bluuebunny/crossref_metadata_2025 huggingface dataset:
Bottom-up Neighbourhood Revitalisation: A Language Approach for Participatory Decision Support
Authors: Arias Ernesto G.
Published: July 2002

Urban planners have become increasingly aware of the importance of implementation in the formulation of policy. This concern has guided on-going efforts of the Urban Simulations and Information Systems Laboratory (SIMLab) at the University of Colorado in developing interactive three-dimensional planning simulations and games. These simulations are used as decision-making tools to help neighbourhood residents to help themselves in making decisions that shape the housing and communities where they live. The present paper describes the conceptual aspects behind the development of these tools, applies the tools to the redevelopment of the Cole neighbourhood in Denver, Colorado, and critiques the limitations of the tools and discusses their future applications.
Lending Jobs to Global Cities: Skilled International Labour Migration, Investment Banking and the City of London
Authors: Beaverstock Jonathan V. , Smith Joanne
Published: July 2002

The agglomeration of skilled international migrants in global cities' financial communities has parallelled the globalisation of financial capital, international markets and deregulation. International workers have clustered in global cities as a response to their geo-economic functions, and in particular those labour market demands created within transnational corporate headquarters. Within this context, this paper will provide a discussion of the significance of skilled international labour migration within a global city: the City of London's transnational investment banking community.
Windows on the City
Authors: Cosgrove Denis
Published: July 2002

Nigel Thrift's strictures against reading technological change and the emergence of information space as evidence for increased urban dehumanisation are well founded. Throughout the Modern period, distrust of the performative aspects of urban life has characterised puritan criticism and is linked to an elevation of textual over visual authority. Human corporeality resists techno-power; it has the capacity to escape sociological categorisation as effectively.
Maternal authoritative and authoritarian attitudes and mother–child interactions and relationships in urban China
Authors: Chen Xinyin , Liu Mowei , Li Boshu , Cen Guozhen , Chen Huichang , Wang Li
Published: July 2002

The purpose of the study was to examine how maternal authoritative and authoritarian attitudes were manifested in mother–child interactions and relationships in Chinese culture. A sample of 2-year-old children and their mothers ( N= 166) in Beijing and Shanghai, People’s Republic of China, participated in the study. Information on maternal child-rearing attitudes and mother–child attachment relationships was obtained from mothers’ reports. Data concerning maternal parenting strategies, child behaviours, and child reactions to maternal reunion were collected from observations. The results indicated that maternal authoritative attitudes were associated with low power parenting strategies, whereas maternal authoritarian attitudes were associated with enforcement and prohibitory strategies. It was also found that maternal attitudes were associated with child behaviours through the mediation of parenting strategies. However, maternal attitudes might contribute uniquely to mother–child relationships, over and above maternal behaviours. The study provided evidence on the internal coherence of authoritative and authoritarian patterns and their differential significance for child development in urban China.
Pulmonary Function in Nonsmokers Following Exposure to Sidestream Cigarette Smoke
Authors: Smith Carr J. , Bombick David W. , Ryan Brett A. , Morton Michael J. , Doolittle David J.
Published: July 2002

Ten healthy male and 10 healthy female, “never-smoking” subjects (ages 21—50) participated in a 5-day environmental room study to determine if an acute exposure to a high level of fresh diluted sidestream smoke (FDSS) would alter pulmonary function. On Monday, Tuesday, Thursday and Friday, the twenty subjects sat in an environmental room for 7.33 hours and were exposed to fi ltered and humidified air. On Wednesday, the twenty subjects were exposed in an environmental room for 7.33 hours to an average respirable suspended particle (RSP) concentration of 179 micrograms per m3 of FDSS generated by machine smoking Kentucky 1R4F reference cigarettes. This level of FDSS is 3.3 times the 95th percentile concentration of workplace environmental tobacco smoke exposure levels previously measured in the US. FVC and FEV1 decreased approximately 1.6% ( p < 0.05) in both males and females after exposure. Similarly, PEF decreased approximately 1.3% ( p < 0.03) following exposure. The observed decrease in pulmonary function was consistent with a “stress” related norepinephrine-induce d alteration in blood flow leading to transient bronchoconstriction. Alternatively, a cholinergic refl ex due to activation of bronchopulmonar y C fibers may have also played a role in the transient bronchoconstriction. These small exposure-related decrements in pulmonary function were reversible.
Digital Songlines: The Use of Modern Communication Technology by an Aboriginal Community in Remote Australia
Authors: Buchtmann Lydia
Published: July 2002

In the mid-1980s the AUSSAT satellite brought television and radio to remote Australia for the first time. There was concern amongst Aboriginal communities that the imposition of mass media without consultation could result in permanent damage to culture and language. However, over the years, the Warlpiri people have adopted modern communication technology including radio, video making, locally produced television, and more recently on-line services. This paper examines why the Warlpiri have adopted modern communication technology and whether there have been social changes as a result. It also looks at the pioneering media work by the Pitjantjatjara people at Ernabella in the far north of South Australia.
Skin Conductance Hypo‐Responding in Recently Abstinent Cocaine Dependent Inpatients
Authors: Killeen Therese K. , Brady Kathleen T.
Published: July 2002

Skin conductance has been used as a measure of physiological arousal in cocaine cue reactivity studies. In this study, skin conductance responses in recently abstinent (average 3.1 ± 1.7 days) cocaine dependent inpatients (N = 30) were assessed. A video depicting individuals preparing and using cocaine was the cue stimulus. Skin conductance responses were not increased by the stimulus cue. Several explanations are explored that may support not using skin conductance as an outcome measure in recently abstinent cocaine dependent patients.
Chemically Induced Myelinopathies
Authors: Gemert Marciavan , Killeen James
Published: July 2002

The diverse, structurally unrelated chemicals that cause toxic myelinopathies have been investigated and can be categorized into two types of primary demyelinators. Some demyelinating chemicals seem to leave intact the myeli-nating cells (oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system), while others damage the myelinating cells as well as the myelin. The significance between the two is that with the myelinating cells still in tact, repair of the myelin sheath can occur. However, if the myelinating cells are destroyed, repair and reversal of the neuropathy may not occur. Histologically, these chemicals produce an edema of the white matter of the brain, and in some cases the peripheral nervous system, that appears spongy by light microscopy. By electron microscopy, vacuoles can be seen in the myelin surrounding axons. These vacuoles are characterized as fluid-filled separations (splitting) of myelin lamellae at the intraperiod line. In some cases these vacuoles can degenerate further to full demyelination, affecting conduction through those axons. Regeneration of the myelin layers can occur, and in some cases occurs at the same time other axons are undergoing toxic demyelination. Several of these chemicals, however, have been shown to increase cerebrospinal fluid pressure in the brain, optic nerve, and spinal cord, and/or intraneuronal pressure in the perineurium surrounding the axons in the peripheral nervous system. This increased pressure has been correlated with decreased conduction capacity through the axon, ischemia to the neuronal tissue from decreased blood flow because of pressure against the blood vessels, and, if unrelieved, permanent axonal damage. Several of these chemicals havebeen shown to inhibit oxidative phosphorylation, while others uncouple oxidative phosphorylation. One chemical appears to inhibit an enzyme critical to cholesterol synthesis, thus destabilizing myelin. Another hypothesis for a mechanism of action may be in the ability of these compounds to alter membrane permeability.
Testing Guidelines for Evaluation of Reproductive and Developmental Toxicity of Food Additives in Females
Authors: Collins Thomas F. X. , Sprando Robert L. , Hansen Deborah L. , Shachelford Mary E. , Welsh John J.
Published: July 2002

The original Redbook issued by the Food and Drug Administration in 1982 pro-vided guidelines for testing the effects of direct food additives and color additives on mothers and developing embryos. The tests included brief teratol-ogy/developmental toxicity studies and longer studies spanning several generations called multigeneration reproduction studies. In 1993, the draft version of Redbook II was made available for public comment. In it, the revised chapter on reproduction and developmental toxicity is the result of extensive literature review and public comment. It includes discussions of the major mani-festations of an effect on the developing organism — death, structural anomaly, altered or retarded growth, and functional deficiency — and evaluation of these manifestations with respect to dosage and other factors. Additional test end-points and developmental landmarks are included and evaluated. The use of the degree of maternal toxicity in assessing fetotoxicity or embryotoxicity is discussed. FDA's revised guidelines for developmental toxicity and multigeneration reproduction studies are described and compared with current EPA draft guidelines and the guidelines issued by the OECD.
Children with word‐finding difficulties‐prevalence, presentation and naming problems
Authors: Dockrell Julie E. , Messer David , George Rachel
Published: July 2002

There is increasing interest from therapists and researchers in children's wordfinding difficulties (WFDs). Word finding difficulties are usually considered to be present when children are able to identify a referent from a set of exemplars, but have difficulty producing the target word when shown a picture or in conversation. Word finding difficulties are associated with a number of conversational forms such as delays in the production of a word, the use of long pauses within phrases, frequent use of place holders (‘uh’, ‘um’, etc.) and the use of circumlocutions. Although interest is being shown in WFDs, most of the data come from relatively small samples with the result that one knows little about the prevalence of the condition, what circumstances exacerbate the condition, the occurrence of associated difficulties and the types of therapy that are employed. To obtain this information a survey was carried out in the SouthEast of England of practitioners working with children who have language difficulties. Twenty‐three per cent of children in language support services were identified as having WFDs. Most respondents used a mixture of formal and informal assessments. It was reported that WFDs were associated with difficulties in grammatical production, word meaning and grammatical comprehension. WFDs in addition were more likely to occur in situations with high processing demands. A variety of intervention strategies were identified. These findings are discussed in relation to current practice and the authors understanding of the condition.
Enhancement of Oxidative Damage to Cultured Cells and Caenorhabditis elegans by Mitochondrial Electron Transport Inhibitors
Authors: Ishiguro Hiroyuki , Yasuda Kayo , Ishii Naoaki , Ihara Kenichi , Ohkubo Tomoichi , Hiyoshi Mineyoshi , Ono Kazuhiro , Senoo‐Matsuda Nanami , Shinohara Osamu , Yosshii Fumihiro , Murakami Masaru , Hartman Philip S. , Tsuda Michio
Published: July 2002

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans ( C. elegans ) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans . In addition the cell damage is different dependent on cell type and organism.
Nuclear Membrane Dynamics and Reassembly in Living Cells: Targeting of an Inner Nuclear Membrane Protein in Interphase and Mitosis
Authors: Ellenberg Jan , Siggia Eric D. , Moreira Jorge E. , Smith Carolyn L. , Presley John F. , Worman Howard J. , Lippincott-Schwartz Jennifer
Published: July 2002

The mechanisms of localization and retention of membrane proteins in the inner nuclear membrane and the fate of this membrane system during mitosis were studied in living cells using the inner nuclear membrane protein, lamin B receptor, fused to green fluorescent protein (LBR–GFP). Photobleaching techniques revealed the majority of LBR–GFP to be completely immobilized in the nuclear envelope (NE) of interphase cells, suggesting a tight binding to heterochromatin and/or lamins. A subpopulation of LBR–GFP within ER membranes, by contrast, was entirely mobile and diffused rapidly and freely (D = 0.41 ± 0.1 μm2/s). High resolution confocal time-lapse imaging in mitotic cells revealed LBR–GFP redistributing into the interconnected ER membrane system in prometaphase, exhibiting the same high mobility and diffusion constant as observed in interphase ER membranes. LBR–GFP rapidly diffused across the cell within the membrane network defined by the ER, suggesting the integrity of the ER was maintained in mitosis, with little or no fragmentation and vesiculation. At the end of mitosis, nuclear membrane reformation coincided with immobilization of LBR–GFP in ER elements at contact sites with chromatin. LBR–GFP–containing ER membranes then wrapped around chromatin over the course of 2–3 min, quickly and efficiently compartmentalizing nuclear material. Expansion of the NE followed over the course of 30–80 min. Thus, selective changes in lateral mobility of LBR–GFP within the ER/NE membrane system form the basis for its localization to the inner nuclear membrane during interphase. Such changes, rather than vesiculation mechanisms, also underlie the redistribution of this molecule during NE disassembly and reformation in mitosis.
Golgi Tubule Traffic and the Effects of Brefeldin A Visualized in Living Cells
Authors: Sciaky Noah , Presley John , Smith Carolyn , Zaal Kristien J.M. , Cole Nelson , Moreira Jorge E. , Terasaki Mark , Siggia Eric , Lippincott-Schwartz Jennifer
Published: July 2002

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein–Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5–10 min until rapidly emptying Golgi contents into the ER within 15–30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.
Integrin α1β1 Mediates a Unique Collagen-dependent Proliferation Pathway In Vivo
Authors: Pozzi Ambra , Wary Kishore K. , Giancotti Filippo G. , Gardner Humphrey A.
Published: July 2002

Activation of integrins upon binding to extracellular matrix proteins is believed to be a crucial step for the regulation of cell survival and proliferation. We have used integrin α1-null mice to investigate the role of this collagen receptor in the regulation of cell growth and survival in vivo. α1-deficient animals, which are viable and fertile, have a hypocellular dermis and a deficiency in dermal fibroblast proliferation as embryos. In vitro analysis of α1-null embryonic fibroblasts has revealed that their proliferation rate is markedly reduced when plated on collagenous substrata, despite normal attachment and spreading. Moreover, on the same collagenous matrices, α1-null fibroblasts fail to recruit and activate the adaptor protein Shc. The failure to activate Shc is accompanied by a downstream deficiency in recruitment of Grb2 and subsequent mitogen-activated protein kinase activation. Taken together with the growth deficiency observed on collagens, this finding indicates that the α1β1 is the sole collagen receptor which can activate the Shc mediated growth pathway. Thus, integrin α1 has a unique role among the collagen receptors in regulating both in vivo and in vitro cell proliferation in collagenous matrices.
Overexpression of Tau Protein Inhibits Kinesin-dependent Trafficking of Vesicles, Mitochondria, and Endoplasmic Reticulum: Implications for Alzheimer's Disease
Authors: Ebneth A. , Godemann R. , Stamer K. , Illenberger S. , Trinczek B. , Mandelkow E.-M. , Mandelkow E.
Published: July 2002

The neuronal microtubule-associated protein tau plays an important role in establishing cell polarity by stabilizing axonal microtubules that serve as tracks for motor-protein–driven transport processes. To investigate the role of tau in intracellular transport, we studied the effects of tau expression in stably transfected CHO cells and differentiated neuroblastoma N2a cells. Tau causes a change in cell shape, retards cell growth, and dramatically alters the distribution of various organelles, known to be transported via microtubule-dependent motor proteins. Mitochondria fail to be transported to peripheral cell compartments and cluster in the vicinity of the microtubule-organizing center. The endoplasmic reticulum becomes less dense and no longer extends to the cell periphery. In differentiated N2a cells, the overexpression of tau leads to the disappearance of mitochondria from the neurites. These effects are caused by tau's binding to microtubules and slowing down intracellular transport by preferential impairment of plus-end–directed transport mediated by kinesin-like motor proteins. Since in Alzheimer's disease tau protein is elevated and mislocalized, these observations point to a possible cause for the gradual degeneration of neurons.
Cytokine-Induced Nuclear Factor Kappa B Activation Promotes the Survival of Developing Neurons
Authors: Middleton Gayle , Hamanoue Makoto , Enokido Yasushi , Wyatt Sean , Pennica Diane , Jaffray Ellis , Hay Ronald T. , Davies Alun M.
Published: July 2002

Ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and interleukin 6 (IL-6) comprise a group of structurally related cytokines that promote the survival of subsets of neurons in the developing peripheral nervous system, but the signaling pathways activated by these cytokines that prevent neuronal apoptosis are unclear. Here, we show that these cytokines activate NF-κB in cytokine-dependent developing sensory neurons. Preventing NF-κB activation with a super-repressor IκB-α protein markedly reduces the number of neurons that survive in the presence of cytokines, but has no effect on the survival response of the same neurons to brain-derived neurotrophic factors (BDNF), an unrelated neurotrophic factor that binds to a different class of receptors. Cytokine-dependent sensory neurons cultured from embryos that lack p65, a transcriptionally active subunit of NF-κB, have a markedly impaired ability to survive in response to cytokines, but respond normally to BDNF. There is increased apoptosis of cytokine- dependent neurons in p65−/− embryos in vivo, resulting in a reduction in the total number of these neurons compared with their numbers in wild-type embryos. These results demonstrate that NF-κB plays a key role in mediating the survival response of developing neurons to cytokines.
Targeting Pyk2 to β1-Integrin–Containing Focal Contacts Rescues Fibronectin-Stimulated Signaling and Haptotactic Motility Defects of Focal Adhesion Kinase–Null Cells
Authors: Klingbeil Candice K. , Hauck Christof R. , Hsia Datsun A. , Jones K.C. , Reider Shannon R. , Schlaepfer David D.
Published: July 2002

Focal adhesion kinase–null (FAK−/−) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK−/− cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK−/− cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a β1-integrin–containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK−/− cells, exhibit elevated FN-stimulated extracellular signal–regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH2-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to β-integrin–containing focal contact sites via interactions mediated by the FAK-CT domain.
UGO1 Encodes an Outer Membrane Protein Required for Mitochondrial Fusion
Authors: Sesaki Hiromi , Jensen Robert E.
Published: July 2002

Membrane fusion plays an important role in controlling the shape, number, and distribution of mitochondria. In the yeast Saccharomyces cerevisiae, the outer membrane protein Fzo1p has been shown to mediate mitochondrial fusion. Using a novel genetic screen, we have isolated new mutants defective in the fusion of their mitochondria. One of these mutants, ugo1, shows several similarities to fzo1 mutants. ugo1 cells contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. ugo1 mutants lose mitochondrial DNA (mtDNA). In zygotes formed by mating two ugo1 cells, mitochondria do not fuse and mix their matrix contents. Fragmentation of mitochondria and loss of mtDNA in ugo1 mutants are rescued by disrupting DNM1, a gene required for mitochondrial division. We find that UGO1 encodes a 58-kD protein located in the mitochondrial outer membrane. Ugo1p appears to contain a single transmembrane segment, with its NH2 terminus facing the cytosol and its COOH terminus in the intermembrane space. Our results suggest that Ugo1p is a new outer membrane component of the mitochondrial fusion machinery.
Cd44 Is a Major E-Selectin Ligand on Human Hematopoietic Progenitor Cells
Authors: Dimitroff Charles J. , Lee Jack Y. , Rafii Shahin , Fuhlbrigge Robert C. , Sackstein Robert
Published: July 2002

E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin–dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin–dependent adhesive interactions that direct homing of human HPC to BM.
An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells
Authors: Belgareh Naïma , Rabut Gwénaël , Baï Siau Wei , van Overbeek Megan , Beaudouin Joël , Daigle Nathalie , Zatsepina Olga V. , Pasteau Fabien , Labas Valérie , Fromont-Racine Micheline , Ellenberg Jan , Doye Valérie
Published: July 2002

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.
Targeting of an abundant cytosolic form of the protein import receptor at Toc159 to the outer chloroplast membrane
Authors: Hiltbrunner Andreas , Bauer Jörg , Vidi Pierre-Alexandre , Infanger Sibylle , Weibel Petra , Hohwy Morten , Kessler Felix
Published: July 2002

Chloroplast biogenesis requires the large-scale import of cytosolically synthesized precursor proteins. A trimeric translocon (Toc complex) containing two homologous GTP-binding proteins (atToc33 and atToc159) and a channel protein (atToc75) facilitates protein translocation across the outer envelope membrane. The mechanisms governing function and assembly of the Toc complex are not yet understood. This study demonstrates that atToc159 and its pea orthologue exist in an abundant, previously unrecognized soluble form, and partition between cytosol-containing soluble fractions and the chloroplast outer membrane. We show that soluble atToc159 binds directly to the cytosolic domain of atToc33 in a homotypic interaction, contributing to the integration of atToc159 into the chloroplast outer membrane. The data suggest that the function of the Toc complex involves switching of atToc159 between a soluble and an integral membrane form.
SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles
Authors: Joseph Jomon , Tan Shyh-Han , Karpova Tatiana S. , McNally James G. , Dasso Mary
Published: July 2002

RanGAP1 was the first documented substrate for conjugation with the ubiquitin-like protein SUMO-1. However, the functional significance of this conjugation has not been fully clarified. We sought to examine RanGAP1 behavior during mitosis. We found that RanGAP1 associates with mitotic spindles and that it is particularly concentrated at foci near kinetochores. Association with kinetochores appeared soon after nuclear envelope breakdown and persisted until late anaphase, but it was lost coincident with nuclear envelope assembly in telophase. A mutant RanGAP1 protein lacking the capacity to be conjugated to SUMO-1 no longer associated with spindles, indicating that conjugation was essential for RanGAP1's mitotic localization. RanBP2, a nuclear pore protein that binds SUMO-1–conjugated RanGAP1 during interphase, colocalized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. This report shows for the first time that SUMO-1 conjugation is required for mitotic localization of RanGAP1, and suggests that a major role of SUMO-1 conjugation to RanGAP1 may be the spatial regulation of the Ran pathway during mitosis.
T Helper Subset Differentiation in the Absence of Invariant Chain
Authors: Brown Daniel R. , Swier Kevin , Moskowitz Naomi H. , Naujokas Marisa F. , Locksley Richard M. , Reiner Steven L.
Published: July 2002

The outcome of murine infection with Leishmania major is regulated by major histocompatibility complex class II–restricted T helper cells. Invariant chain-deficient (Ii −/−) mice have impaired ability to present major histocompatibility complex class II–restricted antigens, and reduced numbers of CD4+ T cells. Despite these deficits, C57BL/6 Ii −/− mice controlled L. major infection comparably to wild-type mice. As assessed by mRNA analysis and in vitro antigen restimulation for IFN-γ, Ii −/− mice had normal induction of Th1 subset differentiation even though antigen-dependent proliferation of their lymph node cells was substantially compromised. In addition, BALB/c Ii −/− mice exhibited a progressive course of infection and Th2 effector cell development that were comparable to that seen in wild-type BALB/c mice. We wished to determine whether this unexpected efficiency of T helper subset induction despite inefficient T cell stimulation could be modeled in vitro. In the presence of rIL-12 or rIL-4 naive parasite-specific transgenic T cells could mature into IFN-γ–or IL-4–secreting T helper cells, respectively, even when antigen presentation was suboptimal or antigen dose was submitogenic. These experiments demonstrate that activation of T helper cells to a threshold required for IL-2 production or proliferation is not required to achieve induction of disease-regulating T helper cell effector functions, and that pathogen-associated secondary activation signals may facilitate the full differentiation of T helper subsets during limiting presentation of antigenic peptides.
Interleukin-4 Protects against a Genetically Linked Lupus-like Autoimmune Syndrome
Authors: Santiago Marie-Laure , Fossati Liliane , Jacquet Chantal , Müller Werner , Izui Shozo , Reininger Luc
Published: July 2002

Interleukin-4 (IL-4) provides support for humoral immune responses through upregulation of T helper (Th) type 2 cell differentiation, but it is not known whether IL-4 promotes antibodymediated autoimmune diseases such as systemic lupus erythematosus (SLE). Here, we show that the constitutive expression of an IL-4 transgene by B cells completely prevents the development of lethal lupus-like glomerulonephritis in the (NZW × C57BL/6.Yaa)F1 murine model of SLE. This was associated with marked changes in the serum levels of IgG subclasses, rather than in the total levels of anti-DNA antibodies, with a lack of IgG3, a decrease of IgG2a, and an increase in IgG1 subclasses, and by a strong reduction in the serum levels of gp70-antigp70 immune complexes. This effect of the transgene appears to result from a modulation of the Th1 versus Th2 autoimmune response, since the protected mice displayed comparably modified IgG2a and IgG3 antibody response against exogenous T cell–dependent antigen, but not against T cell–independent antigens. Thus, IL-4 prevents the development of this lupuslike autoimmune disease, most likely by downregulating the appearance of Th1-mediated IgG subclasses of autoantibodies such as the IgG3 autoantibodies which have been shown to be especially nephritogenic.
4-1BB Costimulatory Signals Preferentially Induce CD8+ T Cell Proliferation and Lead to the Amplification In Vivo of Cytotoxic T Cell Responses
Authors: Shuford Walter W. , Klussman Kerry , Tritchler Douglas D. , Loo Deryk T. , Chalupny Jan , Siadak Anthony W. , Brown T. Joseph , Emswiler John , Raecho Hong , Larsen Christian P. , Pearson Thomas C. , Ledbetter Jeffrey A. , Aruffo Alejandro , Mittler Robert S.
Published: July 2002

The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d–specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-γ production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.
Type I Interferon-mediated Stimulation of T Cells by CpG DNA
Authors: Sun Siquan , Zhang Xiaohong , Tough David F. , Sprent Jonathan
Published: July 2002

Immunostimulatory DNA and oligodeoxynucleotides containing unmethylated CpG motifs (CpG DNA) are strongly stimulatory for B cells and antigen-presenting cells (APCs). We report here that, as manifested by CD69 and B7-2 upregulation, CpG DNA also induces partial activation of T cells, including naive-phenotype T cells, both in vivo and in vitro. Under in vitro conditions, CpG DNA caused activation of T cells in spleen cell suspensions but failed to stimulate highly purified T cells unless these cells were supplemented with APCs. Three lines of evidence suggested that APC-dependent stimulation of T cells by CpG DNA was mediated by type I interferons (IFN-I). First, T cell activation by CpG DNA was undetectable in IFN-IR−/− mice. Second, in contrast to normal T cells, the failure of purified IFN-IR−/− T cells to respond to CpG DNA could not be overcome by adding normal IFN-IR+ APCs. Third, IFN-I (but not IFN-γ) caused the same pattern of partial T cell activation as CpG DNA. Significantly, T cell activation by IFN-I was APC independent. Thus, CpG DNA appeared to stimulate T cells by inducing APCs to synthesize IFN-I, which then acted directly on T cells via IFN-IR. Functional studies suggested that activation of T cells by IFN-I was inhibitory. Thus, exposing normal (but not IFN-IR−/−) T cells to CpG DNA in vivo led to reduced T proliferative responses after TCR ligation in vitro.
Immature Dendritic Cells Phagocytose Apoptotic Cells via αvβ5 and CD36, and Cross-present Antigens to Cytotoxic T Lymphocytes
Authors: Albert Matthew L. , Pearce S.Frieda A. , Francisco Loise M. , Sauter Birthe , Roy Pampa , Silverstein Roy L. , Bhardwaj Nina
Published: July 2002

Dendritic cells, but not macrophages, efficiently phagocytose apoptotic cells and cross-present viral, tumor, and self-antigens to CD8+ T cells. This in vitro pathway corresponds to the in vivo phenomena of cross-priming and cross-tolerance. Here, we demonstrate that phagocytosis of apoptotic cells is restricted to the immature stage of dendritic cell (DC) development, and that this process is accompanied by the expression of a unique profile of receptors, in particular the αvβ5 integrin and CD36. Upon maturation, these receptors and, in turn, the phagocytic capacity of DCs, are downmodulated. Macrophages engulf apoptotic cells more efficiently than DCs, and although they express many receptors that mediate this uptake, they lack the αvβ5 integrin. Furthermore, in contrast to DCs, macrophages fail to cross-present antigenic material contained within the engulfed apoptotic cells. Thus, DCs use unique pathways for the phagocytosis, processing, and presentation of antigen derived from apoptotic cells on class I major histocompatibility complex. We suggest that the αvβ5 integrin plays a critical role in the trafficking of exogenous antigen by immature DCs in this cross-priming pathway.
Interleukin 2, but Not Other Common γ Chain–Binding Cytokines, Can Reverse the Defect in Generation of Cd4 Effector T Cells from Naive T Cells of Aged Mice
Authors: Haynes Laura , Linton Phyllis-Jean , Eaton Sheri M. , Tonkonogy Susan L. , Swain Susan L.
Published: July 2002

Development of effectors from naive CD4 cells occurs in two stages. The early stage involves activation and limited proliferation in response to T cell receptor (TCR) stimulation by antigen and costimulatory antigen presenting cells, whereas the later stage involves proliferation and differentiation in response to growth factors. Using a TCR-transgenic (Tg+) model, we have examined the effect of aging on effector generation and studied the ability of γc signaling cytokines to reverse this effect. Our results indicate that responding naive CD4 cells from aged mice, compared with cells from young mice, make less interleukin (IL)-2, expand poorly between days 3 to 5, and give rise to fewer effectors with a less activated phenotype and reduced ability to produce cytokines. When exogenous IL-2 or other γc signaling cytokines are added during effector generation, the Tg+ cells from both young and aged mice proliferate vigorously. However, IL-4, IL-7, and IL-15 all fail to restore efficient effector production. Only effectors from aged mice generated in the presence of IL-2 are able to produce IL-2 in amounts equivalent to those produced by effectors generated from young mice, suggesting that the effect of aging on IL-2 production is reversible only in the presence of exogenous IL-2.
Immature Thymocytes Undergoing Receptor Rearrangements Are Resistant to an Atm-Dependent Death Pathway Activated in Mature T Cells by Double-Stranded DNA Breaks
Authors: Bhandoola Avinash , Dolnick Benjamin , Fayad Nihal , Nussenzweig Andre , Singer Alfred
Published: July 2002

Immature CD4+CD8+ thymocytes rearrange their T cell receptor (TCR)-α gene locus to generate clonotypic α/β TCR, after which a few cells expressing selectable TCR are signaled to further differentiate into mature T cells. Because of requirements for self-tolerance, immature CD4+CD8+ thymocytes are programmed to die in the thymus in response to a variety of stimuli that do not induce death of mature T cells. We now demonstrate that, in contrast to all previously described stimuli, immature CD4+CD8+ thymocytes are selectively more resistant than mature T cells to apoptotic death induced by DNA intercalating agents. Importantly, we demonstrate that DNA intercalating agents induce double-stranded DNA breaks in both immature thymocytes and mature T cells, but immature thymocytes tolerate these DNA breaks, whereas mature T cells are signaled to die by an Atm-dependent but p53-independent death mechanism. Thus, our results indicate that absence of an Atm-dependent but p53-independent pathway allows immature thymocytes to survive double-stranded DNA breaks. It is likely that the unique ability of immature thymocytes to survive DNA-damaging intercalating agents reflects their tolerance of double-stranded DNA breaks that occur normally during antigen receptor gene rearrangements.
Bone Marrow–Derived Antigen-Presenting Cells Are Required for the Generation of Cytotoxic T Lymphocyte Responses to Viruses and Use Transporter Associated with Antigen Presentation (Tap)-Dependent and -Independent Pathways of Antigen Presentation
Authors: Sigal Luis J. , Rock Kenneth L.
Published: July 2002

Bone marrow (BM)-derived professional antigen-presenting cells (pAPCs) are required for the generation of cytotoxic T lymphocyte (CTL) responses to vaccinia virus and poliovirus. Furthermore, these BM-derived pAPCs require a functional transporter associated with antigen presentation (TAP). In this report we analyze the requirements for BM-derived pAPCs and TAP in the initiation of CTL responses to lymphocytic choriomeningitis virus (LCMV) and influenza virus (Flu). Our results indicate a requirement for BM-derived pAPCs for the CTL responses to these viruses. However, we found that the generation of CTLs to one LCMV epitope (LCMV nucleoprotein 396–404) was dependent on BM-derived pAPCs but, surprisingly, TAP independent. The study of the CTL response to Flu confirmed the existence of this BM-derived pAPC-dependent/TAP-independent CTL response and indicated that the TAP-independent pathway is ∼10–300-fold less efficient than the TAP-dependent pathway.
Anergy in Peripheral Memory Cd4+ T Cells Induced by Low Avidity Engagement of T Cell Receptor
Authors: Mirshahidi Saied , Huang Ching-Tai , Sadegh-Nasseri Scheherazade
Published: July 2002

Induction of tolerance in self-reactive memory T cells is an important process in the prevention of autoimmune responses against peripheral self-antigens in autoimmune diseases. Although naive T cells can readily be tolerized, memory T cells are less susceptible to tolerance induction. Recently, we demonstrated that low avidity engagement of T cell receptor (TCR) by low densities of agonist peptides induced anergy in T cell clones. Since memory T cells are more responsive to lower antigenic stimulation, we hypothesized that a low avidity TCR engagement may induce tolerance in memory T cells. We have explored two antigenic systems in two transgenic mouse models, and have tracked specific T cells that are primed and show memory phenotype. We demonstrate that memory CD4+ T cells can be rendered anergic by presentation of low densities of agonist peptide–major histocompatibility complex complexes in vivo. We rule out other commonly accepted mechanisms for induction of T cell tolerance in vivo, such as deletion, ignorance, or immunosuppression. Anergy is the most likely mechanism because addition of interleukin 2–reversed anergy in specific T cells. Moreover, cytotoxic T lymphocyte antigen (CTLA)-4 plays a critical role in the induction of anergy because we observed that there was increased surface expression of CTLA-4 on anergized T cells, and that injection of anti–CTLA-4 blocking antibody restored anergy in vivo.
Molecular Dissection of Ca2+ Efflux in Immortalized Proximal Tubule Cells
Authors: White Kenneth E. , Gesek Frank A. , Nesbitt Teresa , Drezner Marc K. , Friedman Peter A.
Published: July 2002

Plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger participate in regulating cell function by maintaining proper intracellular Ca2+ concentrations ([Ca2+]i). In renal epithelial cells these proteins have been additionally implicated in cellular calcium absorption. The purpose of the present studies was to determine the Ca2+ extrusion mechanisms in cells derived from the proximal tubule. Homology-based RT-PCR was used to amplify PMCA transcripts from RNA isolated from mouse cell lines originating from the S1, S2, and S3 proximal tubule segments. S1, S2, and S3 cells exhibited only PMCA1 and PMCA4 products. PCR product identity was confirmed by sequence analysis. Northern analysis of proximal tubule cell RNAs revealed appropriate transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4, but were negative for PMCA2 and PMCA3. Western analysis with a monoclonal antibody to PMCA showed that all proximal cell lines expressed a reacting plasma membrane protein of 140 kD, the reported PMCA molecular mass. Na+/Ca2+ exchanger (NCX1) mRNA expression, analyzed by RT-PCR, protein expression by Western analysis, and functional exchange activity were uniformly absent from all proximal tubule cell lines. These observations support the idea that immortalized cells derived from the proximal tubule express PMCA1 and PMCA4, which may serve as the primary mechanism of cellular Ca2+ efflux.
Octameric Stoichiometry of the KATP Channel Complex
Authors: Shyng S.-L. , Nichols C.G.
Published: July 2002

ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits—a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.