bluuebunny/crossref_metadata_2025 huggingface dataset:
Antiretroviral resistance during successful therapy of HIV type 1 infection
Authors: Martinez-Picado J. , DePasquale M. P. , Kartsonis N. , Hanna G. J. , Wong J. , Finzi D. , Rosenberg E. , Günthard H. F. , Sutton L. , Savara A. , Petropoulos C. J. , Hellmann N. , Walker B. D. , Richman D. D. , Siliciano R. , D'Aquila R. T.
Published: July 2002

HIV type 1 (HIV-1) drug resistance mutations were selected during
antiretroviral therapy successfully suppressing plasma HIV-1 RNA to
<50 copies/ml. New resistant mutant subpopulations were identified
by clonal sequencing analyses of viruses cultured from blood cells.
Drug susceptibility tests showed that biological clones of virus with
the mutations acquired during successful therapy had increased
resistance. Each of the five subjects with new resistant mutants had
evidence of some residual virus replication during highly active
antiretroviral therapy (HAART), based on transient episodes of plasma
HIV-1 RNA > 50 copies/ml and virus
env
gene
sequence changes. Each had received a suboptimal regimen before
starting HAART. Antiretroviral-resistant HIV-1 can be selected from
residual virus replication during HAART in the absence of sustained
rebound of plasma HIV-1 RNA.
Neuromelanin biosynthesis is driven by excess cytosolic catecholamines not accumulated by synaptic vesicles
Authors: Sulzer David , Bogulavsky Johanna , Larsen Kristin E. , Behr Gerald , Karatekin Erdem , Kleinman Mark H. , Turro Nicholas , Krantz David , Edwards Robert H. , Greene Lloyd A. , Zecca Luigi
Published: July 2002

Melanin, the pigment in hair, skin, eyes, and feathers, protects
external tissue from damage by UV light. In contrast, neuromelanin (NM)
is found in deep brain regions, specifically in loci that degenerate in
Parkinson's disease. Although this distribution suggests a role for NM
in Parkinson's disease neurodegeneration, the biosynthesis and
function of NM have eluded characterization because of lack of an
experimental system. We induced NM in rat substantia nigra and PC12
cell cultures by exposure to
l
-dihydroxyphenylalanine,
which is rapidly converted to dopamine (DA) in the cytosol. This
pigment was identical to human NM as assessed by paramagnetic resonance
and was localized in double membrane autophagic vacuoles identical to
NM granules of human substantia nigra. NM synthesis was abolished by
adenoviral-mediated overexpression of the synaptic vesicle
catecholamine transporter VMAT2, which decreases cytosolic DA by
increasing vesicular accumulation of neurotransmitter. The NM is in a
stable complex with ferric iron, and NM synthesis was inhibited by the
iron chelator desferrioxamine, indicating that cytosolic DA and
dihydroxyphenylalanine are oxidized by iron-mediated catalysis to
membrane-impermeant quinones and semiquinones. NM synthesis thus
results from excess cytosolic catecholamines not accumulated into
synaptic vesicles. The permanent accumulation of excess catechols,
quinones, and catechol adducts into a membrane-impermeant substance
trapped in organelles may provide an antioxidant mechanism for
catecholamine neurons. However, NM in organelles associated with
secretory pathways may interfere with signaling, as it delays
stimulated neurite outgrowth in PC12 cells.
Functional interaction between c-Abl and the p21-activated protein kinase γ-PAK
Authors: Roig Joan , Tuazon Polygena T. , Zipfel Patricia A. , Pendergast Ann Marie , Traugh Jolinda A.
Published: July 2002

A member of the p21-activated protein kinase (PAK) family, γ-PAK
has cytostatic properties and is activated by cellular stresses such as
hyperosmolarity or DNA damage. We report herein that γ-PAK is
associated
in vivo
with the nonreceptor protein tyrosine
kinase c-Abl. γ-PAK phosphorylates c-Abl on sites located in the
kinase domain, in a region that is implicated in protein–protein
interactions and in subcellular localization. Activation of γ-PAK in
human embryonic kidney 293T cells by cotransfection with constitutively
active Cdc42 induces activation of c-Abl, resulting in increased
phosphotyrosine levels. Cotransfection of c-Abl and γ-PAK elicits
phosphorylation of γ-PAK on tyrosine and down-regulation of γ-PAK
activity, promoting accumulation of inactive γ-PAK. γ-PAK is also
phosphorylated
in vitro
by c-Abl. γ-PAK activity is
regulated by ubiquitination and proteolysis
in vivo
, as
shown by immunoblotting with an anti-ubiquitin antibody in the presence
of proteasome inhibitors. In summary, we describe a functional
interaction between γ-PAK and c-Abl in which γ-PAK stimulates c-Abl
tyrosine kinase activity and c-Abl phosphorylates and down-regulates
γ-PAK, suggesting the existence of a negative feedback loop between
c-Abl and γ-PAK.
Reciprocal subsidies: Dynamic interdependence between terrestrial and aquatic food webs
Authors: Nakano Shigeru , Murakami Masashi
Published: July 2002

Mutual trophic interactions between contiguous habitats have
remained poorly understood despite their potential significance for
community maintenance in ecological landscapes. In a deciduous forest
and stream ecotone, aquatic insect emergence peaked around spring, when
terrestrial invertebrate biomass was low. In contrast, terrestrial
invertebrate input to the stream occurred primarily during summer, when
aquatic invertebrate biomass was nearly at its lowest. Such reciprocal,
across-habitat prey flux alternately subsidized both forest birds and
stream fishes, accounting for 25.6% and 44.0% of the annual total
energy budget of the bird and fish assemblages, respectively. Seasonal
contrasts between allochthonous prey supply and
in situ
prey biomass determine the importance of reciprocal subsidies.
Social Justice: Outcome and Procedure
Authors: Tyler Tom R.
Published: July 2002

A review of recent research demonstrates that people are more willing to accept decisions when they feel that those decisions are made through decision‐making procedures they view as fair. Studies of procedural justice judgements further suggest that people evaluate fairness primarily through criteria that can be provided to all the parties to a conflict: whether there are opportunities to participate; whether the authorities are neutral; the degree to which people trust the motives of the authorities; and whether people are treated with dignity and respect during the process. These findings are optimistic and suggest that authorities have considerable ability to bridge differences and interests and values through the use of fair decision‐making procedures. The limits to the effectiveness of such procedural approaches are also outlined.
On the Nature of Neighbourhood
Authors: Galster George
Published: July 2002

The paper advances the conceptualisation of neighbourhood by specifying it as a bundle of spatially based attributes associated with clusters of residences, sometimes in conjunction with other land uses. There follows a discussion of how this 'composite commodity' definition relates to the planning challenge of spatially bounding neighbourhood. The paper then probes the myriad idiosyncrasies associated with the concept of neighbourhood: cross-attribute variation in durability and ability to be priced, relativistic evaluations of attributes and consumption impacts on attributes. It discusses how, within this new paradigmatic context, neighbourhoods are produced by the same actors that consume them: households, property owners, business people and local government. Finally, consideration is given to various aspects of the origins and nature of neighbourhood change and it is argued that neighbourhood dynamics are rife with social inefficiencies.
Sub-centring and Commuting: Evidence from the San Francisco Bay Area, 1980-90
Authors: Cervero Robert , Wu Kang-Li
Published: July 2002

Like many large US metropolitan areas, the San Francisco Bay Area has experienced rapid suburban employment growth since 1980, much of it concentrated in sub-centres. This paper shows that, contrary to the co-location hypothesis, employment decentralisation has not been associated with shorter average commute distances or durations in the Bay Area. Combining statistics on shifts in modal splits and average vehicle occupancy levels reveals that parallelling the region's sub-centring trend has been a substantial increase in average commute vehicle miles travelled (VMT) per employee between 1980 and 1990. The largest increases occurred in the fastest-growing and most remote suburban centres. Using decomposition analysis, we found that increasing commute distances contributed the most to rising commute VMT per employee, and the distance factor had proportionately the greatest effect on rising commute VMT rates in the most peripheral work centres. Since shifts in commute VMT per employee are thought to be strongly associated with transport externalities, we conclude that the social and environmental implications of the Bay Area's regional growth trends deserve more public policy attention than given to date.
A Synthetic Approach to Estimating the Impacts of Telecommuting on Travel
Authors: Mokhtarian Patricia L.
Published: July 2002

A multiplicative model is proposed as a framework for examining the current knowledge in forecasting the demand for telecommuting and the resulting transport impacts. A running illustrative example (containing a base and a future case) is developed, using plausible values for each factor in the model. The base case suggests that 6.1 per cent of the workforce may be currently telecommuting, with 1.5 per cent doing so on any given day, eliminating at most 1 per cent of total household vehicle-miles travelled. Future reductions could be smaller as commute distances of telecommuters become more average and as the stimulation effect of telecommuting grows. In any event, it is likely that—due to counteracting forces—the aggregate travel impacts will remain relatively flat into the future, even if the amount of telecommuting increases considerably.
Sense of Community and Neighbourhood Form: An Assessment of the Social Doctrine of New Urbanism
Authors: Talen Emily
Published: July 2002

New urbanism, an umbrella term which encompasses 'neotraditional development' as well as 'traditional neighbourhood design', lives by an unswerving belief in the ability of the built environment to create a 'sense of community'. The purpose of this paper is to assess whether the social doctrine of new urbanism can be successfully supported or at least integrated with the social science literature which deals with the question of community formation. Towards this goal, the paper first delineates the social doctrine of new urbanism, and then discusses the conceptual frameworks and empirical findings that either support or contradict the idea that a sense of community will follow the physical form of cities and neighbourhoods generally and new urbanist principles specifically. After laying this groundwork, the remainder of the paper presents an assessment of whether a reconciliation between research and doctrine may be possible, in light of various apparent contradictions between the social claims of new urbanists and the results of research by social scientists. It is concluded that new urbanists need to clarify the meaning of sense of community as it pertains to physical design. Further, it is maintained that while some research supports the idea that resident interaction and sense of community are related to environmental factors, the effectuation of this goal is usually only achieved via some intermediate variable. This latter point leaves open the question of whether any number of other design creeds could produce the same result via a different design philosophy. The need for further research is stressed; this should be focused on investigating the issue more directly.
Pupil Groupings in Primary School Classrooms: Sites for learning and social pedagogy?
Authors: Kutnick Peter , Blatchford Peter , Baines Ed
Published: July 2002

Abstract Studies of teaching and learning within classrooms rarely consider the multilayered effects of social context, especially involving within‐class groupings. Yet, all pupils in classes are placed in some form of grouping throughout their classroom life and this will have an impact on their learning. This article seeks to move forward the understanding of within‐class groupings in real classrooms. Five ‘core themes’ central to pupils' experience of groups in classrooms are identified (group size, group composition, learning tasks, within group interaction and adult presence) and used as a basis to collect information on classroom groupings (using a novel classroom ‘mapping’ survey). Teachers in 187 classrooms from Years 2 and 5 were surveyed and undertook the mapping during normal classroom work time. The most common group size found was the small group of 4‐6 pupils. Other group sizes, including whole classes, individuals, dyads, and triads were less frequent. Most pupil groupings were teacher designated and combined girls and boys of similar ability. Groupings mainly studied English and mathematics, and worked on individuated tasks involving the practice or revision of skills/knowledge. Adults were present with one‐third of groupings—with particular adults interacting with particular types of groupings (low‐ability boys, high‐ability girls, and whole classes). When data from the themes are interrelated, especially group size and composition with learning task and interaction type, many points of concern arise. Findings suggest that teachers may not think strategically about the size and composition of groupings in relation to the tasks assigned. Adult presence is associated with control of knowledge and behaviour, and different types of adults are associated with the support of groupings at different levels of ability. Teachers provide little training for children to develop group work skills, and offer little opportunity for these skills to be practised in the promotion of learning. The findings and discussion combine to identify that the area of ‘social pedagogy of pupil grouping’ is a new and legitimate concern, and that further (and more focused) research in the area should be undertaken.
Sociability and prosocial orientation as predictors of youth adjustment: A seven-year longitudinal study in a Chinese sample
Authors: Chen Xinyin , Liu Mowei , Rubin Kenneth H. , Cen Guo-zhen , Gao Xiangping , Li Dan
Published: July 2002

The purpose of the study was to examine contributions of sociable and prosocial dimensions of social competence in childhood to the prediction of educational attainment and socioemotional adjustment in early adulthood in urban China. A sample of children at 12 years of age participated in the original study, and was followed up seven years later when they graduated from high school. Sociability and prosocial orientation in childhood were assessed by peer evaluations. Data concerning youth adjustment were collected from self-reports of the participants and their parents. The results indicated that sociability and prosocial orientation had differential significance for later adjustment in different areas. Whereas prosocial orientation made unique contributions to the prediction of educational achievement, sociability in the early years was a significant and reliable predictor of later socioemotional adjustment.
Oxidant–antioxidant profiles of platelet rich plasma in smokers
Authors: Gökkusu C. , Ademoglu E. , Tamer S. , Alkan G.
Published: July 2002

Cigarette smoke is a major risk factor for both coronary heart disease and peripheral vascular disease and has been reported to contain many oxidizing agents that lead to generation of free radicals. In this study, we investigated the levels of lipid peroxides (LPO) and antioxidant vitamins (C and E), total thiol content (t‐SH), the activities of superoxide dismutase (SOD), glutathione S‐transferase (GST) and glutathione peroxidase (GPx) in the platelet‐rich plasma (PRP) and plasma of 50 smokers and 30 non‐smokers. Total cholesterol (TC), low density‐cholesterol (LDL‐C), triglyceride (TG) and phospholipid (PL) levels of the plasma were significantly higher (p < 0.001) and high density‐cholesterol (HDL‐C) levels were significantly lower in smokers (p < 0.001) when compared with non‐smokers. In plasma and PRP, LPO levels, GST and SOD activities were found to be increased (p < 0.001) in smokers, whereas GPx activity, vitamin C levels and t‐SH content were found to be decreased. On the other hand, the levels of vitamin E was unchanged in plasma and PRP. The relationships between plasma levels of lipids, LPO and antioxidant systems were also investigated in both groups. A strong positive correlation was found between TC and Vit E (r = 0.5575; p < 0.001), LPO and PL (r = 0.4270; p < 0.01), LPO and GST (r = 0.3770; p < 0.01) and t‐SH and GPx (r = 0.3781; p < 0.01) in smokers. These findings reveal a disturbance of oxidant‐antioxidant balance by free radicals present in cigarette smoke, which may cause reduction in platelet hyperreactivity and endothelial dysfunction in smokers.
A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor
Authors: Fujimura-Kamada Konomi , Nouvet Franklin J. , Michaelis Susan
Published: July 2002

Many secreted bioactive signaling molecules, including the yeast mating pheromones a-factor and α-factor, are initially synthesized as precursors requiring multiple intracellular processing enzymes to generate their mature forms. To identify new gene products involved in the biogenesis of a-factor in Saccharomyces cerevisiae, we carried out a screen for MATa-specific, mating-defective mutants. We have identified a new mutant, ste24, in addition to previously known sterile mutants. During its biogenesis in a wild-type strain, the a-factor precursor undergoes a series of COOH-terminal CAAX modifications, two sequential NH2-terminal cleavage events, and export from the cell. Identification of the a-factor biosynthetic intermediate that accumulates in the ste24 mutant revealed that STE24 is required for the first NH2-terminal proteolytic processing event within the a-factor precursor, which takes place after COOH-terminal CAAX modification is complete. The STE24 gene product contains multiple predicted membrane spans, a zinc metalloprotease motif (HEXXH), and a COOH-terminal ER retrieval signal (KKXX). The HEXXH protease motif is critical for STE24 activity, since STE24 fails to function when conserved residues within this motif are mutated. The identification of Ste24p homologues in a diverse group of organisms, including Escherichia coli, Schizosaccharomyces pombe, Haemophilus influenzae, and Homo sapiens, indicates that Ste24p has been highly conserved throughout evolution. Ste24p and the proteins related to it define a new subfamily of proteins that are likely to function as intracellular, membrane-associated zinc metalloproteases.
Mitotic Spindle Poles are Organized by Structural and Motor Proteins in Addition to Centrosomes
Authors: Gaglio Tirso , Dionne Mary A. , Compton Duane A.
Published: July 2002

The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420–425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.
Phorbol Esters and SDF-1 Induce Rapid Endocytosis and Down Modulation of the Chemokine Receptor CXCR4
Authors: Signoret Natalie , Oldridge Joanne , Pelchen-Matthews Annegret , Klasse Per J. , Tran Thanh , Brass Lawrence F. , Rosenkilde Mette M. , Schwartz Thue W. , Holmes William , Dallas Walt , Luther Michael A. , Wells Timothy N.C. , Hoxie James A. , Marsh Mark
Published: July 2002

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficiency. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechanism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surface expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 undergoes slow constitutive internalization (1.0% of the cell surface pool/min). Addition of phorbol esters increased this endocytosis rate >6-fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 min. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time ∼5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-terminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation through different cellular mechanisms.

SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligand induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Together these studies indicate that endocytosis can regulate the cell-surface expression of CXCR4 and that SDF-1–mediated down regulation of cell-surface coreceptor expression contributes to chemokine-mediated inhibition of HIV infection.
Suppression of Steady-state, but not Stimulus-induced NF-κB Activity Inhibits Alphavirus-induced Apoptosis
Authors: Lin Kuo-I , DiDonato Joseph A. , Hoffmann Alexander , Marie Hardwick J. , Ratan Rajiv R.
Published: July 2002

Recent studies have established cell type– specific, proapoptotic, or antiapoptotic functions for the transcription factor NF-κB. In each of these studies, inhibitors of NF-κB activity have been present before the apoptotic stimulus, and so the role of stimulus- induced NF-κB activation in enhancing or inhibiting survival could not be directly assessed. Sindbis virus, an alphavirus, induces NF-κB activation and apoptosis in cultured cell lines. To address whether Sindbis virus– induced NF-κB activation is required for apoptosis, we used a chimeric Sindbis virus that expresses a superrepressor of NF-κB activity. Complete suppression of virus-induced NF-κB activity neither prevents nor potentiates Sindbis virus–induced apoptosis. In contrast, inhibition of NF-κB activity before infection inhibits Sindbis virus–induced apoptosis. Our results demonstrate that suppression of steady-state, but not stimulus-induced NF-κB activity, regulates expression of gene products required for Sindbis virus–induced death. Furthermore, we show that in the same cell line, NF-κB can be proapoptotic or antiapoptotic depending on the death stimulus. We propose that the role of NF-κB in regulating apoptosis is determined by the death stimulus and by the timing of modulating NF-κB activity relative to the death stimulus.
Small Espin: A Third Actin-bundling Protein and Potential Forked Protein Ortholog in Brush Border Microvilli
Authors: Bartles James R. , Zheng Lili , Li Anli , Wierda Allison , Chen Bin
Published: July 2002

An ∼30-kD isoform of the actin-binding/ bundling protein espin has been discovered in the brush borders of absorptive epithelial cells in rat intestine and kidney. Small espin is identical in sequence to the COOH terminus of the larger (∼110-kD) espin isoform identified in the actin bundles of Sertoli cell–spermatid junctional plaques (Bartles, J.R., A. Wierda, and L. Zheng. 1996. J. Cell Sci. 109:1229–1239), but it contains two unique peptides at its NH2 terminus. Small espin was localized to the parallel actin bundles of brush border microvilli, resisted extraction with Triton X-100, and accumulated in the brush border during enterocyte differentiation/migration along the crypt–villus axis in adults. In transfected BHK fibroblasts, green fluorescent protein–small espin decorated F-actin–containing fibers and appeared to elicit their accumulation and/or bundling. Recombinant small espin bound to skeletal muscle and nonmuscle F-actin with high affinity (Kd = 150 and 50 nM) and cross-linked the filaments into bundles. Sedimentation, gel filtration, and circular dichroism analyses suggested that recombinant small espin was a monomer with an asymmetrical shape and a high percentage of α-helix. Deletion mutagenesis suggested that small espin contained two actin-binding sites in its COOH-terminal 116–amino acid peptide and that the NH2-terminal half of its forked homology peptide was necessary for bundling activity.
The Yeast Dynamin-like Protein, Mgm1p, Functions on the Mitochondrial Outer Membrane to Mediate Mitochondrial Inheritance
Authors: Shepard Kelly A. , Yaffe Michael P.
Published: July 2002

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37°C, while loss of the mitochondrial genome occurred after 4–24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.
The Drosophila Ral GTPase Regulates Developmental Cell Shape Changes through the Jun NH2-terminal Kinase Pathway
Authors: Sawamoto Kazunobu , Winge Per , Koyama Shinya , Hirota Yuki , Yamada Chiharu , Miyao Sachiyo , Yoshikawa Shingo , Jin Ming-hao , Kikuchi Akira , Okano Hideyuki
Published: July 2002

The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRalG20V and DRalS25N, were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRalG20V and DRalS25N act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRalG20V and DRalS25N mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH2-terminal kinase kinase (JNKK) and Jun NH2-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.
Insulin-like Growth Factor II Signaling in Neoplastic Proliferation Is Blocked by Transgenic Expression of the Metalloproteinase Inhibitor Timp-1
Authors: Martin David C. , Fowlkes John L. , Babic Bojana , Khokha Rama
Published: July 2002

Insulin-like growth factor (IGF) II is overexpressed in many human cancers and is reactivated by, and crucial for viral oncogene (SV40 T antigen, [TAg])–induced tumorigenesis in several tumor models. Using a double transgenic murine hepatic tumor model, we demonstrate that tissue inhibitor of metalloproteinase 1 (TIMP-1) blocks liver hyperplasia during tumor development, despite TAg-mediated reactivation of IGF-II. Because the activity of IGFs is controlled by IGF-binding proteins (IGFBPs), we investigated whether TIMP-1 overexpression altered the IGFBP status in the transgenic liver. Ligand blotting showed that IGFBP-3 protein levels were increased in TIMP-1–overexpressing double transgenic littermates, whereas IGFBP-3 mRNA levels were not different, suggesting that TIMP-1 affects IGFBP-3 at a posttranscriptional level. IGFBP-3 proteolysis assays demonstrated that IGFBP-3 degradation was lower in TIMP-1–overexpressing livers, and zymography showed that matrix metalloproteinases (MMPs) were present in the liver homogenates and were capable of degrading IGFBP-3. As a consequence of reduced IGFBP-3 proteolysis and elevated IGFBP-3 protein levels, dissociable IGF-II levels were significantly lower in TIMP-1–overexpressing animals. This decrease in bioavailable IGF-II ultimately resulted in diminished IGF-I receptor signaling in vivo as evidenced by diminished receptor kinase activity and decreased tyrosine phosphorylation of the IGF-I receptor downstream effectors, insulin receptor substrate 1 (IRS-1), extracellular signal regulatory kinase (Erk)-1, and Erk-2. Together, these results provide evidence that TIMP-1 inhibits liver hyperplasia, an early event in TAg-mediated tumorigenesis, by reducing the activity of the tumor-inducing mitogen, IGF-II. These data implicate the control of MMP-mediated degradation of IGFBPs as a novel therapy for controlling IGF bioavailability in cancer.
Function of α3β1–Tetraspanin Protein Complexes in Tumor Cell Invasion. Evidence for the Role of the Complexes in Production of Matrix Metalloproteinase 2 (Mmp-2)
Authors: Sugiura Tsuyoshi , Berditchevski Fedor
Published: July 2002

Tumor cell migration through the three- dimensional extracellular matrix (ECM) environment is an important part of the metastatic process. We have analyzed a role played by the integrin–tetraspanin protein complexes in invasive migration by culturing MDA-MB-231 cells within Matrigel. Using time-lapse video recording, we demonstrated that the Matrigel-embedded cells remain round and exhibit only limited ability for migration by extending short, highly dynamic pseudopodia. The α3β1–tetraspanin protein complexes were clustered on the thin microvilli-like protrusions extending from both the main cell body and pseudopodia. Ligation of the α3β1–tetraspanin protein complexes with monoclonal antibodies specifically stimulates production of matrix metalloproteinase 2 (MMP-2) and induces formation of long invasive protrusions within Matrigel. Accordingly, treatment with the monoclonal antibodies to various tetraspanin proteins and to the α3 integrin subunit increases invasive potential of the MDA-MB-231 cells in the Matrigel-penetration assay. A specific inhibitor of phosphoinositide 3-kinase (PI3K), LY294002, negated the effect of the monoclonal antibodies on the morphology of the Matrigel-embedded cells and on production of MMP-2. Interestingly, broad-spectrum inhibitors of protein tyrosine kinases (genistein) and protein tyrosine phosphatases (orthovanadate), and actin filament stabilizing compound (jasplakinolide), also block protrusive activity of the Matrigel-embedded cells but have no effect on the production of MMP-2. These results indicate that α3β1–tetraspanin protein complexes may control invasive migration of tumor cells by using at least two PI3K-dependent signaling mechanisms: through rearrangement of the actin cytoskeleton and by modulating the MMP-2 production.
Role for Drs2p, a P-Type Atpase and Potential Aminophospholipid Translocase, in Yeast Late Golgi Function
Authors: Chen Chih-Ying , Ingram Michael F. , Rosal Peter H. , Graham Todd R.
Published: July 2002

ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Δ synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Δ mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro–α-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Δ cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.
Blood Platelets Are Assembled Principally at the Ends of Proplatelet Processes Produced by Differentiated Megakaryocytes
Authors: Italiano Joseph E. , Lecine Patrick , Shivdasani Ramesh A. , Hartwig John H.
Published: July 2002

Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.
Cell Elongation Induces Laminin α2 Chain Expression in Mouse Embryonic Mesenchymal Cells
Authors: Relan Nand K. , Yang Yan , Beqaj Safedin , Miner Jeffrey H. , Schuger Lucia
Published: July 2002

Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) α2 chain not present in round mesenchymal cells. LM α2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM β1 and γ1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM α2. Dy/dy mice express very low levels of LM α2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM α-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM α2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.
A Cell-Free System for Regulated Exocytosis in Pc12 Cells
Authors: Avery Julia , Ellis Darren J. , Lang Thorsten , Holroyd Phillip , Riedel Dietmar , Henderson Robert M. , Edwardson J. Michael , Jahn Reinhard
Published: July 2002

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca2+ at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca2+-dependent manner dur- ing 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimula- tion. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.
Reciprocal Role of ERK and Nf-κb Pathways in Survival and Activation of Osteoclasts
Authors: Miyazaki Tsuyoshi , Katagiri Hideki , Kanegae Yumi , Takayanagi Hiroshi , Sawada Yasuhiro , Yamamoto Aiichiro , Pando Mattew P. , Asano Tomoichiro , Verma Inder M. , Oda Hiromi , Nakamura Kozo , Tanaka Sakae
Published: July 2002

To examine the role of mitogen-activated protein kinase and nuclear factor kappa B (NF-κB) pathways on osteoclast survival and activation, we constructed adenovirus vectors carrying various mutants of signaling molecules: dominant negative Ras (RasDN), constitutively active MEK1 (MEKCA), dominant negative IκB kinase 2 (IKKDN), and constitutively active IKK2 (IKKCA). Inhibiting ERK activity by RasDN overexpression rapidly induced the apoptosis of osteoclast-like cells (OCLs) formed in vitro, whereas ERK activation after the introduction of MEKCA remarkably lengthened their survival by preventing spontaneous apoptosis. Neither inhibition nor activation of ERK affected the bone-resorbing activity of OCLs. Inhibition of NF-κB pathway with IKKDN virus suppressed the pit-forming activity of OCLs and NF-κB activation by IKKCA expression upregulated it without affecting their survival. Interleukin 1α (IL-1α) strongly induced ERK activation as well as NF-κB activation. RasDN virus partially inhibited ERK activation, and OCL survival promoted by IL-1α. Inhibiting NF-κB activation by IKKDN virus significantly suppressed the pit-forming activity enhanced by IL-1α. These results indicate that ERK and NF-κB regulate different aspects of osteoclast activation: ERK is responsible for osteoclast survival, whereas NF-κB regulates osteoclast activation for bone resorption.
Reduced Loading of Intracellular Ca2+ Stores and Downregulation of Capacitative Ca2+Influx in Bcl-2–Overexpressing Cells
Authors: Pinton Paolo , Ferrari Davide , Magalhães Paulo , Schulze-Osthoff Klaus , Di Virgilio Francesco , Pozzan Tullio , Rizzuto Rosario
Published: July 2002

The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca2+-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca2+ homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca2+ leak (while leaving Ca2+ accumulation unaffected), hence reducing the steady-state [Ca2+] levels. As a direct consequence, the [Ca2+] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca2+ influx activated by Ca2+ store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca2+-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca2+ homeostasis may contribute to the protective role of this oncogene against programed cell death.
Three Regions within Acta Promote Arp2/3 Complex-Mediated Actin Nucleation and Listeria monocytogenes Motility
Authors: Skoble Justin , Portnoy Daniel A. , Welch Matthew D.
Published: July 2002

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH2-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer–binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell–cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.
Atrophin-1, the Dentato-Rubral and Pallido-Luysian Atrophy Gene Product, Interacts with Eto/Mtg8 in the Nuclear Matrix and Represses Transcription
Authors: Wood Jonathan D. , Nucifora Frederick C. , Duan Kui , Zhang Chuanyi , Wang Jianxiang , Kim Yujin , Schilling Gabriele , Sacchi Nicoletta , Liu Johnson M. , Ross Christopher A.
Published: July 2002

Dentato-rubral and pallido-luysian atrophy (DRPLA) is one of the family of neurodegenerative diseases caused by expansion of a polyglutamine tract. The drpla gene product, atrophin-1, is widely expressed, has no known function or activity, and is found in both the nuclear and cytoplasmic compartments of neurons. Truncated fragments of atrophin-1 accumulate in neuronal nuclei in a transgenic mouse model of DRPLA, and may underlie the disease phenotype.

Using the yeast two-hybrid system, we identified ETO/MTG8, a component of nuclear receptor corepressor complexes, as an atrophin-1–interacting protein. When cotransfected into Neuro-2a cells, atrophin-1 and ETO/MTG8 colocalize in discrete nuclear structures that contain endogenous mSin3A and histone deacetylases. These structures are sodium dodecyl sulfate–soluble and associated with the nuclear matrix. Cotransfection of ETO/MTG8 with atrophin-1 recruits atrophin-1 to the nuclear matrix, while atrophin-1 and ETO/MTG8 cofractionate in nuclear matrix preparations from brains of DRPLA transgenic mice. Furthermore, in a cell transfection–based assay, atrophin-1 represses transcription. Together, these results suggest that atrophin-1 associates with nuclear receptor corepressor complexes and is involved in transcriptional regulation.

Emerging links between disease-associated polyglutamine proteins, nuclear receptors, translocation-leukemia proteins, and the nuclear matrix may have important repercussions for the pathobiology of this family of neurodegenerative disorders.
Two Intermembrane Space Tim Complexes Interact with Different Domains of Tim23p during Its Import into Mitochondria
Authors: Davis Alison J. , Sepuri Naresh B. , Holder Jason , Johnson Arthur E. , Jensen Robert E.
Published: July 2002

Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p–Tim13p and Tim9p–Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p–Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH2-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p–Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p–Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.