bluuebunny/crossref_metadata_2025 huggingface dataset:
The
Pseudomonas syringae
Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants
Authors: Alfano James R. , Charkowski Amy O. , Deng Wen-Ling , Badel Jorge L. , Petnicki-Ocwieja Tanja , van Dijk Karin , Collmer Alan
Published: July 2002

The plant pathogenic bacterium
Pseudomonas syringae
is divided into pathovars differing in host specificity, with
P. syringae
pv.
syringae
(
Psy
) and
P. syringae
pv.
tomato
(
Pto
) representing particularly divergent pathovars.
P. syringae hrp
/
hrc
genes encode a type III protein secretion system that appears to translocate Avr and Hop effector proteins into plant cells. DNA sequence analysis of the
hrp
/
hrc
regions in
Psy
61,
Psy
B728a, and
Pto
DC3000 has revealed a Hrp pathogenicity island (Pai) with a tripartite mosaic structure. The
hrp
/
hrc
gene cluster is conserved in all three strains and is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus (CEL). The EELs begin 3 nt downstream of the stop codon of
hrpK
and end, after 2.5–7.3 kb of dissimilar intervening DNA with tRNA
Leu
–
queA–tgt
sequences that are also found in
Pseudomonas aeruginosa
but without linkage to any Hrp Pai sequences. The EELs encode diverse putative effectors, including HopPsyA (HrmA) in
Psy
61 and proteins similar to AvrPphE and the AvrB/AvrC/AvrPphC and AvrBsT/AvrRxv/YopJ protein families in
Psy
B728a. The EELs also contain mobile genetic element sequences and have a G + C content significantly lower than the rest of the Hrp Pai or the
P. syringae
genome. The CEL carries at least seven ORFs that are conserved between
Psy
B728a and
Pto
DC3000. Deletion of the
Pto
DC3000 EEL slightly reduces bacterial growth in tomato, whereas deletion of a large portion of the CEL strongly reduces growth and abolishes pathogenicity in tomato.
Essential Psychology for Environmental Policy Making
Authors: Vlek Charles
Published: July 2002

In this article major environmental problems and their different levels and global spheres of impact are surveyed. Environmental exploitation is discussed as an inherent characteristic of free market economies under limited cognitive‐motivational inclinations of individual actors. A conceptual modelling of environmental problem solving is presented, which comprises the commons dilemma paradigm as well as a needs‐opportunities‐abilities (NOA) model of consumer behaviour, a categorization of human needs, a simple taxonomy of behavioural processes, and seven strategies for behaviour change. Six lines of psychological research are identified, ranging from environmental attitudes to environmental policy‐decision support systems. An ecological critique of mainstream psychology is summarized and some suggestions are made to resolve this. Conclusions are drawn about needed research, policy making, and international diplomacy.
Redintegration and the Useful Lifetime of the Verbal Memory Representation
Authors: Schweickert Richard , Chen Shengbao , Poirier Marie
Published: July 2002

Factors affecting immediate serial recall of individual items can be classified according to whether they influence the degradation of the representation, or influence the ability to redintegrate a degraded representation. For recall of entire lists, factors can be classified according to whether or not they influence the rate at which items are recalled. We review experiments in order to classify the factors of serial position, word length, word frequency, and lexicality. We propose that the two classification systems coincide, at least for those factors whose classification is known for both schemes. We end by considering whether probability of recall of an entire list might be predicted from probability of recall of individual items.
Inclusion or Exclusion? The Role of Housing Subsidies and Benefits
Authors: Hills John
Published: July 2002

This paper explores the reasons why the UK has ended up with such an extensive system of intervention in the housing market, particularly through providing subsidised council and housing association housing, and through Housing Benefit. The most important justification which remains valid is to enable those who would not otherwise be able to do so to afford to live in housing of acceptable quality, and to do so while avoiding the area polarisation which would result from the operation of the free market. However, the way in which social housing and subsidies operate in Britain today often reinforces rather than counters social polarisation and divisions. Looking ahead, as society becomes more affluent, including in an optimistic scenario those with low incomes, these problems may become even more acute as housing quality and consumer choice become more important. Systems of rationed provision with little tenant choice or control and no trade-off between cost and quality or location will look increasingly outmoded. The paper explores what could be done to reinvent the way in which we subsidise housing.
The Spatial Pattern of Land Values in Jakarta
Authors: Han Sun Sheng , Basuki Ann
Published: July 2002

Rapid urban development and globalisation have brought dynamic changes to large cities in the developing countries. Yet understanding of the changes has been handicapped by the lack of data and systematic attempts. By using the latest release of land-value data in Jakarta, this paper provides insights into the spatial structure of land values in a typical Third World metropolis. Land values vary drastically from one kelurahan to another, with most of the expensive land parcels in central Jakarta. However, in central Jakarta one could also easily find cheap land parcels whose values were lower than the lowest land values of some parcels in non-central regions of the city, reflecting the mixture of slums and skyscrapers in central Jakarta. Land values were not distributed evenly in the non-central regions. Rather, they were more expensive in west and south Jakarta than in north and east Jakarta. Spatial variables, especially distance to the central business district, were important in shaping land-value patterns in Jakarta, but the explanatory power of distance declined over time. The findings of this paper will not only be useful for an understanding of spatial land-value patterns in Asian cities, but will also be beneficial for investors and policy-makers in their decision-making processes.
Spatial Mismatch or Automobile Mismatch? An Examination of Race, Residence and Commuting in US Metropolitan Areas
Authors: Taylor Brian D. , Ong Paul M.
Published: July 2002

This paper uses data from the metropolitan samples of the American Housing Survey in 1977-78 and 1985 to examine the commute patterns of whites, blacks and Hispanics in US metropolitan areas, with a particular focus on the commutes of workers living in predominantly minority residential areas. Overall, the commute patterns of white and minority workers appear to be converging rather than diverging over time, even among low-skilled workers. Contrary to the spatial mismatch hypothesis, black and Hispanic workers living in minority areas had both shorter commutes and commutes that increased more slowly between 1977-78 and 1985 compared to workers in other areas. Further, a longitudinal analysis shows that the average commute times of non-moving minority workers in predominantly minority areas decreased during the study period. We find no evidence in these commuting data to support the spatial mismatch hypothesis.
The Rent Gap Re-examined
Authors: Clark Eric
Published: July 2002

Rent gap theory is re-examined in view of Bourassa's recent critique. The critique is found to be flawed on three accounts. It equates an abstract concept (actual land rent) with a concrete event (contract rent); it seeks the relevance of the rent gap at times of redevelopment; and it diminishes rent gap theory to an equation for calculating the profitability of redevelopment. In this re-examination, it is argued that: (1) actual land rent is not equal to contract rent; (2) the significance of the rent gap extends over a longer period prior to redevelopment; and (3) rent gap theory should not be reduced to a break-even point in real estate accounting. It is a political economic theory of uneven development on the urban scale and as such cannot be divorced from the societal relations and power struggles involved in the creation and capture of values in the built environment.
Outsourcing, R&D and the Pattern of Intra-metropolitan Location: The Electronics Industries of Madrid
Authors: Suarez-Villa Luis , Rama Ruth
Published: July 2002

The relationship between intra-metropolitan location, research and development (R&D) and the outsourcing of production is analysed to determine differences in, and the supportive character of, intra-metropolitan areas for high-technology production. Madrid's electronics industries have shown a remarkable resilience, in the face of extensive economic restructuring and the change in trade regimes that has accompanied continental integration. Much of the strength of the electronics sector may have been due to the metropolitan area's support for outsourcing, and the creative specialisation that it facilitated. The distribution of manufacturing establishments, their local sourcing characteristics, industrial district or agglomeration effects, and the role of R&D are evaluated to determine the intra-metropolitan context of production. Extensive statistical analyses of the relationship between R&D and productive performance, with various indicators of capital, costs, revenues and scale, are undertaken with establishment-level survey data. These analyses provide conclusive evidence of the influence of intra-metropolitan location on R&D and internal organisation.
Sustainable Cities or Cities that Contribute to Sustainable Development?
Authors: Satterthwaite David
Published: July 2002

This paper outlines a framework for assessing the environmental performance of cities in regard to the meeting of sustainable development goals. It also considers how the environmental goals fit with the social, economic and political goals of sustainable development and the kinds of national framework and international context needed to encourage city-based consumers, enterprises and governments to progress towards their achievement. In a final section, it considers the extent to which the recommendations of the Habitat II Conference helped to encourage national governments and city and municipal authorities in this direction.
Modelling Sustainable Home-ownership: Demographics or Economics?
Authors: Meen Geoffrey
Published: July 2002

According to recent official projections, 4.4 million new households will be formed in England between 1991 and 2016. These estimates are based on a detailed analysis of demographic trends, but take no account of the effect of potential changes in economic conditions, particularly on the owner-occupancy rate. In this paper, we propose a new approach to modelling sustainable owner-occupancy which places less emphasis on demographics, but considers in more detail the role of market mechanisms through changes in house prices and rates of new construction.
Black Minority Ethnic Concentration, Segregation and Dispersal in Britain
Authors: Phillips Deborah
Published: July 2002

This paper examines the post-war migration and settlement in Britain of black minority ethnic groups originating from countries such as India, Pakistan, Bangladesh and the West Indies. The processes underlying the pattern of minority ethnic concentration and segregation over the past four decades are reviewed and provide a framework for interpreting the uneven pattern of deconcentration and dispersal evident over the past 10-15 years. The paper draws on evidence from the labour market and the housing market to argue that there are forces for both minority ethnic inclusion and exclusion from competition for economic rewards and social status in Britain. These forces, it is argued, produce different outcomes for different groups and a variable experience within minority ethnic groups according to generation, gender and class. A picture of fragmented social and spatial change emerges, with those of Indian origin in particular following a different trajectory from other black minority ethnic groups.
Sample Selection Bias in Models of Commuting Time
Authors: Cooke Thomas J. , Ross Stephen L.
Published: July 2002

This research conceptualises, measures and evaluates the effects of sample selection bias on models of commuting time. Data are drawn from the Public Use Microdata Sample of the 1990 US Census for the Boston metropolitan area. The major finding of the analysis is that the process that determines entry into employment introduces sample selection bias into the estimates of commuting-time models. The degree of sample selection bias observed differs by race/ethnicity and gender on such key variables as marital and parental status and reliance on public transport, because the influence of these variables on employment differs by race/ethnicity and gender. These variables are important for evaluating both the spatial mismatch and the spatial entrapment hypotheses and therefore the contribution of previous analyses should be reconsidered.
Krugman's Core-Periphery Model with Heterogeneous Quality of Land
Authors: Lanaspa Luis Fernando , Sanz Fernando
Published: July 2002

In 1991, Krugman developed a general equilibrium model with two sectors and two regions, from which two patterns of industrial location could be deduced endogenously, namely symmetric dispersion at 50 per cent and total concentration. In this paper, we present a very simple extension to this original model. The consideration of regions with different agricultural populations, reflecting a different quality of land, allows us to deduce asymmetric distributions of the manufacturing sector. These asymmetric distributions complete and enrich the casuistry derived from the original approach, drawing it closer to the observable world.
Sympathy in the context of mother-child and teacher-child relationships
Authors: Kienbaum Jutta , Volland Cordelia , Ulich Dieter
Published: July 2002

Two studies were carried out to investigate relations between socialisation influences, person variables, and sympathy, as well as prosocial behaviour in 5-year-old preschool children. Specifically, we were interested in the interactions between child characteristics (sex, inhibition toward strangers) and the socialisation practices of child care teachers (Study I) and mothers’ caregiving style (Study II). Participants in Study I were 105 five-year-old children who were confronted with the simulated distress of a puppet; 25 teachers were observed while interacting with the children during free play, and 93 parents rated their child’s inhibition. Participants in Study II were 79 five-year-old children and their mothers. As in Study I, the children’s reactions to distress were observed. The mothers rated their child’s inhibition and participated in an interview to assess the quality of their caregiving style. Positive, albeit weak, relations occurred between child care teachers’ warmth and children’s sympathetic-prosocial reactions to distress; no direct effects emerged for maternal behaviour. Negative, albeit weak, associations were found between inhibition and sympathetic-prosocial reactions. These relations improved when interactions between sex, inhibition, and the caregiving style of the teachers (but not of the mothers) were taken into account. The results are discussed with regard to the context-specificity of socialisation.
Secrecy in Middle Childhood
Authors: Watson Alan J. , Valtin Renate
Published: July 2002

We investigated children’s understanding of the nature of secrets as an expression of their knowledge of the intentions of others. The sample of 200 was drawn from 5-, 6-, 8-, 10- and 12-year-old children (40 from each age group), with equal numbers of males and females, and equal numbers from Berlin and Sydney. In structured interviews we tested the children’s understanding of four kinds of secrets: guilty, innocent, dangerous, and embarrassing. Differences across age were found for some secrets (e.g. telling mother guilty and dangerous secrets) but not for others (e.g. telling mother innocent and embarrassing secrets), and for some aspects of understanding mothers’ and friends’ intentions. Some gender and cultural differences were found. Findings are interpreted in terms of children’s understanding of the intentions of others and the emergence of peer solidarity and conscious trust.
Salivary cortisol during opiate dependence and withdrawal
Authors: Bearn J. , Buntwal N. , Papadopoulos A. , Checkley S.
Published: July 2002

Seventeen inpatients (14 men, three women) with opiate dependence and polysubstance misuse participated in a longitudinal study of salivary cortisol secretion during and after lofexidine detoxification treatment. Both opiate withdrawal symptoms and salivary cortisol were measured every morning for up to 25 days. Results were compared with a control group of 10 normal volunteers. There was an 80% reduction in withdrawal symptom severity between the first 12 days and the subsequent 12 days of treatment. Salivary cortisol fell from a mean of 22.3 nm/l over days 1–12 to 18.5 nm/l during days 13–25, a reduction of 17%. Salivary cortisol concentration correlated significantly with withdrawal symptom severity. Salivary cortisol levels remained significantly higher than controls for the duration of the study. The study supports a role for hypothalamo–pituitary–adrenal (HPA) activation in opiate withdrawal. The contribution of persistant dysregulation of the HPA, found in this study, to the vulnerability for relapse after abstinence has been achieved, is discussed.
Prospore Membrane Formation Defines a Developmentally Regulated Branch of the Secretory Pathway in Yeast
Authors: Neiman Aaron M.
Published: July 2002

Spore formation in yeast is an unusual form of cell division in which the daughter cells are formed within the mother cell cytoplasm. This division requires the de novo synthesis of a membrane compartment, termed the prospore membrane, which engulfs the daughter nuclei. The effect of mutations in late-acting genes on sporulation was investigated. Mutation of SEC1, SEC4, or SEC8 blocked spore formation, and electron microscopic analysis of the sec4-8 mutant indicated that this inability to produce spores was caused by a failure to form the prospore membrane. The soluble NSF attachment protein 25 (SNAP-25) homologue SEC9, by contrast, was not required for sporulation. The absence of a requirement for SEC9 was shown to be due to the sporulation-specific induction of a second, previously undescribed, SNAP-25 homologue, termed SPO20. These results define a developmentally regulated branch of the secretory pathway and suggest that spore morphogenesis in yeast proceeds by the targeting and fusion of secretory vesicles to form new plasma membranes in the interior of the mother cell. Consistent with this model, the extracellular proteins Gas1p and Cts1p were localized to an internal compartment in sporulating cells. Spore formation in yeast may be a useful model for understanding secretion-driven cell division events in a variety of plant and animal systems.
A Role for Giantin in Docking COPI Vesicles to Golgi Membranes
Authors: Sönnichsen Birte , Lowe Martin , Levine Tim , Jämsä Eija , Dirac-Svejstrup Barbara , Warren Graham
Published: July 2002

We have previously shown that p115, a vesicle docking protein, binds to two proteins (p130 and p400) in detergent extracts of Golgi membranes. p130 was identified as GM130, a Golgi matrix protein, and was shown to act as a membrane receptor for p115. p400 has now been identified as giantin, a Golgi membrane protein with most of its mass projecting into the cytoplasm. Giantin is found on COPI vesicles and pretreatment with antibodies inhibits both the binding of p115 and the docking of these vesicles with Golgi membranes. In contrast, GM130 is depleted from COPI vesicles and inhibition of the GM130 on Golgi membranes, using either antibodies or an NH2-terminal GM130 peptide, inhibits p115 binding and vesicle docking. Together these results suggest that COPI vesicles are docked by giantin on the COPI vesicles and GM130 on Golgi membranes with p115 providing a bridge.
Changes in Chromosomal Localization of Heterochromatin-binding Proteins during the Cell Cycle in Drosophila
Authors: Suso Platero J. , Csink Amy K. , Quintanilla Adrian , Henikoff Steven
Published: July 2002

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brownDominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.
Dynamin-mediated Internalization of Caveolae
Authors: Henley John R. , Krueger Eugene W.A. , Oswald Barbara J. , McNiven Mark A.
Published: July 2002

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.
Changes in the Balance of Phosphoinositide 3-Kinase/Protein Kinase B (Akt) and the Mitogen-activated Protein Kinases (ERK/p38MAPK) Determine a Phenotype of Visceral and Vascular Smooth Muscle Cells
Authors: Hayashi Ken'ichiro , Takahashi Masanori , Kimura Kazuhiro , Nishida Wataru , Saga Hiroshi , Sobue Kenji
Published: July 2002

The molecular mechanisms behind phenotypic modulation of smooth muscle cells (SMCs) remain unclear. In our recent paper, we reported the establishment of novel culture system of gizzard SMCs (Hayashi, K., H. Saga, Y. Chimori, K. Kimura, Y. Yamanaka, and K. Sobue. 1998. J. Biol. Chem. 273: 28860–28867), in which insulin-like growth factor-I (IGF-I) was the most potent for maintaining the differentiated SMC phenotype, and IGF-I triggered the phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB(Akt)) pathway. Here, we investigated the signaling pathways involved in de-differentiation of gizzard SMCs induced by PDGF-BB, bFGF, and EGF. In contrast to the IGF-I–triggered pathway, PDGF-BB, bFGF, and EGF coordinately activated ERK and p38MAPK pathways. Further, the forced expression of active forms of MEK1 and MKK6, which are the upstream kinases of ERK and p38MAPK, respectively, induced de-differentiation even when SMCs were stimulated with IGF-I. Among three growth factors, PDGF-BB only triggered the PI3-K/PKB(Akt) pathway in addition to the ERK and p38MAPK pathways. When the ERK and p38MAPK pathways were simultaneously blocked by their specific inhibitors or an active form of either PI3-K or PKB(Akt) was transfected, PDGF-BB in turn initiated to maintain the differentiated SMC phenotype. We applied these findings to vascular SMCs, and demonstrated the possibility that the same signaling pathways might be involved in regulating the vascular SMC phenotype. These results suggest that changes in the balance between the PI3-K/PKB(Akt) pathway and the ERK and p38MAPK pathways would determine phenotypes of visceral and vascular SMCs. We further reported that SMCs cotransfected with active forms of MEK1 and MKK6 secreted a nondialyzable, heat-labile protein factor(s) which induced de-differentiation of surrounding normal SMCs.
A Molecular Mechanism of Integrin Crosstalk: αvβ3 Suppression of Calcium/Calmodulin-dependent Protein Kinase II Regulates α5β1 Function
Authors: Blystone Scott D. , Slater Suzanne E. , Williams Matthew P. , Crow Michael T. , Brown Eric J.
Published: July 2002

Many cells express more than one integrin receptor for extracellular matrix, and in vivo these receptors may be simultaneously engaged. Ligation of one integrin may influence the behavior of others on the cell, a phenomenon we have called integrin crosstalk. Ligation of the integrin αvβ3 inhibits both phagocytosis and migration mediated by α5β1 on the same cell, and the β3 cytoplasmic tail is necessary and sufficient for this regulation of α5β1. Ligation of α5β1 activates the calcium- and calmodulin-dependent protein kinase II (CamKII). This activation is required for α5β1-mediated phagocytosis and migration. Simultaneous ligation of αvβ3 or expression of a chimeric molecule with a free β3 cytoplasmic tail prevents α5β1-mediated activation of CamKII. Expression of a constitutively active CamKII restores α5β1 functions blocked by αvβ3-initiated integrin crosstalk. Thus, αvβ3 inhibition of α5β1 activation of CamKII is required for its role in integrin crosstalk. Structure-function analysis of the β3 cytoplasmic tail demonstrates a requirement for Ser752 in β3-mediated suppression of CamKII activation, while crosstalk is independent of Tyr747 and Tyr759, implicating Ser752, but not β3 tyrosine phosphorylation in initiation of the αvβ3 signal for integrin crosstalk.
α-Dystroglycan Is a Laminin Receptor Involved in Extracellular Matrix Assembly on Myotubes and Muscle Cell Viability
Authors: Montanaro Federica , Lindenbaum Michael , Carbonetto Salvatore
Published: July 2002

α-Dystroglycan (α-DG) is a laminin-binding protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes from two resulting clones (11F and 11E) had at least a 40–50% and 80–90% reduction, respectively, in α-DG but normal or near normal levels of α-sarcoglycan, integrin β1 subunit, acetylcholine receptors (AChRs), and muscle-specific kinase (MuSK) when compared with parental C2 cells or three clones (11A, 9B, and 10C) which went through the same transfection and selection procedures but expressed normal levels of α-DG. Antisense DG-expressing myoblasts proliferate at the same rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with condensed chromatin and more nuclei labeled by the TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as would be expected with necrosis. Unlike parental C2 myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with α-DG on their surfaces but far fewer LN/α-DG plaques were seen after LN addition to 11F or 11E myotubes. These results suggest that α-DG is a functional LN receptor in situ which is required for deposition of LN on the cell and, further, implicate α-DG in the maintenance of myotube viability.
A novel role for FGF and extracellular signal–regulated kinase in gap junction–mediated intercellular communication in the lens
Authors: Le Anh-Chi N. , Musil Linda S.
Published: July 2002

Gap junction–mediated intercellular coupling is higher in the equatorial region of the lens than at either pole, a property believed to be essential for lens transparency. We show that fibroblast growth factor (FGF) upregulates gap junctional intercellular dye transfer in primary cultures of embryonic chick lens cells without detectably increasing either gap junction protein (connexin) synthesis or assembly. Insulin and insulin-like growth factor 1, as potent as FGF in inducing lens cell differentiation, had no effect on gap junctions. FGF induced sustained activation of extracellular signal–regulated kinase (ERK) in lens cells, an event necessary and sufficient to increase gap junctional coupling. We also identify vitreous humor as an in vivo source of an FGF-like intercellular communication-promoting activity and show that FGF-induced ERK activation in the intact lens is higher in the equatorial region than in polar and core fibers. These findings support a model in which regional differences in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function. Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.
A Gip1p–Glc7p phosphatase complex regulates septin organization and spore wall formation
Authors: Tachikawa Hiroyuki , Bloecher Andrew , Tatchell Kelly , Neiman Aaron M.
Published: July 2002

Sporulation of Saccharomyces cerevisiae is a developmental process in which a single cell is converted into four haploid spores. GIP1, encoding a developmentally regulated protein phosphatase 1 interacting protein, is required for spore formation. Here we show that GIP1 and the protein phosphatase 1 encoded by GLC7 play essential roles in spore development. The gip1Δ mutant undergoes meiosis and prospore membrane formation normally, but is specifically defective in spore wall synthesis. We demonstrate that in wild-type cells, distinct layers of the spore wall are deposited in a specific temporal order, and that gip1Δ cells display a discrete arrest at the onset of spore wall deposition. Localization studies revealed that Gip1p and Glc7p colocalize with the septins in structures underlying the growing prospore membranes. Interestingly, in the gip1Δ mutant, not only is Glc7p localization altered, but septins are also delocalized. Similar phenotypes were observed in a glc7–136 mutant, which expresses a Glc7p defective in interacting with Gip1p. These results indicate that a Gip1p–Glc7p phosphatase complex is required for proper septin organization and initiation of spore wall formation during sporulation.
BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations
Authors: Ye Qinong , Hu Yan-Fen , Zhong Hongjun , Nye Anne C. , Belmont Andrew S. , Li Rong
Published: July 2002

The breast cancer susceptibility gene BRCA1 encodes a protein that has been implicated in multiple nuclear functions, including transcription and DNA repair. The multifunctional nature of BRCA1 has raised the possibility that the polypeptide may regulate various nuclear processes via a common underlying mechanism such as chromatin remodeling. However, to date, no direct evidence exists in mammalian cells for BRCA1-mediated changes in either local or large-scale chromatin structure. Here we show that targeting BRCA1 to an amplified, lac operator–containing chromosome region in the mammalian genome results in large-scale chromatin decondensation. This unfolding activity is independently conferred by three subdomains within the transactivation domain of BRCA1, namely activation domain 1, and the two BRCA1 COOH terminus (BRCT) repeats. In addition, we demonstrate a similar chromatin unfolding activity associated with the transactivation domains of E2F1 and tumor suppressor p53. However, unlike E2F1 and p53, BRCT-mediated chromatin unfolding is not accompanied by histone hyperacetylation. Cancer-predisposing mutations of BRCA1 display an allele-specific effect on chromatin unfolding: 5′ mutations that result in gross truncation of the protein abolish the chromatin unfolding activity, whereas those in the 3′ region of the gene markedly enhance this activity. A novel cofactor of BRCA1 (COBRA1) is recruited to the chromosome site by the first BRCT repeat of BRCA1, and is itself sufficient to induce chromatin unfolding. BRCA1 mutations that enhance chromatin unfolding also increase its affinity for, and recruitment of, COBRA1. These results indicate that reorganization of higher levels of chromatin structure is an important regulated step in BRCA1-mediated nuclear functions.
Intrinsic and extrinsic pathway signaling during neuronal apoptosis
Authors: Putcha Girish V. , Harris Charles A. , Moulder Krista L. , Easton Rachael M. , Thompson Craig B. , Johnson Eugene M.
Published: July 2002

Trophic factor deprivation (TFD)-induced apoptosis in sympathetic neurons requires macromolecular synthesis–dependent BAX translocation, cytochrome c (cyt c) release, and caspase activation. Here, we report the contributions of other intrinsic and extrinsic pathway signals to these processes. Sympathetic neurons expressed all antiapoptotic BCL-2 proteins examined, yet expressed only certain BH3-only and multidomain proapoptotic BCL-2 family members. All coexpressed proapoptotic proteins did not, however, exhibit functional redundancy or compensatory expression, at least in the Bax−/−, Bak−/−, Bim−/−, Bid−/−, and Bad−/− neurons examined. Although the subcellular distribution or posttranslational modification of certain BCL-2 proteins changed with TFD, neither transcriptional nor posttranslational mechanisms regulated the expression or subcellular localization of BID, BAD, or BAK in this paradigm. Despite modest induction of Fas and FasL expression, Fas-mediated signaling did not contribute to TFD-induced apoptosis in sympathetic neurons. Similar findings were obtained with K+ withdrawal–induced apoptosis in cerebellar granule neurons, a model for activity-dependent neuronal survival in the CNS. Thus, expression alone does not guarantee functional redundancy (or compensation) among BCL-2 family members, and, at least in some cells, extrinsic pathway signaling and certain BH3-only proteins (i.e., BID and BAD) do not contribute to BAX-dependent cyt c release or apoptosis caused by TFD.
PEX11 promotes peroxisome division independently of peroxisome metabolism
Authors: Li Xiaoling , Gould Stephen J.
Published: July 2002

The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species. It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. We also observed that mouse cells lacking PEX11β display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11β−/− mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism.
Functional Inactivation in the Whole Population of Human Vγ9/Vδ2 T Lymphocytes Induced By a Nonpeptidic Antagonist
Authors: Bürk Martin R. , Carena Ilaria , Donda Alena , Mariani Francesca , Mori Lucia , De Libero Gennaro
Published: July 2002

Nonpeptidic compounds stimulate human T cells bearing the TCR-γδ in the absence of major histocompatibility complex restriction. We report that one of these ligands, 2,3-diphosphoglyceric acid (DPG), which induces expansion of Vγ9/Vδ T cells ex vivo, antagonizes the same cell population after repetitive activation. Stimulation with DPG results in partial early protein tyrosine phosphorylation and a prolonged, but reversible, state of unresponsiveness to agonist ligands in Vγ9/Vδ2, but not in other T cells. These findings show that TCR antagonism is a general phenomenon of T cells. However, in contrast to the clonal specificity of altered peptides antagonizing αβ T cells, all the tested Vγ9/Vδ2 polyclonal cell lines and clones become unresponsive, a fact that may be relevant for the regulation of their response in vivo.
pH-dependent Perforation of Macrophage Phagosomes by Listeriolysin O from Listeria monocytogenes
Authors: Beauregard Kathryn E. , Lee Kyung-Dall , Collier R. John , Swanson Joel A.
Published: July 2002

The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles. Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow–derived macrophages. Perforation was observed when acidic vacuoles containing wild-type L. monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol. These changes were not seen with LLO-deficient mutants. Perforation occurred at acidic vacuolar pH (4.9–6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1. We conclude that acidic pH facilitates LLO activity in vivo.
B Lymphocytes Differentially Use the Rel and Nuclear Factor κB1 (NF-κB1) Transcription Factors to Regulate Cell Cycle Progression and Apoptosis in Quiescent and Mitogen-activated Cells
Authors: Grumont Raelene J. , Rourke Ian J. , O'Reilly Lorraine A. , Strasser Andreas , Miyake Kensuke , Sha William , Gerondakis Steve
Published: July 2002

Rel and nuclear factor (NF)-κB1, two members of the Rel/NF-κB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-κB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1−/− mice, the level of apoptosis in cultures of quiescent nfkb1−/−, but not c-rel−/−, B cells is higher. The failure of c-rel−/− or nfkb1−/− B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel−/− and nfkb1−/− B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-κB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-κB–dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel−/− B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-κB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-κB factors to control cell cycle progression and prevent apoptosis.
B Cells Directly Tolerize CD8+ T Cells
Authors: Bennett Sally R.M. , Carbone Francis R. , Toy Tracey , Miller Jacques F.A.P. , Heath William R.
Published: July 2002

This report investigates the response of CD8+ T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the Kb-restricted ovalbumin (OVA) determinant OVA257–264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro–activated CD8+ T cells from the Kb-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8+ T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.
B Cell Development in the Spleen Takes Place in Discrete Steps and Is Determined by the Quality of B Cell Receptor–Derived Signals
Authors: Loder By Florienne , Mutschler Bettina , Ray Robert J. , Paige Christopher J. , Sideras Paschalis , Torres Raul , Lamers Marinus C. , Carsetti Rita
Published: July 2002

Only mature B lymphocytes can enter the lymphoid follicles of spleen and lymph nodes and thus efficiently participate in the immune response. Mature, long-lived B lymphocytes derive from short-lived precursors generated in the bone marrow. We show that selection into the mature pool is an active process and takes place in the spleen. Two populations of splenic B cells were identified as precursors for mature B cells. Transitional B cells of type 1 (T1) are recent immigrants from the bone marrow. They develop into the transitional B cells of type 2 (T2), which are cycling and found exclusively in the primary follicles of the spleen. Mature B cells can be generated from T1 or T2 B cells.
Chronic Restraint Stress Promotes Lymphocyte Apoptosis by Modulating Cd95 Expression
Authors: Yin Deling , Tuthill David , Mufson R. Allan , Shi Yufang
Published: July 2002

Depending on the duration and severity, psychological tension and physical stress can enhance or suppress the immune system in both humans and animals. Although it is well established that stress alters the release of various hormones and neurotransmitters, the mechanisms by which stress affects immune responses remain elusive. We report here that mice subjected to chronic 12-hour daily physical restraint for two days exhibited a significant reduction in splenocytes, a process likely mediated by apoptosis as demonstrated by the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling assay. CD95 (Fas/APO-1) expression in splenic lymphocytes of stressed mice was substantially increased. Interestingly, Fas-immunoglobulin fusion protein and blocking antibodies against CD95 ligand inhibit stress-induced reduction in lymphocytes. The stress-induced changes in CD95 expression and lymphocyte number could be blocked by naltrexone or naloxone, specific opioid receptor antagonists, indicating a pivotal role of endogenous opioids in this process. In addition, the reduction of splenocytes in this model system seems to be independent of the hypothalamo-pituitary-adrenal axis, as both adrenalectomized and sham-operated mice exhibited similar responses to chronic stress. Moreover, chronic physical restraint failed to induce a decrease in lymphocyte numbers in CD95-deficient (Faslpr/lpr) mice. Therefore, stress modulates the immune system through CD95-mediated apoptosis dependent on endogenous opioids.
The Association of ASAP1, an ADP Ribosylation Factor-GTPase Activating Protein, with Focal Adhesion Kinase Contributes to the Process of Focal Adhesion Assembly
Authors: Liu Yunhao , Loijens Joost C. , Martin Karen H. , Karginov Andrei V. , Parsons J. Thomas
Published: July 2002

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src. Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). GlutathioneS-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.
The Merck Gene Index browser: an extensible data integration system for gene finding, gene characterization and EST data mining.
Authors: Eckman B A , Aaronson J S , Borkowski J A , Bailey W J , Elliston K O , Williamson A R , Blevins R A
Published: July 2002

MOTIVATION: To make effective use of the vast amounts of expressed sequence tag (EST) sequence data generated by the Merck-sponsored EST project and other similar efforts, sequences must be organized into gene classes, and scientists must be able to 'mine' the gene class data in the context of related genomic data. RESULTS: This paper presents the Merck Gene Index browser, an easily extensible, World Wide Web-based system for mining the Merck Gene Index (MGI) and related genomic data. The MGI is a non-redundant set of clones and sequences, each representing a distinct gene, constructed from all high-quality 3' EST sequences generated by the Merck-sponsored EST project. The MGI browser integrates data from a variety of sources and storage formats, both local and remote, using an eclectic integration strategy, including a federation of relational databases, a local data warehouse and simple hypertext links. Data currently integrated include: LENS cDNA clone and EST data, dbEST protein and non-EST nucleic acid similarity data, WashU sequence chromatograms. Entrez sequence and Medline entries, and UniGene gene clusters. Flatfile sequence data are accessed using the Bioapps server, an internally developed client-server system that supports generic sequence analysis applications. Browser data are retrieved and formatted by means of the Bioinformatics Data Integration Toolkit (B-DIT), a new suite of Perl routines.