bluuebunny/crossref_metadata_2025 huggingface dataset:
Seceding from Responsibility? Secession Movements in Los Angeles
Authors: Boudreau Julie-Anne , Keil Roger
Published: July 2002

This paper seeks to understand why secession movements gained momentum in Los Angeles and what their effect will be on regional governance. A brief discussion of liberal theories of secession demonstrates that they cannot explain secession movements at the urban scale, as they are exclusively focused on cases of nationalist secession from a nation-state. Furthermore, liberal theories of secession offer normative arguments on the right to secede. Following a change in California legislation granting municipalities the right to secede, the secessionist debate in Los Angeles is not so much concerned with normative issues, but more with devising an effective and revenue-neutral process for secession. Using a threefold theoretical approach based on theories of secession, regulation theory and theories of state rescaling, and theories of social movements, this paper argues that the 'political opportunity structures' provided by globalisation and the prevalent neo-conservatism, might explain how secession movements in Los Angeles were able to mobilise large efforts to their cause. We hold that globalisation has forced cities to re-open the debate on size and governance. Secession movements have been very successful in raising public awareness on the issue. Their strength lies mostly in their populism, well in tune with the prevalent wave of neo-conservatism. These movements use arguments well grounded in Southern California's complex history of regional fragmentation and consolidation. As a social project of the Right, they offer secession as a potential 'solution' to the problems of urban governance in the age of globalisation, in a context of simultaneous consolidation.
Persistent Polarisation Post-Apartheid? Progress towards Urban Integration in Cape Town
Authors: Turok Ivan
Published: July 2002

The paper examines the progress made since 1994 to reduce the deep social and spatial divisions in South African cities, focusing on the impact of current development trends in Cape Town. Basic public services are gradually being extended to the historically neglected townships. However, the character of economic and social development differs markedly across the city. Private-sector investment and jobs continue to be concentrated in the affluent north and west, while low-income subsidised housing is focused on the poorer south-east. Institutional practices and market forces are tending to reinforce spatial divisions, with costly consequences for the poor majority of the population and for the wider urban economy and society. The broad statements from policy-makers about the need for urban integration are not being translated into consistent action, apparently because of a lack of political will and institutional capacity. The new Unicity authority provides scope in principle for a more coherent approach, based in part on a strategic framework to manage land development in the interests of the whole city, and a more proactive approach towards building a stronger employment base in the south-east.
The Definition of Housing Market Areas and Strategic Planning
Authors: Jones Colin
Published: July 2002

The purpose of this paper is to develop the concept of a housing market area, thereby provide insights into our understanding of the sub-regional structures of the housing system and hence propose a framework for strategic planning research in the housing market. To tackle this task, it is first necessary to consider the criteria for a local housing market area. The paper begins by reviewing previous definitions by both academics and planners, and their underlying assumptions. Based on the principle of spatial arbitrage, a local housing market area is defined by reference to criteria linked to migration self-containment. Parallels are drawn with the principles that have been applied to define a system of spatial labour markets using travel-to-work areas. This spatial arbitrage definition is tested empirically by developing a system of housing market areas for west central Scotland using data derived from the Land Registry. Finally, the paper draws together the empirical findings to discuss the nature of spatial housing markets and the implications for strategic planning. It argues that the establishment of a geography of functional housing market areas represents the first step in the development of systematic planning research.
Knowledge Spillovers and Reasons for the Concentration of Innovative SMEs
Authors: Simmie James
Published: July 2002

This paper reports the findings of a survey of innovative firms in the South East of England undertaken during 1999/2000. It is argued that in order to explain the observed concentration of innovation in a limited number of cities both in the US and in Europe it is necessary to consider both local knowledge spillovers and international knowledge transfers. Knowledge and experience are argued to be important in overcoming the uncertainties that characterise the innovation process. The survey reported here shows that two main categories of knowledge are important for SMEs. These are first mainly supply-side local knowledge spillovers from suppliers, collaborators and HEIs. Secondly, they are often demand-side national and international knowledge transfers from clients, customers and their international distributors. Cities rich in both local capacity and international connections are usually the locations for high levels of innovation among the firms based in them.
Knowledge-based Development: Policy and Planning Implications for Cities
Authors: Knight Richard V.
Published: July 2002

The paper will present a new approach to city development focusing on knowledge-based development. The paper will present a conceptual framework and methodology for defining and assessing a city's knowledge base, i.e. its knowledge resources, knowledge cultures, core competencies and their local and global linkages. The approach draws from the experience of US cities and is being elaborated while undertaking strategic diagnostics of knowledge-based development in several cities for the cities of Amsterdam and Delft, then for the EC-FAST MONITOR study on the Future of European Cities and presently by the city of Vienna. The paper will present key findings from studies of the transformation from production to knowledge in several types and sizes of cities including New York and Cleveland in the US and Amsterdam, Delft, Lille, Lyon, Barcelona, Genoa, Milan and Vienna in Europe. Implications of knowledge-based development for cities and regions will be summarised and the need for a policy framework for cities at the Community level will be explored. Some guiding principles for city development in an open and increasingly knowledge-based global society will be outlined.
Urban Experiments Limited Revisited: Urban Policy Comes Full Circle?
Authors: Wilks-Heeg Stuart
Published: July 2002

This article assesses the role of urban policy as a testing-ground for various policy experiments. In particular, it considers how the failure and controversy stemming from the property-led regeneration experiments of the 1980s have recently led to a major re-organisation of urban policy which appears to return to many of the principles embodied in the 1977 White Paper on the Inner Cities, itself a response to the failed experiments of the 1970s. This apparent circularity of policy is explained with reference to tensions which exist within and between the material, political and ideological contexts in which policy initiatives operate. As a result, urban policy is highly unstable, resulting in an alternation between periods of experimentation and periods of managerialist re-organisation.
Comparing South African and Latin American Experience: Migration and Housing Mobility in Soweto
Authors: Gilbert Alan , Crankshaw Owen
Published: July 2002

Given similar levels of economic development and inequality, it seems strange that more effort has not been made by South Africans to learn from the earlier experience of urbanisation in countries such as Brazil, Chile and Colombia. The paper shows that there are problems in drawing comparisons about migration and residential movement in Latin America and South Africa, but that there are growing signs of similarity. Some of these similarities were apparent even before the demise of apartheid, a policy that was never consistent and changed many times. The paper observes that South African migrants are becoming more like Latin Americans in demonstrating a growing attachment to the city. Many Sowetans' lives do not fit the model of the 'circular' African migrant; many have lived in Johannesburg for a long time. Semi-permanent residence means that residential movement within the city is increasingly similar to that typical in many Latin American cities. To judge from Latin American experience, the dismantling of formal apartheid is unlikely to reduce levels of social segregation in Johannesburg. Rather, class divisions will be just as effective as apartheid in guaranteeing residential segregation.
A Capital Theory of School Effectiveness and Improvement [1]
Authors: Hargreaves David H.
Published: July 2002

Abstract A new theory of school effectiveness and improvement is outlined, based on the master concepts of intellectual capital, social capital and leverage, linked with the conventional concept of institutional outputs. Each master concept is defined in terms of two subsidiary concepts. Twelve specifically educational concepts are set within this framework to provide the theory. It is proposed that, through a simplified model, the range and fertility of the theory can be exemplified and tested in three specific cases—the changing nature of school effectiveness and improvement in knowledge economies, citizenship education and teacher effectiveness.
Linkages Between Causal Ascriptions, Emotion, and Behaviour
Authors: Caprara Gian Vittorio , Pastorelli Concetta , Weiner Bernard
Published: July 2002

Italian schoolboys between the ages of 9 and 10 participated in three experiments guided by attribution theory as conceptualised by Weiner (1985, 1986). In Experiment 1, following teacher-emotional feedback of anger or sympathy for failure, attributional inferences regarding low ability or lack of effort as the cause of that failure were rated. In Experiment 2, controllable and uncontrollable causes of a social transgression were given, and children rated the anticipated anger of the “victim” and their intention to withhold or reveal the cause. In Experiment 3, effects of perceived causality and related emotions of anger and sympathy (pity) with regard to helping behaviour were investigated. Results replicate previous US findings and largely support the established linkages between attribution-emotion-action as proposed by Weiner (1986).
Vocational Training and Career Development in Germany: Results from a Longitudinal Study
Authors: Heinz Walter R. , Kelle Udo , Witzel Andreas , Zinn Jens
Published: July 2002

The present paper presents empirical results from a German panel study which collected longitudinal data regarding the job entry of young adults in six of the top training occupations in the service and technical-industrial sectors. The data clearly demonstrate the influence of gender and social origin on the access to training in particular occupations. Furthermore, the existence of gender and occupation-specific patterns of career development is demonstrated. However, the apprenticeship system also provides mobility opportunities which depend on the specific training occupation. Moving along a certain occupational pathway results in an interplay between the structural opportunities and constraints of occupational contexts, on the one hand, and the young workers’ aspirations and orientations, on the other. These orientations and aspirations were investigated with qualitative methods which helped to identify different modes of biographical action orientations of young workers.
Recognition of Adverse and Nonadverse Effects in Toxicity Studies
Authors: Lewis Richard W. , Billington Richard , Debryune Eric , Gamer Armin , Lang B. , Carpanini Francis
Published: July 2002

One of the most important quantitative outputs from toxicity studies is identification of the highest exposure level (dose or concentration) that does not cause treatment related effects that could be considered relevant to human health risk assessment. A review of regulatory and other scientifi c literature and of current practices has revealed a lack of consistency in definition and application of frequently used terms such as No Observed Effect Level (NOEL), No Observed Adverse Effect Level (NOAEL), adverse effect, biologically significant effect, or toxicologically significant effect. Moreover, no coherent criteria were found that could be used to guide consistent interpretation of toxicity studies, including the recognition and differentiation between adverse and nonadverse effects. This presentation will address these issues identified first by proposing a standard set of definitions for key terms such as NOEL and NOAEL that are frequently used to describe the overall outcome of a toxicity study. Second, a coherent framework is outlined that can assist the toxicologist in arriving at consistent study interpretation. This structured process involves two main steps. In the first, the toxicologist must decide whether differences from control values are treatment related or if they are chance deviations. In the second step, only those differences judged to be effects are further evaluated in order to discriminate between those that are adverse and those that are not. For each step, criteria are described that can be used to make consistent judgments. In differentiating an effect from a chance finding, consideration is given inter alia to dose response, spurious measurements in individual parameters, the precision of the measurement under evaluation, ranges of natural variation and the overall biological plausibility of the observation. In discriminating between the adverse and the non-adverse effect consideration is given to: whether the effect is an adaptive response, whether it is transient, the magnitude of the effect, its association with effects in other related endpoints, whether it is a precursor to a more signifi cant effect, whether it has an effect on the overall function of the organism, whether it is a specific effect on an organ or organ system or secondary to general toxicity or whether the effect is a predictable consequence of the experimental model. In interpreting complex studies it is recognised that a weight of the evidence approach, combining the criteria outlined here to reach an overall judgment, is the optimal way of applying the process. It is believed that the use of such a scheme will help to improve the consistency of study interpretation that is the foundation of hazard and risk assessment.
Clinical Review of Inhalants
Authors: Brouette Thomas , Anton Raymond
Published: July 2002

While the rate of inhalant abuse continues to rise in this country, it remains one of the least studied or discussed groups of abused substances. This review focuses on the current knowledge of the epidemiology, pharmacology, and sequela of inhalant abuse. We will discuss three groups of inhalants: volatile solvents, nitrous oxide, and nitrites. We will then conclude by proposing means by which inhalant abuse may be prevented and treated.
Correlates Of Social Development In Children With Communication Disorders: The Concurrent Predictive Value Of Verbal Short‐Term Memory Span
Authors: Donlan Chris , Masters Judy
Published: July 2002

Linguistic and cognitive correlates of social skill were examined in a clinically selected sample of children with communication disorders. Thirty‐two children aged between 5 and 10 years were assessed using a questionnaire‐based sociability scale. Specially designed measures of verbal and visuo‐spatial short‐term memory span were administered, as well as standardized tests of grammatical and lexical comprehension. Multiple regression analyses revealed strong prediction of social skill from STM measures but not from comprehension scores. The prediction was based on a striking negative correlation between serial word span and sociability. This word‐span measure could offer a useful tool in the assessment of children with communication disorders. However, attention is drawn to the substantial number of children in the sample whose patterns of cognitive, linguistic and social skill are not clearly differentiated. It is suggested that assessment should aim to specify as clearly as possible individual patterns of strength and weakness and avoid unwarranted categorization.
Spindle Assembly and Mitosis without Centrosomes in Parthenogenetic Sciara Embryos
Authors: de Saint Phalle Brigitte , Sullivan William
Published: July 2002

In Sciara, unfertilized embryos initiate parthenogenetic development without centrosomes. By comparing these embryos with normal fertilized embryos, spindle assembly and other microtubule-based events can be examined in the presence and absence of centrosomes. In both cases, functional mitotic spindles are formed that successfully proceed through anaphase and telophase, forming two daughter nuclei separated by a midbody. The spindles assembled without centrosomes are anastral, and it is likely that their microtubules are nucleated at or near the chromosomes. These spindles undergo anaphase B and successfully segregate sister chromosomes. However, without centrosomes the distance between the daughter nuclei in the next interphase is greatly reduced. This suggests that centrosomes are required to maintain nuclear spacing during the telophase to interphase transition. As in Drosophila, the initial embryonic divisions of Sciara are synchronous and syncytial. The nuclei in fertilized centrosome-bearing embryos maintain an even distribution as they divide and migrate to the cortex. In contrast, as division proceeds in embryos lacking centrosomes, nuclei collide and form large irregularly shaped nuclear clusters. These nuclei are not evenly distributed and never successfully migrate to the cortex. This phenotype is probably a direct result of a failure to form astral microtubules in parthenogenetic embryos lacking centrosomes. These results indicate that the primary function of centrosomes is to provide astral microtubules for proper nuclear spacing and migration during the syncytial divisions. Fertilized Sciara embryos produce a large population of centrosomes not associated with nuclei. These free centrosomes do not form spindles or migrate to the cortex and replicate at a significantly reduced rate. This suggests that the centrosome must maintain a proper association with the nucleus for migration and normal replication to occur.
Annexins in Cell Membrane Dynamics
Authors: Babiychuk Eduard B. , Draeger Annette
Published: July 2002

The sarcolemma of smooth muscle cells is composed of alternating stiff actin-binding, and flexible caveolar domains. In addition to these stable macrodomains, the plasma membrane contains dynamic glycosphingolipid- and cholesterol-enriched microdomains, which act as sorting posts for specific proteins and are involved in membrane trafficking and signal transduction. We demonstrate that these lipid rafts are neither periodically organized nor exclusively confined to the actin attachment sites or caveolar regions. Changes in the Ca2+ concentration that are affected during smooth muscle contraction lead to important structural rearrangements within the sarcolemma, which can be attributed to members of the annexin protein family. We show that the associations of annexins II, V, and VI with smooth muscle microsomal membranes exhibit a high degree of Ca2+ sensitivity, and that the extraction of annexins II and VI by detergent is prevented by elevated Ca2+ concentrations. Annexin VI participates in the formation of a reversible, membrane–cytoskeleton complex (Babiychuk, E.B., R.J. Palstra, J. Schaller, U. Kämpfer, and A. Draeger. 1999. J. Biol. Chem. 274:35191–35195). Annexin II promotes the Ca2+-dependent association of lipid raft microdomains, whereas annexin V interacts with glycerophospholipid microcompartments. These interactions bring about a new configuration of membrane-bound constituents, with potentially important consequences for signaling events and Ca2+ flux.
Alterations at the Intercalated Disk Associated with the Absence of Muscle Lim Protein
Authors: Ehler Elisabeth , Horowits Robert , Zuppinger Christian , Price Robert L. , Perriard Evelyne , Leu Martin , Caroni Pico , Sussman Mark , Eppenberger Hans M. , Perriard Jean-Claude
Published: July 2002

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain– containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.
Restricted Accumulation of Phosphatidylinositol 3-Kinase Products in a Plasmalemmal Subdomain during Fcγ Receptor-Mediated Phagocytosis
Authors: Marshall John G. , Booth James W. , Stambolic Vuk , Mak Tak , Balla Tamas , Schreiber Alan D. , Meyer Tobias , Grinstein Sergio
Published: July 2002

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3′ phosphoinositides (3′PIs) in macrophages during the course of phagocytosis. The presence of 3′PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3′PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3′PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3′PI at the cup. 3′PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3′PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.
Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor
Authors: Swindle C. Scott , Tran Kien T. , Johnson Terry D. , Banerjee Pallab , Mayes Anne M. , Griffith Linda , Wells Alan
Published: July 2002

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal–regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-“tethered” ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of “insoluble” growth factor ligands and a novel mode of activation for growth factor receptors.
The NEDD8 system is essential for cell cycle progression and morphogenetic pathway in mice
Authors: Tateishi Keisuke , Omata Masao , Tanaka Keiji , Chiba Tomoki
Published: July 2002

NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)–like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3−/− mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57Kip2. These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. β-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3−/− cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.
Activation of mammalian Chk1 during DNA replication arrest
Authors: Feijoo Carmen , Hall-Jackson Clare , Wu Rong , Jenkins David , Leitch Jane , Gilbert David M. , Smythe Carl
Published: July 2002

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.
Activated platelets mediate inflammatory signaling by regulated interleukin 1β synthesis
Authors: Lindemann Stephan , Tolley Neal D. , Dixon Dan A. , McIntyre Thomas M. , Prescott Stephen M. , Zimmerman Guy A. , Weyrich Andrew S.
Published: July 2002

Platelets release preformed mediators and generate eicosanoids that regulate acute hemostasis and inflammation, but these anucleate cytoplasts are not thought to synthesize proteins or cytokines, or to influence inflammatory responses over time. Interrogation of an arrayed cDNA library demonstrated that quiescent platelets contain many messenger RNAs, one of which codes for interleukin 1β precursor (pro–IL-1β). Unexpectedly, the mRNA for IL-1β and many other transcripts are constitutively present in polysomes, providing a mechanism for rapid synthesis. Platelet activation induces rapid and sustained synthesis of pro–IL-1β protein, a response that is abolished by translational inhibitors. A portion of the IL-1β is shed in its mature form in membrane microvesicles, and induces adhesiveness of human endothelial cells for neutrophils. Signal-dependent synthesis of an active cytokine over several hours indicates that platelets may have previously unrecognized roles in inflammation and vascular injury. Inhibition of β3 integrin engagement markedly attenuated the synthesis of IL-1β, identifying a new link between the coagulation and inflammatory cascades, and suggesting that antithrombotic therapies may also have novel antiinflammatory effects.
Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex
Authors: Sprong Hein , Degroote Sophie , Claessens Tijs , van Drunen Judith , Oorschot Viola , Westerink Ben H.C. , Hirabayashi Yoshio , Klumperman Judith , van der Sluijs Peter , van Meer Gerrit
Published: July 2002

A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.
Unequal Death in T Helper Cell (Th)1 and Th2 Effectors: Th1, but not Th2, Effectors Undergo Rapid Fas/FasL-mediated Apoptosis
Authors: Zhang Xiaohong , Brunner Thomas , Carter Laura , Dutton Richard W. , Rogers Paul , Bradley Linda , Sato Takaaki , Reed John C. , Green Douglas , Swain Susan L.
Published: July 2002

T helper cell (Th) 1, but not Th2, effectors undergo rapid Fas/Fas ligand (FasL)-mediated, activation-induced cell death upon restimulation with antigen. Unequal apoptosis is also observed without restimulation, after a longer lag period. Both effectors undergo delayed apoptosis induced by a non–Fas-mediated pathway. When Th1 and Th2 effectors are co-cultured, Th2 effectors survive preferentially, suggesting the responsible factor(s) is intrinsic to each population. Both Th1 and Th2 effectors express Fas and FasL, but only Th2 effectors express high levels of FAP-1, a Fas-associated phosphatase that may act to inhibit Fas signaling. The rapid death of Th1 effectors leading to selective Th2 survival provides a novel mechanism for differential regulation of the two subsets.
Inhibition of T-tropic HIV Strains by Selective Antagonization of the Chemokine Receptor CXCR4
Authors: Schols Dominique , Struyf Sofie , Damme Jo Van , Esté José A. , Henson Geoffrey , Clercq Erik De
Published: July 2002

Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1α or MIP-1β, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell–derived factor 1α, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1α, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists.
Phenotypic and Functional Separation of Memory and Effector Human CD8+ T Cells
Authors: Hamann Dörte , Baars Paul A. , Rep Martin H.G. , Hooibrink Berend , Kerkhof-Garde Susana R. , Klein Michèl R. , Lier René A.W. van
Published: July 2002

Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA−CD45R0+ cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon γ, tumor necrosis factor α, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27− population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-γ and TNF-α. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27− cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.
Defective Acidification in Human Breast Tumor Cells and Implications for Chemotherapy
Authors: Altan Nihal , Chen Yu , Schindler Melvin , Simon Sanford M.
Published: July 2002

Multidrug resistance (MDR) is a significant problem in the treatment of cancer. Chemotherapeutic drugs distribute through the cyto- and nucleoplasm of drug-sensitive cells but are excluded from the nucleus in drug-resistant cells, concentrating in cytoplasmic organelles. Weak base chemotherapeutic drugs (e.g., anthracyclines and vinca alkaloids) should concentrate in acidic organelles. This report presents a quantification of the pH for identified compartments of the MCF-7 human breast tumor cell line and demonstrates that (a) the chemotherapeutic Adriamycin concentrates in acidified organelles of drug-resistant but not drug-sensitive cells; (b) the lysosomes and recycling endosomes are not acidified in drug-sensitive cells; (c) the cytosol of drug-sensitive cells is 0.4 pH units more acidic than the cytosol of resistant cells; and (d) disrupting the acidification of the organelles of resistant cells with monensin, bafilomycin A1, or concanamycin A is sufficient to change the Adriamycin distribution to that found in drug-sensitive cells, rendering the cell vulnerable once again to chemotherapy. These results suggest that acidification of organelles is causally related to drug resistance and is consistent with the hypothesis that sequestration of drugs in acidic organelles and subsequent extrusion from the cell through the secretory pathways contribute to chemotherapeutic resistance.
T Cell–dependent Immune Response in C1q-deficient Mice: Defective Interferon γ Production by Antigen-specific T Cells
Authors: Cutler Antony J. , Botto Marina , van Essen Dominic , Rivi Roberta , Davies Kevin A. , Gray David , Walport Mark J.
Published: July 2002

The role of the classical complement pathway in humoral immune responses was investigated in gene-targeted C1q-deficient mice (C1qA−/−). Production of antigen-specific immunoglobulin (Ig)G2a and IgG3 in primary and secondary responses to T cell–dependent antigen was significantly reduced, whereas IgM, IgG1, and IgG2b responses were similar in control and C1qA−/− mice. Despite abnormal humoral responses, B cells from C1qA−/− mice proliferated normally to a number of stimuli in vitro. Immune complex localization to follicular dendritic cells within splenic follicles was lacking in C1qA−/− mice. The precursor frequency of antigen-specific T cells was similar in C1qA−/− and wild-type mice. However, analysis of cytokine production by primed T cells in response to keyhole limpet hemocyanin revealed a significant reduction in interferon-γ production in C1qA−/− mice compared with control mice, whereas interleukin 4 secretion was equivalent. These data suggest that the classical pathway of complement may influence the cytokine profile of antigen-specific T lymphocytes and the subsequent immune response.
Conversion of Membrane-bound Fas(CD95) Ligand to Its Soluble Form Is Associated with Downregulation of Its Proapoptotic Activity and Loss of Liver Toxicity
Authors: Schneider Pascal , Holler Nils , Bodmer Jean-Luc , Hahne Michael , Frei Karl , Fontana Adriano , Tschopp Jürg
Published: July 2002

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-α undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-α) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.
Increased Susceptibility of  Thymocytes to Apoptosis in Mice Lacking AIM, a Novel Murine Macrophage-derived Soluble Factor Belonging to the Scavenger Receptor Cysteine-rich Domain Superfamily
Authors: Miyazaki Toru , Hirokami Yumiko , Matsuhashi Nobuyuki , Takatsuka Hisakazu , Naito Makoto
Published: July 2002

Apoptosis of cells must be regulated both positively and negatively in response to a variety of stimuli in the body. Various environmental stresses are known to initiate apoptosis via differential signal transduction cascades. However, induction of signals that may inhibit apoptosis is poorly understood, although a number of intracellular molecules that mediate inhibition of apoptosis have been identified. Here we present a novel murine macrophage-specific 54-kD secreted protein which inhibits apoptosis (termed AIM, for apoptosis inhibitor expressed by macrophages). AIM belongs to the macrophage scavenger receptor cysteine-rich domain superfamily (SRCR-SF), members of which share a highly homologous conserved cysteine-rich domain. In AIM-deficient mice, the thymocyte numbers were diminished to half those in wild-type mice, and CD4/CD8 double-positive (DP) thymocytes were strikingly more susceptible to apoptosis induced by both dexamethasone and irradiation in vivo. Recombinant AIM protein significantly inhibited cell death of DP thymocytes in response to a variety of stimuli in vitro. These results indicate that in the thymus, AIM functions in trans to induce resistance to apoptosis within DP cells, and thus supports the viability of DP thymocytes before thymic selection.
In Vivo–Activated Cd4 T Cells Upregulate Cxc Chemokine Receptor 5 and Reprogram Their Response to Lymphoid Chemokines
Authors: Ansel K. Mark , McHeyzer-Williams Louise J. , Ngo Vu N. , McHeyzer-Williams Michael G. , Cyster Jason G.
Published: July 2002

Migration of antigen-activated CD4 T cells to B cell areas of lymphoid tissues is important for mounting T cell–dependent antibody responses. Here we show that CXC chemokine receptor (CXCR)5, the receptor for B lymphocyte chemoattractant (BLC), is upregulated on antigen-specific CD4 T cells in vivo when animals are immunized under conditions that promote T cell migration to follicles. In situ hybridization of secondary follicles for BLC showed high expression in mantle zones and low expression in germinal centers. When tested directly ex vivo, CXCR5hi T cells exhibited a vigorous chemotactic response to BLC. At the same time, the CXCR5hi cells showed reduced responsiveness to the T zone chemokines, Epstein-Barr virus–induced molecule 1 (EBI-1) ligand chemokine (ELC) and secondary lymphoid tissue chemokine (SLC). After adoptive transfer, CXCR5hi CD4 T cells did not migrate to follicles, indicating that additional changes may occur after immunization that help direct T cells to follicles. To further explore whether T cells could acquire an intrinsic ability to migrate to follicles, CD4−CD8− double negative (DN) T cells from MRL-lpr mice were studied. These T cells normally accumulate within follicles of MRL-lpr mice. Upon transfer to wild-type recipients, DN T cells migrated to follicle proximal regions in all secondary lymphoid tissues. Taken together, our findings indicate that reprogramming of responsiveness to constitutively expressed lymphoid tissue chemokines plays an important role in T cell migration to the B cell compartment of lymphoid tissues.
Aberrant High Expression of B Lymphocyte Chemokine (Blc/Cxcl13) by C11b+Cd11c+ Dendritic Cells in Murine Lupus and Preferential Chemotaxis of B1 Cells towards Blc
Authors: Ishikawa Sho , Sato Taku , Abe Masaaki , Nagai Shigenori , Onai Nobuyuki , Yoneyama Hiroyuki , Zhang Yan-yun , Suzuki Takuji , Hashimoto Shin-ichi , Shirai Toshikazu , Lipp Martin , Matsushima Kouji
Published: July 2002

We observed here that the expression of B lymphocyte chemokine (BLC/CXCL13) was markedly enhanced in the thymus and kidney in aged (NZB × NZW)F1 (BWF1) mice developing lupus nephritis, but not in similarly aged NZB and NZW mice. BLC-positive cells were present in the cellular infiltrates in the target organs with a reticular pattern of staining. CD11b+CD11c+ dendritic cells were increased in the thymus and spleen in aged BWF1 mice and identified as the major cell source for BLC. CD4+ T cells as well as B cells were dramatically increased in the thymus in aged BWF1 mice, whereas no increase was observed in aged NZB and NZW mice. B1/B2 ratio in the thymus was significantly higher than those in the spleen and peripheral blood in aged BWF1 mice. Interestingly, BLC showed preferential chemotactic activity for B1 cells derived from several mouse strains, including nonautoimmune mice. Cell surface CXCR5 expression on B1 cells was significantly higher than that on B2 cells. Thus, aberrant high expression of BLC by myeloid dendritic cells in the target organs in aged BWF1 mice may play a pivotal role in breaking immune tolerance in the thymus and in recruiting autoantibody-producing B cells in the development of murine lupus.
Hepatocyte Nuclear Factor 1α Controls the Expression of Terminal Complement Genes
Authors: Pontoglio Marco , Pausa Mario , Doyen Antonia , Viollet Benoit , Yaniv Moshe , Tedesco Francesco
Published: July 2002

The terminal components of the complement system contribute to host defense by forming the multiprotein membrane attack complex (MAC) which is responsible for cell lysis and several noncytotoxic effects. Most of the complement proteins are synthesized in the liver, but the mechanisms controlling their tissue-specific expression have not been elucidated. In this study we show that mice lacking the hepatic transcription factor hepatocyte nuclear factor 1α (HNF1α) fail to transcribe C5 and C8A complement genes. In addition, mRNAs encoding for several other terminal complement components or subunits are expressed at lower levels, including C8β, C8γ, and C9. We next used a reconstitution assay involving human sera with selective complement deficiencies to assess mouse complement activity. Sera from HNF1α-deficient mice showed negligible hemolytic activity of both C5 and C8α-γ subunits. The activity of C8β was severely affected despite only a 50% reduction in C8β mRNA levels in the liver. This is reminiscent of C8α-γ–deficient patients who accumulate extremely low levels of the C8β subunit.

Our results demonstrate that HNF1α plays a key role in the expression of C5 and C8A genes, two terminal complement component genes that are essential for the assembly of MAC as a result of complement activation.