggests that there are mechanisms to regulate β-catenin signaling in addition to controlling its level of accumulation.
Golgi clusters and vesicles mediate mitotic inheritance independently of the endoplasmic reticulum
Authors: Jokitalo Eija , Cabrera-Poch Noemi , Warren Graham , Shima David T.
Published: July 2002

We have examined the fate of Golgi membranes during mitotic inheritance in animal cells using four-dimensional fluorescence microscopy, serial section reconstruction of electron micrographs, and peroxidase cytochemistry to track the fate of a Golgi enzyme fused to horseradish peroxidase. All three approaches show that partitioning of Golgi membranes is mediated by Golgi clusters that persist throughout mitosis, together with shed vesicles that are often found associated with spindle microtubules. We have been unable to find evidence that Golgi membranes fuse during the later phases of mitosis with the endoplasmic reticulum (ER) as a strategy for Golgi partitioning (Zaal, K.J., C.L. Smith, R.S. Polishchuk, N. Altan, N.B. Cole, J. Ellenberg, K. Hirschberg, J.F. Presley, T.H. Roberts, E. Siggia, et al. 1999. Cell. 99:589–601) and suggest that these results, in part, are the consequence of slow or abortive folding of GFP–Golgi chimeras in the ER. Furthermore, we show that accurate partitioning is accomplished early in mitosis, by a process of cytoplasmic redistribution of Golgi fragments and vesicles yielding a balance of Golgi membranes on either side of the metaphase plate before cell division.
The importin-β binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro
Authors: Huber Jochen , Dickmanns Achim , Lührmann Reinhard
Published: July 2002

The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-β binding (IBB) domain and a COOH-terminal m3G-cap–binding region that bears no structural similarity to known import adaptors like importin-α (impα). Here, we show that recombinant snurportin1 and importin-β (impβ) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impα–dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1–impβ, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impβ and a snurportin1 mutant containing an impα IBB domain (IBBimpα), nuclear U1 snRNP import was Ran dependent. Furthermore, β-galactosidase (βGal) containing a snurportin1 IBB domain, but not IBBimpα-βGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impβ with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions.
Dynamics of human DNA topoisomerases IIα and IIβ in living cells
Authors: Christensen Morten O. , Larsen Morten K. , Barthelmes Hans Ullrich , Hock Robert , Andersen Claus L. , Kjeldsen Eigil , Knudsen Birgitta R. , Westergaard Ole , Boege Fritz , Mielke Christian
Published: July 2002

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (α and β) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIα and IIβ behaved similarly in interphase but differently in mitosis, where only topo IIα was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIα was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.
Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes
Authors: Peng Renwang , Gallwitz Dieter
Published: July 2002

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.
Survival of Mature CD4 T Lymphocytes Is Dependent on Major Histocompatibility Complex Class II–expressing Dendritic Cells
Authors: Brocker Thomas
Published: July 2002

Thymic T cell development is controlled by T cell receptor (TCR)–major histocompatibility complex (MHC) interactions, whereas a further dependence of peripheral mature T cells on TCR–MHC contact has not been described so far. To study this question, CD4 T cell survival was surveyed in mice lacking MHC class II expression and in mice expressing MHC class II exclusively on dendritic cells. Since neither of these mice positively select CD4 T cells in the thymus, they were grafted with MHC class II–positive embryonic thymic tissue, which had been depleted of bone marrow derived cells. Although the thymus grafts in both hosts were repopulated with host origin thymocytes of identical phenotype and numbers, an accumulation of CD4+ T cells in peripheral lymphoid organs could only be observed in mice expressing MHC class II on dendritic cells, but not in mice that were completely MHC class II deficient. As assessed by histology, the accumulating peripheral CD4 T cells were found to be in close contact with MHC class II+ dendritic cells, suggesting that CD4 T cells need peripheral MHC class II expression for survival and that class II+ dendritic cells might play an important role for the longevity of CD4 T cells.
Activated Ras Signals Developmental Progression of Recombinase-activating Gene (RAG)-deficient Pro-B Lymphocytes
Authors: Shaw Albert C. , Swat Wojciech , Ferrini Roger , Davidson Laurie , Alt Frederick W.
Published: July 2002

To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras–RAG). Similar to the effects of an immunoglobulin (Ig) μ heavy chain (HC) transgene, activated Ras caused progression of RAG1–deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of λ5, RAG2, and germline κ locus transcripts. However, these Ras–RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig μ HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cells induces developmental progression by activating both differentiation and survival signals.
Different Mismatch Repair Deficiencies All Have the Same Effects on Somatic Hypermutation
Authors: Kim Nayun , Bozek Grazyna , Lo James C. , Storb Ursula
Published: July 2002

Somatic hypermutation of Ig genes is probably dependent on transcription of the target gene via a mutator factor associated with the RNA polymerase (Storb, U., E.L. Klotz, J. Hackett, Jr., K. Kage, G. Bozek, and T.E. Martin. 1998. J. Exp. Med. 188:689–698). It is also probable that some form of DNA repair is involved in the mutation process. It was shown that the nucleotide excision repair proteins were not required, nor were mismatch repair (MMR) proteins. However, certain changes in mutation patterns and frequency of point mutations were observed in Msh2 (MutS homologue) and Pms2 (MutL homologue) MMR-deficient mice (for review see Kim, N., and U. Storb. 1998. J. Exp. Med. 187:1729–1733). These data were obtained from endogenous immunoglobulin (Ig) genes and were presumably influenced by selection of B cells whose Ig genes had undergone certain mutations. In this study, we have analyzed somatic hypermutation in two MutL types of MMR deficiencies, Pms2 and Mlh1. The mutation target was a nonselectable Ig-κ gene with an artificial insert in the V region. We found that both Pms2- and Mlh1-deficient mice can somatically hypermutate the Ig test gene at approximately twofold reduced frequencies. Furthermore, highly mutated sequences are almost absent. Together with the finding of genome instability in the germinal center B cells, these observations support the conclusion, previously reached for Msh2 mice, that MMR-deficient B cells undergoing somatic hypermutation have a short life span. Pms2- and Mlh1-deficient mice also resemble Msh2-deficient mice with respect to preferential targeting of G and C nucleotides. Thus, it appears that the different MMR proteins do not have unique functions with respect to somatic hypermutation. Several intrinsic characteristics of somatic hypermutation remain unaltered in the MMR-deficient mice: a preference for targeting A over T, a strand bias, mutational hot spots, and hypermutability of the artificial insert are all seen in the unselectable Ig gene. This implies that the MMR proteins are not required for and most likely are not involved in the primary step of introducing the mutations. Instead, they are recruited to repair certain somatic point mutations, presumably soon after these are created.
Caspase Activation Is Required for T Cell Proliferation
Authors: Kennedy Norman J. , Kataoka Takao , Tschopp Jürg , Budd Ralph C.
Published: July 2002

Triggering of Fas (CD95) by its ligand (FasL) rapidly induces cell death via recruitment of the adaptor protein Fas-associated death domain (FADD), resulting in activation of a caspase cascade. It was thus surprising that T lymphocytes deficient in FADD were reported recently to be not only resistant to FasL-mediated apoptosis, but also defective in their proliferative capacity. This finding suggested potentially dual roles of cell growth and death for Fas and possibly other death receptors. We report here that CD3-induced proliferation and interleukin 2 production by human T cells are blocked by inhibitors of caspase activity. This is paralleled by rapid cleavage of caspase-8 after CD3 stimulation, but no detectable processing of caspase-3 during the same interval. The caspase contribution to T cell activation may occur via TCR-mediated upregulation of FasL, as Fas-Fc blocked T cell proliferation, whereas soluble FasL augmented CD3-induced proliferation. These findings extend the role of death receptors to the promotion of T cell growth in a caspase-dependent manner.
Cd47 Ligation Selectively Downregulates Human Interleukin 12 Production
Authors: Armant Myriam , Avice Marie-Noëlle , Hermann Patrice , Rubio Manuel , Kiniwa Mamoru , Delespesse Guy , Sarfati Marika
Published: July 2002

Interleukin (IL)-12 plays a key role not only in protective innate and adaptive T helper cell type 1 (Th1) responses but also in chronic inflammatory diseases. We report here that engagement of CD47 by either monoclonal antibody, its natural ligand thrombospondin (TSP), or 4N1K (a peptide of the COOH-terminal domain of TSP selectively binding CD47) inhibits IL-12 release by monocytes. The suppression occurred after T cell–dependent or –independent stimulation of monocytes and was selective for IL-12 inasmuch as the production of tumor necrosis factor (TNF)-α, IL-1, IL-6, and granulocyte/macrophage colony-stimulating factor was not inhibited. CD47 ligation did not alter transforming growth factor (TGF)-β and IL-10 production, and the suppressive effect on IL-12 was not due to autocrine secretion of TGF-β or IL-10. The IL-12 inhibition was not mediated by Fcγ receptor ligation, did not require extracellular Ca2+ influx, but was reversed by two phosphoinositide 3-kinase inhibitors (wortmannin and Ly294002). Thus, engagement of CD47 on monocytes by TSP, which transiently accumulates at the inflammatory site, is a novel and unexplored pathway to selectively downregulate IL-12 response. The pathway may be relevant in limiting the duration and intensity of the inflammatory response, and in developing novel therapeutic strategies for Th1-mediated diseases.
Developmental Switches in Chemokine Response Profiles during B Cell Differentiation and Maturation
Authors: Bowman Edward P. , Campbell James J. , Soler Dulce , Dong Zengjun , Manlongat Natasha , Picarella Dominic , Hardy Richard R. , Butcher Eugene C.
Published: July 2002

Developing B cells undergo dramatic changes in their responses to chemoattractant cytokines (chemokines) and in expression of chemokine receptors. Bone marrow pre–pro-B cells (AA4.1+/natural killer 1.1− Fraction A cells) and cells capable of generating pro-B colonies in the presence of interleukin 7 and flt3 ligand migrate to thymus-expressed chemokine (TECK), a response lost in later stages of B cell development. B cell–attracting chemokine 1 (BCA-1) responses correlate with CXC chemokine receptor (CXCR)5 expression, are first displayed by a pro-B cell subset, are lost in pre-B cells, and then are regained just before and after egress from the marrow. All peripheral B cell subsets, including follicular and germinal center as well as marginal zone and peritoneal B1 B cells, respond to BCA-1, implying that responsiveness to this follicular chemokine is not sufficient to predict follicle localization. Responses to the CC chemokine receptor (CCR)7 ligands secondary lymphoid tissue chemoattractant (SLC) and macrophage inflammatory protein (MIP)-3β, implicated in homing to lymphoid tissues, are upregulated before B cell exit from the marrow, but increase further in the periphery and are shared by all peripheral B cells. In contrast, responsiveness to MIP-3α and expression of CCR6 are acquired only after emigration to the periphery and during maturation into the recirculating B cell pool. Chemotaxis to stromal cell–derived factor 1α is observed at all stages of B cell differentiation. Thus, unique patterns of chemokine responses may help define developing B cell populations and direct their maturation in the marrow and migration to the periphery.
Molecular Cloning and Biological Characterization of a Novel Murine Lymphoid Growth Factor
Authors: Sims John E. , Williams Douglas E. , Morrissey Philip J. , Garka Kirsten , Foxworthe Diane , Price Virginia , Friend Sherree L. , Farr Andrew , Bedell Mary A. , Jenkins Nancy A. , Copeland Neal G. , Grabstein Kenneth , Paxton Raymond J.
Published: July 2002

Using a bioassay consisting of the proliferation of a murine B cell line, a cDNA of a gene whose product supports the growth of that cell line was isolated from a thymic stromal cell line. This factor, termed thymic stromal lymphopoietin (TSLP), is a protein of 140 amino acids. The gene encoding TSLP was mapped to murine chromosome 18. Purified recombinant TSLP supported the growth of pre-B cell colonies in vitro, but had no myelopoietic activity. TSLP had comitogenic activity for fetal thymocytes, but was not as potent as interleukin 7 in lobe submersion cultures. Injection of TSLP into neonatal mice induced the expansion of B220+BP-1+ pre-B cells.
Interleukin-18 Regulates Acute Graft-Versus-Host Disease by Enhancing Fas-mediated Donor T Cell Apoptosis
Authors: Reddy Pavan , Teshima Takanori , Kukuruga Mark , Ordemann Rainer , Liu Chen , Lowler Kathy , Ferrara James L.M.
Published: July 2002

Interleukin (IL)-18 is a recently discovered cytokine that modulates both T helper type 1 (Th1) and Th2 responses. IL-18 is elevated during acute graft-versus-host disease (GVHD). We investigated the role of IL-18 in this disorder using a well characterized murine bone marrow transplantation (BMT) model (B6 → B6D2F1). Surprisingly, blockade of IL-18 accelerated acute GVHD-related mortality. In contrast, administration of IL-18 reduced serum tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS) levels, decreased intestinal histopathology, and resulted in significantly improved survival (75 vs. 15%, P < 0.001). Administration of IL-18 attenuated early donor T cell expansion and was associated with increased Fas expression and greater apoptosis of donor T cells. The administration of IL-18 no longer protected BMT recipients from GVHD when Fas deficient (lpr) mice were used as donors. IL-18 also lost its ability to protect against acute GVHD when interferon (IFN)-γ knockout mice were used as donors. Together, these results demonstrate that IL-18 regulates acute GVHD by inducing enhanced Fas-mediated apoptosis of donor T cells early after BMT, and donor IFN-γ is critical for this protective effect.
The Multiple Immune-Evasion Genes of Murine Cytomegalovirus Are Not Redundant
Authors: Kavanagh Daniel G. , Gold Marielle C. , Wagner Markus , Koszinowski Ulrich H. , Hill Ann B.
Published: July 2002

Both human cytomegaloviruses (HCMVs) and murine cytomegaloviruses (MCMVs) encode multiple genes that interfere with antigen presentation by major histocompatibility complex (MHC) class I, and thus protect infected targets from lysis by virus-specific cytotoxic T lymphocytes (CTLs). HCMV has been shown to encode four such genes and MCMV to encode two. MCMV m152 blocks the export of class I from a pre-Golgi compartment, and MCMV m6 directs class I to the lysosome for degradation. A third MCMV gene, m4, encodes a glycoprotein which is expressed at the cell surface in association with class I. Here we here show that m4 is a CTL-evasion gene which, unlike previously described immune-evasion genes, inhibited CTLs without blocking class I surface expression. m152 was necessary to block antigen presentation to both Kb- and Db-restricted CTL clones, while m4 was necessary to block presentation only to Kb-restricted clones. m152 caused complete retention of Db, but only partial retention of Kb, in a pre-Golgi compartment. Thus, while m152 effectively inhibited Db-restricted CTLs, m4 was required to completely inhibit Kb-restricted CTLs. We propose that cytomegaloviruses encode multiple immune-evasion genes in order to cope with the diversity of class I molecules in outbred host populations.
B-1a B Cells that Link the Innate and Adaptive Immune Responses Are Lacking in the Absence of the Spleen
Authors: Wardemann Hedda , Boehm Thomas , Dear Neil , Carsetti Rita
Published: July 2002

Splenectomized individuals are prone to overwhelming infections with encapsulated bacteria and splenectomy of mice increases susceptibility to streptococcal infections, yet the exact mechanism by which the spleen protects against such infections is unknown. Using congenitally asplenic mice as a model, we show that the spleen is essential for the generation of B-1a cells, a B cell population that cooperates with the innate immune system to control early bacterial and viral growth. Splenectomy of wild-type mice further demonstrated that the spleen is also important for the survival of B-1a cells. Transfer experiments demonstrate that lack of these cells, as opposed to the absence of the spleen per se, is associated with an inability to mount a rapid immune response against streptococcal polysaccharides. Thus, absence of the spleen and the associated increased susceptibility to streptococcal infections is correlated with lack of B-1a B cells. These findings reveal a hitherto unknown role of the spleen in generating and maintaining the B-1a B cell pool.
Aging Leads to Disturbed Homeostasis of Memory Phenotype CD8+ Cells
Authors: Zhang Xiaohong , Fujii Hideki , Kishimoto Hidehiro , LeRoy Eric , Surh Charles D. , Sprent Jonathan
Published: July 2002

Examining the rate of in vivo T cell turnover (proliferation) in aged mice revealed a marked reduction in turnover at the level of memory-phenotype CD44hi CD8+ cells relative to young mice. Based on adoptive transfer experiments, the reduced turnover of aged CD44hi CD8+ cells reflected an inhibitory influence of the aged host environment. Aged CD44hi CD8+ cells also showed poor in vivo responses to IL-15 and IL-15–inducing agents, but responded well to IL-15 in vitro. Two mechanisms could account for the reduced turnover of aged CD44hi CD8+ cells in vivo. First, aging was associated with a prominent and selective increase in Bcl-2 expression in CD44hi CD8+ cells. Hence, the reduced turnover of aged CD44hi CD8+ cells may in part reflect the antiproliferative effect of enhanced Bcl-2 expression. Second, the impaired in vivo response of aged CD44hi CD8+ cells to IL-15 correlated with increased serum levels of type I interferons (IFN-I) and was largely reversed by injection of anti–IFN-I antibody. Hence the selective reduction in the turnover of aged CD44hi CD8+ cells in vivo may reflect the combined inhibitory effects of enhanced Bcl-2 expression and high IFN-I levels.
Pivotal Role of Dendritic Cell–derived CXCL10 in the Retention of T Helper Cell 1 Lymphocytes in Secondary Lymph Nodes
Authors: Yoneyama Hiroyuki , Narumi Shosaku , Zhang Yanyun , Murai Masako , Baggiolini Marco , Lanzavecchia Antonio , Ichida Takafumi , Asakura Hitoshi , Matsushima Kouji
Published: July 2002

Various immune diseases are considered to be regulated by the balance of T helper (Th)1 and Th2 subsets. Although Th lymphocytes are believed to be generated in draining lymph nodes (LNs), in vivo Th cell behaviors during Th1/Th2 polarization are largely unexplored. Using a murine granulomatous liver disease model induced by Propionibacterium acnes, we show that retention of Th1 cells in the LNs is controlled by a chemokine, CXCL10/interferon (IFN) inducible protein 10 produced by mature dendritic cells (DCs). Hepatic LN DCs preferentially produced CXCL10 to attract 5′-bromo-2′-deoxyuridine (BrdU)+CD4+ T cells and form clusters with IFN-γ–producing CD4+ T cells by day 7 after antigen challenge. Blockade of CXCL10 dramatically altered the distribution of cluster-forming BrdU+CD4+ T cells. BrdU+CD4+ T cells in the hepatic LNs were selectively diminished while those in the circulation were significantly increased by treatment with anti-CXCL10 monoclonal antibody. This was accompanied by accelerated infiltration of memory T cells into the periphery of hepatic granuloma sites, most of them were in cell cycle and further produced higher amount of IFN-γ leading to exacerbation of liver injury. Thus, mature DC-derived CXCL10 is pivotal to retain Th1 lymphocytes within T cell areas of draining LNs and optimize the Th1-mediated immune responses.
Activation of Shaker Potassium Channels
Authors: Schoppa N.E. , Sigworth F.J.
Published: July 2002

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker's symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e0), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e0). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker's activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.
Mechanism of Peptide-induced Mast Cell Degranulation
Authors: Lorenz Dorothea , Wiesner Burkhard , Zipper Josef , Winkler Anett , Krause Eberhard , Beyermann Michael , Lindau Manfred , Bienert Michael
Published: July 2002

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription–-PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100–500 s at concentrations of substance P from 50 to 5 μM). Degranulation in response to intracellularly applied substance P was inhibited by GDPβS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein α subunits subsequent to their translocation across the plasma membrane.
Reversible Ca Gradients between the Subplasmalemma and Cytosol Differentially Activate Ca-dependent Cl Currents
Authors: Machaca Khaled , Criss Hartzell H.
Published: July 2002

Xenopus oocytes express several different Ca-activated Cl currents that have different waveforms and biophysical properties. We compared the stimulation of Ca-activated Cl currents measured by two-microelectrode voltage clamp with the Ca transients measured in the same cell by confocal microscopy and Ca-sensitive fluorophores. The purpose was to determine how the amplitude and/or spatio-temporal features of the Ca signal might explain how these different Cl currents were activated by Ca. Because Ca release from stores was voltage independent, whereas Ca influx depended upon the electrochemical driving force, we were able to separately assess the contribution of Ca from these two sources. We were surprised to find that Ca signals measured with a cytosolic Ca-sensitive dye, dextran-conjugated Ca-green-1, correlated poorly with Cl currents. This suggested that Cl channels located at the plasma membrane and the Ca-sensitive dye located in the bulk cytosol were sensing different [Ca]. This was true despite Ca measurement in a confocal slice very close to the plasma membrane. In contrast, a membrane-targeted Ca-sensitive dye (Ca-green-C18) reported a Ca signal that correlated much more closely with the Cl currents. We hypothesize that very local, transient, reversible Ca gradients develop between the subplasmalemmal space and the bulk cytosol. [Ca] is higher near the plasma membrane when Ca is provided by Ca influx, whereas the gradient is reversed when Ca is released from stores, because Ca efflux across the plasma membrane is faster than diffusion of Ca from the bulk cytosol to the subplasmalemmal space. Because dissipation of the gradients is accelerated by inhibition of Ca sequestration into the endoplasmic reticulum with thapsigargin, we conclude that [Ca] in the bulk cytosol declines slowly partly due to futile recycling of Ca through the endoplasmic reticulum.
Effects of Ultraviolet Modification on the Gating Energetics of Cyclic Nucleotide–Gated Channels
Authors: Middendorf Thomas R. , Aldrich Richard W.
Published: July 2002

Middendorf et al. (Middendorf, T.R., R.W. Aldrich, and D.A. Baylor. 2000. J. Gen. Physiol. 116:227–252) showed that ultraviolet light decreases the current through cloned cyclic nucleotide–gated channels from bovine retina activated by high concentrations of cGMP. Here we probe the mechanism of the current reduction. The channels' open probability before irradiation, Po(0), determined the sign of the change in current amplitude that occurred upon irradiation. UV always decreased the current through channels with high initial open probabilities [Po(0) > 0.3]. Manipulations that promoted channel opening antagonized the current reduction by UV. In contrast, UV always increased the current through channels with low initial open probabilities [Po(0) ≤ 0.02], and the magnitude of the current increase varied inversely with Po(0). The dual effects of UV on channel currents and the correlation of both effects with Po(0) suggest that the channels contain two distinct classes of UV target residues whose photochemical modification exerts opposing effects on channel gating. We present a simple model based on this idea that accounts quantitatively for the UV effects on the currents and provides estimates for the photochemical quantum yields and free energy costs of modifying the UV targets. Simulations indicate that UV modification may be used to produce and quantify large changes in channel gating energetics in regimes where the associated changes in open probability are not measurable by existing techniques.
Polynomial bounds for rings of invariants
Authors: Derksen Harm
Published: July 2002

Hilbert proved that invariant rings are finitely generated for linearly reductive groups acting rationally on a finite dimensional vector space. Popov gave an explicit upper bound for the smallest integer

d
d

such that the invariants of degree

≤
d

\leq d

generate the invariant ring. This bound has factorial growth. In this paper we will give a bound which depends only polynomially on the input data.
A proof of the trace theorem of Sobolev spaces on Lipschitz domains
Authors: Ding Zhonghai
Published: July 2002

A complete proof of the trace theorem of Sobolev spaces on Lipschitz domains has not appeared in the literature yet. The purpose of this paper is to give a complete proof of the trace theorem of Sobolev spaces on Lipschitz domains by taking advantage of the intrinsic norm on

H

s

(
∂
Ω
)

H^{s}(\partial \Omega )

. It is proved that the trace operator is a linear bounded operator from

H

s

(
Ω
)

H^{s}(\Omega )

to

H

s
−

1
2

(
∂
Ω
)

H^{s-\frac {1}{2}}(\partial \Omega )

for

1
2

>
s
>

3
2

\frac {1}{2}>s>\frac {3}{2}

.
Hyers-Ulam-Rassias stability of Jensen’s equation and its application
Authors: Jung Soon-Mo
Published: July 2002

The Hyers-Ulam-Rassias stability for the Jensen functional equation is investigated, and the result is applied to the study of an asymptotic behavior of the additive mappings; more precisely, the following asymptotic property shall be proved: Let

X
X

and

Y
Y

be a real normed space and a real Banach space, respectively. A mapping

f
:
X
→
Y

f: X \rightarrow Y

satisfying

f
(
0
)
=
0

f(0)=0

is additive if and only if

‖
2
f

[
(
x
+
y
)

/

2
]

−
f
(
x
)
−
f
(
y
)
‖

→
0

\left \| 2f\left [ (x+y)/2 \right ] - f(x) - f(y) \right \| \rightarrow 0

as

‖
x
‖
+
‖
y
‖
→
∞

\| x \| + \| y \| \rightarrow \infty

.
Estimates for functions of the Laplace operator on homogeneous trees
Authors: Cowling Michael , Meda Stefano , Setti Alberto
Published: July 2002

In this paper, we study the heat equation on a homogeneous graph, relative to the natural (nearest–neighbour) Laplacian. We find pointwise estimates for the heat and resolvent kernels, and the

L

p

−

L

q

L^{p}-L^{q}

mapping properties of the corresponding operators.
Harmonic Bergman Functions on Half-Spaces
Authors: Ramey Wade , Yi HeungSu
Published: July 2002

We study harmonic Bergman functions on the upper half-space of

R

n

\mathbf {R}^n

. Among our main results are: The Bergman projection is bounded for the range

1
>
p
>
∞

1> p > \infty

; certain nonorthogonal projections are bounded for the range

1
≤
p
>
∞

1\leq p > \infty

; the dual space of the Bergman

L
1

L^1

-space is the harmonic Bloch space modulo constants; harmonic conjugation is bounded on the Bergman spaces for the range

1
≤
p
>
∞

1\leq p > \infty

; the Bergman norm is equivalent to a “normal derivative norm” as well as to a “tangential derivative norm”.
Numerical solution of the scalar double-well problem allowing microstructure
Authors: Carstensen Carsten , Plecháč Petr
Published: July 2002

The direct numerical solution of a non-convex variational problem (

P
P

) typically faces the difficulty of the finite element approximation of rapid oscillations. Although the oscillatory discrete minimisers are properly related to corresponding Young measures and describe real physical phenomena, they are costly and difficult to compute. In this work, we treat the scalar double-well problem by numerical solution of the relaxed problem (

R
P

RP

) leading to a (degenerate) convex minimisation problem. The problem (

R
P

RP

) has a minimiser

u
u

and a related stress field

σ
=
D

W

∗
∗

(
∇

u

)

\sigma = DW^{**}(\nabla {u})

which is known to coincide with the stress field obtained by solving (

P
P

) in a generalised sense involving Young measures. If

u
h

u_h

is a finite element solution,

σ
h

:=
D

W

∗
∗

(
∇

u

h

)

\sigma _h:= D W^{**}(\nabla {u}_h)

is the related discrete stress field. We prove a priori and a posteriori estimates for

σ
−

σ
h

\sigma -\sigma _h

in

L

4

/

3

(
Ω
)

L^{4/3}(\Omega )

and weaker weighted estimates for

∇

u

−
∇

u

h

\nabla {u}-\nabla {u}_h

. The a posteriori estimate indicates an adaptive scheme for automatic mesh refinements as illustrated in numerical experiments.
The Diophantine equation 𝑥⁴+1=𝐷𝑦²
Authors: Cohn J.
Published: July 2002

An effective method is derived for solving the equation of the title in positive integers

x
x

and

y
y

for given

D
D

completely, and is carried out for all

D
>
100000

D>100000

. If

D
D

is of the form

m
4

+
1

m^4+1

, then there is the solution

x
=
m

x=m

,

y
=
1

y=1

; in the above range, except for

D
=
70258

D=70258

with solution

x
=
261

x=261

,

y
=
257

y=257

, there are no other solutions.
Maximum excursion and stopping time record-holders for the problem: Computational results
Authors: e Silva Tomás
Published: July 2002

This paper presents some results concerning the search for initial values to the so-called

3
x
+
1

3x+1

problem which give rise either to function iterates that attain a maximum value higher than all function iterates for all smaller initial values, or which have a stopping time higher than those of all smaller initial values. Our computational results suggest that for an initial value of 

n
n

, the maximum value of the function iterates is bounded from above by

n
2

f
(
n
)

n^2 f(n)

, with

f
(
n
)

f(n)

either a constant or a very slowly increasing function of 

n
n

. As a by-product of this (exhaustive) search, which was performed up to

n
=
3
⋅

2

53

≈
2.702
⋅

10

16

n=3 \cdot 2^{53}\approx 2.702 \cdot 10^{16}

, the

3
x
+
1

3x+1

conjecture was verified up to that same number.
Quantum affine algebras at roots of unity
Authors: Chari Vyjayanthi , Pressley Andrew
Published: July 2002

Let

U

q

(

g

^

)

U_{q}(\hat {\mathfrak {g}})

be the quantized universal enveloping algebra of the affine Lie algebra

g

^

\hat {\mathfrak {g}}

associated to a finite-dimensional complex simple Lie algebra

g

\mathfrak {g}

, and let

U

q

r
e
s

(

g

^

)

U_{q}^{\mathrm {res}}(\hat {\mathfrak {g}})

be the

C

[
q
,

q

−
1

]

\mathbb {C}[q,q^{-1}]

-subalgebra of

U

q

(

g

^

)

U_{q}(\hat {\mathfrak {g}})

generated by the

q
q

-divided powers of the Chevalley generators. Let

U

ϵ

r
e
s

(

g

^

)

U_{\epsilon }^{\mathrm {res}}(\hat {\mathfrak {g}})

be the Hopf algebra obtained from

U

q

r
e
s

(

g

^

)

U_{q}^{\mathrm {res}}(\hat {\mathfrak {g}})

by specialising

q
q

to a non-zero complex number

ϵ
\epsilon

of odd order. We classify the finite-dimensional irreducible representations of

U

ϵ

r
e
s

(

g

^

)

U_{\epsilon }^{\mathrm {res}}(\hat {\mathfrak {g}})

in terms of highest weights. We also give a “factorisation” theorem for such representations: namely, any finite-dimensional irreducible representation of

U

ϵ

r
e
s

(

g

^

)

U_{\epsilon }^{\mathrm {res}}(\hat {\mathfrak {g}})

is isomorphic to a tensor product of two representations, one factor being the pull-back of a representation of

g

^

\hat {\mathfrak {g}}

by Lusztig’s Frobenius homomorphism
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="ModifyingAbove normal upper F normal r With caret Subscript epsilon Baseline colon upper U Subscript epsilon Superscript normal r normal e normal s Baseline left-parenthesis ModifyingAbove German g With caret right-parenthesis right-arrow upper U left-parenthesis ModifyingAbove German g With caret right-parenthesis">

F
r

^

ϵ

:

U

ϵ

r
e
s

(

g

^

)
→
U
(

g

^

)

\hat {\mathrm {Fr}}_{\epsilon }:U_{\epsilon }^{\mathrm {res}}(\hat {\mathfrak {g}})\to U(\hat {\mathfrak {g}})

, the other factor being an irreducible representation of the Frobenius kernel. Finally, we give a concrete construction of all of the finite-dimensional irreducible representations of

U

ϵ

r
e
s

(

s
l

^

2

)

U_{\epsilon }^{\mathrm {res}}(\hat {sl}_{2})

. The proofs make use of several interesting new identities in

U

q

(

g

^

)

U_{q}(\hat {\mathfrak {g}})

.
Systematic genomic screening and analysis of mRNA in untranslated regions and mRNA precursors: combining experimental and computational approaches.
Authors: Dandekar T , Beyer K , Bork P , Kenealy M R , Pantopoulos K , Hentze M , Sonntag-Buck V , Flouriot G , Gannon F , Schreiber S
Published: July 2002

MOTIVATION: The untranslated regions (UTRs) of mRNA upstream (5'UTR) and downstream (3'UTR) of the open reading frame, as well as the mRNA precursor, carry important regulatory sequences. To reveal unidentified regulatory signals, we combine information from experiments with computational approaches. Depending on available knowledge, three different strategies are employed. RESULTS: Searching with a consensus template, new RNAs with regulatory RNA elements can be identified in genomic screens. By this approach, we identify new candidate regulatory motifs resembling iron-responsive elements in the 5'UTRs of HemA, FepB and FrdB mRNA from Escherichia coli. If an RNA element is not yet defined, it may be analyzed by combining results from SELEX (selective enrichment of ligands by exponential amplification) and a search of databases from RNA or genomic sequences. A cleavage stimulating factor (CstF) binding element 3 of the polyadenylation site in the mRNA precursor serves as a test example. Alternatively, the regulatory RNA element may be found by studying different RNA foldings and their correlation with simple experimental tests. We delineate a novel instability element in the 3'UTR of the estrogen receptor mRNA in this way. AVAILABILITY: Strategy, methods and programs are available on request from T.Dandekar. CONTACT: dandekar@embl-heidelberg.de
Oriloc: prediction of replication boundaries in unannotated
bacterial chromosomes
Authors: Frank A. C. , Lobry J. R.
Published: July 2002

Summary: A program called Oriloc has been developed for the prediction of bacterial replication origins. The method builds on the fact that there are compositional asymmetries between the leading and the lagging strand for replication. The program works with unannotated sequences in fasta format and therefore uses glimmer 2.0 outputs to discriminate between codon positions so as to increase the signal/noise ratio.

Availability: The ANSI C source code is freely available for academic use at ftp://pbil.univ-lyon1.fr/pub/logiciel/oriloc/oriloc.c.

Contact: lobry@biomserv.univ-lyon1.fr

Supplementary information: ftp://pbil.univ-lyon1.fr/pub/logiciel/oriloc/oriloc.ps

* To whom correspondence should be addressed.
THE EFFECT OF JOB MOBILITY ON ACADEMIC SALARIES
Authors: Barbezat Debra A. , Hughes James W.
Published: July 2002

The hypothesis that individuals increase future earnings by investing in themselves through job change has considerable empirical support. To date, this hypothesis has not been tested on the academic labor market. Using a national faculty survey that includes respondents' complete work history, the authors estimate the impact of job mobility on faculty salaries. The rate of job mobility among faculty members appears low compared to that of other workers. Salaries overall are not significantly related to the number of academic jobs held until the number of jobs reaches four, and then the effect is negative. An examination by gender reveals that women incur a salary penalty of 8% in moving to the second job.
Two distinct action mechanisms of immunophilin–ligand complexes for the blockade of T‐cell activation
Authors: Matsuda Satoshi , Shibasaki Futoshi , Takehana Kenji , Mori Hiroaki , Nishida Eisuke , Koyasu Shigeo
Published: July 2002

The immunosuppressive effects of cyclosporin A (CsA) and FK506 are mediated through binding to immunophilins. Here we show that FK506–FKBP complex suppresses the activation of JNK and p38 pathways at a level upstream of mitogen‐activated protein kinase (MAPK) kinase kinase (MAPKK‐K) besides the calcineurin–NFAT pathway. A238L, a viral gene product that binds to immunophilin, also blocks activation of both pathways. In contrast, direct inhibitors of calcineurin, Cabin 1 and FR901725, suppress the activation of NFAT but not the JNK or p38 pathway. We further demonstrate that co‐expression of a constitutively active NFAT and a constitutively active MEKK1 renders the interleukin‐2 promoter in Jurkat T lymphocytes resistant to CsA and FK506, whereas Jurkat cells expressing a constitutively active NFAT alone are still sensitive to CsA or FK506. Therefore, CsA and FK506 exert their immunosuppressive effects through targeting both the calcineurin‐dependent NFAT pathway and calcineurin‐independent activation pathway for JNK and p38.
The polarized epithelia‐specific μ1B‐adaptin complements μ1A‐deficiency in fibroblasts
Authors: Eskelinen Eeva‐Liisa , Meyer Christoph , Ohno Hiroshi , von Figura Kurt , Schu Peter
Published: July 2002

The heterotetrameric AP‐1A adaptor complex of clathrin‐coated vesicles is ubiquitously expressed. The μ1‐adaptin subunit of the complex exists as the ubiquitous μ1A and the polarized epithelia‐specific μ1B, which are 80% identical. In polarized epithelia, μ1B is incorporated into the AP‐1B complex, which is required for basolateral plasma membrane sorting of the low‐density lipoprotein receptor. Binding of AP‐1B to subdomains of the trans‐Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed μ1B in μ1A‐deficient fibroblasts to test for μ1B function in non‐polarized cells. AP‐1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP‐1B restored the AP‐1A‐dependent sorting of mannose 6‐phosphate receptors between endosomes and the TGN. This demonstrates that μ1A and μ1B do have overlapping sorting functions and indicates that AP‐1A and AP‐1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.