bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubule and Motor-Dependent Endocytic Vesicle Sorting in Vitro
Authors: Bananis Eustratios , Murray John W. , Stockert Richard J. , Satir Peter , Wolkoff Allan W.
Published: July 2002

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor–ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by ∼300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end–directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5′-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 μM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.
Growth Cone Collapse through Coincident Loss of Actin Bundles and Leading Edge Actin without Actin Depolymerization
Authors: Zhou Feng-quan , Cohan Christopher S.
Published: July 2002

Repulsive guidance cues can either collapse the whole growth cone to arrest neurite outgrowth or cause asymmetric collapse leading to growth cone turning. How signals from repulsive cues are translated by growth cones into this morphological change through rearranging the cytoskeleton is unclear. We examined three factors that are able to induce the collapse of extending Helisoma growth cones in conditioned medium, including serotonin, myosin light chain kinase inhibitor, and phorbol ester. To study the cytoskeletal events contributing to collapse, we cultured Helisoma growth cones on polylysine in which lamellipodial collapse was prevented by substrate adhesion. We found that all three factors that induced collapse of extending growth cones also caused actin bundle loss in polylysine-attached growth cones without loss of actin meshwork. In addition, actin bundle loss correlated with specific filamentous actin redistribution away from the leading edge that is characteristic of repulsive factors. Finally, we provide direct evidence using time-lapse studies of extending growth cones that actin bundle loss paralleled collapse. Taken together, these results suggest that actin bundles could be a common cytoskeletal target of various collapsing factors, which may use different signaling pathways that converge to induce growth cone collapse.
Regulation of presynaptic phosphatidylinositol 4,5-biphosphate by neuronal activity
Authors: Micheva Kristina D. , Holz Ronald W. , Smith Stephen J.
Published: July 2002

Phosphatidylinositol 4,5-biphosphate (PIP2) has been implicated in a variety of cellular processes, including synaptic vesicle recycling. However, little is known about the spatial distribution of this phospholipid in neurons and its dynamics. In this study, we have focused on these questions by transiently expressing the phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain fused to green fluorescent protein (GFP) in cultured hippocampal neurons. This PH domain binds specifically and with high affinity to PIP2. Live confocal imaging revealed that in resting cells, PH-GFP is localized predominantly on the plasma membrane. Interestingly, no association of PH-GFP with synaptic vesicles in quiescent neurons was observed, indicating the absence of detectable PIP2 on mature synaptic vesicles. Electrical stimulation of hippocampal neurons resulted in a decrease of the PH-GFP signal at the plasma membrane, most probably due to a PLC-mediated hydrolysis of PIP2. This was accompanied in the majority of presynaptic terminals by a marked increase in the cytoplasmic PH-GFP signal, localized most probably on freshly endocytosed membranes. Further investigation revealed that the increase in PH-GFP signal was dependent on the activation of N-methyl-D-aspartate receptors and the consequent production of nitric oxide (NO). Thus, PIP2 in the presynaptic terminal appears to be regulated by postsynaptic activity via a retrograde action of NO.
A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae
Authors: Hoepfner Dominic , van den Berg Marlene , Philippsen Peter , Tabak Henk F. , Hettema Ewald H.
Published: July 2002

In vivo time-lapse microscopy reveals that the number of peroxisomes in Saccharomyces cerevisiae cells is fairly constant and that a subset of the organelles are targeted and segregated to the bud in a highly ordered, vectorial process. The dynamin-like protein Vps1p controls the number of peroxisomes, since in a vps1Δ mutant only one or two giant peroxisomes remain. Analogous to the function of other dynamin-related proteins, Vps1p may be involved in a membrane fission event that is required for the regulation of peroxisome abundance. We found that efficient segregation of peroxisomes from mother to bud is dependent on the actin cytoskeleton, and active movement of peroxisomes along actin filaments is driven by the class V myosin motor protein, Myo2p: (a) peroxisomal dynamics always paralleled the polarity of the actin cytoskeleton, (b) double labeling of peroxisomes and actin cables revealed a close association between both, (c) depolymerization of the actin cytoskeleton abolished all peroxisomal movements, and (d) in cells containing thermosensitive alleles of MYO2, all peroxisome movement immediately stopped at the nonpermissive temperature. In addition, time-lapse videos showing peroxisome movement in wild-type and vps1Δ cells suggest the existence of various levels of control involved in the partitioning of peroxisomes.
Evolutionary perspective on innate immune recognition
Authors: Mushegian Arcady , Medzhitov Ruslan
Published: July 2002

Analysis of human and Drosophila genomes demonstrates an ancient origin of innate immunity and the diversity of the mechanisms of innate immune recognition.
Mouse Ly-49A Interrupts Early Signaling Events in Natural Killer Cell Cytotoxicity and Functionally Associates with the SHP-1 Tyrosine Phosphatase
Authors: Nakamura Mary C. , Niemi Eréne C. , Fisher Mark J. , Shultz Leonard D. , Seaman William E. , Ryan James C.
Published: July 2002

The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1–deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.
The Human Immunodeficiency Virus Type 1 (HIV-1) Vpu Protein Interferes with an Early Step in the Biosynthesis of Major Histocompatibility Complex (MHC) Class I Molecules
Authors: Kerkau Thomas , Bacik Igor , Bennink Jack R. , Yewdell Jonathan W. , Hünig Thomas , Schimpl Anneliese , Schubert Ulrich
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a small integral membrane phosphoprotein with two established functions: degradation of the viral coreceptor CD4 in the endoplasmic reticulum (ER) and augmentation of virus particle release from the plasma membrane of HIV-1–infected cells. We show here that Vpu is also largely responsible for the previously observed decrease in the expression of major histocompatibility complex (MHC) class I molecules on the surface of HIV-1–infected cells. Cells infected with HIV-1 isolates that fail to express Vpu, or that express genetically modified forms of Vpu that no longer induce CD4 degradation, exhibit little downregulation of MHC class I molecules. The effect of Vpu on class I biogenesis was analyzed in more detail using a Vpu-expressing recombinant vaccinia virus (VV). VV-expressed Vpu induces the rapid loss of newly synthesized endogenous or VV-expressed class I heavy chains in the ER, detectable either biochemically or by reduced cell surface expression. This effect is of similar rapidity and magnitude as the VV-expressed Vpu-induced degradation of CD4. Vpu had no discernible effects on cell surface expression of VV-expressed mouse CD54, demonstrating the selectivity of its effects on CD4 and class I heavy chains. VVexpressed Vpu does not detectably affect class I molecules that have been exported from the ER. The detrimental effects of Vpu on class I molecules could be distinguished from those caused by VV-expressed herpes virus protein ICP47, which acts by decreasing the supply of cytosolic peptides to class I molecules, indicating that Vpu functions in a distinct manner from ICP47. Based on these findings, we propose that Vpu-induced downregulation of class I molecules may be an important factor in the evolutionary selection of the HIV-1–specific vpu gene by contributing to the inability of CD8+ T cells to eradicate HIV-1 from infected individuals.
Altered Proliferative Response by T Lymphocytes of Ly-6A (Sca-1) Null Mice
Authors: Stanford William L. , Haque Salma , Alexander Robert , Liu Xuemei , Latour Anne M. , Snodgrass H. Ralph , Koller Beverly H. , Flood Patrick M.
Published: July 2002

Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A–deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A–deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.
Inhibition of Virus Attachment to CD4+ Target Cells Is a Major Mechanism of T Cell Line–adapted HIV-1 Neutralization
Authors: Ugolini Sophie , Mondor Isabelle , Parren Paul W.H.I. , Burton Dennis R. , Tilley Shermaine A. , Klasse Per Johan , Sattentau Quentin J.
Published: July 2002

Antibody-mediated neutralization of human immunodeficiency virus type–1 (HIV-1) is thought to function by at least two distinct mechanisms: inhibition of virus–receptor binding, and interference with events after binding, such as virus–cell membrane fusion. Here we show, by the use of a novel virus–cell binding assay, that soluble CD4 and monoclonal antibodies to all confirmed glycoprotein (gp)120 neutralizing epitopes, including the CD4 binding site and the V2 and V3 loops, inhibit the adsorption of two T cell line–adapted HIV-1 viruses to CD4+ cells. A correlation between the inhibition of virus binding and virus neutralization was observed for soluble CD4 and all anti-gp120 antibodies, indicating that this is a major mechanism of HIV neutralization. By contrast, antibodies specific for regions of gp120 other than the CD4 binding site showed little or no inhibition of either soluble gp120 binding to CD4+ cells or soluble CD4 binding to HIV-infected cells, implying that this effect is specific to the virion–cell interaction. However, inhibition of HIV-1 attachment to cells is not a universal mechanism of neutralization, since an anti-gp41 antibody did not inhibit virus–cell binding at neutralizing concentrations, implying activity after virus–cell binding.
Lyn, Jak2, and Raf-1 Kinases Are Critical for the Antiapoptotic Effect of Interleukin 5, whereas only Raf-1 Kinase Is Essential for Eosinophil Activation and Degranulation
Authors: Pazdrak Konrad , Olszewska-Pazdrak Barbara , Stafford Susan , Garofalo Roberto P. , Alam Rafeul
Published: July 2002

Interleukin (IL)-5 has been shown to activate many signaling molecules in eosinophils, but their functional relevance remains unknown. We have examined the functional relevance of Lyn, Jak2, and Raf-1 kinases in eosinophil survival, upregulation of adhesion molecules and degranulation. To this goal we used Lyn and Raf-1 antisense (AS) oligodeoxynucleotides (ODN) to inhibit the expression of these proteins and tyrphostin AG490 to specifically block the activation of Jak2. We have demonstrated that all three kinases are important for IL-5– induced suppression of eosinophil apoptosis. However, Lyn and Jak2 tyrosine kinases are not important for the upregulation of CD11b and the secretion of eosinophil cationic protein. In contrast, Raf-1 kinase is critical for both these functions. This is the first identification of specific signaling molecules responsible for three important functions of eosinophils. We have established a central role for Raf-1 kinase in regulating eosinophil survival, expression of β2 integrins and degranulation. Further, there appears to be a dissociation between two receptor-associated tyrosine kinases, i.e., Lyn and Jak2, and the activation of Raf-1 kinase. The delineation of the functional relevance of signaling molecules will help design therapeutic approaches targeting specific eosinophil function.
Trophoblast Class I Major Histocompatibility Complex (MHC) Products Are Resistant to Rapid Degradation Imposed by the Human Cytomegalovirus (HCMV) Gene Products US2 and US11
Authors: Schust Danny J. , Tortorella Domenico , Seebach Jörg , Phan Cindy , Ploegh Hidde L.
Published: July 2002

US11 and US2 encode gene products expressed early in the replicative cycle of human cytomegalovirus (HCMV), which cause dislocation of human and murine major histocompatibility complex (MHC) class I molecules from the lumen of the endoplasmic reticulum to the cytosol, where the class I heavy chains are rapidly degraded. Human histocompatibility leukocyte antigens (HLA)-C and HLA-G are uniquely resistant to the effects of both US11 and US2 in a human trophoblast cell line as well as in porcine endothelial cells stably transfected with human class I genes. Dislocation and degradation of MHC class I heavy chains do not appear to involve cell type–specific factors, as US11 and US2 are fully active in this xenogeneic model. Importantly, trophoblasts HLA-G and HLA-C possess unique characteristics that allow their escape from HCMV-associated MHC class I degradation. Trophoblast class I molecules could serve not only to block recognition by natural killer cells, but also to guide virus-specific HLA-C– and possibly HLA-G–restricted cytotoxic T-lymphocytes to their targets.
The Vav–Rac1 Pathway in Cytotoxic Lymphocytes Regulates the Generation of Cell-mediated Killing
Authors: Billadeau Daniel D. , Brumbaugh Kathryn M. , Dick Christopher J. , Schoon Renee A. , Bustelo Xose R. , Leibson Paul J.
Published: July 2002

The Rac1 guanine nucleotide exchange factor, Vav, is activated in hematopoietic cells in response to a large variety of stimuli. The downstream signaling events derived from Vav have been primarily characterized as leading to transcription or transformation. However, we report here that Vav and Rac1 in natural killer (NK) cells regulate the development of cell-mediated killing. There is a rapid increase in Vav tyrosine phosphorylation during the development of antibody-dependent cellular cytotoxicity and natural killing. In addition, overexpression of Vav, but not of a mutant lacking exchange factor activity, enhances both forms of killing by NK cells. Furthermore, dominant-negative Rac1 inhibits the development of NK cell–mediated cytotoxicity by two mechanisms: (a) conjugate formation between NK cells and target cells is decreased; and (b) those NK cells that do form conjugates have decreased ability to polarize their granules toward the target cell. Therefore, our results suggest that in addition to participating in the regulation of transcription, Vav and Rac1 are pivotal regulators of adhesion, granule exocytosis, and cellular cytotoxicity.
Lineage-specific Requirement for Signal Transducer and Activator of  Transcription (Stat)4 in Interferon γ Production from CD4+ Versus CD8+ T Cells
Authors: Carter Laura L. , Murphy Kenneth M.
Published: July 2002

CD4+ and CD8+ T cells exhibit important differences in their major effector functions. CD8+ T cells provide protection against pathogens through cytolytic activity, whereas CD4+ T cells exert important regulatory activity through production of cytokines. However, both lineages can produce interferon (IFN)-γ, which can contribute to protective immunity. Here we show that CD4+ and CD8+ T cells differ in their regulation of IFN-γ production. Both lineages require signal transducer and activator of transcription (Stat)4 activation for IFN-γ induced by interleukin (IL)-12/IL-18 signaling, but only CD4+ T cells require Stat4 for IFN-γ induction via the TCR pathway. In response to antigen, CD8+ T cells can produce IFN-γ independently of IL-12, whereas CD4+ T cells require IL-12 and Stat4 activation. Thus, there is a lineage-specific requirement for Stat4 activation in antigen-induced IFN-γ production based on differences in TCR signaling between CD4+ and CD8+ T cells.
Role of the Scavenger Receptor MARCO in Alveolar Macrophage Binding of Unopsonized Environmental Particles
Authors: Palecanda Aiyappa , Paulauskis Joseph , Al-Mutairi Eiman , Imrich Amy , Qin Guozhong , Suzuki Hiroshi , Kodama Tatsuhiko , Tryggvason Karl , Koziel Henry , Kobzik Lester
Published: July 2002

Alveolar macrophages (AMs) avidly bind and ingest unopsonized environmental particles and bacteria through scavenger-type receptors (SRs). AMs from mice with a genetic deletion of the major macrophage SR (types AI and AII; SR−/−) showed no decrease in particle binding compared with SR+/+ mice, suggesting that other SRs are involved. To identify these receptors, we generated a monoclonal antibody (mAb), PAL-1, that inhibits hamster AM binding of unopsonized particles (TiO2, Fe2O3, and latex beads; 66 ± 5, 77 ± 2, and 85 ± 2% inhibition, respectively, measured by flow cytometry). This antibody identifies a protein of ∼70 kD on the AM surface (immunoprecipitation) that is expressed by AMs and other macrophages in situ. A cDNA clone encoding the mAb PAL-1–reactive protein isolated by means of COS cell expression was found to be 84 and 77% homologous to mouse and human scavenger receptor MARCO mRNA, respectively. Transfection of COS cells with MARCO cDNA conferred mAb-inhibitable TiO2 binding. Hamster MARCO also mediates AM binding of unopsonized bacteria (67 ± 5 and 47 ± 4% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PAL-1). A polyclonal antibody to human MARCO identified the expected ∼70-kD band on Western blots of lysates of normal bronchoalveolar lavage (BAL) cells (>90% AMs) and showed strong immunolabeling of human AMs in BAL cytocentrifuge preparations and within lung tissue specimens. In normal mouse AMs, the anti-MARCO mAb ED31 also showed immunoreactivity and inhibited binding of unopsonized particles (e.g., TiO2 ∼40%) and bacteria. The novel function of binding unopsonized environmental dusts and pathogens suggests an important role for MARCO in the lungs' response to inhaled particles.
Severe B Cell Deficiency in Mice Lacking the Tec Kinase Family Members Tec and Btk
Authors: Ellmeier Wilfried , Jung Steffen , Sunshine Mary Jean , Hatam Farah , Xu Yang , Baltimore David , Mano Hiroyuki , Littman Dan R.
Published: July 2002

The cytoplasmic protein tyrosine kinase Tec has been proposed to have important functions in hematopoiesis and lymphocyte signal transduction. Here we show that Tec-deficient mice developed normally and had no major phenotypic alterations of the immune system. To reveal potential compensatory roles of other Tec kinases such as Bruton's tyrosine kinase (Btk), Tec/Btk double-deficient mice were generated. These mice exhibited a block at the B220+CD43+ stage of B cell development and displayed a severe reduction of peripheral B cell numbers, particularly immunoglobulin (Ig)MloIgDhi B cells. Although Tec/Btknull mice were able to form germinal centers, the response to T cell–dependent antigens was impaired. Thus, Tec and Btk together have an important role both during B cell development and in the generation and/or function of the peripheral B cell pool. The ability of Tec to compensate for Btk may also explain phenotypic differences in X-linked immunodeficiency (xid) mice compared with human X-linked agammaglobulinemia (XLA) patients.
Induction of M3-Restricted Cytotoxic T Lymphocyte Responses by N-Formylated Peptides Derived from Mycobacterium tuberculosis
Authors: Chun Taehoon , Serbina Natalya V. , Nolt Dawn , Wang Bin , Chiu Nancy M. , Flynn JoAnne L. , Wang Chyung-Ru
Published: July 2002

Major histocompatibility complex (MHC) class I–restricted CD8+ T cells play a critical role in the protective immunity against Mycobacterium tuberculosis (Mtb). However, only a few Mtb peptides recognized by MHC class Ia–restricted CD8+ T cells have been identified. Information on epitopes recognized by class Ib–restricted T cells is even more limited. M3 is an MHC class Ib molecule that preferentially presents N-formylated peptides to CD8+ T cells. Because bacteria initiate protein synthesis with N-formyl methionine, the unique binding specificity of M3 makes it especially suitable for presenting these particular bacterial epitopes. We have scanned the full sequence of the Mtb genome for NH2-terminal peptides that share features with other M3-binding peptides. Synthetic peptides corresponding to these sequences were tested for their ability to bind to M3 in an immunofluorescence-based peptide-binding assay. Four of the N-formylated Mtb peptides were able to elicit cytotoxic T lymphocytes (CTLs) from mice immunized with peptide-coated splenocytes. The Mtb peptide–specific, M3-restricted CTLs lysed the Mtb-infected macrophages effectively, suggesting that these N-formylated Mtb peptides are presented as the naturally processed epitopes by Mtb-infected cells. Furthermore, T cells from Mtb-infected lungs, spleen, and lymph nodes responded to N-formylated Mtb peptides in an M3-restricted manner. Taken together, our data suggest that M3-restricted T cells may participate in the immune response to Mtb.
Gntb-A, a Novel Sh2d1a-Associated Surface Molecule Contributing to the Inability of Natural Killer Cells to Kill Epstein-Barr Virus–Infected B Cells in X-Linked Lymphoproliferative Disease
Authors: Bottino Cristina , Falco Michela , Parolini Silvia , Marcenaro Emanuela , Augugliaro Raffaella , Sivori Simona , Landi Elena , Biassoni Roberto , Notarangelo Luigi D. , Moretta Lorenzo , Moretta Alessandro
Published: July 2002

In humans, natural killer (NK) cell function is regulated by a series of receptors and coreceptors with either triggering or inhibitory activity. Here we describe a novel 60-kD glycoprotein, termed NTB-A, that is expressed by all human NK, T, and B lymphocytes. Monoclonal antibody (mAb)-mediated cross-linking of NTB-A results in the induction of NK-mediated cytotoxicity. Similar to 2B4 (CD244) functioning as a coreceptor in the NK cell activation, NTB-A also triggers cytolytic activity only in NK cells expressing high surface densities of natural cytotoxicity receptors. This suggests that also NTB-A may function as a coreceptor in the process of NK cell activation. Molecular cloning of the cDNA coding for NTB-A molecule revealed a novel member of the immunoglobulin superfamily belonging to the CD2 subfamily. NTB-A is characterized, in its extracellular portion, by a distal V-type and a proximal C2-type domain and by a cytoplasmic portion containing three tyrosine-based motifs. NTB-A undergoes tyrosine phosphorylation and associates with the Src homology 2 domain–containing protein (SH2D1A) as well as with SH2 domain–containing phosphatases (SHPs). Importantly, analysis of NK cells derived from patients with X-linked lymphoproliferative disease (XLP) showed that the lack of SH2D1A protein profoundly affects the function not only of 2B4 but also of NTB-A. Thus, in XLP-NK cells, NTB-A mediates inhibitory rather than activating signals. These inhibitory signals are induced by the interaction of NTB-A with still undefined ligands expressed on Epstein-Barr virus (EBV)-infected target cells. Moreover, mAb-mediated masking of NTB-A can partially revert this inhibitory effect while a maximal recovery of target cell lysis can be obtained when both 2B4 and NTB-A are simultaneously masked. Thus, the altered function of NTB-A appears to play an important role in the inability of XLP-NK cells to kill EBV-infected target cells.
Early Thymocyte Development Is Regulated by Modulation of E2a Protein Activity
Authors: Engel Isaac , Johns Carol , Bain Gretchen , Rivera Richard R. , Murre Cornelis
Published: July 2002

The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase–dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.
Inhibition of Methylcholanthrene-induced Carcinogenesis by an Interferon γ Receptor–dependent Foreign Body Reaction
Authors: Qin Zhihai , Kim Hye-Jung , Hemme Jens , Blankenstein Thomas
Published: July 2002

The foreign body reaction is one of the oldest host defense mechanisms against tissue damage which involves inflammation, scarring, and encapsulation. The chemical carcinogen methylcholanthrene (MCA) induces fibrosarcoma and tissue damage in parallel at the injection site. Tumor development induced by MCA but not due to p53-deficiency is increased in interferon-γ receptor (IFN-γR)–deficient mice. In the absence of IFN-γR, MCA diffusion and DNA damage of surrounding cells is increased. Locally produced IFN-γ induces the formation of a fibrotic capsule. Encapsulated MCA can persist virtually life-long in mice without inducing tumors. Together, the foreign body reaction against MCA prevents malignant transformation, probably by reducing DNA damage. This mechanism is more efficient in the presence of IFN-γR. Our results indicates that inflammation and scarring, both suspected to contribute to malignancy, prevent cancer in certain situations.
On the Molecular Basis of Ion Permeation in the Epithelial Na+ Channel
Authors: Kellenberger Stephan , Hoffmann-Pochon Nicole , Gautschi Ivan , Schneeberger Estelle , Schild Laurent
Published: July 2002

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two α, one β, and one γ homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of β and γ subunits, which precedes the second putative transmembrane α helix (M2). A residue in the α subunit (αS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of α, β, and γ subunits (αG587, βG529, γS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of βG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported αS589 mutations. We conclude that the residues αG587 to αS589 and homologous residues in the β and γ subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.
Voltage-Dependent Structural Interactions in the Shaker K+ Channel
Authors: Tiwari-Woodruff Seema K. , Lin Meng-chin A. , Schulteis Christine T. , Papazian Diane M.
Published: July 2002

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K+ channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D.M. Papazian. 1997. Biophys. J. 72:1489–1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K+ channels.
Biophysical and Molecular Mechanisms Underlying the Modulation of Heteromeric Kir4.1–Kir5.1 Channels by Co2 and Ph
Authors: Yang Zhenjiang , Xu Haoxing , Cui Ningren , Qu Zhiqiang , Chanchevalap Sengthong , Shen Wangzhen , Jiang Chun
Published: July 2002

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4.1–Kir5.1 were studied in inside-out patches. These Kir4.1–Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (Popen) ∼ 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of Popen without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at ∼1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1–Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline Popen and reduced channel sensitivity to intracellular protons. In the presence of 10 μM PIP2, the Kir4.1–Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1–Kir5.1. In excised patches, interestingly, the Kir4.1–Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.
Allosteric Regulation of Na/Ca Exchange Current by Cytosolic Ca in Intact Cardiac Myocytes
Authors: Weber Christopher R. , Ginsburg Kenneth S. , Philipson Kenneth D. , Shannon Thomas R. , Bers Donald M.
Published: July 2002

The cardiac sarcolemmal Na-Ca exchanger (NCX) is allosterically regulated by [Ca]i such that when [Ca]i is low, NCX current (INCX) deactivates. In this study, we used membrane potential (Em) and INCX to control Ca entry into and Ca efflux from intact cardiac myocytes to investigate whether this allosteric regulation (Ca activation) occurs with [Ca]i in the physiological range. In the absence of Ca activation, the electrochemical effect of increasing [Ca]i would be to increase inward INCX (Ca efflux) and to decrease outward INCX. On the other hand, Ca activation would increase INCX in both directions. Thus, we attributed [Ca]i-dependent increases in outward INCX to allosteric regulation. Ca activation of INCX was observed in ferret myocytes but not in wild-type mouse myocytes, suggesting that Ca regulation of NCX may be species dependent. We also studied transgenic mouse myocytes overexpressing either normal canine NCX or this same canine NCX lacking Ca regulation (Δ680–685). Animals with the normal canine NCX transgene showed Ca activation, whereas animals with the mutant transgene did not, confirming the role of this region in the process. In native ferret cells and in mice with expressed canine NCX, allosteric regulation by Ca occurs under physiological conditions (KmCaAct = 125 ± 16 nM SEM ≈ resting [Ca]i). This, along with the observation that no delay was observed between measured [Ca]i and activation of INCX under our conditions, suggests that beat to beat changes in NCX function can occur in vivo. These changes in the INCX activation state may influence SR Ca load and resting [Ca]i, helping to fine tune Ca influx and efflux from cells under both normal and pathophysiological conditions. Our failure to observe Ca activation in mouse myocytes may be due to either the extent of Ca regulation or to a difference in KmCaAct from other species. Model predictions for Ca activation, on which our estimates of KmCaAct are based, confirm that Ca activation strongly influences outward INCX, explaining why it increases rather than declines with increasing [Ca]i.
Selective Open-Channel Block of Shaker (Kv1) Potassium Channels by S-Nitrosodithiothreitol (Sndtt)
Authors: Brock Mathew W. , Mathes Chris , Gilly William F.
Published: July 2002

Large quaternary ammonium (QA) ions block voltage-gated K+ (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH2CH(OH)CH(OH)CH2SNO) produces qualitatively similar “open-channel block” in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na+ or Ca2+ channels. Inactivation-removed ShakerB (ShBΔ) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose–response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (Kd2 = 0.14 mM) is lower than that of the first molecule (Kd1 = 0.67 mM), indicating cooperativity. The half-blocking concentration (K1/2) is ∼0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about −0.3 e0) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (τ ≈ 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1–1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.
Pressing the ‘send’ key — preferential journal access in developing countries
Authors: Silver Keith
Published: July 2002

Arguably the most striking recent departure in online publishing has been the succession of initiatives designed to provide free or reduced‐rate journal access to the developing world. This article examines the motives behind some of these campaigns and probes the difficulties associated with supplying scientific information equitably, productively, and to an appropriate readership in developing or transitional countries. It considers the strengths and weaknesses of the main solutions currently on offer, while advocating a more unified approach based on the three C's of co‐ordination, comprehensiveness, and clarity.
Journal of Zhejiang University (SCIENCE): a new journal for the 21st century
Authors: Yuehong (Helen) Zhang
Published: July 2002

The first issue of a quarterly international scientific journal published in English by Zhejiang University, in the People's Republic of China, appeared early in 2000. The background to its establishment, its aims and objectives, and its progress to date are described.
Authors and e‐delivery
Authors: Dorner Jane
Published: July 2002

I describe a number of the e‐book formats currently available and point out some of the implications of these new delivery mechanisms for authors' rights and traditional cultural values.
Book reviews
Authors: Fraser Ron , Mabe Michael , Watkinson Anthony
Published: July 2002

The Web of Knowledge: A Festschrift in Honor of Eugene Garfield edited by Blaise Cronin and Helen Barsky Atkins ASIS Monograph Series, Information Today, Medford, NJ, 2000. Pp. 565. Hardback, $49.50. ISBN 1‐57387‐099‐4

A Century of Science Publishing: A Collection of Essays edited by Einar H. Frederiksson IOS Press (Nieuwe Hemweg 6B, 1013 BG Amsterdam), 2001, Pp. 320. Paperback, $38/Eur40/£26. ISBN 1‐58603‐148
Simple algebraic and semialgebraic groups over real closed fields
Authors: Peterzil Ya’acov , Pillay Anand , Starchenko Sergei
Published: July 2002

We continue the investigation of infinite, definably simple groups which are definable in o-minimal structures. InDefinably simple groups in o-minimal structures, we showed that every such group is a semialgebraic group over a real closed field. Our main result here, stated in a model theoretic language, is that every such group is either bi-interpretable with an algebraically closed field of characteristic zero (when the group is stable) or with a real closed field (when the group is unstable). It follows that every abstract isomorphism between two unstable groups as above is a composition of a semialgebraic map with a field isomorphism. We discuss connections to theorems of Freudenthal, Borel-Tits and Weisfeiler on automorphisms of real Lie groups and simple algebraic groups over real closed fields.
Density of cubic field discriminants
Authors: Roberts David
Published: July 2002

In this paper we give a conjectural refinement of the Davenport-Heilbronn theorem on the density of cubic field discriminants. Our refinement is plausible theoretically and agrees very well with computational data.
Composite wavelet bases for operator equations
Authors: Dahmen Wolfgang , Schneider Reinhold
Published: July 2002

This paper is concerned with the construction of biorthogonal wavelet bases defined on a union of parametric images of the unit

n
n

-cube. These bases are to satisfy certain requirements imposed by applications to a class of operator equations acting on such domains. This covers also elliptic boundary value problems, although this study is primarily motivated by our previous analysis of wavelet methods for pseudo-differential equations with special emphasis on boundary integral equations. In this case it is natural to model the boundary surface as a union of parametric images of the unit cube. It will be shown how to construct wavelet bases on the surface which are composed of wavelet bases defined on each surface patch. Here the relevant properties are the validity of norm equivalences in certain ranges of Sobolev scales, as well as appropriate moment conditions.
The Arason invariant and mod 2 algebraic cycles
Authors: Esnault Hélène , Kahn Bruno , Levine Marc , Viehweg Eckart
Published: July 2002

Letkkbe a field,XXoverkka smooth variety with function fieldKKandEEa quadratic vector bundle overXX. Assuming that the generic fibreqqofEEis inI3K⊂W(K)I^3K\subset W(K), we compute the image of its Arason invariant\[<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="e cubed left-parenthesis q right-parenthesis element-of upper H Superscript 0 Baseline left-parenthesis upper X comma script upper H Subscript ModifyingAbove normal e With acute normal t Superscript 3 Baseline left-parenthesis double-struck upper Z slash 2 right-parenthesis right-parenthesis">e3(q)∈H0(X,He´t3(Z/2))e^3(q)\in H^0(X,{\mathcal H}_{\mathrm {\acute {e}t}}^3({\mathbb Z}/2))\]inCH2(X)/2CH^2(X)/2by thed2d_2differential of the Bloch-Ogus spectral sequence. This gives an obstruction toe3(q)e^3(q)being a global cohomology class.
On spaces with periodic cohomology
Authors: Adem Alejandro , Smith Jeff
Published: July 2002

We define a generalized notion of cohomological periodicity for a connected CW-complex

X
X

, and show that it is equivalent to the existence of an oriented spherical fibration over

X
X

with total space homotopy equivalent to a finite dimensional complex. As applications we characterize discrete groups which can act freely and properly on some

R

n

×

S

m

\mathbb R^n\times \mathbb S^m

, show that every rank two

p
p

-group acts freely on a homotopy product of two spheres and construct exotic free actions of many simple groups on such spaces.
On pairs of metrics invariant under a cocompact action of a group
Authors: Krat S.
Published: July 2002

Consider two intrinsic metrics invariant under the same cocompact action of an abelian group. Assume that the ratio of the distances tends to one as the distances grow to infinity. Then it is known (due to D. Burago) that the difference between the metric functions is uniformly bounded. We will prove an analog of this result for hyperbolic groups, as well as a partial generalization of this result for the Heisenberg group: a word metric on the Heisenberg group lies within bounded GH distance from its asymptotic cone.
METATOOL: for studying metabolic networks.
Authors: Pfeiffer T , S√°nchez-Valdenebro I , Nu√±o J C , Montero F , Schuster S
Published: July 2002

MOTIVATION: To reconstruct metabolic pathways from biochemical and/or genome sequence data, the stoichiometric and thermodynamic feasibility of the pathways has to be tested. This is achieved by characterizing the admissible region of flux distributions in steady state. This region is spanned by what can be called a convex basis. The concept of 'elementary flux modes' provides a mathematical tool to define all metabolic routes that are feasible in a given metabolic network. In addition, we define 'enzyme subsets' to be groups of enzymes that operate together in fixed flux proportions in all steady states of the system. RESULTS: Algorithms for computing the convex basis and elementary modes developed earlier are briefly reviewed. A newly developed algorithm for detecting all enzyme subsets in a given network is presented. All of these algorithms have been implemented in a novel computer program named METATOOL, whose features are outlined here. The algorithms are illustrated by an example taken from sugar metabolism. AVAILABILITY: METATOOL is available from ftp://bmsdarwin.brookes.ac. uk/pub/software/ibmpc/metatool. SUPPLEMENTARY INFORMATION: http://www. biologie.hu-berlin.de/biophysics/Theory/tpfeiffer/metatoo l.html
Earliest pages of bioinformatics
Authors: Trifonov Edward N.
Published: July 2002

This review is a brief outline of the chronology and essence of early events in bioinformatics, covering the period from 1869 (discovery of DNA by Miescher) to 1980–1981 (beginning of massive sequencing). For the purpose of this review, bioinformatics is understood as a chapter of molecular biology dealing with the amino acid and nucleotide sequences and with the information they carry.

Contact: edward.trifonov@weizmann.ac.il