bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement of the Chemokine Receptor CXCR3 for Acute Allograft Rejection
Authors: Hancock Wayne W. , Lu Bao , Gao Wei , Csizmadia Vilmos , Faia Kerrie , King Jennifer A. , Smiley Stephen T. , Ling Mai , Gerard Norma P. , Gerard Craig
Published: July 2002

Chemokines provide signals for activation and recruitment of effector cells into sites of inflammation, acting via specific G protein–coupled receptors. However, in vitro data demonstrating the presence of multiple ligands for a given chemokine receptor, and often multiple receptors for a given chemokine, have led to concerns of biologic redundancy. Here we show that acute cardiac allograft rejection is accompanied by progressive intragraft production of the chemokines interferon (IFN)-γ–inducible protein of 10 kD (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T cell α chemoattractant (I-TAC), and by infiltration of activated T cells bearing the corresponding chemokine receptor, CXCR3. We used three in vivo models to demonstrate a role for CXCR3 in the development of transplant rejection. First, CXCR3-deficient (CXCR3−/−) mice showed profound resistance to development of acute allograft rejection. Second, CXCR3−/− allograft recipients treated with a brief, subtherapeutic course of cyclosporin A maintained their allografts permanently and without evidence of chronic rejection. Third, CXCR+/+ mice treated with an anti-CXCR3 monoclonal antibody showed prolongation of allograft survival, even if begun after the onset of rejection. Taken in conjunction with our findings of CXCR3 expression in rejecting human cardiac allografts, we conclude that CXCR3 plays a key role in T cell activation, recruitment, and allograft destruction.
Melanocyte Destruction after Antigen-Specific Immunotherapy of Melanoma
Authors: Yee Cassian , Thompson John A. , Roche Patrick , Byrd David R. , Lee Peter P. , Piepkorn Michael , Kenyon Karla , Davis Mark M. , Riddell Stanley R. , Greenberg Philip D.
Published: July 2002

Current strategies for the immunotherapy of melanoma include augmentation of the immune response to tumor antigens represented by melanosomal proteins such as tyrosinase, gp100, and MART-1. The possibility that intentional targeting of tumor antigens representing normal proteins can result in autoimmune toxicity has been postulated but never demonstrated previously in humans. In this study, we describe a patient with metastatic melanoma who developed inflammatory lesions circumscribing pigmented areas of skin after an infusion of MART-1–specific CD8+ T cell clones. Analysis of the infiltrating lymphocytes in skin and tumor biopsies using T cell–specific peptide–major histocompatibility complex tetramers demonstrated a localized predominance of MART-1–specific CD8+ T cells (>28% of all CD8 T cells) that was identical to the infused clones (as confirmed by sequencing of the complementarity-determining region 3). In contrast to skin biopsies obtained from the patient before T cell infusion, postinfusion biopsies demonstrated loss of MART-1 expression, evidence of melanocyte damage, and the complete absence of melanocytes in affected regions of the skin. This study provides, for the first time, direct evidence in humans that antigen-specific immunotherapy can target not only antigen-positive tumor cells in vivo but also normal tissues expressing the shared tumor antigen.
Bltr Mediates Leukotriene B4–Induced Chemotaxis and Adhesion and Plays a Dominant Role in Eosinophil Accumulation in a Murine Model of Peritonitis
Authors: Tager Andrew M. , Dufour Jennifer H. , Goodarzi Katayoon , Bercury Scott D. , von Andrian Ulrich H. , Luster Andrew D.
Published: July 2002

Leukotriene B4 (LTB4) is a potent chemoattractant active on multiple leukocytes, including neutrophils, macrophages, and eosinophils, and is implicated in the pathogenesis of a variety of inflammatory processes. A seven transmembrane–spanning, G protein–coupled receptor, called BLTR (LTB4 receptor), has recently been identified as an LTB4 receptor. To determine if BLTR is the sole receptor mediating LTB4-induced leukocyte activation and to determine the role of LTB4 and BLTR in regulating leukocyte function in inflammation in vivo, we generated a BLTR-deficient mouse by targeted gene disruption. This mouse reveals that BLTR alone is responsible for LTB4-mediated leukocyte calcium flux, chemotaxis, and firm adhesion to endothelium in vivo. Furthermore, despite the apparent functional redundancy with other chemoattractant–receptor pairs in vitro, LTB4 and BLTR play an important role in the recruitment and/or retention of leukocytes, particularly eosinophils, to the inflamed peritoneum in vivo. These studies demonstrate that BLTR is the key receptor that mediates LTB4-induced leukocyte activation and establishes a model to decipher the functional roles of BLTR and LTB4 in vivo.
Efficient Identification of Novel Hla-A*0201–Presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen Prame by Proteasome-Mediated Digestion Analysis
Authors: Kessler Jan H. , Beekman Nico J. , Bres-Vloemans Sandra A. , Verdijk Pauline , van Veelen Peter A. , Kloosterman-Joosten Antoinette M. , Vissers Debby C.J. , ten Bosch George J.A. , Kester Michel G.D. , Sijts Alice , Drijfhout Jan Wouter , Ossendorp Ferry , Offringa Rienk , Melief Cornelis J.M.
Published: July 2002

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.
Pivotal Role of Signal Transducer and Activator of Transcription (Stat)4 and Stat6 in the Innate Immune Response during Sepsis
Authors: Matsukawa Akihiro , Kaplan Mark H. , Hogaboam Cory M. , Lukacs Nicholas W. , Kunkel Steven L.
Published: July 2002

Signal transducer and activator of transcription (Stat)4 and Stat6 are transcription factors that provide type 1 and type 2 response, respectively. Here, we explored the role of Stat4 and Stat6 in innate immunity during septic peritonitis. Stat4−/− and Stat6−/− mice were resistant to the lethality compared with wild-type (WT) mice. At the mechanistic level, bacterial levels in Stat6−/− mice were much lower than in WT mice, which was associated with increased peritoneal levels of interleukin (IL)-12, tumor necrosis factor (TNF)-α, macrophage-derived chemokine (MDC), and C10, known to enhance bacterial clearance. In Stat4−/− mice, hepatic inflammation and injury during sepsis were significantly ameliorated without affecting local responses. This event was associated with increased hepatic levels of IL-10 and IL-13, while decreasing those of macrophage inflammatory protein (MIP)-2 and KC. Sepsis-induced renal injury was also abrogated in Stat4−/− mice, which was accompanied by decreased renal levels of MIP-2 and KC without altering IL-10 and IL-13 levels. Thus, Stat6−/− and Stat4−/− mice appeared to be resistant to septic peritonitis by enhancing local bacterial clearance and modulating systemic organ damage, respectively, via balancing cytokine responses. These results clearly highlight an important role of local type 1 and systemic type 2 cytokine response in protective immunity during sepsis, which can be regulated by Stat proteins.
The Inhibition of Arginase by Nω-Hydroxy-l-Arginine Controls the Growth of Leishmania Inside Macrophages
Authors: Iniesta Virginia , Gómez-Nieto L. Carlos , Corraliza Inés
Published: July 2002

Polyamine synthesis from l-ornithine is essential for Leishmania growth. We have investigated the dependence of Leishmania infection on arginase, which generates l-ornithine, in macrophages from BALB/c, C57BL/6, and nitric oxide synthase II (NOS II)-deficient mouse strains. We have found that Nω-hydroxy-l-arginine (LOHA), a physiological inhibitor of arginase, controls cellular infection and also specifically inhibits arginase activity from Leishmania major and Leishmania infantum parasites. The effect was proportional to the course of infection, concentration dependent up to 100 μM, and achieved without an increase in nitrite levels of culture supernatants. Moreover, when the l-arginine metabolism of macrophages is diverted towards ornithine generation by interleukin 4–induced arginase I, parasite growth is promoted. This effect can be reversed by LOHA. Inhibition of NOS II by NG-methyl-l-arginine (LNMMA) restores the killing obtained in the presence of interferon (IFN)-γ plus lipolysaccharide (LPS), whereas the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl (carboxy-PTIO) was without effect. However, exogenous l-ornithine almost completely inhibits parasite killing when added in the presence of LOHA to macrophages from NOS II–deficient mice or to BALB/c-infected cells activated with IFN-γ plus LPS. These results suggest that LOHA is an effector molecule involved in the control of Leishmania infection. In addition, macrophage arginase I induction by T helper cell type 2 cytokines could be a mechanism used by parasites to spread inside the host.
Ligand-independent Signaling Functions for the B Lymphocyte Antigen Receptor and Their Role in Positive Selection during B Lymphopoiesis
Authors: Bannish Gregory , Fuentes-Pananá Ezequiel M. , Cambier John C. , Pear Warren S. , Monroe John G.
Published: July 2002

Signal transduction through the B cell antigen receptor (BCR) is determined by a balance of positive and negative regulators. This balance is shifted by aggregation that results from binding to extracellular ligand. Aggregation of the BCR is necessary for eliciting negative selection or activation by BCR-expressing B cells. However, ligand-independent signaling through intermediate and mature forms of the BCR has been postulated to regulate B cell development and peripheral homeostasis. To address the importance of ligand-independent BCR signaling functions and their regulation during B cell development, we have designed a model that allows us to isolate the basal signaling functions of immunoglobulin (Ig)α/Igβ-containing BCR complexes from those that are dependent upon ligand-mediated aggregation. In vivo, we find that basal signaling is sufficient to facilitate pro-B → pre-B cell transition and to generate immature/mature peripheral B cells. The ability to generate basal signals and to drive developmental progression were both dependent on plasma membrane association of Igα/Igβ complexes and intact immunoregulatory tyrosine activation motifs (ITAM), thereby establishing a correlation between these processes. We believe that these studies are the first to directly demonstrate biologically relevant basal signaling through the BCR where the ability to interact with both conventional as well as nonconventional extracellular ligands is eliminated.
Synergism of Cytotoxic T Lymphocyte–Associated Antigen 4 Blockade and Depletion of Cd25+ Regulatory T Cells in Antitumor Therapy Reveals Alternative Pathways for Suppression of Autoreactive Cytotoxic T Lymphocyte Responses
Authors: Sutmuller Roger P.M. , van Duivenvoorde Leonie M. , van Elsas Andrea , Schumacher Ton N.M. , Wildenberg Manon E. , Allison James P. , Toes Rene E.M. , Offringa Rienk , Melief Cornelis J.M.
Published: July 2002

Therapeutic efficacy of a tumor cell–based vaccine against experimental B16 melanoma requires the disruption of either of two immunoregulatory mechanisms that control autoreactive T cell responses: the cytotoxic T lymphocyte–associated antigen (CTLA)-4 pathway or the CD25+ regulatory T (Treg) cells. Combination of CTLA-4 blockade and depletion of CD25+ Treg cells results in maximal tumor rejection. Efficacy of the antitumor therapy correlates with the extent of autoimmune skin depigmentation as well as with the frequency of tyrosinase-related protein 2180–188–specific CTLs detected in the periphery. Furthermore, tumor rejection is dependent on the CD8+ T cell subset. Our data demonstrate that the CTL response against melanoma antigens is an important component of the therapeutic antitumor response and that the reactivity of these CTLs can be augmented through interference with immunoregulatory mechanisms. The synergism in the effects of CTLA-4 blockade and depletion of CD25+ Treg cells indicates that CD25+ Treg cells and CTLA-4 signaling represent two alternative pathways for suppression of autoreactive T cell immunity. Simultaneous intervention with both regulatory mechanisms is therefore a promising concept for the induction of therapeutic antitumor immunity.
A Trypanosoma cruzi Small Surface Molecule Provides the First Immunological Evidence that Chagas' Disease Is Due to a Single Parasite Lineage
Authors: Di Noia Javier M. , Buscaglia Carlos A. , De Marchi Claudia R. , Almeida Igor C. , Frasch Alberto C.C.
Published: July 2002

Chagas' disease is a major health and economic problem caused by the protozoan Trypanosoma cruzi. Multiple independently evolving clones define a complex parasite population that can be arranged into two broad genetic lineages termed T. cruzi I and II. These lineages have different evolutionary origin and display distinct ecological and biological traits. Here we describe a novel molecule termed TSSA for trypomastigote small surface antigen that provides the first immunological marker allowing discrimination between lineages. TSSA is a surface, glycosylphosphatidyl inositol (GPI)-anchored mucin-like protein, highly antigenic during the infection. TSSA sequences from different parasite isolates reveal a population dimorphism that perfectly matches with the two T. cruzi lineages. Interestingly, this dimorphism is restricted to the central region of the molecule, which comprises the immunodominant B cell epitopes. This sequence variability has a major impact on TSSA antigenicity, leading to no immunological cross-reactivity between both isoforms for antibodies present either in immunization or infection sera. Furthermore, the absolute seroprevalence for TSSA in confirmed Chagasic patients is restricted to T. cruzi II isoform, strongly suggesting that human infections are due to this particular subgroup. Even though association of T. cruzi II with Chagas' disease has been proposed based on molecular markers, this is the first immunological evidence supporting this hypothesis. The implications of these results for the future research on Chagas' disease could be envisaged.
Lipid Raft Microdomains
Authors: Bavari Sina , Bosio Catharine M. , Wiegand Elizabeth , Ruthel Gordon , Will Amy B. , Geisbert Thomas W. , Hevey Michael , Schmaljohn Connie , Schmaljohn Alan , Aman M. Javad
Published: July 2002

Spatiotemporal aspects of filovirus entry and release are poorly understood. Lipid rafts act as functional platforms for multiple cellular signaling and trafficking processes. Here, we report the compartmentalization of Ebola and Marburg viral proteins within lipid rafts during viral assembly and budding. Filoviruses released from infected cells incorporated raft-associated molecules, suggesting that viral exit occurs at the rafts. Ectopic expression of Ebola matrix protein and glycoprotein supported raft-dependent release of filamentous, virus-like particles (VLPs), strikingly similar to live virus as revealed by electron microscopy. Our findings also revealed that the entry of filoviruses requires functional rafts, identifying rafts as the site of virus attack. The identification of rafts as the gateway for the entry and exit of filoviruses and raft-dependent generation of VLPs have important implications for development of therapeutics and vaccination strategies against infections with Ebola and Marburg viruses.
Virus-induced Interferon α Production by a Dendritic Cell Subset in the Absence of Feedback Signaling In Vivo
Authors: Barchet Winfried , Cella Marina , Odermatt Bernhard , Asselin-Paturel Carine , Colonna Marco , Kalinke Ulrich
Published: July 2002

An effective type I interferon (IFN-α/β) response is critical for the control of many viral infections. Here we show that in vesicular stomatitis virus (VSV)-infected mouse embryonic fibroblasts (MEFs) the production of IFN-α is dependent on type I IFN receptor (IFNAR) triggering, whereas in infected mice early IFN-α production is IFNAR independent. In VSV-infected mice type I IFN is produced by few cells located in the marginal zone of the spleen. Unlike other dendritic cell (DC) subsets, FACS®-sorted CD11cintCD11b−GR-1+ DCs show high IFN-α expression, irrespective of whether they were isolated from VSV-infected IFNAR-competent or -deficient mice. Thus, VSV preferentially activates a specialized DC subset presumably located in the marginal zone to produce high-level IFN-α largely independent of IFNAR feedback signaling.
Contribution of Sialic Acid to the Voltage Dependence of Sodium Channel Gating
Authors: Bennett Eric , Urcan Mary S. , Tinkle Sally S. , Koszowski Adam G. , Levinson Simon R.
Published: July 2002

A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium channels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line (lec2). The steady-state half-activation voltages (Va) of channels under each condition of reduced sialylation were ∼10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10 mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for channels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements.
Equilibrium of Phosphointermediates of Sodium and Potassium Ion Transport Adenosine Triphosphatase
Authors: Suzuki Kuniaki , Post Robert L.
Published: July 2002

Sodium and potassium ion transport adenosine triphosphatase accepts and donates a phosphate group in the course of its reaction sequence. The phosphorylated enzyme has two principal reactive states, E1P and E2P. E1P is formed reversibly from ATP in the presence of Na+ and is precursor to E2P, which equilibrates with Pi in the presence of K+. We studied equilibrium between these states at 4°C and the effect of Na+ on it. To optimize the reaction system we used a Hofmeister effect, replacing the usual anion, chloride, with a chaotropic anion, usually nitrate. We phosphorylated enzyme from canine kidney with [32P]ATP. We estimated interconversion rate constants for the reaction E1P ⇌ E2P and their ratio. To estimate rate constants we terminated phosphorylation and observed decay kinetics. We observed E1P or E2P selectively by adding K+ or ADP respectively. K+ dephosphorylates E2P leaving E1P as observable species; ADP dephosphorylates E1P leaving E2P as observable species. We fitted a 2-pool model comprising two reactive species or a twin 2-pool model, comprising a pair of independent 2-pool models, to the data and obtained interconversion and hydrolysis rate constants for each state. Replacing Na+ with Tris+ or lysine+ did not change the ratio of interconversion rate constants between E1P and E2P. Thus Na+ binds about equally strongly to E1P and E2P. This conclusion is consistent with a model of Pedemonte (1988. J. Theor. Biol. 134:165–182.). We found that Na+ affected another equilibrium, that of transphosphorylation between ATP·dephosphoenzyme and ADP·E1P. We used the reactions and model of Pickart and Jencks (1982. J. Biol. Chem. 257:5319–5322.) to generate and fit data. Decreasing the concentration of Na+ 10-fold shifted the equilibrium constant 10-fold favoring ADP·E1P over ATP·dephosphoenzyme. Thus Na+ can dissociate from E1P·Na3. Furthermore, we found two characteristics of Hofmeister effects on this enzyme.
Adenosine Triphosphate–dependent Asymmetry of Anion Permeation in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel
Authors: Linsdell Paul , Hanrahan John W.
Published: July 2002

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a tightly regulated channel that mediates the passive diffusion of Cl− ions. Here we show, using macroscopic current recording from excised membrane patches, that CFTR also shows significant, but highly asymmetrical, permeability to a broad range of large organic anions. Thus, all large organic anions tested were permeant when present in the intracellular solution under biionic conditions (PX/PCl = 0.048–0.25), whereas most were not measurably permeant when present in the extracellular solution. This asymmetry was not observed for smaller anions. ATPase inhibitors that “lock” CFTR channels in the open state (pyrophosphate, 5′-adenylylimidodiphosphate) disrupted the asymmetry of large anion permeation by allowing their influx from the extracellular solution, which suggests that ATP hydrolysis is required to maintain asymmetric permeability. The ability of CFTR to allow efflux of large organic anions represents a novel function of CFTR. Loss of this function may contribute to the pleiotropic symptoms seen in cystic fibrosis.
Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells
Authors: Ishikawa Toru , Marunaka Yoshinori , Rotin Daniela
Published: July 2002

The epithelial Na+ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na+ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na+ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na+ conductance was Li+ > Na+ >> K+ = N-methyl-d-glucamine+ (NMDG+). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na+ channel current, was found to be ∼5 and 8 pS when Na+ and Li+ were used as a charge carrier, respectively. K+ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (Po), was increased by membrane hyperpolarization. Both whole-cell Na+ current and conductance were saturated with increased extracellular Na+ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nPo) was significantly decreased when cytosolic Na+ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na+ conductance (with Li+ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca2+ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca2+ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca2+ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca2+ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca2+ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na+ and Ca2+.
Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel
Authors: Fyfe Gregor K. , Canessa Cecilia M.
Published: July 2002

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of α with either wild-type β or γ subunits and α with carboxy-terminal truncated β (βT) or γ (γT) subunits in Xenopus laevis oocytes. αβ and αβT channels (9.6 and 8.7 pS, respectively, with 150 mM Li+) were found to be constitutively open. Only upon inclusion of 1 μM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (Po) for αβ was 0.54 and for αβT was 0.50. In comparison, αγ and αγT channels exhibited different kinetics: αγ channels (6.7 pS in Li+) had either long open times with short closings, resulting in a high Po (0.78), or short openings with long closed times, resulting in a low Po (0.16). The mean Po for all αγ channels was 0.48. αγT (6.6 pS in Li+) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean Po of 0.6, similar to that of αγ. Inclusion of 0.1 μM amiloride in the pipette solution reduced the mean open time of αγT to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of αβ, αβT (1 μM amiloride), and αγT (0.1 μM amiloride) channels. αβ and αβT had similar blocking and unblocking rate constants, whereas the unblocking rate constant for αγT was 10-fold slower than αβT. Our results indicate that subunit composition of ENaC is a main determinant of Po. In addition, channel kinetics and Po are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the γ subunit affects channel kinetics but not Po.
Single Channel Properties of P2X2 Purinoceptors
Authors: Ding Shinghua , Sachs Frederick
Published: July 2002

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.
Role of Calcium Permeation in Dihydropyridine Receptor Function
Authors: Dirksen Robert T. , Beam Kurt G.
Published: July 2002

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca2+ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809–2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca2+ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca2+ currents at a few potentials (from −20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca2+ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca2+ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: τact = 30.7 ± 1.9 ms, n = 11; CEIIIK: τact = 2.9 ± 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca2+ transients with parameters of voltage dependence (V0.5 = 6.5 ± 3.2 mV and k = 9.3 ± 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125–147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca2+ transients at any potential. Thus, high Ca2+ permeation is required for cardiac-type excitation–contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to −50 mV (Qoff) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Qoff was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 ± 2.0%, n = 6). The increase in Qoff for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Qoff may reflect a voltage sensor movement required for activation of L-type Ca2+ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.
Swelling-Induced, Cftr-Independent Atp Release from a Human Epithelial Cell Line
Authors: Hazama Akihiro , Shimizu Takahiro , Ando-Akatsuka Yuhko , Hayashi Seiji , Tanaka Shoko , Maeno Emi , Okada Yasunobu
Published: July 2002

To examine a possible relation between the swelling-induced ATP release pathway and the volume-sensitive Cl− channel, we measured the extracellular concentration of ATP released upon osmotic swelling and whole-cell volume-sensitive Cl− currents in a human epithelial cell line, Intestine 407, which lacks expression of cystic fibrosis transmembrane conductance regulator (CFTR). Significant release of ATP was observed within several minutes after a hypotonic challenge (56–80% osmolality) by the luciferin/luciferase assay. A carboxylate analogue Cl− channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, suppressed ATP release in a concentration-dependent manner with a half-maximal inhibition concentration of 6.3 μM. However, swelling-induced ATP release was not affected by a stilbene-derivative Cl− channel blocker, 4-acetamido-4′-isothiocyanostilbene at 100 μM. Glibenclamide (500 μM) and arachidonic acid (100 μM), which are known to block volume-sensitive outwardly rectifying (VSOR) Cl− channels, were also ineffective in inhibiting the swelling-induced ATP release. Gd3+, a putative blocker of stretch-activated channels, inhibited swelling-induced ATP release in a concentration-dependent manner, whereas the trivalent lanthanide failed to inhibit VSOR Cl− currents. Upon osmotic swelling, the local ATP concentration in the immediate vicinity of the cell surface was found to reach ∼13 μM by a biosensor technique using P2X2 receptors expressed in PC12 cells. We have raised antibodies that inhibit swelling-induced ATP release from Intestine 407 cells. Earlier treatment with the antibodies almost completely suppressed swelling-induced ATP release, whereas the activity of VSOR Cl− channel was not affected by pretreatment with the antibodies. Taking the above results together, the following conclusions were reached: first, in a CFTR-lacking human epithelial cell line, osmotic swelling induces ATP release and increases the cell surface ATP concentration over 10 μM, which is high enough to stimulate purinergic receptors; second, the pathway of ATP release is distinct from the pore of the volume-sensitive outwardly rectifying Cl− channel; and third, the ATP release is not a prerequisite to activation of the Cl− channel.
Rapid Activation of the Cardiac Ryanodine Receptor by Submillisecond Calcium Stimuli
Authors: Zahradníková A. , Zahradník I. , Györke I. , Györke S.
Published: July 2002

The local control concept of excitation–contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca2+entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (<0.5 ms) Ca2+elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca2+signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca2+generated by flash photolysis of DM-nitrophen. Application of such Ca2+spikes (amplitude ∼10–30 μM, duration ∼0.1–0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope ∼2.5) with spike amplitude. The time constants of RyR activation were 0.07–0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a powern∼ 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca2+-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca2+triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca2+ions in accordance with the tetrameric organization of the channel protein.
The Role of Steady Phosphodiesterase Activity in the Kinetics and Sensitivity of the Light-Adapted Salamander Rod Photoresponse
Authors: Nikonov S. , Lamb T.D. , Pugh E.N.
Published: July 2002

We investigated the kinetics and sensitivity of photocurrent responses of salamander rods, both in darkness and during adaptation to steady backgrounds producing 20–3,000 photoisomerizations per second, using suction pipet recordings. The most intense backgrounds suppressed 80% of the circulating dark current and decreased the flash sensitivity ∼30-fold. To investigate the underlying transduction mechanism, we expressed the responses as a fraction of the steady level of cGMP-activated current recorded in the background. The fractional responses to flashes of any fixed intensity began rising along a common trajectory, regardless of background intensity. We interpret these invariant initial trajectories to indicate that, at these background intensities, light adaptation does not alter the gain of any of the amplifying steps of phototransduction. For subsaturating flashes of fixed intensity, the fractional responses obtained on backgrounds of different intensity were found to “peel off” from their common initial trajectory in a background-dependent manner: the more intense the background, the earlier the time of peeling off. This behavior is consistent with a background-induced reduction in the effective lifetime of at least one of the three major integrating steps in phototransduction; i.e., an acceleration of one or more of the following: (1) the inactivation of activated rhodopsin (R*); (2) the inactivation of activated phosphodiesterase (E*, representing the complex Gα–PDE of phosphodiesterase with the transducin α-subunit); or (3) the hydrolysis of cGMP, with rate constant β. Our measurements show that, over the range of background intensities we used, β increased on average to ∼20 times its dark-adapted value; and our theoretical analysis indicates that this increase in β is the primary mechanism underlying the measured shortening of time-to-peak of the dim-flash response and the decrease in sensitivity of the fractional response.
Intracellular Mg2+ Enhances the Function of Bk-Type Ca2+-Activated K+ Channels
Authors: Shi Jingyi , Cui Jianmin
Published: July 2002

BK channels modulate neurotransmitter release due to their activation by voltage and Ca2+. Intracellular Mg2+ also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg2+ blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg2+ activates mslo1 BK channels independently of Ca2+ and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca2+ binding sites in the tail, is not activated by Mg2+. However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg2+ sensitivity similar to mslo1 channels, indicating that Mg2+ sites differ from Ca2+ sites. We discovered that Mg2+ also binds to Ca2+ sites and competitively inhibits Ca2+-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg2+ under physiological conditions is to enhance BK channel function.
Crisis and transition: the economics of scholarly communication
Authors: Houghton John W.
Published: July 2002

This paper explores the scholarly communication ‘industry’ using a product systems approach, scopes and sizes the Australian scholarly communication product system, and examines the economics behind its operation. We find that the set of interrelationships between the market characteristics of information and cost structures, patterns of demand and acquisition practices, and the forces encouraging increasing scholarly output go a long way towards explaining the operation of the system and the genesis of the crisis. Based on our economic analysis of the system, we outline broad market conforming and market distorting approaches to alleviating the crisis.
The links in the information chain
Authors: Jones Simon
Published: July 2002

Thanks to the World Wide Web, users now expect to link easily between disparate information services. It is no longer enough to study raw data in one database, search for relevant published articles in another, and look at the full articles in yet another. Users want to move seamlessly between all these sources, in order to follow lines of research efficiently and accurately. What are the problems in building links using data resources which were not designed for this purpose? This article presents examples drawn from recent work in The Dialog Corporation as well as from the European Union‐funded ADLIB project, and examines the key issues of scalability and the interaction between different types of publishers.
A remark on Gelfand-Kirillov dimension
Authors: Smith S. , Zhang James
Published: July 2002

Let

A
A

be a finitely generated non-PI Ore domain and

Q
Q

the quotient division algebra of

A
A

. If

C
C

is the center of

Q
Q

, then

GKdim
⁡
C
≤
GKdim
⁡
A
−
2

\operatorname {GKdim} C\leq \operatorname {GKdim} A-2

.
On hypercyclic operators on Banach spaces
Authors: Bernal-González Luis
Published: July 2002

We provide in this paper a direct and constructive proof of the following fact: for a Banach space

X
X

there are bounded linear operators having hypercyclic vectors if and only if

X
X

is separable and dim

X
=
∞

\, X = \infty

. This is a special case of a recent result, which in turn solves a problem proposed by S. Rolewicz.
On the number of solutions of an algebraic equation on the curve 𝑦=𝑒^{𝑥}+sin𝑥,𝑥>0, and a consequence for o-minimal structures
Authors: Gwoździewicz Janusz , Kurdyka Krzysztof , Parusiński Adam
Published: July 2002

We prove that every polynomial

P
(
x
,
y
)

P(x,y)

of degree

d
d

has at most

2
(
d
+
2

)

12

2(d+2)^{12}

zeros on the curve

y
=

e

x

+
sin
⁡
(
x
)
,

x
>
0

y=e^{x}+\sin (x),\quad x>0

. As a consequence we deduce that the existence of a uniform bound for the number of zeros of polynomials of a fixed degree on an analytic curve does not imply that this curve belongs to an o-minimal structure.
On zeros of characters of finite groups
Authors: Chillag David
Published: July 2002

We present several results connecting the number of conjugacy classes of a finite group on which an irreducible character vanishes, and the size of some centralizer of an element. For example, we show that if

G
G

is a finite group such that

G
≠

G
′

≠
G

G\ne G’\ne G

, then

G
G

has an element

x
x

, such that

|

C
G

(
x
)

|

≤
2
m

|C_G(x)|\le 2m

, where

m
m

is the maximal number of zeros in a row of the character table of

G
G

. Dual results connecting the number of irreducible characters which are zero on a fixed conjugacy class, and the degree of some irreducible character, are included too. For example, the dual of the above result is the following: Let

G
G

be a finite group such that

1
≠
Z
(
G
)
≠

Z
2

(
G
)

1\ne Z(G)\ne Z_2(G)

; then

G
G

has an irreducible character

χ
\chi

such that

|

G

|

χ
2

(
1
)

≤
2
m

\frac {|G|}{\chi ^2(1)}\le 2m

, where

m
m

is the maximal number of zeros in a column of the character table of

G
G

.
Estimates for the Green function of a general Sturm-Liouville operator and their applications
Authors: Chernyavskaya N. , Shuster L.
Published: July 2002

For a general Sturm-Liouville operator with nonnegative coefficients, we obtain two-sided estimates for the Green function, sharp by order on the diagonal.
Good ideals in Gorenstein local rings
Authors: Goto Shiro , Iai Sin-Ichiro , Watanabe Kei-ichi
Published: July 2002

Let

I
I

be an

m

\mathfrak {m}

-primary ideal in a Gorenstein local ring (

A
A

,

m

\mathfrak {m}

) with

dim
⁡
A
=
d

\dim A = d

, and assume that

I
I

contains a parameter ideal

Q
Q

in

A
A

as a reduction. We say that

I
I

is a good ideal in

A
A

if

G
=

∑

n
≥
0

I

n

/

I

n
+
1

G = \sum _{n \geq 0} I^{n}/I^{n+1}

is a Gorenstein ring with

a

(
G
)
=
1
−
d

\mathrm {a} (G) = 1 - d

. The associated graded ring

G
G

of

I
I

is a Gorenstein ring with

a

(
G
)
=
−
d

\mathrm {a}(G) = -d

if and only if

I
=
Q

I = Q

. Hence good ideals in our sense are good ones next to the parameter ideals

Q
Q

in

A
A

. A basic theory of good ideals is developed in this paper. We have that

I
I

is a good ideal in

A
A

if and only if

I

2

=
Q
I

I^{2} = QI

and

I
=
Q
:
I

I = Q : I

. First a criterion for finite-dimensional Gorenstein graded algebras

A
A

over fields

k
k

to have nonempty sets

X

A

\mathcal {X}_{A}

of good ideals will be given. Second in the case where

d
=
1

d = 1

we will give a correspondence theorem between the set

X

A

\mathcal {X}_{A}

and the set

Y

A

\mathcal {Y}_{A}

of certain overrings of

A
A

. A characterization of good ideals in the case where

d
=
2

d = 2

will be given in terms of the goodness in their powers. Thanks to Kato’s Riemann-Roch theorem, we are able to classify the good ideals in two-dimensional Gorenstein rational local rings. As a conclusion we will show that the structure of the set

X

A

\mathcal {X}_{A}

of good ideals in

A
A

heavily depends on

d
=
dim
⁡
A

d = \dim A

. The set

X

A

\mathcal {X}_{A}

may be empty if

d
≤
2

d \leq 2

, while

X

A

\mathcal {X}_{A}

is necessarily infinite if

d
≥
3

d \geq 3

and

A
A

contains a field. To analyze this phenomenon we shall explore monomial good ideals in the polynomial ring

k
[

X

1

,

X

2

,

X

3

]

k[X_{1},X_{2},X_{3}]

in three variables over a field

k
k

. Examples are given to illustrate the theorems.
Whitney’s extension problem for multivariate 𝐶^{1,𝜔}-functions
Authors: Brudnyi Yuri , Shvartsman Pavel
Published: July 2002

We prove that the trace of the space

C

1
,
ω

(

R

n

)

C^{1,\omega }({\mathbb R}^n)

to an arbitrary closed subset

X
⊂

R

n

X\subset {\mathbb R}^n

is characterized by the following “finiteness” property. A function

f
:
X
→

R

f:X\rightarrow {\mathbb R}

belongs to the trace space if and only if the restriction

f

|

Y

f|_Y

to an arbitrary subset

Y
⊂
X

Y\subset X

consisting of at most

3
⋅

2

n
−
1

3\cdot 2^{n-1}

can be extended to a function

f
Y

∈

C

1
,
ω

(

R

n

)

f_Y\in C^{1,\omega }({\mathbb R}^n)

such that
\[
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="sup left-brace double-vertical-bar f Subscript upper Y Baseline double-vertical-bar Subscript upper C Sub Superscript 1 comma omega Subscript Baseline colon upper Y subset-of upper X comma c a r d upper Y less-than-or-equal-to 3 dot 2 Superscript n minus 1 Baseline right-brace greater-than normal infinity period">

sup
{
‖

f
Y

‖

C

1
,
ω

:
 
Y
⊂
X
,
 
card
⁡
Y
≤
3
⋅

2

n
−
1

}
>
∞
.

\sup \{\|f_Y\|_{C^{1,\omega }}:~Y\subset X, ~\operatorname {card} Y\le 3\cdot 2^{n-1}\}>\infty .

\]
The constant

3
⋅

2

n
−
1

3\cdot 2^{n-1}

is sharp.

The proof is based on a Lipschitz selection result which is interesting in its own right.
Some New Homogeneous Einstein Metrics on Symmetric Spaces
Authors: Kerr Megan
Published: July 2002

We classify homogeneous Einstein metrics on compact irreducible symmetric spaces. In particular, we consider symmetric spaces with rank

(
M
)
>
1

(M)> 1

, not isometric to a compact Lie group. Whenever there exists a closed proper subgroup

G
G

of Isom

(
M
)

(M)

acting transitively on

M
M

we find all

G
G

-homogeneous (non-symmetric) Einstein metrics on

M
M

.