bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure and activity of multiple nitrifying bacterial populations co‐existing in a biofilm
Authors: Gieseke Armin , Bjerrum Lotte , Wagner Michael , Amann Rudolf
Published: June 2003

A biofilm from a nitrifying pilot‐scale sequencing batch reactor was investigated for effects of varying process conditions on its microscale activity and structure. Microsensor measurements of oxygen, substrates and products of nitrification were applied under incubation at different ammonium and oxygen concentrations which reflected various situations during a treatment cycle. A high net N loss was observed under high ammonium (HA) concentrations in contrast to low ones. Additionally, results indicated inhibition of nitrite‐oxidizing bacteria (NOB), but not of ammonia‐oxidizing bacteria (AOB) by free ammonia under HA conditions. Diversity, spatial distribution, and abundance of nitrifying bacteria as analysed by fluorescence in situ hybridization (FISH) revealed six different nitrifying populations with heterogeneous distributions. Nitrosococcus mobilis formed conspicuous microcolonies locally surrounded by cells of the dominating N. europaea/eutropha‐related AOB population. A third less abundant population was affiliated to N. oligotropha. Nitrite‐oxidizing bacteria of the genera Nitrobacter and Nitrospira (with at least two distinct populations) showed a large scale heterogeneity in their distribution. Nitrospira spp. were also found in deeper inactive layers where they might persist rather than thrive, and act as seed population when detached. Results of functional and structural analyses are discussed with respect to specific niches of individual populations in this system.
The gene cassette metagenome is a basic resource for bacterial genome evolution
Authors: Holmes Andrew J. , Gillings Michael R. , Nield Blair S. , Mabbutt Bridget C. , Nevalainen K. M. Helena , Stokes H. W.
Published: June 2003

Lateral gene transfer has been proposed as a fundamental process underlying bacterial diversity. Transposons, plasmids and phage are widespread and have been shown to significantly contribute to lateral gene transfer. However, the processes by which disparate genes are assembled and integrated into the host regulatory network to yield new phenotypes are poorly known. Recent discoveries about the integron/gene cassette system indicate it has the potential to play a role in this process. Gene cassettes are small mobile elements typically consisting of a promoterless orf and a recombination site. Integrons are capable of acquisition and re‐arrangement of gene cassettes and of the expression of their associated genes. The potential of the integron/gene cassette system is thus largely determined by the diversity contained within the cassette pool and the rate at which integrons sample this pool. We show here using a polymerase chain reaction (PCR) approach by which the environmental gene cassette (EGC) metagenome can be directly sampled that this metagenome contains both protein‐coding and non‐protein coding genes. Environmental gene cassette‐associated recombination sites showed greater diversity than previously seen in integron arrays. Class 1 integrons were shown to be capable of accessing this gene pool through tests of recombinational activity with a representative range of EGCs. We propose that gene cassettes represent a vast, prepackaged genetic resource that could be thought of as a metagenomic template for bacterial evolution.
Rice seedling whole exudates and extracted alkylresorcinols induce stress‐response in Escherichia coli biosensors
Authors: Miché Lucie , Belkin Shimshon , Rozen Rachel , Balandreau Jacques
Published: June 2003

A set of Escherichia coli sensor strains was used to evaluate the stress exerted on surrounding bacteria by germinating rice seed exudates. These biosensor strains contain Vibrio fischeri luxCDABE genes fused to the promoters of different genes involved in bacterial responses to environmental stresses. They provided clear evidence for a stress exerted by rice exudates, as shown by the induction of the universal stress protein gene uspA as well as genes of the heat shock regulon, grpE, lon and dnaK. The oxidative stress gene katG, and the post‐transcriptional ompF regulator encoded by micF were also activated. The lack of derepression of recA, uvrA and alkA indicated that damage to the DNA was not induced in the E. coli strains tested. Interestingly, resorcinolic lipids extracted from rice root seedlings induced the same promoters as whole exudates, suggesting that these compounds may contribute to the stress exerted by seedling exudates. The results obtained with E. coli biosensors thus indicate that, in vivo, exudates may also exert a selective pressure on root‐colonizing bacteria.
Glutamine synthetase from the marine cyanobacteria Prochlorococcus spp.: characterization, phylogeny and response to nutrient limitation
Authors: El Alaoui Sabah , Diez Jesús , Toribio Fermín , Gómez‐Baena Guadalupe , Dufresne Alexis , García‐Fernández Jose M.
Published: June 2003

The regulation of glutamine synthetase (EC 6.3.1.2) from Prochlorococcus was previously shown to exhibit unusual features: it is not upregulated by nitrogen starvation and it is not inactivated by darkness (El Alaoui et al. (2001) Appl Environ Microbiol 67: 2202–2207). These are probably caused by adaptations to oligotrophic environments, as confirmed in this work by the marked decrease in the enzymatic activity when cultures were subjected to iron or phosphorus starvation. In order to further understand the adaptive features of ammonium assimilation in this cyanobacterium, glutamine synthetase was purified from two Prochlorococcus strains: PCC 9511 (high‐light adapted) and SS120 (low‐light adapted). We obtained ∼ 100‐fold purified samples of glutamine synthetase electrophoretically homogeneous, with a yield of ∼ 30%. The estimated molecular mass of the subunits was roughly the same for both strains: 48.3 kDa. The apparent Km constants for the biosynthetic activity were 0.30 mM for ammonium, 1.29 mM for glutamate and 1.35 mM for ATP; the optimum pH was 8.0. Optimal temperature was surprisingly high (55°C). Phylogenetic analysis of glnA from three Prochlorococcus strains (MED4, MIT9313 and SS120) showed they group closely with marine Synechococcus isolates, in good agreement with other studies based on 16 S RNA sequences. All of our results suggest that the structure and kinetics of glutamine synthetase in Prochlorococcus have not been significantly modified during the evolution within the cyanobacterial radiation, in sharp contrast with its regulatory properties.
Early Diagnosis of Alzheimer's Disease: Clinical and Economic Benefits
Authors: Leifer Bennett P.
Published: June 2003

An estimated four million individuals in the United States have Alzheimer's disease (AD). This number is expected to more than triple by mid‐century. Primary care physicians have a key role in evaluating older patients for early signs of dementia and in initiating treatment that can significantly retard its progression over the maximum period of time. That role and its challenges will inevitably grow along with the expected increase in the population aged 65 and older. The tendency for physicians to dismiss memory complaints as normal aging must be replaced by awareness of the need to assess and possibly intervene. Early intervention is the optimal strategy, not only because the patient's level of function will be preserved for a longer period, but also because community‐dwelling patients with AD incur less societal cost than those who require long‐term institutional placement. Institutionalization contributes heavily to the annual cost of care for AD in the United States, which is estimated to be $100 billion annually.
Vascular Dementia: Distinguishing Characteristics, Treatment, and Prevention
Authors: Román Gustavo C.
Published: June 2003

Vascular dementia (VaD) is the second‐most‐common cause of dementia in the elderly, after Alzheimer's disease (AD). VaD is defined as loss of cognitive function resulting from ischemic, hypoperfusive, or hemorrhagic brain lesions due to cerebrovascular disease or cardiovascular pathology. Diagnosis requires the following criteria: cognitive loss, often predominantly subcortical; vascular brain lesions demonstrated by imaging; a temporal link between stroke and dementia; and exclusion of other causes of dementia. Poststroke VaD may be caused by large‐vessel disease with multiple strokes (multiinfarct dementia) or by a single stroke (strategic stroke VaD). A common form is subcortical ischemic VaD caused by small‐vessel occlusions with multiple lacunas and by hypoperfusive lesions resulting from stenosis of medullary arterioles, as in Binswanger's disease. Unlike with AD, in VaD, executive dysfunction is commonly seen, but memory impairment is mild or may not even be present. The cholinesterase inhibitors used for AD are also useful in VaD. Prevention strategies should focus on reduction of stroke and cardiovascular disease, with attention to control of risk factors such as hypertension, diabetes mellitus, hypercholesterolemia, and hyperhomocysteinemia.
Medical Management of Advanced Dementia
Authors: Tariot Pierre N.
Published: June 2003

A cure for Alzheimer's disease (AD) is still far off, and clinicians face the burden of caring for patients at all stages of dementia for the foreseeable future. Those with advanced disease suffer neurological symptoms and signs that include incontinence; problems with gait and mobility; marked cognitive, language, and functional impairment; and in about 90% of patients, significant behavior problems. Dementia precludes the ability to initiate meaningful activities or social interactions. Whether patients are resident in the community or living in a nursing home, this composite reflects a highly complex medical and neuropsychiatric management challenge. Predictable medical conditions also must be addressed (i.e., those that accompany dementia, such as parkinsonism, and those that are prevalent in any aging population, such as hypertension).

Clinicians can better address these problems with awareness of current treatment options. Placebo‐controlled trials of some psychotropic agents have shown modest favorable effects on behavior problems. Use of acetylcholinesterase inhibitors (AChEIs) to treat cognitive impairment and secondary behavioral symptoms derives primarily from results of placebo‐controlled clinical trials. Trials in patients with moderate to severe AD, outpatients as well as nursing home residents, show overall effects similar to those seen in outpatients with milder dementia. Treatment with AChEIs may delay institutional placement. Memantine has shown benefit in trials in moderate to severe dementia, although it is not yet approved in the United States. Emerging data have expanded physicians' ability to use pharmacotherapy in patients with advanced dementia. Physicians need to enact the principle that something can be done for our afflicted parents and grandparents.
Too Many Voices, “Too Problematic to Be Plausible”: Representing Multiple Responses to Local Economic Development Strategies?
Authors: Jayne Mark
Published: June 2003

Recent writing around urban political economy has highlighted the difficulty of representing multiple responses to local economic development strategies. It has been argued that it is conceptually important but nonetheless problematic to account for the multiplicity of individual, business, institutional, community, social and other groups' involvement in, interpretation of, or consumption of dominant city images and urban regeneration projects. This paper attempts such a project, presenting empirical case material from the UK city of Stoke-on-Trent, utilising the ethnographic grid–group theory conceived by Mary Douglas, and later developed with Aaron Wildavsky into a ‘cultural theory’ of political and economic change.
The Shopping Mall as an Emergent Public Space in Turkey
Authors: Erkip Feyzan
Published: June 2003

The shopping mall as a part of the recent transformations in Turkish urban lifestyle is the focus of this research. Characteristics of the Turkish way of using shopping malls, and their social and spatial consequences, are investigated and analyzed through a case study in Ankara, the capital of Turkey. The field survey was carried out in Bilkent Shopping Center, a newly built shopping mall in a suburban area which was also established recently as a high-income housing settlement. This shopping mall is an appropriate example of spatial transformations under the influence of global forces, which may also give clues about changes in urban lifestyle. A field survey was carried out through user surveys, and various observations are used to enrich the analysis. The results indicate that the shopping mall as a postmodern site matched the changing shopping and consumption requirements of Turkish urban citizens. The development of the shopping mall turns out to be timely for the Turkish urban citizen searching for modernity through new identity components in consumption patterns. Some benefit from this development more than others, for example, working women, indicating the process of feminization of the flaneur. However, these sites simultaneously produce a new arena of negotiation and conflict as well, creating new forms of exclusion—particularly for the urban poor. Although malls appear more public and democratic than the streets for the time being, the potential for segregation is implicit in their private character.
Extending Geodemographic Classification: A New Regional Prototype
Authors: Debenham James , Clarke Graham , Stillwell John
Published: June 2003

Geodemographic systems currently provide classifications of small areas based primarily on their demographic and socioeconomic characteristics. In this paper the authors aim to demonstrate the value of extending geodemographics by including new sets of variables. These have been selected to represent further dimensions of demand, workplace-based characteristics, and indicators reflecting the interaction between areas. A prototype regional system is constructed that is based on postal sectors in Yorkshire and the Humber classified into nine distinctive clusters.
Boundary Keeping and Access to Gaharu among Kenyah Forest Users
Authors: Wollenberg Eva K
Published: June 2003

As people living near forests in many parts of the world receive recognition of resource-management rights, questions arise about where forest boundaries should be set and who should legitimately receive these rights. Drawing on research conducted among forest-dwelling Kenyah communities in Kalimantan, Indonesia, during 1995 to 1998, I show that the realization of resource rights must be understood in the social context of how boundaries are interpreted and negotiated. Access to and control over forest resources is as much a matter of boundary keeping as of boundary setting. The analysis shows that boundary keepers assessed whether someone should be given access based on the potential user's entitlement, identity, and the potential for exchange. Understanding the ‘fuzziness’ of how seemingly clear boundary rules are applied should provide a more realistic picture of how groups gain and control access to resources in practice.
Transportation Planning for Disasters: An Accessibility Approach
Authors: Chang Stephanie E
Published: June 2003

A methodology is developed in this paper for evaluating and enhancing the performance of urban transportation systems in the aftermath of a disaster. A performance measure is proposed for assessing overall and distributional impacts based on the concept of accessibility. The methodology is demonstrated with two case studies. The first focuses on passenger railroad disruption in the catastrophic 1995 Kobe earthquake in Japan. An accessibility measure is applied to evaluate the loss of transportation service and, in particular, its attendant spatial disparities. Alternative repair and restoration strategies are investigated and shown, in hindsight, to be preferable to the actual sequence of repairs. The second case study focuses on the potential loss of road and highway transportation in Seattle, WA, after a hypothetical earthquake. A refined accessibility measure is applied that accounts for postdisaster detouring and congestion. Again, this brings attention to spatial disparities in loss and the spatial implications of alternative restoration priorities. It further demonstrates that restoration strategies should be designed from a systems perspective. The limitations and broader applicability of the approach are discussed.
Territorial Inequality, Regional Productivity, and Industrial Change in Postcommunism: Regional Transformations in Slovakia
Authors: Smith Adrian
Published: June 2003

In this paper the author examines the development and causes of territorial inequalities in the postcommunist economies of East–Central Europe (ECE), with a specific focus on the Slovak Republic (Slovakia). He begins by examining the extent of regional economic differentiation in ECE, within the context of the European Union (EU) enlargement process, and argues that an EU of twenty-five or twenty-seven states will face enormous territorial inequality. Through a specific examination of the experience of Slovakia he then goes on to examine some of the determinants and trajectories of these territorial inequalities. Specifically, he focuses on the contribution of relative regional industrial productivity trends in the second half of the 1990s to the endurance of territorial disparities. A decomposition of regional productivity and employment across industrial sectors is provided to examine some of the reasons for the comparative performance of Slovakia's regional economies. Attention is paid to the role of key strategic inward investment projects in the upgrading of particular regional industrial sectors. In contrast, other forms of global integration through, for example, outward processing of clothing for the EU market have, in other regions, been unable to stimulate a positive upgrading of regional productivity. Indeed, it is argued that the challenge for transitional economies such as Slovakia in the early part of the 21st century is how to manage the process of increasing, yet geographically uneven, global and European economic integration at the same time as ensuring that territorial and social inequalities are reduced.
Governance for Sustainability: Towards a ‘Thick’ Analysis of Environmental Decisionmaking
Authors: Adger W Neil , Brown Katrina , Fairbrass Jenny , Jordan Andrew , Paavola Jouni , Rosendo Sergio , Seyfang Gill
Published: June 2003

Environmental decisions made by individuals, civil society, and the state involve questions of economic efficiency, environmental effectiveness, equity, and political legitimacy. These four criteria are constitutive of the economic, social, and environmental dimensions of sustainable development, which has become the dominant rhetorical device of environmental governance. We discuss the tendency for disciplinary research to focus on particular subsets of the four criteria, and argue that such a practice promotes solutions that do not acknowledge the dynamics of scale and the heterogeneity of institutional contexts. We advocate an interdisciplinary framework for the analysis of environmental decisionmaking that seeks to identify legitimate and context-sensitive institutional solutions producing equitable, efficient, and effective outcomes. We demonstrate the usefulness of our approach by using it to examine decisions concerning contested nature conservation and multiple-use commons in the management of Hickling Broad in Norfolk in the United Kingdom. We conclude that interdisciplinary approaches enable the generalisation and transfer of lessons in a way that respects the specifics and context of the issue at hand.
Network Structure of an Industrial Cluster: Electronics in Toronto
Authors: Britton John N H
Published: June 2003

The literature on the theory of regional industrial success, including that focused on regional innovation systems, provides the conceptual foundation for this exploration of the extent to which firms in clusters of advanced technology industry depend on interregional sources for a wide variety of knowledge inputs to support innovation. The substantive focus is the electronics cluster of the Toronto region, Canada's largest manufacturing center. A small, stratified sample of establishments drawn from this cluster is used to verify the importance of external sources of material inputs, and other knowledge sources, and the strength of distant market connections. Interregional and local collaboration vary in importance as a result of scale-dependent resource differences between firms and in response to choices associated with foreign rather than domestic ownership. The results support the rejection of simple models of clusters and learning regions in which internal connections are privileged over interregional and international transactions operating either between or within firms.
ADP ribosylation factor 6 is activated and controls membrane delivery during phagocytosis in macrophages
Authors: Niedergang Florence , Colucci-Guyon Emma , Dubois Thierry , Raposo Graça , Chavrier Philippe
Published: June 2003

Engulfment of particles by phagocytes is induced by their interaction with specific receptors on the cell surface, which leads to actin polymerization and the extension of membrane protrusions to form a closed phagosome. Membrane delivery from internal pools is considered to play an important role in pseudopod extension during phagocytosis. Here, we report that endogenous ADP ribosylation factor 6 (ARF6), a small GTP-binding protein, undergoes a sharp and transient activation in macrophages when phagocytosis was initiated via receptors for the Fc portion of immunoglobulins (FcRs). A dominant-negative mutant of ARF6 (T27N mutation) dramatically affected FcR-mediated phagocytosis. Expression of ARF6-T27N lead to a reduction in the focal delivery of vesicle-associated membrane protein 3+ endosomal recycling membranes at phagocytosis sites, whereas actin polymerization was unimpaired. This resulted in an early blockade in pseudopod extension and accumulation of intracellular vesicles, as observed by electron microscopy. We conclude that ARF6 is a major regulator of membrane recycling during phagocytosis.
Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation
Authors: Sztalryd Carole , Xu Guoheng , Dorward Heidi , Tansey John T. , Contreras Juan A. , Kimmel Alan R. , Londos Constantine
Published: June 2003

Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the β-adrenergic receptor–adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A–phosphorylated HSL and perilipin A.
Adhesion-independent mechanism for suppression of tumor cell invasion by E-cadherin
Authors: Wong Alice S.T. , Gumbiner Barry M.
Published: June 2003

Loss of E-cadherin expression or function in tumors leads to a more invasive phenotype. In this study, we investigated whether the invasion suppressor activity of E-cadherin is mediated directly by tighter physical cell adhesion, indirectly by sequestering β-catenin and thus antagonizing β-catenin/T cell factor (TCF) signaling, or by other signaling pathways. To distinguish mechanisms, we expressed wild-type E-cadherin and various E-cadherin mutants in invasive E-cadherin–negative human breast (MDA-MB-231) and prostate (TSU-Pr1) epithelial carcinoma cell lines using a tetracycline-inducible system. Our data confirm that E-cadherin inhibits human mammary and prostate tumor cell invasion. We find that adhesion is neither necessary nor sufficient for suppressing cancer invasion. Rather, the invasion suppressor signal is mediated through the β-catenin–binding domain of the E-cadherin cytoplasmic tail but not through the p120ctn-binding domain. β-catenin depletion also results in invasion suppression. However, alteration in the β-catenin/TCF transcriptional regulation of target genes is not required for the invasion suppressor activity of E-cadherin, suggesting the involvement of other β-catenin–binding proteins.
Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast
Authors: Dong Yuqing , Pruyne David , Bretscher Anthony
Published: June 2003

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1–5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways.
The localization and phosphorylation of p47 are important for Golgi disassembly–assembly during the cell cycle
Authors: Uchiyama Keiji , Jokitalo Eija , Lindman Mervi , Jackman Mark , Kano Fumi , Murata Masayuki , Zhang Xiaodong , Kondo Hisao
Published: June 2003

In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly–reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.
Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane
Authors: Umebayashi Kyohei , Nakano Akihiko
Published: June 2003

It was known that the uptake of tryptophan is reduced in the yeast erg6 mutant, which is defective in a late step of ergosterol biosynthesis. Here, we show that this is because the high affinity tryptophan permease Tat2p is not targeted to the plasma membrane. In wild-type cells, the plasma membrane localization of Tat2p is regulated by the external tryptophan concentration. Tat2p is transported from the Golgi apparatus to the vacuole at high tryptophan, and to the plasma membrane at low tryptophan. However, in the erg6 mutant, Tat2p is missorted to the vacuole at low tryptophan. The plasma membrane targeting of Tat2p is dependent on detergent-insoluble membrane domains, suggesting that sterol affects the sorting through the organization of lipid rafts. The erg6 mutation also caused missorting to the multivesicular body pathway in late endosomes. Thus, sterol composition is crucial for protein sorting late in the secretory pathway. Tat2p is subject to polyubiquitination, which acts as a vacuolar-targeting signal, and the inhibition of this process suppresses the Tat2p sorting defects of the erg6 mutant. The sorting mechanisms of Tat2p that depend on both sterol and ubiquitin will be discussed.
Tumors Acquire Inhibitor of Apoptosis Protein (IAP)-mediated Apoptosis Resistance through Altered Specificity of Cytosolic Proteolysis
Authors: Hong Xu , Lei Lu , Glas Rickard
Published: June 2003

Many tumors overexpress members of the inhibitor of apoptosis protein (IAP) family. IAPs contribute to tumor cell apoptosis resistance by the inhibition of caspases, and are degraded by the proteasome to allow further progression of apoptosis. Here we show that tumor cells can alter the specificity of cytosolic proteolysis in order to acquire apoptosis resistance, which promotes formation of rapidly growing tumors. Survival of tumor cells with low proteasomal activity can occur in the presence of high expression of Tri-peptidyl-peptidase II (TPP II), a large subtilisin-like peptidase that complements proteasomal activity. We find that this state leaves tumor cells unable of effectively degrading IAPs, and that cells in this state form rapidly growing tumors in vivo. We also find, in studies of apoptosis resistant cells derived from large in vivo tumors, that these have acquired an altered peptidase activity, with up-regulation of TPP II activity and decreased proteasomal activity. Importantly, we find that growth of subcutaneous tumors is limited by maintenance of the apoptosis resistant phenotype. The apoptosis resistant phenotype was reversed by increased expression of Smac/DIABLO, an antagonist of IAP molecules. Our data suggest a reversible mechanism in regulation of apoptosis resistance that drives tumor progression in vivo. These data are relevant in relation to the multitude of therapy-resistant clinical tumors that have increased levels of IAP molecules.
Transforming Growth Factor β Blocks Tec Kinase Phosphorylation, Ca2+ Influx, and NFATc Translocation Causing Inhibition of T Cell Differentiation
Authors: Chen Chang-Hung , Seguin-Devaux Carole , Burke Nancy A. , Oriss Timothy B. , Watkins Simon C. , Clipstone Neil , Ray Anuradha
Published: June 2003

Transforming growth factor (TGF)-β inhibits T cell proliferation and differentiation. TGF-β has been shown to inhibit the expression of transcription factors such as GATA-3 and T-bet that play important roles in T cell differentiation. Here we show that TGF-β inhibits T cell differentiation at a more proximal step. An early event during T cell activation is increased intracellular calcium levels. Calcium influx in activated T cells and the subsequent activation of transcription factors such as NFATc, events essential for T cell differentiation, are modulated by the Tec kinases that are downstream of the T cell receptor and CD28. We show that in stimulated CD4+ T cells, TGF-β inhibits phosphorylation and activation of the Tec kinase Itk, increase in intracellular Ca2+ levels, NFATc translocation, and activation of the mitogen-activated protein kinase ERK that together regulate T cell differentiation. Our studies suggest that by inhibiting Itk, and consequently Ca2+ influx, TGF-β limits T cell differentiation along both the Th1 and Th2 lineages.
Multistep Nature of Microvascular Recruitment of Ex Vivo–expanded Embryonic Endothelial Progenitor Cells during Tumor Angiogenesis
Authors: Vajkoczy Peter , Blum Sabine , Lamparter Mathias , Mailhammer Reinhard , Erber Ralph , Engelhardt Britta , Vestweber Dietmar , Hatzopoulos Antonis K.
Published: June 2003

Tissue neovascularization involves recruitment of circulating endothelial progenitor cells that originate in the bone marrow. Here, we show that a class of embryonic endothelial progenitor cells (Tie-2+, c-Kit+, Sca-1+, and Flk-1−/low), which were isolated at E7.5 of mouse development at the onset of vasculogenesis, retain their ability to contribute to tumor angiogenesis in the adult. Using intravital fluorescence videomicroscopy, we further defined the multistep process of embryonic endothelial progenitor cell (eEPC) homing and incorporation. Circulating eEPCs are specifically arrested in “hot spots” within the tumor microvasculature, extravasate into the interstitium, form multicellular clusters, and incorporate into functional vascular networks. Expression analysis and in vivo blocking experiments provide evidence that the initial cell arrest of eEPC homing is mediated by E- and P-selectin and P-selectin glycoprotein ligand 1. This paper provides the first in vivo insights into the mechanisms of endothelial progenitor cell recruitment and, thus, indicates novel ways to interfere with pathological neovascularization.
Bruton's Tyrosine Kinase Is Required For Lipopolysaccharide-induced Tumor Necrosis Factor α Production
Authors: Horwood Nicole J. , Mahon Tara , McDaid John P. , Campbell Jamie , Mano Hiroyuki , Brennan Fionula M. , Webster David , Foxwell Brian M.J.
Published: June 2003

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)α production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNFα production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNFα production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNFα production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNFα production. We examined the role of Btk in TNFα production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNFα mRNA via the 3′ untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNFα production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNFα production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.
B7DC/PDL2 Promotes Tumor Immunity by a PD-1–independent Mechanism
Authors: Liu Xingluo , Gao Jian Xin , Wen Jing , Yin Lijie , Li Ou , Zuo Tao , Gajewski Thomas F. , Fu Yang-Xin , Zheng Pan , Liu Yang
Published: June 2003

B7H1 (PDL1) and B7DC (PDL2) are two new members of the B7 family that can interact with PD-1, a putative negative regulator for immune function. Recent studies have provided evidence for inhibitory functions of both members via PD-1. Meanwhile, compelling evidence exists for costimulatory function of both members. Here we demonstrate that expression of B7DC on the tumor cells promotes CD8 T cell–mediated rejection of tumor cells, at both the induction and effector phase of antitumor immunity. Moreover, B7DC binds to PD-1(−/−) cells and enhances T cell killing in a PD-1–independent mechanism. Our results demonstrate a novel pathway for B7DC to promote tumor immunity and may reconcile the apparently contradictory findings on the function of B7DC.
Fractalkine Preferentially Mediates Arrest and Migration of CD16+ Monocytes
Authors: Ancuta Petronela , Rao Ravi , Moses Ashlee , Mehle Andrew , Shaw Sunil K. , Luscinskas F. William , Gabuzda Dana
Published: June 2003

CD16+ monocytes represent 5–10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16− monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16− monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendo-thelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1α (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface–expressed FKN under flow with higher frequency compared with CD16− monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.
Differential Requirement for Rel/Nuclear Factor κB Family Members in Natural Killer T Cell Development
Authors: Sivakumar Vallabhapurapu , Hammond Kirsten J.L. , Howells Norma , Pfeffer Klaus , Weih Falk
Published: June 2003

Natural killer T (NKT) cells have been implicated in diverse immune responses ranging from suppression of autoimmunity to tumor rejection. Thymus-dependent NKT cells are positively selected by the major histocompatibility complex class I–like molecule CD1d, but the molecular events downstream of CD1d are still poorly understood. Here, we show that distinct members of the Rel/nuclear factor (NF)-κB family of transcription factors were required in both hematopoietic and nonhematopoietic cells for normal development of thymic NKT cells. Activation of NF-κB via the classical IκBα-regulated pathway was required in a cell autonomous manner for the transition of NK-1.1–negative precursors that express the TCR Vα14-Jα18 chain to mature NK-1.1–positive NKT cells. The Rel/NF-κB family member RelB, on the other hand, had to be expressed in radiation resistant thymic stromal cells for the generation of early NK-1.1–negative NKT precursors. Moreover, NF-κB–inducing kinase (NIK) was required for both constitutive thymic DNA binding of RelB and the specific induction of RelB complexes in vitro. Thus, distinct Rel/NF-κB family members in hematopoietic and nonhematopoietic cells regulate NKT cell development with a unique requirement for NIK-mediated activation of RelB in thymic stroma.
NIK-dependent RelB Activation Defines a Unique Signaling Pathway for the Development of Vα14i NKT Cells
Authors: Elewaut Dirk , Shaikh Raziya B. , Hammond Kirsten J. L. , De Winter Hilde , Leishman Andrew J. , Sidobre Stephane , Turovskaya Olga , Prigozy Theodore I. , Ma Lisa , Banks Theresa A. , Lo David , Ware Carl F. , Cheroutre Hilde , Kronenberg Mitchell
Published: June 2003

A defect in RelB, a member of the Rel/nuclear factor (NF)-κB family of transcription factors, affects antigen presenting cells and the formation of lymphoid organs, but its role in T lymphocyte differentiation is not well characterized. Here, we show that RelB deficiency in mice leads to a selective decrease of NKT cells. RelB must be expressed in an irradiation-resistant host cell that can be CD1d negative, indicating that the RelB expressing cell does not contribute directly to the positive selection of CD1d-dependent NKT cells. Like RelB-deficient mice, aly/aly mice with a mutation for the NF-κB–inducing kinase (NIK), have reduced NKT cell numbers. An analysis of NK1.1 and CD44 expression on NKT cells in the thymus of aly/aly mice reveals a late block in development. In vitro, we show that NIK is necessary for RelB activation upon triggering of surface receptors. This link between NIK and RelB was further demonstrated in vivo by analyzing RelB+/− × aly/+ compound heterozygous mice. After stimulation with α-GalCer, an antigen recognized by NKT cells, these compound heterozygotes had reduced responses compared with either RelB+/− or aly/+ mice. These data illustrate the complex interplay between hemopoietic and nonhemopoietic cell types for the development of NKT cells, and they demonstrate the unique requirement of NKT cells for a signaling pathway mediated by NIK activation of RelB in a thymic stromal cell.
Unraveling a Revealing Paradox
Authors: Bosselut Remy , Guinter Terry I. , Sharrow Susan O. , Singer Alfred
Published: June 2003

The mechanism by which T cell receptor specificity determines the outcome of the CD4/CD8 lineage decision in the thymus is not known. An important clue is the fact that major histocompatibility complex (MHC)-I–signaled thymocytes paradoxically appear as CD4+8lo transitional cells during their differentiation into CD8+ T cells. Lineage commitment is generally thought to occur at the CD4+8+ (double positive) stage of differentiation and to result in silencing of the opposite coreceptor gene. From this perspective, the appearance of MHC-I–signaled thymocytes as CD4+8lo cells would be due to effects on CD8 surface protein expression, not CD8 gene expression. But contrary to this perspective, this study demonstrates that MHC-I–signaled thymocytes appear as CD4+8lo cells because of transient down-regulation of CD8 gene expression, not because of changes in CD8 surface protein expression or distribution. This study also demonstrates that initial cessation of CD8 gene expression in MHC-I–signaled thymocytes is not necessarily indicative of commitment to the CD4+ T cell lineage, as such thymocytes retain the potential to differentiate into CD8+ T cells. These results challenge classical concepts of lineage commitment but fulfill predictions of the kinetic signaling model.
Memory CD8+ T Cells Are Required for Protection from Persistent Hepatitis C Virus Infection
Authors: Shoukry Naglaa H. , Grakoui Arash , Houghton Michael , Chien David Y. , Ghrayeb John , Reimann Keith A. , Walker Christopher M.
Published: June 2003

Few hepatitis C virus (HCV) infections resolve spontaneously but those that do appear to afford protective immunity. Second infections are usually shorter in duration and are less likely to persist but mechanisms of virus control in immune individuals have not been identified. In this study we investigated whether memory helper and/or cytotoxic T lymphocytes provide protection in chimpanzees serially reinfected with the virus. Clearance of the first infection took 3–4 mo and coincided with the delayed onset of CD4+ and CD8+ T cell responses. High frequencies of memory T cells targeting multiple HCV proteins were stable over 7 yr of follow-up. Animals were infected for a second time to assess the protective role of memory T cells. In contrast to the prolonged course of the first infection, viremia was terminated within 14 d. Control of this second infection was kinetically linked to rapid acquisition of virus-specific cytolytic activity by liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in blood. The importance of memory CD8+ T cells in control of HCV infection was confirmed by antibody-mediated depletion of this lymphocyte subset before a third infection. Virus replication was prolonged despite the presence of memory CD4+ T helper cells primed by the two prior infections and was not terminated until HCV-specific CD8+ T cells recovered in the liver. These experiments demonstrate an essential role for memory CD8+ T cells in long-term protection from chronic hepatitis C.
Stimulus-dependent Deacylation of Bacterial Lipopolysaccharide by Dendritic Cells
Authors: Lu Mingfang , Zhang Mei , Kitchens Richard L. , Fosmire Susan , Takashima Akira , Munford Robert S.
Published: June 2003

We describe here a previously unrecognized property of dendritic cells (DCs), the ability to deacylate the lipid A moiety of gram-negative bacterial LPSs. Both immature DCs of the XS52 cell line and bone marrow–derived DCs produce acyloxyacyl hydrolase, an enzyme that detoxifies LPS by selectively removing the secondary acyl chains from lipid A. Acyloxyacyl hydrolase expression decreased when DCs were incubated with IL-4, IL-1β, TNFα, and an agonistic CD40 antibody (maturation cocktail), and increased after treatment with LPS, CpG oligodeoxynucleotides, or a gram-positive bacterium (Micococcus luteus). Maturation cocktail treatment also diminished, whereas LPS treatment enhanced or maintained the cells' ability to kill Escherichia coli, deacylate LPS, and degrade bacterial protein. Enzymatic deacylation of LPS is an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist.
H2AX Is Required for Recombination Between Immunoglobulin Switch Regions but Not for Intra-Switch Region Recombination or Somatic Hypermutation
Authors: Reina-San-Martin Bernardo , Difilippantonio Simone , Hanitsch Leif , Masilamani Revati F. , Nussenzweig André , Nussenzweig Michel C.
Published: June 2003

Changes in chromatin structure induced by posttranslational modifications of histones are important regulators of genomic function. Phosphorylation of histone H2AX promotes DNA repair and helps maintain genomic stability. Although B cells lacking H2AX show impaired class switch recombination (CSR), the precise role of H2AX in CSR and somatic hypermutation (SHM) has not been defined. We show that H2AX is not required for SHM, suggesting that the processing of DNA lesions leading to SHM is fundamentally different from CSR. Impaired CSR in H2AX−/− B cells is not due to alterations in switch region transcription, accessibility, or aberrant joining. In the absence of H2AX, short-range intra-switch region recombination proceeds normally while long-range inter-switch region recombination is impaired. Our results suggest a role for H2AX in regulating the higher order chromatin remodeling that facilitates switch region synapsis.