bluuebunny/crossref_metadata_2025 huggingface dataset:
A combination of three distinct trafficking signals mediates axonal targeting and presynaptic clustering of GAD65
Authors: Kanaani Jamil , El-Husseini Alaa El-Din , Aguilera-Moreno Andrea , Diacovo Julia M. , Bredt David S. , Baekkeskov Steinunn
Published: September 2002

The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the γ-aminobutyric acid–synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH2-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1–23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH2-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters.
Does size matter?
Authors: Wells William A.
Published: September 2002

For 40 years, the debate has raged. Do mammalian cells monitor cell size when deciding whether to divide? More recent models suggest an indirect solution, but the field is far from reaching a final verdict.
Uncoupling of Proliferative Potential and Gain of Effector Function by CD8+ T Cells Responding to Self-Antigens
Authors: Hernández Javier , Aung Sandra , Marquardt Kristi , Sherman Linda A.
Published: September 2002

Professional antigen-presenting cells (APCs) are capable of transporting self-antigens from peripheral tissues to secondary lymphoid organs where they are presented to potentially autoreactive CD8+ T cells. In the absence of an inflammatory response, this results in immune tolerance. The presence of activated, antigen-specific CD4+ T cells converts this tolerogenic encounter into an immunogenic one by promoting extensive proliferation of CD8+ T cells and their development into effectors. Surprisingly, activation of APCs with an agonistic antibody specific for CD40 could not substitute for CD4+ help in this task. Anti-CD40 induced recruitment of dendritic cells expressing high levels of B7 costimulatory molecules into the lymph nodes, which in turn, greatly enhanced activation and expansion of CD8+ T cells. However, these activated CD8+ cells did not demonstrate effector function. We conclude that proliferative potential and gain of effector function are separable events in the differentiation program of CD8+ T cells.
Surface Cathepsin B Protects Cytotoxic Lymphocytes from Self-destruction after Degranulation
Authors: Balaji Kithiganahalli N. , Schaschke Norbert , Machleidt Werner , Catalfamo Marta , Henkart Pierre A.
Published: September 2002

The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B–specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B–specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.
The Hematopoietic System–specific Minor Histocompatibility Antigen HA-1 Shows Aberrant Expression in Epithelial Cancer Cells
Authors: Klein Christoph A. , Wilke Martina , Pool Jos , Vermeulen Corine , Blokland Els , Burghart Elke , Krostina Sabine , Wendler Nicole , Passlick Bernward , Riethmüeller Gert , Goulmy Els
Published: September 2002

Allogeneic stem cell transplantation (SCT) can induce curative graft-versus-tumor reactions in patients with hematological malignancies and solid tumors. The graft-versus-tumor reaction after human histocompatibility leukocyte antigen (HLA)-identical SCT is mediated by alloimmune donor T cells specific for polymorphic minor histocompatibility antigens (mHags). Among these, the mHag HA-1 was found to be restricted to the hematopoietic system. Here, we report on the HA-1 ribonucleic acid expression by microdissected carcinoma tissues and by single disseminated tumor cells isolated from patients with various epithelial tumors. The HA-1 peptide is molecularly defined, as it forms an immunogenic peptide ligand with HLA-A2 on the cell membrane of carcinoma cell lines. HA-1–specific cytotoxic T cells lyse epithelial tumor cell lines in vitro, whereas normal epithelial cells are not recognized. Thus, HA-1–specific immunotherapy combined with HLA-identical allogeneic SCT may now be feasible for patients with HA-1+ carcinomas.
Functional Inactivation of CXC Chemokine Receptor 4–mediated Responses through SOCS3 Up-regulation
Authors: Soriano Silvia F. , Hernanz-Falcón Patricia , Rodríguez-Frade José Miguel , de Ana Ana Martín , Garzón Ruth , Carvalho-Pinto Carla , Vila-Coro Antonio J. , Zaballos Angel , Balomenos Dimitrios , Martínez-A. Carlos , Mellado Mario
Published: September 2002

Hematopoietic cell growth, differentiation, and chemotactic responses require coordinated action between cytokines and chemokines. Cytokines promote receptor oligomerization, followed by Janus kinase (JAK) kinase activation, signal transducers and transactivators of transcription (STAT) nuclear translocation, and transcription of cytokine-responsive genes. These include genes that encode a family of negative regulators of cytokine signaling, the suppressors of cytokine signaling (SOCS) proteins. After binding their specific receptors, chemokines trigger receptor dimerization and activate the JAK/STAT pathway. We show that SOCS3 overexpression or up-regulation, stimulated by a cytokine such as growth hormone, impairs the response to CXCL12, measured by Ca2+ flux and chemotaxis in vitro and in vivo. This effect is mediated by SOCS3 binding to the CXC chemokine receptor 4 receptor, blocking JAK/STAT and Gαi pathways, without interfering with cell surface chemokine receptor expression. The data provide clear evidence for signaling cross-talk between cytokine and chemokine responses in building a functional immune system.
Accumulation of Immature Langerhans Cells in Human Lymph Nodes Draining Chronically Inflamed Skin
Authors: Geissmann F. , Dieu-Nosjean M.C. , Dezutter C. , Valladeau J. , Kayal S. , Leborgne M. , Brousse N. , Saeland S. , Davoust J.
Published: September 2002

The coordinated migration and maturation of dendritic cells (DCs) such as intraepithelial Langerhans cells (LCs) is considered critical for T cell priming in response to inflammation in the periphery. However, little is known about the role of inflammatory mediators for LC maturation and recruitment to lymph nodes in vivo. Here we show in human dermatopathic lymphadenitis (DL), which features an expanded population of LCs in one draining lymph node associated with inflammatory lesions in its tributary skin area, that the Langerin/CD207+ LCs constitute a predominant population of immature DCs, which express CD1a, and CD68, but not CD83, CD86, and DC–lysosomal-associated membrane protein (LAMP)/CD208. Using LC-type cells generated in vitro in the presence of transforming growth factor (TGF)-β1, we further found that tumor necrosis factor (TNF)-α, as a prototype proinflammatory factor, and a variety of inflammatory stimuli and bacterial products, increase Langerin expression and Langerin dependent Birbeck granules formation in cell which nevertheless lack costimulatory molecules, DC–LAMP/CD208 and potent T cell stimulatory activity but express CCR7 and respond to the lymph node homing chemokines CCL19 and CCL21. This indicates that LC migration and maturation can be independently regulated events. We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation. Immature LCs might regulate immune responses during chronic inflammation.
Phenotype, Localization, and Mechanism of Suppression of CD4+CD25+ Human Thymocytes
Authors: Annunziato Francesco , Cosmi Lorenzo , Liotta Francesco , Lazzeri Elena , Manetti Roberto , Vanini Vittorio , Romagnani Paola , Maggi Enrico , Romagnani Sergio
Published: September 2002

Phenotypic markers, localization, functional activities, and mechanisms of action in vitro of CD4+CD25+ T cells, purified from postnatal human thymuses, were investigated. These cells showed poor or no proliferation in mixed lymphocyte culture (MLC), and suppressed in a dose-dependent fashion the proliferative response to allogeneic stimulation of CD4+CD25− thymocytes. Virtually all CD4+CD25+ thymocytes constitutively expressed cytoplasmic T lymphocyte antigen (CTLA)-4, surface tumor necrosis factor type 2 receptor (TNFR2), and CCR8. They prevalently localized to perivascular areas of fibrous septa and responded to the chemoattractant activity of CCL1/I-309, which was found to be produced by either thymic medullary macrophages or fibrous septa epithelial cells. After polyclonal activation, CD4+CD25+ thymocytes did not produce the cytokines interleukin (IL)-2, IL-4, IL-5, IL-13, interferon γ, and only a very few produced IL-10, but all they expressed on their surface CTLA-4 and the majority of them also transforming growth factor (TGF)-β1. The suppressive activity of these cells was contact dependent and associated with the lack of IL-2 receptor (IL-2R) α-chain (CD25) expression in target cells. Such a suppressive activity was partially inhibited by either anti–CTLA-4 or anti–TGF-β1, and was completely blocked by a mixture of these monoclonal antibodies, which were also able to restore in target T cells the expression of IL-2R α-chain and, therefore, their responsiveness to IL-2. These data demonstrate that CD4+CD25+ human thymocytes represent a population of regulatory cells that migrate in response to the chemokine CCL1/I-309 and exert their suppressive function via the inhibition of IL-2R α-chain in target T cells, induced by the combined activity of CTLA-4 and membrane TGF-β1.
Blocking of HIV-1 Infection by Targeting CD4 to Nonraft Membrane Domains
Authors: del Real Gustavo , Jiménez-Baranda Sonia , Lacalle Rosa Ana , Mira Emilia , Lucas Pilar , Gómez-Moutón Concepción , Carrera Ana C. , Martínez-A. Carlos , Mañes Santos
Published: September 2002

Human immunodeficiency virus (HIV)-1 infection depends on multiple lateral interactions between the viral envelope and host cell receptors. Previous studies have suggested that these interactions are possible because HIV-1 receptors CD4, CXCR4, and CCR5 partition in cholesterol-enriched membrane raft domains. We generated CD4 partitioning mutants by substituting or deleting CD4 transmembrane and cytoplasmic domains and the CD4 ectodomain was unaltered. We report that all CD4 mutants that retain raft partitioning mediate HIV-1 entry and CD4-induced Lck activation independently of their transmembrane and cytoplasmic domains. Conversely, CD4 ectodomain targeting to a nonraft membrane fraction results in a CD4 receptor with severely diminished capacity to mediate Lck activation or HIV-1 entry, although this mutant binds gp120 as well as CD4wt. In addition, the nonraft CD4 mutant inhibits HIV-1 X4 and R5 entry in a CD4+ cell line. These results not only indicate that HIV-1 exploits host membrane raft domains as cell entry sites, but also suggest new strategies for preventing HIV-1 infection.
Analysis of Protease Activity in Live Antigen-presenting Cells Shows Regulation of the Phagosomal Proteolytic Contents During Dendritic Cell Activation
Authors: Lennon-Duménil Ana-Maria , Bakker Arnold H. , Maehr René , Fiebiger Edda , Overkleeft Herman S. , Rosemblatt Mario , Ploegh Hidde L. , Lagaudrière-Gesbert Cécile
Published: September 2002

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow–derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte.
Receptor-mediated Immunoglobulin G Transport Across Mucosal Barriers in Adult Life
Authors: Spiekermann Gerburg M. , Finn Patricia W. , Ward E. Sally , Dumont Jennifer , Dickinson Bonny L. , Blumberg Richard S. , Lencer Wayne I.
Published: September 2002

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain the immunoglobulins (Ig)G and secretory IgA (sIgA) that function together in host defense. Exactly how IgG crosses epithelial barriers to function in mucosal immunity remains unknown. Here, we test the idea that the MHC class I–related Fc-receptor, FcRn, transports IgG across the mucosal surface of the human and mouse lung from lumen to serosa. We find that bronchial epithelial cells of the human, nonhuman primate, and mouse, express FcRn in adult-life, and demonstrate FcRn-dependent absorption of a bioactive Fc-fusion protein across the respiratory epithelium of the mouse in vivo. Thus, IgG, like dimeric IgA, can cross epithelial barriers by receptor-mediated transcytosis in adult animals. These data show that mucosal surfaces that express FcRn reabsorb IgG and explain a mechanism by which IgG may act in immune surveillance to retrieve lumenal antigens for processing in the lamina propria or systemically.
Blockade of HIV-1 Infection of New World Monkey Cells Occurs Primarily at the Stage of Virus Entry
Authors: LaBonte Jason A. , Babcock Gregory J. , Patel Trushar , Sodroski Joseph
Published: September 2002

HIV-1 naturally infects chimpanzees and humans, but does not infect Old World monkeys because of replication blocks that occur after virus entry into the cell. To understand the species-specific restrictions operating on HIV-1 infection, the ability of HIV-1 to infect the cells of New World monkeys was examined. Primary cells derived from common marmosets and squirrel monkeys support every phase of HIV-1 replication with the exception of virus entry. Efficient HIV-1 entry typically requires binding of the viral envelope glycoproteins and host cell receptors, CD4 and either CCR5 or CXCR4 chemokine receptors. HIV-1 did not detectably bind or utilize squirrel monkey CD4 for entry, and marmoset CD4 was also very inefficient compared with human CD4. A marmoset CD4 variant, in which residues 48 and 59 were altered to the amino acids found in human CD4, supported HIV-1 entry efficiently. The CXCR4 molecules of both marmosets and squirrel monkeys supported HIV-1 infection, but the CCR5 proteins of both species were only marginally functional. These results demonstrate that the CD4 and CCR5 proteins of New World monkeys represent the major restriction against HIV-1 replication in these primates. Directed adaptation of the HIV-1 envelope glycoproteins to common marmoset receptors might allow the development of New World monkey models of HIV-1 infection.
Targeting Mucosal Sites by Polymeric Immunoglobulin Receptor-directed Peptides
Authors: White Kendra D. , Capra J. Donald
Published: September 2002

Polymeric immunoglobulins provide first line humoral defense at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR) on mucosal and glandular epithelial cells. Previous studies from our laboratory suggested that amino acids 402–410 of the Cα3 domain of dimeric IgA (dIgA) represented a potential binding site for the pIgR. Here by binding human secretory component to overlapping decapeptides of Cα3, we confirm these residues and also uncover an additional site. Furthermore, we show that the Cα3 motif appears to be sufficient to direct transport of green fluorescent protein through the pIgR-specific cellular transcytosis system. An alternative approach identified phage peptides, selected from a library by the in vitro Madin Darby Canine Kidney transcytosis assay, for pIgR-mediated transport through epithelial cells. Some transcytosis-selected peptides map to the same 402–410 pIgR-binding Cα3 site. Further in vivo studies document that at least one of these peptides is transported in a rat model measuring hepatic bile transport. In addition to identifying small peptides that are both bound and transported by the pIgR, this study provides evidence that the pIgR-mediated mucosal secretion system may represent a means of targeting small molecule therapeutics and genes to mucosal epithelial cells.
Extracellular Protons Regulate the Extracellular Cation Selectivity of the Sodium Pump
Authors: Milanick Mark A. , Arnett Krista L.
Published: September 2002

The effects of 0.3–10 nM extracellular protons (pH 9.5–8.0) on ouabain-sensitive rubidium influx were determined in 4,4′-diisocyanostilbene-2, 2′-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump (∼5–10 mM). The Kd for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the Kd for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like.
IRK1 Inward Rectifier K+ Channels Exhibit No Intrinsic Rectification
Authors: Guo Donglin , Lu Zhe
Published: September 2002

In intact cells the depolarization-induced outward IRK1 currents undergo profound relaxation so that the steady-state macroscopic I-V curve exhibits strong inward rectification. A modest degree of rectification persists after the membrane patches were perfused with artificial solutions devoid of Mg2+ and polyamines, which has been interpreted as a reflection of intrinsic channel gating and led to the view that inward rectification results from enhancement of the intrinsic gating by intracellular cations rather than simple pore block. Furthermore, IRK1 exhibits significant extracellular K+-sensitive relaxation of its inward current, a feature that has been likened to the C-type inactivation observed in the voltage-activated Shaker K+ channels. We found that both these current relaxations can be accounted for by impurities in some common constituents of recording solutions, such as residual hydroxyethylpiperazine in HEPES and ethylenediamine in EDTA. Therefore, inherently, IRK1 channels are essentially ohmic at the macroscopic level, and the voltage jump–induced current relaxations do not reflect IRK1 gating but the unusually high affinity of its pore for cations. Furthermore, our study helps define the optimal experimental conditions for studying IRK1.
Bistatic Reverberation in Shallow Water: Modelling and Data Comparison
Authors: Li Feng-Hua  , Liu Jian-Jun
Published: September 2002

Bistatic and monostatic reverberation data were recorded in the 2001 Asian Sea International Acoustic Experiment (ASIAEX). A model based on the normal mode theory has been developed to calculate bistatic bottom reverberation in shallow water and to explain the recorded data. The comparisons between the monostatic and bistatic reverberation data are discussed, and the comparisons between model predictions and measured bistatic reverberation data are also presented. The numerical and experimental results show that the numerical predictions from the bistatic reverberation model fit the experimental data well, and the long-range bistatic reverberation with a time delay can be approximately expressed by the monostatic reverberation data.
Cross-Linking CD98 Promotes Integrin-like Signaling and Anchorage-independent Growth
Authors: Rintoul Robert C. , Buttery Robert C. , Mackinnon Alison C , Wong Weng Sie , Mosher Deane , Haslett Christopher , Sethi Tariq
Published: September 2002

CD98, an early marker of T-cell activation, is an important regulator of integrin-mediated adhesion events. Previous studies suggest that CD98 is coupled to both cellular activation and transformation and is involved in the pathogenesis of viral infection, inflammatory disease, and cancer. Understanding of the molecular mechanisms underlying CD98 activity may have far-reaching practical applications in the development of novel therapeutic strategies in these disease states. Using small cell lung cancer cell lines, which are nonadherent, nonpolarized, and highly express CD98, we show that, in vitro, under physiological conditions, CD98 is constitutively associated with β1 integrins regardless of activation status. Cross-linking CD98 with the monoclonal antibody 4F2 stimulated phosphatidylinositol (PI) 3-kinase, PI(3,4,5)P3, and protein kinase B in the absence of integrin ligation or extracellular matrix engagement. Furthermore, cross-linking CD98 promoted anchorage-independent growth. Using fibroblasts derived from β1 integrin null stem cells (GD25), wild-type GD25β1, or GD25 cells expressing a mutation preventing β1 integrin-dependent FAK phosphorylation, we demonstrate that a functional β1 integrin is required for CD98 signaling. We propose that by cross-linking CD98, it acts as a “molecular facilitator” in the plasma membrane, clustering β1 integrins to form high-density complexes. This results in integrin activation, integrin-like signaling, and anchorage-independent growth. Activation of PI 3-kinase may, in part, explain cellular transformation seen on overexpressing CD98. These results may provide a paradigm for events involved in such diverse processes as inflammation and viral-induced cell fusion.
Structure of the Golgi and Distribution of Reporter Molecules at 20°C Reveals the Complexity of the Exit Compartments
Authors: Ladinsky Mark S. , Wu Christine C. , McIntosh Shane , McIntosh J. Richard , Howell Kathryn E.
Published: September 2002

Incubating cells at 20°C blocks transport out of the Golgi complex and amplifies the exit compartments. We have used the 20°C block, followed by EM tomography and serial section reconstruction, to study the structure of Golgi exit sites in NRK cells. The dominant feature of Golgi structure in temperature-blocked cells is the presence of large bulging domains on the three trans-most cisternae. These domains extend laterally from the stack and are continuous with “cisternal” domains that maintain normal thickness and alignment with the other stacked Golgi cisternae. The bulging domains do not resemble the perpendicularly extending tubules associated with the trans-cisternae of control cells. Such tubules are completely absent in temperature-blocked cells. The three cisternae with bulging domains can be identified as trans by their association with specialized ER and the presence of clathrin-coated buds on the trans-most cisterna only. Immunogold labeling and immunoblots show a significant degradation of a medial- and a trans-Golgi marker with no evidence for their redistribution within the Golgi or to other organelles. These data suggest that exit from the Golgi occurs directly from three trans-cisternae and that specialized ER plays a significant role in trans-Golgi function.
The MEK Kinase Ssk2p Promotes Actin Cytoskeleton Recovery After Osmotic Stress
Authors: Yuzyuk Tatiana , Foehr Marissa , Amberg David C.
Published: September 2002

Saccharomyces cerevisiae adapts to osmotic stress through the activation of a conserved high-osmolarity growth (HOG) mitogen-activated protein (MAP) kinase pathway. Transmission through the HOG pathway is very well understood, yet other aspects of the cellular response to osmotic stress remain poorly understood, most notably regulation of actin organization. The actin cytoskeleton rapidly disassembles in response to osmotic insult and is induced to reassemble only after osmotic balance with the environment is reestablished. Here, we show that one of three MEK kinases of the HOG pathway, Ssk2p, is specialized to facilitate actin cytoskeleton reassembly after osmotic stress. Within minutes of cells' experiencing osmotic stress or catastrophic disassembly of the actin cytoskeleton through latrunculin A treatment, Ssk2p concentrates in the neck of budding yeast cells and concurrently forms a 1:1 complex with actin. These observations suggest that Ssk2p has a novel, previously undescribed function in sensing damage to the actin cytoskeleton. We also describe a second function for Ssk2p in facilitating reassembly of a polarized actin cytoskeleton at the end of the cell cycle, a prerequisite for efficient cell cycle completion. Loss of Ssk2p, its kinase activity, or its ability to localize and interact with actin led to delays in actin recovery and a resulting delay in cell cycle completion. These unique capabilities of Ssk2p are activated by a novel mechanism that does not involve known components of the HOG pathway.
Downstream E-Box–mediated Regulation of the Human Telomerase Reverse Transcriptase (hTERT) Gene Transcription: Evidence for an Endogenous Mechanism of Transcriptional Repression
Authors: Horikawa Izumi , Cable P. LouAnn , Mazur Sharlyn J. , Appella Ettore , Afshari Cynthia A. , Barrett J. Carl
Published: September 2002

Regulation of the hTERT gene encoding the telomerase catalytic subunit plays an important role in human cell senescence, immortalization, and carcinogenesis. By examining the activity of various deleted or mutated hTERT promoter fragments, we show that an E-box element downstream of the transcription initiation site is critical to differential hTERT transcription between the telomerase/hTERT-positive renal cell carcinoma cell line (RCC23) and its telomerase/hTERT-negative counterpart containing a transferred, normal chromosome 3 (RCC23+3). This E-box element mediated repression of hTERT transcription in RCC23+3 but not in RCC23. A copy number–dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23+3. Endogenous expression levels of c-Myc or Mad1, which could activate or repress hTERT transcription when overexpressed, did not account for the differential hTERT transcription. Gel mobility shift assays identified the upstream stimulatory factors (USFs) as a major E-box–binding protein complex in both RCC23 and RCC23+3 and, importantly, detected an RCC23+3-specific, E-box–binding factor that was distinct from the USF and Myc/Mad families. The E-box–mediated repression was also active in normal human fibroblasts and epithelial cells and inactive in some, but not all, telomerase/hTERT-positive cancer cells. These findings provide evidence for an endogenous, repressive mechanism that actively functions in telomerase/hTERT-negative normal cells and becomes defective during carcinogenic processes, e.g., by an inactivation of the telomerase repressor gene on chromosome 3.
Crp79p, Like Mex67p, Is an Auxiliary mRNA Export Factor inSchizosaccharomyces pombe
Authors: Thakurta Anjan G. , Whalen William A. , Yoon Jin Ho , Bharathi Anekella , Kozak Libor , Whiteford Craig , Love Dona C. , Hanover John A. , Dhar Ravi
Published: September 2002

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p inSchizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)+RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.
Cell Stress and MEKK1-mediated c-Jun Activation Modulate NFκB Activity and Cell Viability
Authors: Sánchez-Pérez Isabel , Benitah Salvador Aznar , Martı́nez-Gomariz Montserrat , Lacal Juan Carlos , Perona Rosario
Published: September 2002

Chemotherapeutic agents such as cisplatin induce persistent activation of N-terminal c-Jun Kinase, which in turn mediates induction of apoptosis. By using a common MAPK Kinase, MEKK1, cisplatin also activates the survival transcription factor NFκB. We have found a cross-talk between c-Jun expression and NFκB transcriptional activation in response to cisplatin. Fibroblast derived from c-jun knock out mice are more resistant to cisplatin-induced cell death, and this survival advantage is mediated by upregulation of NFκB-dependent transcription and expression of MIAP3. This process can be reverted by ectopic expression of c-Jun in c-jun−/−fibroblasts, which decreases p65 transcriptional activity back to normal levels. Negative regulation of NFκB-dependent transcription by c-jun contributes to cisplatin-induced cell death, which suggests that inhibition of NFκB may potentiate the antineoplastic effect of conventional chemotherapeutic agents.
Organizational Diversity among Distinct Glycoprotein Endoplasmic Reticulum-associated Degradation Programs
Authors: Cabral Christopher M. , Liu Yan , Moremen Kelley W. , Sifers Richard N.
Published: September 2002

Protein folding and quality control in the early secretory pathway function as posttranslational checkpoints in eukaryote gene expression. Herein, an aberrant form of the hepatic secretory protein α1-antitrypsin was stably expressed in a human embryonic kidney cell line to elucidate the mechanisms by which glycoprotein endoplasmic reticulum-associated degradation (GERAD) is administered in cells from higher eukaryotes. After biosynthesis, genetic variant PI Z underwent alternative phases of secretion and degradation, the latter of which was mediated by the proteasome. Degradation required release from calnexin- and asparagine-linked oligosaccharide modification by endoplasmic reticulum mannosidase I, the latter of which occurred as PI Z was bound to the molecular chaperone grp78/BiP. That a distinct GERAD program operates in human embryonic kidney cells was supported by the extent of PI Z secretion, apparent lack of polymerization, inability of calnexin to participate in the degradation process, and sequestration of the glycoprotein folding sensor UDP-glucose:glycoprotein glucosyltransferase in the Golgi complex. Because UDP-glucose:glycoprotein glucosyltransferase sustains calnexin binding, its altered distribution is consistent with a GERAD program that hinders the reentry of substrates into the calnexin cycle, allowing grp78/BiP to partner with a lectin, other than calnexin, in the recognition of a two-component GERAD signal to facilitate substrate recruitment. How the processing of a mutant protein, rather than the mutation itself, can contribute to disease pathogenesis, is discussed.
The Septation Apparatus, an Autonomous System in Budding Yeast
Authors: Roh Dong-Hyun , Bowers Blair , Schmidt Martin , Cabib Enrico
Published: September 2002

Actomyosin ring contraction and chitin primary septum deposition are interdependent processes in cell division of budding yeast. By fusing Myo1p, as representative of the contractile ring, and Chs2p for the primary septum, to different fluorescent proteins we show herein that the two processes proceed essentially at the same location and simultaneously. Chs2p differs from Myo1p in that it reflects the changes in shape of the plasma membrane to which it is attached and in that it is packed after its action into visible endocytic vesicles for its disposal. To ascertain whether this highly coordinated system could function independently of other cell cycle events, we reexamined the septum-like structures made by the septin mutant cdc3 at various sites on the cell cortex at the nonpermissive temperature. With the fluorescent fusion proteins mentioned above, we observed that incdc3 at 37°C both Myo1p and Chs2p colocalize at different spots of the cell cortex. A contraction of the Myo1p patch could also be detected, as well as that of a Chs2p patch, with subsequent appearance of vesicles. Furthermore, the septin Cdc12p, fused with yellow or cyan fluorescent protein, also colocalized with Myo1p and Chs2p at the aberrant locations. The formation of delocalized septa did not require nuclear division. We conclude that the septation apparatus, composed of septins, contractile ring, and the chitin synthase II system, can function at ectopic locations autonomously and independently of cell division, and that it can recruit the other elements necessary for the formation of secondary septa.
Multiple Functions of Sterols in Yeast Endocytosis
Authors: Heese-Peck Antje , Pichler Harald , Zanolari Bettina , Watanabe Reika , Daum Günther , Riezman Howard
Published: September 2002

Sterols are essential factors for endocytosis in animals and yeast. To investigate the sterol structural requirements for yeast endocytosis, we created a variety of ergΔ mutants, each accumulating a distinct set of sterols different from ergosterol. Mutant erg2Δerg6Δ anderg3Δerg6Δ cells exhibit a strong internalization defect of the α-factor receptor (Ste2p). Specific sterol structures are necessary for pheromone-dependent receptor hyperphosphorylation, a prerequisite for internalization. The lack of phosphorylation is not due to a defect in Ste2p localization or in ligand–receptor interaction. Contrary to most known endocytic factors, sterols seem to function in internalization independently of actin. Furthermore, sterol structures are required at a postinternalization step of endocytosis. ergΔ cells were able to take up the membrane marker FM4-64, but exhibited defects in FM4-64 movement through endosomal compartments to the vacuole. Therefore, there are at least two roles for sterols in endocytosis. Based on sterol analysis, the sterol structural requirements for these two processes were different, suggesting that sterols may have distinct functions at different places in the endocytic pathway. Interestingly, sterol structures unable to support endocytosis allowed transport of the glycosylphosphatidylinositol-anchored protein Gas1p from the endoplasmic reticulum to Golgi compartment.
Dealing with errors in interactive sequencing by hybridization
Authors: Phan Vinhthuy T. , Skiena Steven S.
Published: September 2002

Motivation: A realistic approach to sequencing by hybridization must deal with realistic sequencing errors. The results of such a method can surely be applied to similar sequencing tasks.

Results: We provide the first algorithms for interactive sequencing by hybridization which are robust in the presence of hybridization errors. Under a strong error model allowing both positive and negative hybridization errors without repeated queries, we demonstrate accurate and efficient reconstruction with error rates up to 7%. Under the weaker traditional error model of Shamir and Tsur (Proceedings of the Fifth International Conference on Computational Molecular Biology (RECOMB-01) , pp 269–277, 2000), we obtain accurate reconstructions with up to 20% false negative hybridization errors. Finally, we establish theoretical bounds on the performance of the sequential probing algorithm of Skiena and Sundaram (J. Comput. Biol. , 2, 333–353, 1995) under the strong error model.

Availability: Freely available upon request.

Contact: skiena@cs.sunysb.edu
Enrichment of regulatory signals in conserved non-coding
genomic sequence
Authors: Levy Samuel , Hannenhalli Sridhar , Workman Christopher
Published: September 2002

Motivation: Whole genome shotgun sequencing strategies generate sequence data prior to the application of assembly methodologies that result in contiguous sequence. Sequence reads can be employed to indicate regions of conservation between closely related species for which only one genome has been assembled. Consequently, by using pairwise sequence alignments methods it is possible to identify novel, non-repetitive, conserved segments in non-coding sequence that exist between the assembled human genome and mouse whole genome shotgun sequencing fragments. Conserved non-coding regions identify potentially functional DNA that could be involved in transcriptional regulation.

Results: Local sequence alignment methods were applied employing mouse fragments and the assembled human genome. In addition, transcription factor binding sites were detected by aligning their corresponding positional weight matrices to the sequence regions. These methods were applied to a set of transcripts corresponding to 502 genes associated with a variety of different human diseases taken from the Online Mendelian Inheritance in Man database. Using statistical arguments we have shown that conserved non-coding segments contain an enrichment of transcription factor binding sites when compared to the sequence background in which the conserved segments are located. This enrichment of binding sites was not observed in coding sequence. Conserved non-coding segments are not extensively repeated in the genome and therefore their identification provides a rapid means of finding genes with related conserved regions, and consequently potentially related regulatory mechanism. Conserved segments in upstream regions are found to contain binding sites that are co-localized in a manner consistent with experimentally known transcription factor pairwise co-occurrences and afford the identification of novel co-occurring Transcription Factor (TF) pairs. This study provides a methodology and more evidence to suggest that conserved non-coding regions are biologically significant since they contain a statistical enrichment of regulatory signals and pairs of signals that enable the construction of regulatory models for human genes.

Contact: samuel.levy@celera.com

* To whom correspondence should be addressed.
Predicting class II MHC/peptide multi-level binding with an
iterative stepwise discriminant analysis meta-algorithm
Authors: Mallios R. R.
Published: September 2002

Motivation: Predicting peptides that bind to both Major Histocompatibility Complex (MHC) molecules and T cell receptors provides crucial information for vaccine development. An agretope is that portion of a peptide that interacts with an MHC molecule. The identification and prediction of agretopes is the first step towards vaccine design.

Results: An iterative stepwise discriminant analysis meta-algorithm is utilized to derive a quantitative motif for classifying potential agretopes as high-, moderate- or non-binders for HLA-DR1, a class II MHC molecule. A large molecular online database provides the input for this data-driven algorithm. The model correctly classifies over 85% of the peptides in the database.

Availability: Stepwise discriminant analysis software is available commercially in SPSS and BMDP statistical software packages. Peptides known to bind MHC molecules can be downloaded from http://wehih.wehi.edu.au/mhcpep/. Peptides known not to bind HLA-DR1 are available from the author upon request.

Contact: ronna@ucsfresno.edu
Determining the minimum number of types necessary to represent
the sizes of protein atoms
Authors: Tsai Jerry , Voss Neil , Gerstein Mark
Published: September 2002

Motivation: Traditionally, for packing calculations people have collected atoms together into a number of distinct ‘types’. These, in fact, often represent a heavy atom and its associated hydrogens (i.e. a united atom). Also, atom typing is usually done according to basic chemistry, giving rise to 20–30 protein atom types, such as carbonyl carbons, methyl groups, and hydroxyl groups. No one has yet investigated how similar in packing these chemically derived types are. Here we address this question in detail, using Voronoi volume calculations on a set of high-resolution crystal structures.

Results: We perform a rigorous clustering analysis with cross-validation on tens of thousands of atom volumes and attempt to compile them into types based purely on packing. From our analysis, we are able to determine a ‘minimal’ set of 18 atom types that most efficiently represent the spectrum of packing in proteins. Furthermore, we are able to uncover a number of inconsistencies in traditional chemical typing schemes, where differently typed atoms have almost the same effective size. In particular, we find that tetrahedral carbons with two hydrogens are almost identical in size to many aromatic carbons with a single hydrogen.

Availability: Programs available from http://geometry.molmovdb.org.

Contact: JerryTsai@TAMU.edu; neil.voss@yale.edu; Mark.Gerstein@yale.edu

Supplementary Information: Available at http://geometry.molmovdb.org.

* To whom correspondence should be addressed.
Comprehensive comparison between locations of orthologous genes
on archaeal and bacterial genomes
Authors: Horimoto Katsuhisa , Fukuchi Satoshi , Mori Kentaro
Published: September 2002

Motivation: Following an extensive search for orthologous genes between the complete genomes from archaea and bacteria, the spatial association of the orthologs has been investigated in terms of synteny, the conservation of the order of neighboring genes. However, the relationships between the relative locations of remote orthologs over entire genomes have not been shown.

Results: Comprehensive comparisons between the locations of orthologs on nineteen archaeal and bacterial genomes are presented by the location to location correspondence based on the gene–location distance. When the two genomes are rotated such that a pair of orthologs with the shortest distance is set in the same angle, a statistically significant number of orthologs maintain their relative locations between the genomes. Even by the short distances at the 5% significance level, the rotations are restricted within a narrow range, suggesting an intrinsic angle for realizing similar locations between the orthologs in each genome pair. Furthermore, the rotations in the restricted range agree with the replication origin and terminus sites for the analyzed genomes where such sites are known. The relationship between location-maintained orthologs and gene function is also discussed.

Contact: horimoto@post.saga-med.ac.jp

* To whom correspondence should be addressed.
The HMMTOP transmembrane topology prediction server
Authors: Tusnády Gábor
   E. , Simon István
Published: September 2002

Summary: The HMMTOP transmembrane topology prediction server predicts both the localization of helical transmembrane segments and the topology of transmembrane proteins. Recently, several improvements have been introduced to the original method. Now, the user is allowed to submit additional information about segment localization to enhance the prediction power. This option improves the prediction accuracy as well as helps the interpretation of experimental results, i.e. in epitope insertion experiments.

Availability: HMMTOP 2.0 is freely available to non-commercial users at http://www.enzim.hu/hmmtop. Source code is also available upon request to academic users.

Contact: tusi@enzim.hu

* To whom correspondence should be addressed.
VISTRAJ: exploring protein conformational space
Authors: Salama John J. , Feldman Howard J. , Hogue Christopher W. V.
Published: September 2002

Summary: VISTRAJ is an application which allows 3D visualization, manipulation and editing of protein conformational space using probabilistic maps of this space called ‘trajectory distributions’. Trajectory distributions serve as input to FOLDTRAJ which samples protein structures based on the represented conformational space. VISTRAJ also allows FOLDTRAJ to be used as a tool for homology model creation, and structures may be generated containing post-translationally modified amino acids.

Availability: Binaries are freely available for non-profit use as part of the FOLDTRAJ package at ftp://ftp.mshri.on.ca/pub/TraDES/foldtraj/.

Contact: hogue@mshri.on.ca

Supplementary Information: The Trajectory Directed Ensemble Sampling (TraDES) web site can be visited at http://bioinfo.mshri.on.ca/trades/. The web-based version of INITTRAJ/FOLDTRAJ may be accessed at http://foldtraj.mshri.on.ca/.

* To whom correspondence should be addressed.