bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Early Cytokine Responses and an Interleukin (IL)-6–dependent Pathway of Endogenous Glucocorticoid Induction during Murine Cytomegalovirus Infection
Authors: Ruzek Melanie C. , Miller Andrew H. , Opal Steven M. , Pearce Bradley D. , Biron Christine A.
Published: July 2002

Early infection with murine cytomegalovirus (MCMV) induces circulating levels of interleukin (IL)-12, interferon (IFN)-γ, and tumor necrosis factor (TNF). Studies presented here further characterize these responses by defining kinetics and extending evaluation to include IL-1, IL-6, and glucocorticoids. IL-12 p40, IFN-γ, TNF, IL-1α, and IL-6 were shown to be increased, but IL-1β was undetectable, in serum of MCMV-infected mice. The IL-12 p40, IFN-γ, TNF, and IL-6 responses were dramatic with peak levels reaching >150–10,000 pg/ml at 32–40 h after infection and rapidly declining thereafter. Glucocorticoid induction, peaking at 36 h and reaching 30-fold increases above control values, accompanied the cytokine responses. Mice with cytokine deficiencies or neutralized cytokine function demonstrated that IL-6 was the pivotal mediator of the glucocorticoid response, with IL-1 contributing to IL-6 production. The IL-6 requirement appeared to be specific for virus-type stimuli as the synthetic analogue of viral nucleic acid, polyinosinic-polycytidylic acid, also induced IL-6–dependent glucocorticoid release, but treatments with the bacterial product lipopolysaccharide and a non-immune physical restraint stressor elicited IL-6–independent responses. Collectively, the results identify IL-6 as a primary mediator of glucocorticoid induction, and elucidate specific pathways of interactions between immune and neuroendocrine systems during viral infection.
Selective Inhibition of Ii-dependent Antigen Presentation by Helicobacter pylori Toxin VacA
Authors: Molinari Maurizio , Salio Mariolina , Galli Carmela , Norais Nathalie , Rappuoli Rino , Lanzavecchia Antonio , Montecucco Cesare
Published: July 2002

A major virulence factor in the stomach chronic infection by Helicobacter pylori is a protein toxin (VacA), which alters cell membrane trafficking of late endosomal/prelysosomal compartments. Its role in the chronic infection established by H. pylori is unknown. To test the possibility that VacA alters antigen processing taking place in prelysosomal compartments, we have used the well-established model of antigen processing and presentation consisting of tetanus toxoid–specific human (CD4+) T cells stimulated by autologous antigen-pulsed Epstein-Barr virus-transformed B cells. We found that VacA interferes with proteolytic processing of tetanus toxin and toxoid and specifically inhibits the Ii-dependent pathway of antigen presentation mediated by newly synthesized major histocompatibility complex (MHC) class II, while leaving unaffected the presentation pathway dependent on recycling MHC class II. The results presented here suggest that VacA may contribute to the persistence of H. pylori by interfering with protective immunity and that this toxin is a new useful tool in the study of the different pathways of antigen presentation.
Neonatal Exposure to a Self-Peptide–Immunoglobulin Chimera Circumvents the Use of Adjuvant and Confers Resistance to Autoimmune Disease by a Novel Mechanism Involving Interleukin 4 Lymph Node Deviation and Interferon γ–mediated Splenic Anergy
Authors: Min Booki , Legge Kevin L. , Pack Christopher , Zaghouani Habib
Published: July 2002

Induction of neonatal T cell tolerance to soluble antigens requires the use of incomplete Freund's adjuvant (IFA). The side effects that could be associated with IFA and the ill-defined mechanism underlying neonatal tolerance are setbacks for this otherwise attractive strategy for prevention of T cell–mediated autoimmune diseases. Presumably, IFA contributes a slow antigen release and induction of cytokines influential in T cell differentiation. Immunoglobulins (Igs) have long half-lives and could induce cytokine secretion by binding to Fc receptors on target cells. Our hypothesis was that peptide delivery by Igs may circumvent the use of IFA and induce neonatal tolerance that could confer resistance to autoimmunity. To address this issue we used the proteolipid protein (PLP) sequence 139–151 (hereafter referred to as PLP1), which is encephalitogenic and induces experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. PLP1 was expressed on an Ig, and the resulting Ig–PLP1 chimera when injected in saline into newborn mice confers resistance to EAE induction later in life. Mice injected with Ig–PLP1 at birth and challenged as adults with PLP1 developed T cell proliferation in the lymph node but not in the spleen, whereas control mice injected with Ig–W, the parental Ig not including PLP1, developed T cell responses in both lymphoid organs. The lymph node T cells from Ig–PLP1 recipient mice were deviated and produced interleukin (IL)-4 instead of IL-2, whereas the spleen cells, although nonproliferative, produced IL-2 but not interferon (IFN)-γ. Exogenous IFN-γ, as well as IL-12, restored splenic proliferation in an antigen specific manner. IL-12–rescued T cells continued to secrete IL-2 and regained the ability to produce IFN-γ. In vivo, administration of anti–IL-4 antibody or IL-12 restored disease severity. Therefore, adjuvant-free induced neonatal tolerance prevents autoimmunity by an organ-specific regulation of T cells that involves both immune deviation and a new form of cytokine- dependent T cell anergy.
Interleukin 3 Prevents Delayed Neuronal Death in the Hippocampal CA1 Field
Authors: Wen Tong-Chun , Tanaka Junya , Peng Hui , Desaki Junzo , Matsuda Seiji , Maeda Nobuji , Fujita Hiroko , Sato Kohji , Sakanaka Masahiro
Published: July 2002

In the central nervous system, interleukin (IL)-3 has been shown to exert a trophic action only on septal cholinergic neurons in vitro and in vivo, but a widespread distribution of IL-3 receptor (IL-3R) in the brain does not conform to such a selective central action of the ligand. Moreover, the mechanism(s) underlying the neurotrophic action of IL-3 has not been elucidated, although an erythroleukemic cell line is known to enter apoptosis after IL-3 starvation possibly due to a rapid decrease in Bcl-2 expression. This in vivo study focused on whether IL-3 rescued noncholinergic hippocampal neurons from lethal ischemic damage by modulating the expression of Bcl-xL, a Bcl-2 family protein produced in the mature brain. 7-d IL-3 infusion into the lateral ventricle of gerbils with transient forebrain ischemia prevented significantly hippocampal CA1 neuron death and ischemia-induced learning disability. TUNEL (terminal deoxynucleotidyltransferase–mediated 2′-deoxyuridine 5′-triphosphate-biotin nick end labeling) staining revealed that IL-3 infusion caused a significant reduction in the number of CA1 neurons exhibiting DNA fragmentation 7 d after ischemia. The neuroprotective action of IL-3 appeared to be mediated by a postischemic transient upregulation of the IL-3R α subunit in the hippocampal CA1 field where IL-3Rα was barely detectable under normal conditions. In situ hybridization histochemistry and immunoblot analysis demonstrated that Bcl-xL mRNA expression, even though upregulated transiently in CA1 pyramidal neurons after ischemia, did not lead to the production of Bcl-xL protein in ischemic gerbils infused with vehicle. However, IL-3 infusion prevented the decrease in Bcl-xL protein expression in the CA1 field of ischemic gerbils. Subsequent in vitro experiments showed that IL-3 induced the expression of Bcl-xL mRNA and protein in cultured neurons with IL-3Rα and attenuated neuronal damage caused by a free radical–producing agent FeSO4. These findings suggest that IL-3 prevents delayed neuronal death in the hippocampal CA1 field through a receptor-mediated expression of Bcl-xL protein, which is known to facilitate neuron survival. Since IL-3Rα in the hippocampal CA1 region, even though upregulated in response to ischemic insult, is much less intensely expressed than that in the CA3 region tolerant to ischemia, the paucity of IL-3R interacting with the ligand may account for the vulnerability of CA1 neurons to ischemia.
SLP-65: A New Signaling Component in B Lymphocytes which Requires Expression of the Antigen Receptor for Phosphorylation
Authors: Wienands Jürgen , Schweikert Jutta , Wollscheid Bernd , Jumaa Hassan , Nielsen Peter J. , Reth Michael
Published: July 2002

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-α/Ig-β heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.
Abnormal Chemokine-Induced Responses of Immature and Mature Hematopoietic Cells from Motheaten Mice Implicate the Protein Tyrosine Phosphatase Shp-1 in Chemokine Responses
Authors: Kim Chang H. , Qu Cheng-Kui , Hangoc Giao , Cooper Scott , Anzai Naoyuki , Feng Gen-Sheng , Broxmeyer Hal E.
Published: July 2002

Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (mev/mev) mice that were deficient in SHP-1. Chemotactic responses to stromal call–derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from mev/mev versus wild-type (+/+) mice. SDF-1–dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in mev/mev versus +/+ cells. In contrast, immature subsets of mev/mev bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in mev/mev T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities.
Mycobacterium tuberculosis Expresses a Novel Ph-Dependent Divalent Cation Transporter Belonging to the Nramp Family
Authors: Agranoff Daniel , Monahan Irene M. , Mangan Joseph A. , Butcher Philip D. , Krishna Sanjeev
Published: July 2002

Mammalian natural resistance–associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe2+, Mn2+, Zn2+, and Cu2+. Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp).

RNA encoding Mramp induces ∼20-fold increases in 65Zn2+ and 55Fe2+ uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated 65Zn2+ and 55Fe2+ transport is abolished by an excess of Mn2+ and Cu2+, confirming that Mramp interacts with a broad range of divalent transition metal cations.

Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe2+ and Cu2+ between <1 and 5 μM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5–70 μM Cu2+. M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp.

Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.
Selection and Expansion of CD8α/α1 T Cell Receptor α/β1 Intestinal Intraepithelial Lymphocytes in the Absence of Both Classical Major Histocompatibility Complex Class I and Nonclassical Cd1 Molecules
Authors: Park Se-Ho , Guy-Grand Delphine , Lemonnier François A. , Wang Chyung-Ru , Bendelac Albert , Jabri Bana
Published: July 2002

Intestinal intraepithelial lymphocytes (IELs) in mice include two main subsets of TCR-α/β1 cells which differ functionally and ontogenically from each other. One expresses the CD8α/α homodimer, whereas the other expresses the CD8α/β heterodimer. Although the presence of all CD8+TCR-α/β1 IELs is dependent on β2-microglobulin molecules, the nature of the major histocompatibility complex (MHC) class I molecules recognized by the CD8α/α and the CD8α/β1 subsets has remained elusive. Using mutant mice lacking the expression of both H2-Kb and H2-Db, we show that the CD8α/β1TCR-α/β1 subset is dependent on K or D molecules, whereas the CD8α/α1TCR-α/β1 subset is independent of classical MHC class I molecules. Furthermore, the CD8α/α1 cells are conserved in mice lacking expression of CD1, a nonclassical MHC class I–like molecule previously proposed to be a potential ligand for IELs. Using transporter associated with antigen processing (TAP)-deficient mice, this cell population can be further separated into a TAP-dependent and a TAP-independent subset, suggesting either the recognition of two nonclassical MHC-like molecules, only one of which is TAP dependent, or the involvement of a single nonclassical MHC-like molecule that is only partially TAP dependent. These findings demonstrate that CD8α/β1TCR-α/β1 IELs are restricted by H-2K and H-2D molecules, whereas the unusual subset of CD8α/α1TCR-α/β1 resident IELs recognize nonclassical MHC class I–like molecules that are distinct from CD1.
Cd1-Reactive Natural Killer T Cells Are Required for Development of Systemic Tolerance through an Immune-Privileged Site
Authors: Sonoda Koh-Hei , Exley Mark , Snapper Scott , Balk Steven P. , Stein-Streilein Joan
Published: July 2002

Systemic tolerance can be elicited by introducing antigen into an immune-privileged site, such as the eye, or directly into the blood. Both routes of immunization result in a selective deficiency of systemic delayed type hypersensitivity. Although the experimental animal model of anterior chamber–associated immune deviation (ACAID) occurs in most mouse strains, ACAID cannot be induced in several mutant mouse strains that are coincidentally deficient in natural killer T (NKT) cells. Therefore, this model for immune-privileged site–mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance. The following data show that CD1-reactive NKT cells are required for the development of systemic tolerance induced via the eye as follows: (a) CD1 knockout mice were unable to develop ACAID unless they were reconstituted with NKT cells together with CD1+ antigen-presenting cells; (b) specific antibody depletion of NKT cells in vivo abrogated the development of ACAID; and (c) anti-CD1 monoclonal antibody treatment of wild-type mice prevented ACAID development. Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes. A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.
Modulation of Susceptibility to HIV-1 Infection by the Cytotoxic T Lymphocyte Antigen 4 Costimulatory Molecule
Authors: Riley James L. , Schlienger Katia , Blair Patrick J. , Carreno Beatriz , Craighead Nancy , Kim Daniel , Carroll Richard G. , June Carl H.
Published: July 2002

CD4 T cells activated in vitro by anti-CD3/28–coated beads are resistant to infection by CC chemokine receptor 5 (CCR5)-dependent HIV-1 isolates. In vivo, antigen-presenting cells (APCs) activate CD4 T cells in part by signaling through the T cell receptor and CD28, yet cells stimulated in this manner are susceptible to HIV-1 infection. We show that cytotoxic T lymphocyte antigen 4 (CTLA-4) engagement counteracts the CD28 antiviral effects, and that the ratio of CTLA-4 to CD28 engagement determines the susceptibility of HIV-1 infection. Furthermore, unopposed CTLA-4 signaling provided by CD28 blockade promotes vigorous HIV-1 replication, despite minimal T cell proliferation. Finally, CTLA-4 antibodies decrease the susceptibility of antigen-activated CD4 T cells to HIV, suggesting a potential approach to prevent or limit viral spread in HIV-1–infected individuals.
Molecular Basis for Hematopoietic/Mesenchymal Interaction during Initiation of Peyer's Patch Organogenesis
Authors: Honda Kenya , Nakano Hiroyasu , Yoshida Hisahiro , Nishikawa Satomi , Rennert Paul , Ikuta Koichi , Tamechika Masakatsu , Yamaguchi Kazuhito , Fukumoto Tetsuo , Chiba Tsutomu , Nishikawa Shin-Ichi
Published: July 2002

Mice deficient in lymphotoxin β receptor (LTβR) or interleukin 7 receptor α (IL-7Rα) lack Peyer's patches (PPs). Deficiency in CXC chemokine receptor 5 (CXCR5) also severely affects the development of PPs. A molecular network involving these three signaling pathways has been implicated in PP organogenesis, but it remains unclear how they are connected during this process. We have shown that PP organogenesis is initiated at sites containing IL-7Rα+ lymphoid cells and vascular cell adhesion molecule (VCAM)-1/intercellular adhesion molecule (ICAM)-1 expressing nonlymphoid elements. Here we characterize these lymphoid and nonlymphoid components in terms of chemokine signals. The lymphoid population expresses CXCR5 and has a strong chemotactic response to B lymphocyte chemoattractant (BLC). Importantly, chemokines produced by VCAM-1+ICAM-1+ nonlymphoid cells mediate the recruitment of lymphoid cells. Furthermore, we show that these VCAM-1+ICAM-1+ cells are mesenchymal cells that are activated by lymphoid cells through the LTβR to express adhesion molecules and chemokines. Thus, promotion of PP development relies on mutual interaction between mesenchymal and lymphoid cells.
Expression of the Serpin Serine Protease Inhibitor 6 Protects Dendritic Cells from Cytotoxic T Lymphocyte–Induced Apoptosis
Authors: Medema Jan Paul , Schuurhuis Danita H. , Rea Delphine , van Tongeren Joost , de Jong Joan , Bres Sandra A. , Laban Sandra , Toes René E.M. , Toebes Mireille , Schumacher Ton N.M. , Bladergroen Bellinda A. , Ossendorp Ferry , Kummer J. Alain , Melief Cornelis J.M. , Offringa Rienk
Published: July 2002

Dendritic cells (DCs) play a central role in the immune system as they drive activation of T lymphocytes by cognate interactions. However, as DCs express high levels of major histocompatibility complex class I, this intimate contact may also result in elimination of DCs by activated cytotoxic T lymphocytes (CTLs) and thereby limit induction of immunity. We show here that immature DCs are indeed susceptible to CTL-induced killing, but become resistant upon maturation with anti-CD40 or lipopolysaccharide. Protection is achieved by expression of serine protease inhibitor (SPI)-6, a member of the serpin family that specifically inactivates granzyme B and thereby blocks CTL-induced apoptosis. Anti-CD40 and LPS-induced SPI-6 expression is sustained for long periods of time, suggesting a role for SPI-6 in the longevity of DCs. Importantly, T helper 1 cells, which mature DCs and boost CTL immunity, induce SPI-6 expression and subsequent DC resistance. In contrast, T helper 2 cells neither induce SPI-6 nor convey protection, despite the fact that they trigger DC maturation with comparable efficiency. Our data identify SPI-6 as a novel marker for DC function, which protects DCs against CTL-induced apoptosis.
Reprogramming of the Macrophage Transcriptome in Response to Interferon-γ and Mycobacterium tuberculosis
Authors: Ehrt Sabine , Schnappinger Dirk , Bekiranov Stefan , Drenkow Jörg , Shi Shuangping , Gingeras Thomas R. , Gaasterland Terry , Schoolnik Gary , Nathan Carl
Published: July 2002

Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-γ (IFN-γ) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription–dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-γ and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-γ exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-γ more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.
Regulation of the Cardiac Na+-Ca2+ Exchanger by the Endogenous XIP Region
Authors: Matsuoka Satoshi , Nicoll Debora A. , He Zhaoping , Philipson Kenneth D.
Published: July 2002

The cardiac sarcolemmal Na+-Ca2+ exchanger is modulated by intrinsic regulatory mechanisms. A large intracellular loop of the exchanger participates in the regulatory responses. We have proposed (Li, Z., D.A. Nicoll, A. Collins, D.W. Hilgemann, A.G. Filoteo, J.T. Penniston, J.N. Weiss, J.M. Tomich, and K.D. Philipson. 1991. J. Biol. Chem. 266:1014–1020) that a segment of the large intracellular loop, the endogenous XIP region, has an autoregulatory role in exchanger function. We now test this hypothesis by mutational analysis of the XIP region. Nine XIP-region mutants were expressed in Xenopus oocytes and all displayed altered regulatory properties. The major alteration was in a regulatory mechanism known as Na+-dependent inactivation. This inactivation is manifested as a partial decay in outward Na+-Ca2+ exchange current after application of Na+ to the intracellular surface of a giant excised patch. Two mutant phenotypes were observed. In group 1 mutants, inactivation was markedly accelerated; in group 2 mutants, inactivation was completely eliminated. All mutants had normal Na+ affinities. Regulation of the exchanger by nontransported, intracellular Ca2+ was also modified by the XIP-region mutations. Binding of Ca2+ to the intracellular loop activates exchange activity and also decreases Na+-dependent inactivation. XIP-region mutants were all still regulated by Ca2+. However, the apparent affinity of the group 1 mutants for regulatory Ca2+ was decreased. The responses of all mutant exchangers to Ca2+ application or removal were markedly accelerated. Na+-dependent inactivation and regulation by Ca2+ are interrelated and are not completely independent processes. We conclude that the endogenous XIP region is primarily involved in movement of the exchanger into and out of the Na+-induced inactivated state, but that the XIP region is also involved in regulation by Ca2+.
Protease Modulation of the Activity of the Epithelial Sodium Channel Expressed in Xenopus Oocytes
Authors: Chraïbi Ahmed , Vallet Véronique , Firsov Dmitri , Hess Solange Kharoubi , Horisberger Jean-Daniel
Published: July 2002

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits α, β, and γ of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 μg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (∼20-fold) with the channel obtained by coexpression of the α subunit of Xenopus ENaC with the β and γ subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-γS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.
The Diphtheria Toxin Channel-forming T Domain Translocates Its Own NH2-Terminal Region Across Planar Bilayers
Authors: Senzel Lisa , Huynh Paul D. , Jakes Karen S. , John Collier R. , Finkelstein Alan
Published: July 2002

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1–201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322–378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH2 terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH2-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH2 terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH2-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH2 terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH2 terminus be translocated across the membrane from the cis to the trans side, even though the NH2 terminus is >100 residues removed from the channel-forming part of the molecule.
Nonindependent K+ Movement through the Pore in IRK1 Potassium Channels
Authors: Stampe Per , Arreola Jorge , Pérez-Cornejo Patricia , Begenisich Ted
Published: July 2002

We measured unidirectional K+ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K+ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n′, of ∼2.2 and was constant over a voltage range from −50 to −25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n′ value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K+ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K+ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current–voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K+ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K+ concentration, and may differ in the number of K+ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.
A Mutation Linked with Bartter's Syndrome Locks Kir 1.1a (Romk1) Channels in a Closed State
Authors: Flagg Thomas P. , Tate Margaret , Merot Jean , Welling Paul A.
Published: July 2002

Mutations in the inward rectifying renal K+ channel, Kir 1.1a (ROMK), have been linked with Bartter's syndrome, a familial salt-wasting nephropathy. One disease-causing mutation removes the last 60 amino acids (332–391), implicating a previously unappreciated domain, the extreme COOH terminus, as a necessary functional element. Consistent with this hypothesis, truncated channels (Kir 1.1a 331X) are nonfunctional. In the present study, the roles of this domain were systematically evaluated. When coexpressed with wild-type subunits, Kir 1.1a 331X exerted a negative effect, demonstrating that the mutant channel is synthesized and capable of oligomerization. Plasmalemma localization of Kir 1.1a 331X green fluorescent protein (GFP) fusion construct was indistinguishable from the GFP–wild-type channel, demonstrating that mutant channels are expressed on the oocyte plasma membrane in a nonconductive or locked-closed conformation. Incremental reconstruction of the COOH terminus identified amino acids 332–351 as the critical residues for restoring channel activity and uncovered the nature of the functional defect. Mutant channels that are truncated at the extreme boundary of the required domain (Kir 1.1a 351X) display marked inactivation behavior characterized by frequent occupancy in a long-lived closed state. A critical analysis of the Kir 1.1a 331X dominant negative effect suggests a molecular mechanism underlying the aberrant closed-state stabilization. Coexpression of different doses of mutant with wild-type subunits produced an intermediate dominant negative effect, whereas incorporation of a single mutant into a tetrameric concatemer conferred a complete dominant negative effect. This identifies the extreme COOH terminus as an important subunit interaction domain, controlling the efficiency of oligomerization. Collectively, these observations provide a mechanistic basis for the loss of function in one particular Bartter's-causing mutation and identify a structural element that controls open-state occupancy and determines subunit oligomerization. Based on the overlapping functions of this domain, we speculate that intersubunit interactions within the COOH terminus may regulate the energetics of channel opening.
Gap Junctional Coupling in Lenses from α8 Connexin Knockout Mice
Authors: Baldo George J. , Gong Xiaohua , Martinez-Wittinghan Francisco J. , Kumar Nalin M. , Gilula Norton B. , Mathias Richard T.
Published: July 2002

Lens fiber cell gap junctions contain α3 (Cx46) and α8 (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either α3 or α8 connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from α3 connexin knockout lenses. Here, we present original data from α8 connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell–cell contact of ∼1 S/cm2, which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is ∼0.4 S/cm2, and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/−) was ∼70% of normal and 30–40% of the normal for both −/− lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of α3 +/− lenses. In contrast, the coupling of MF in the α8 +/− lenses was the same as normal. Moreover, no coupling was detected in the MF of α3 −/− lenses. Together, these results suggest that α3 connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and α3 connexin −/− lenses. However, in α8 −/− lenses, the pure α3 connexin junctions did not gate closed in the response to acidification. Since α3 connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears α8 connexin regulates the gating of α3 connexin. Both connexins are clearly important to lens physiology as lenses null for either connexin lose transparency. Gap junctions in the MF survive for the lifetime of the organism without protein turnover. It appears that α3 connexin provides the long-term communication in MF. Gap junctions in DF may be physiologically regulated since they are capable of gating when the cytoplasm is acidified. It appears α8 connexin is required for gating in DF.
Promises and challenges of electronic journals: academic libraries surveyed
Authors: Chu Heting
Published: July 2002

In spring 1997, a questionnaire was mailed to 95 major academic libraries in the United States to explore how serials librarians are responding to the boom of electronic journals (e‐journals). Fifty librarians (52.6%) answered the survey with one follow‐up. The study found that over 70% of the libraries that returned the survey had participated in e‐journal‐related activities. Questions relating to ‘access’ were consistently ranked high in explaining why librarians in major academic libraries think e‐journals are valuable, yet ‘cost’ seems to be a critical factor in determining e‐journal adoption. For most of the libraries surveyed, the figure for e‐journals appears modest, although some institutions claimed to have more than 1,100 titles in their e‐journal collections.
Copyright Clearance for the Digital Library: a practical guide
Authors: Gadd Elizabeth
Published: July 2002

Provides a practical guide to gaining copyright clearance for making electronic copies of journal articles based on experience gained on the eLib project, Project ACORN. Includes tips on identifying and contacting copyright owners, elements to include in letters of approach, chase tactics, and dealing with refusals and charges.
Hermite multipliers and pseudo-multipliers
Authors: Epperson Jay
Published: July 2002

We prove a multiplier theorem for the Hermite-Triebel-Lizorkin spaces introduced by Epperson in [Studia Math. 114 (1995), 87–103]. This extends Thangavelu’s theorem [Revist. Mat. Ibero 3 (1987), 1–24; Math. Notes, vol. 42, 1993] on Hermite multipliers for

L
p

L^p

spaces. We also prove an

L
p

L^p

boundedness result for a class of Hermite pseudo-multipliers.
On the 𝑟-rank Artin Conjecture
Authors: Pappalardi Francesco
Published: July 2002

We assume the generalized Riemann hypothesis and prove an asymptotic formula for the number of primes for which

F

p
∗

\mathbb {F}_p^*

can be generated by

r
r

given multiplicatively independent numbers. In the case when the

r
r

given numbers are primes, we express the density as an Euler product and apply this to a conjecture of Brown–Zassenhaus (J. Number Theory 3 (1971), 306–309). Finally, in some examples, we compare the densities approximated with the natural densities calculated with primes up to

9
⋅

10
4

9\cdot 10^4

.
Preconditioning in H(𝑑𝑖𝑣) and applications
Authors: Arnold Douglas , Falk Richard , Winther R.
Published: July 2002

We consider the solution of the system of linear algebraic equations which arises from the finite element discretization of boundary value problems associated to the differential operator I

−

g
r
a
d

⁡
div

- \operatorname {\mathbf {grad}}\operatorname {div}

. The natural setting for such problems is in the Hilbert space H

(
div
)

(\operatorname {div})

and the variational formulation is based on the inner product in H

(
div
)

(\operatorname {div})

. We show how to construct preconditioners for these equations using both domain decomposition and multigrid techniques. These preconditioners are shown to be spectrally equivalent to the inverse of the operator. As a consequence, they may be used to precondition iterative methods so that any given error reduction may be achieved in a finite number of iterations, with the number independent of the mesh discretization. We describe applications of these results to the efficient solution of mixed and least squares finite element approximations of elliptic boundary value problems.
On 𝜙-amicable pairs
Authors: Cohen Graeme , te Riele Herman
Published: July 2002

Let

ϕ
(
n
)

\phi (n)

denote Euler’s totient function, i.e., the number of positive integers 

>
n

>n

and prime to

n
n

. We study pairs of positive integers

(

a

0

,

a

1

)

(a_{0},a_{1})

with

a

0

≤

a

1

a_{0}\le a_{1}

such that

ϕ
(

a

0

)
=
ϕ
(

a

1

)
=
(

a

0

+

a

1

)

/

k

\phi (a_{0})=\phi (a_{1})=(a_{0}+a_{1})/k

for some integer

k
≥
1

k\ge 1

. We call these numbers

ϕ
\phi

–amicable pairs with multiplier

k
k

, analogously to Carmichael’s multiply amicable pairs for the

σ
\sigma

–function (which sums all the divisors of

n
n

). We have computed all the

ϕ
\phi

–amicable pairs with larger member

≤

10

9

\le 10^{9}

and found

812
812

pairs for which the greatest common divisor is squarefree. With any such pair infinitely many other

ϕ
\phi

–amicable pairs can be associated. Among these

812
812

pairs there are

499
499

so-called primitive

ϕ
\phi

–amicable pairs. We present a table of the

58
58

primitive

ϕ
\phi

–amicable pairs for which the larger member does not exceed

10

6

10^{6}

. Next,

ϕ
\phi

–amicable pairs with a given prime structure are studied. It is proved that a relatively prime

ϕ
\phi

–amicable pair has at least twelve distinct prime factors and that, with the exception of the pair

(
4
,
6
)

(4,6)

, if one member of a

ϕ
\phi

–amicable pair has two distinct prime factors, then the other has at least four distinct prime factors. Finally, analogies with construction methods for the classical amicable numbers are shown; application of these methods yields another 79 primitive

ϕ
\phi

–amicable pairs with larger member

>

10

9

>10^{9}

, the largest pair consisting of two 46-digit numbers.
The second largest prime divisor of an odd perfect number exceeds ten thousand
Authors: Iannucci Douglas
Published: July 2002

Let

σ
(
n
)

\sigma (n)

denote the sum of positive divisors of the natural number

n
n

. Such a number is said to be perfect if

σ
(
n
)
=
2
n

\sigma (n)=2n

. It is well known that a number is even and perfect if and only if it has the form

2

p
−
1

(

2
p

−
1
)

2^{p-1} (2^p-1)

where

2
p

−
1

2^p-1

is prime.

No odd perfect numbers are known, nor has any proof of their nonexistence ever been given. In the meantime, much work has been done in establishing conditions necessary for their existence. One class of necessary conditions would be lower bounds for the distinct prime divisors of an odd perfect number.

For example, Cohen and Hagis have shown that the largest prime divisor of an odd perfect number must exceed

10
6

10^6

, and Hagis showed that the second largest must exceed

10
3

10^3

. In this paper, we improve the latter bound. In particular, we prove the statement in the title of this paper.
Cayley-Bacharach theorems and conjectures
Authors: Eisenbud David , Green Mark , Harris Joe
Published: July 2002

A theorem of Pappus of Alexandria, proved in the fourth century A.D., began a long development in algebraic geometry. In its changing expressions one can see reflected the changing concerns of the field, from synthetic geometry to projective plane curves to Riemann surfaces to the modern development of schemes and duality. We survey this development historically and use it to motivate a brief treatment of a part of duality theory. We then explain one of the modern developments arising from it, a series of conjectures about the linear conditions imposed by a set of points in projective space on the forms that vanish on them. We give a proof of the conjectures in a new special case.
Local Rankin-Selberg convolutions for 𝐺𝐿_{𝑛}: Explicit conductor formula
Authors: Bushnell Colin , Henniart Guy , Kutzko Philip
Published: July 2002

Let

F
F

be a non-Archimedean local field and

n

1

n_{1}

,

n

2

n_{2}

positive integers. For

i
=
1
,
2

i=1,2

, let

G

i

=

G
L

n

i

(
F
)

G_{i}=\mathrm {GL}_{n_{i}}(F)

and let

π

i

\pi _{i}

be an irreducible supercuspidal representation of

G

i

G_{i}

. Jacquet, Piatetskii-Shapiro and Shalika have defined a local constant

ε
(

π

1

×

π

2

,
s
,
ψ
)

\varepsilon (\pi _{1}\times \pi _{2},s,\psi )

to the

π

i

\pi _{i}

and an additive character

ψ
\psi

of

F
F

. This object is of central importance in the study of the local Langlands conjecture. It takes the form

ε
(

π

1

×

π

2

,
s
,
ψ
)
=

q

−
f
s

ε
(

π

1

×

π

2

,
0
,
ψ
)
,

\begin{equation*}\varepsilon (\pi _{1}\times \pi _{2},s,\psi ) = q^{-fs}\varepsilon (\pi _{1} \times \pi _{2},0,\psi ), \end{equation*}

where

f
=
f
(

π

1

×

π

2

,
ψ
)

f=f(\pi _{1}\times \pi _{2},\psi )

is an integer. The irreducible supercuspidal representations of

G
=

G
L

n

(
F
)

G=\mathrm {GL}_{n}(F)

have been described explicitly by Bushnell and Kutzko, via induction from open, compact mod centre, subgroups of

G
G

. This paper gives an explicit formula for

f
(

π

1

×

π

2

,
ψ
)

f(\pi _{1} \times \pi _{2},\psi )

in terms of the inducing data for the

π

i

\pi _{i}

. It uses, on the one hand, the alternative approach to the local constant due to Shahidi, and, on the other, the general theory of types along with powerful existence theorems for types in

G
L

(
n
)

\mathrm {GL}(n)

, developed by Bushnell and Kutzko.
Grothendieck’s theorem on non-abelian 𝐻² and local-global principles
Authors: Flicker Yuval , Scheiderer Claus , Sujatha R.
Published: July 2002

A theorem of Grothendieck asserts that over a perfect field

k
k

of cohomological dimension one, all non-abelian

H

2

H^{2}

-cohomology sets of algebraic groups are trivial. The purpose of this paper is to establish a formally real generalization of this theorem. The generalization — to the context of perfect fields of virtual cohomological dimension one — takes the form of a local-global principle for the

H

2

H^{2}

-sets with respect to the orderings of the field. This principle asserts in particular that an element in

H

2

H^{2}

is neutral precisely when it is neutral in the real closure with respect to every ordering in a dense subset of the real spectrum of

k
k

. Our techniques provide a new proof of Grothendieck’s original theorem. An application to homogeneous spaces over

k
k

is also given.
Nonlocal finiteness of a 𝑊-graph
Authors: Lusztig George
Published: July 2002

It is shown that the

W
W

-graph of an affine Weyl group of type

B

2

B_{2}

(as defined by Kazhdan and Lusztig in Representations of Coxeter groups and Hecke algebras, Invent. Math. 53 (1979), 165–184) is not locally finite.
Repression and Activation Domains of Rme1p Structurally Overlap, but Differ in Genetic Requirements
Authors: Blumental-Perry Anna , Li Weishi , Simchen Giora , Mitchell Aaron P.
Published: July 2002

Rme1p, a repressor of meiosis in the yeast Saccharomyces cerevisiae, acts as both a transcriptional repressor and activator. Rme1p is a zinc-finger protein with no other homology to any protein of known function. The C-terminal DNA binding domain of Rme1p is essential for function. We find that mutations and progressive deletions in all three zinc fingers can be rescued by fusion ofRME1 to the DNA binding domain of another protein. Thus, structural integrity of the zinc fingers is not required for the Rme1p-mediated effects on transcription. Using a series of mutant Rme1 proteins, we have characterized domains responsible for repression and activation. We find that the minimal transcriptional repression and activation domains completely overlap and lie in an 88-amino-acid N-terminal segment (aa 61–148). An additional transcriptional effector determinant lies in the first 31 amino acids of the protein. Notwithstanding the complete overlap between repression and activation domains of Rme1p, we demonstrated a functional difference between repression and activation: Rgr1p and Sin4p are absolutely required for repression but dispensable for activation.
Comprehensive, comprehensible, distributed and intelligent databases: current status.
Authors: Frishman D , Heumann K , Lesk A , Mewes H W
Published: July 2002

MOTIVATION: It is only a matter of time until a user will see not many but one integrated database of information for molecular biology. Is this true? Is it a good thing? Why will it happen? Where are we now? What developments are fostering and what developments are impeding progress towards this end? SUPPLEMENTARY INFORMATION: A list of WWW resources devoted to database issues in molecular biology is available at http://www.mips.biochem.mpg.de CONTACT: frishman@mips.biochem.mpg.de
BLAST Search Updater: a notification system for new database
matches
Authors: Boone Matthew , Upton Chris
Published: July 2002

Summary: BLAST Search Updater (BSU) can help molecular biologists perform large numbers of BLAST searches routinely and screen the results for novel matches. The search result files (HTML format) and delta files (novel matches) are WWW viewable and BSU also emails users alignments of new matches.

Availability: BSU runs on UNIX machines and is freely available to academic users at http://athena.bioc.uvic.ca/genomes/. Additional figures and description are also available.

Contact: cupton@uvic.ca

To whom correspondence should be addressed.