bluuebunny/crossref_metadata_2025 huggingface dataset:
Senescent Impairment in Synergistic Cytokine Pathways That Provide Rapid Cardioprotection in the Rat Heart
Authors: Xaymardan Munira , Zheng Jingang , Duignan Inga , Chin Andrew , Holm Jacquelyne M. , Ballard Victoria L.T. , Edelberg Jay M.
Published: March 2004

Pretreatment of rodent hearts with platelet-derived growth factor (PDGF)–AB decreases myocardial injury after coronary occlusion. However, PDGF-AB cardioprotection is diminished in older animals, suggesting that downstream elements mediating and/or synergizing the actions of PDGF-AB may be limited in aging cardiac vasculature. In vitro PDGF-AB induced vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 expression in 4-mo-old rat cardiac endothelial cells, but not in 24-mo-old heart cells. In vivo injection of young hearts with PDGF-AB increased densities of microvessels staining for VEGF and its receptor, Flk-1, and Ang-2 and its receptor, Tie-2, as well as PDGF receptor (PDGFR)–α. In older hearts, PDGF-AB–mediated induction was primarily limited to PDGFR-α. Studies in a murine cardiac transplantation model demonstrated that synergist interactions of PDGF-AB plus VEGF plus Ang-2 (PVA) provided an immediate restoration of senescent cardiac vascular function. Moreover, PVA injection in young rat hearts, but not PDGF-AB alone or other cytokine combinations, at the time of coronary occlusion suppressed acute myocardial cell death by >50%. However, PVA also reduced the extent of myocardial infarction with an age-associated cardioprotective benefit (4-mo-old with 45% reduction vs. 24-mo-old with 24%; P < 0.05). These studies showed that synergistic cytokine pathways augmenting the actions of PDGF-AB are limited in older hearts, suggesting that strategies based on these interactions may provide age-dependent clinical cardiovascular benefit.
Innate Immune Surveillance of Spontaneous B Cell Lymphomas by Natural Killer Cells and γδ T Cells
Authors: Street Shayna E.A. , Hayakawa Yoshihiro , Zhan Yifan , Lew Andrew M. , MacGregor Duncan , Jamieson Amanda M. , Diefenbach Andreas , Yagita Hideo , Godfrey Dale I. , Smyth Mark J.
Published: March 2004

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking β-2 microglobulin (β2m; and thus MHC class I, CD1d, and CD16) and/or perforin, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perforin gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked β2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/β2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perforin, but not IFN-γ, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1+ and γδTCR+ T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and γδTCR+ T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.
Pax5 Activates Immunoglobulin Heavy Chain V to DJ Rearrangement in Transgenic Thymocytes
Authors: Hsu Lih-Yun , Liang Hong-Erh , Johnson Kristen , Kang Chulho , Schlissel Mark S.
Published: March 2004

Mice deficient for the B cell–restricted transcription factor Pax5 show a defect in the VH to DJH rearrangement step of immunoglobulin heavy chain gene assembly even though the expression of the V(D)J recombinase is not diminished in Pax5−/− pro–B cells. To investigate whether Pax5 is limiting for VH to DJH rearrangement, we generated transgenic mice which express Pax5 in developing thymocytes. We show that enforced expression of Pax5 in thymocytes results in a partial block in T cell development due to defective pre-TCR signaling in β-selection. Moreover, our results demonstrate that expression of Pax5 in early thymocytes is sufficient to induce VH to DJH rearrangements in CD4+CD8+ T cells and lead us to suggest that Pax5 may play a direct role in the lineage-specific regulation of immunoglobulin heavy chain gene rearrangement.
Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly inSaccharomyces cerevisiae
Authors: Reggiori Fulvio , Wang Chao-Wen , Nair Usha , Shintani Takahiro , Abeliovich Hagai , Klionsky Daniel J.
Published: March 2004

The Cvt pathway is a biosynthetic transport route for a distinct subset of resident yeast vacuolar hydrolases, whereas macroautophagy is a nonspecific degradative mechanism that allows cell survival during starvation. Yet, these two vacuolar trafficking pathways share a number of identical molecular components and are morphologically very similar. For example, one of the hallmarks of both pathways is the formation of double-membrane cytosolic vesicles that sequester cargo before vacuolar delivery. The origin of the vesicle membrane has been controversial and various lines of evidence have implicated essentially all compartments of the endomembrane system. Despite the analogies between the Cvt pathway and autophagy, earlier work has suggested that the origin of the engulfing vesicle membranes is different; the endoplasmic reticulum is proposed to be required only for autophagy. In contrast, in this study we demonstrate that the endoplasmic reticulum and/or Golgi complex, but not endosomal compartments, play an important role for both yeast transport routes. Along these lines, we demonstrate that Berkeley bodies, a structure generated from the Golgi complex in sec7 cells, are immunolabeled with Atg8, a structural component of autophagosomes. Finally, we also show that none of the yeast t-SNAREs are located at the preautophagosomal structure, the presumed site of double-membrane vesicle formation. Based on our results, we propose two models for Cvt vesicle biogenesis.
Tyrosine Phosphorylation of Sprouty Proteins Regulates Their Ability to Inhibit Growth Factor Signaling: A Dual Feedback Loop
Authors: Mason Jacqueline M. , Morrison Debra J. , Bassit Bhramdeo , Dimri Manjari , Band Hamid , Licht Jonathan D. , Gross Isabelle
Published: March 2004

Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Here, we report that Sprouty proteins become tyrosine phosphorylated after growth factor treatment. We identified Tyr55 as a key residue for Sprouty2 phosphorylation and showed that phosphorylation was required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augmented signaling. We found that tyrosine phosphorylation of Sprouty2 affected neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation was necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl was required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 was dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, we showed that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling.
Genetic Dissection of Meiotic Cytokinesis inDrosophilaMales
Authors: Giansanti Maria Grazia , Farkas Rebecca M. , Bonaccorsi Silvia , Lindsley Dan L. , Wakimoto Barbara T. , Fuller Margaret T. , Gatti Maurizio
Published: March 2004

We have used Drosophila male meiosis as a model system for genetic dissection of the cytokinesis mechanism. Drosophila mutants defective in meiotic cytokinesis can be easily identified by their multinucleate spermatids. Moreover, the large size of meiotic spindles allows characterization of mutant phenotypes with exquisite cytological resolution. We have screened a collection of 1955 homozygous mutant male sterile lines for those with multinucleate spermatids, and thereby identified mutations in 19 genes required for cytokinesis. These include 16 novel loci and three genes, diaphanous, four wheel drive, and pebble, already known to be involved in Drosophila cytokinesis. To define the primary defects leading to failure of cytokinesis, we analyzed meiotic divisions in male mutants for each of these 19 genes. Examination of preparations stained for tubulin, anillin, KLP3A, and F-actin revealed discrete defects in the components of the cytokinetic apparatus, suggesting that these genes act at four major points in a stepwise pathway for cytokinesis. Our results also indicated that the central spindle and the contractile ring are interdependent structures that interact throughout cytokinesis. Moreover, our genetic and cytological analyses provide further evidence for a cell type-specific control of Drosophila cytokinesis, suggesting that several genes required for meiotic cytokinesis in males are not required for mitotic cytokinesis.
The Role and Regulation of the preRC Component Cdc6 in the Initiation of Premeiotic DNA Replication
Authors: Ofir Yaara , Sagee Shira , Guttmann-Raviv Noga , Pnueli Lilach , Kassir Yona
Published: March 2004

In all eukaryotes, the initiation of DNA replication is regulated by the ordered assembly of DNA/protein complexes on origins of DNA replication. In this report, we examine the role of Cdc6, a component of the prereplication complex, in the initiation of premeiotic DNA replication in budding yeast. We show that in the meiotic cycle, Cdc6 is required for DNA synthesis and sporulation. Moreover, similarly to the regulation in the mitotic cell cycle, Cdc6 is specifically degraded upon entry into the meiotic S phase. By contrast, chromatin-immunoprecipitation analysis reveals that the origin-bound Cdc6 is stable throughout the meiotic cycle. Preliminary evidence suggests that this protection reflects a change in chromatin structure that occurs in meiosis. Using the cdc28-degron allele, we show that depletion of Cdc28 leads to stabilization of Cdc6 in the mitotic cycle, but not in the meiotic cycle. We show physical association between Cdc6 and the meiosis-specific hCDK2 homolog Ime2. These results suggest that under meiotic conditions, Ime2, rather than Cdc28, regulates the stability of Cdc6. Chromatin-immunoprecipitation analysis reveals that similarly to the mitotic cell cycle, Mcm2 binds origins in G1 and meiotic S phases, and at the end of the second meiotic division, it is gradually removed from chromatin.
The Chemokine Receptor D6 Constitutively Traffics to and from the Cell Surface to Internalize and Degrade Chemokines
Authors: Weber Michele , Blair Emma , Simpson Clare V. , O'Hara Maureen , Blackburn Paul E. , Rot Antal , Graham Gerard J. , Nibbs Robert J.B.
Published: March 2004

The D6 heptahelical membrane protein, expressed by lymphatic endothelial cells, is able to bind with high affinity to multiple proinflammatory CC chemokines. However, this binding does not allow D6 to couple to the signaling pathways activated by typical chemokine receptors such as CC-chemokine receptor-5 (CCR5). Here, we show that D6, like CCR5, can rapidly internalize chemokines. However, D6-internalized chemokines are more effectively retained intracellularly because they more readily dissociate from the receptor during vesicle acidification. These chemokines are then degraded while the receptor recycles to the cell surface. Interestingly, D6-mediated chemokine internalization occurs without bringing about a reduction in cell surface D6 levels. This is possible because unlike CCR5, D6 is predominantly localized in recycling endosomes capable of trafficking to and from the cell surface in the absence of ligand. When chemokine is present, it can enter the cells associated with D6 already destined for internalization. By this mechanism, D6 can target chemokines for degradation without the necessity for cell signaling, and without desensitizing the cell to subsequent chemokine exposure.
Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis
Authors: Fan Guo-Huang , Lapierre Lynne A. , Goldenring James R. , Sai Jiqing , Richmond Ann
Published: March 2004

Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for ∼2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129–512), the N-terminal–truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129–512). Moreover, expression of the myosin Vb tail reduced CXCR2- and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine.
Identification of Novel Principles of Keratin Filament Network Turnover in Living Cells
Authors: Windoffer Reinhard , Wöll Stefan , Strnad Pavel , Leube Rudolf E.
Published: March 2004

It is generally assumed that turnover of the keratin filament system occurs by exchange of subunits along its entire length throughout the cytoplasm. We now present evidence that a circumscribed submembranous compartment is actually the main site for network replenishment. This conclusion is based on the following observations in living cells synthesizing fluorescent keratin polypeptides: 1) Small keratin granules originate in close proximity to the plasma membrane and move toward the cell center in a continuous motion while elongating into flexible rod-like fragments that fuse with each other and integrate into the peripheral KF network. 2) Recurrence of fluorescence after photobleaching is first seen in the cell periphery where keratin filaments are born that translocate subsequently as part of the network toward the cell center. 3) Partial keratin network reformation after orthovanadate-induced disruption is restricted to a distinct peripheral zone in which either keratin granules or keratin filaments are transiently formed. These findings extend earlier investigations of mitotic cells in which de novo keratin network formation was shown to originate from the cell cortex. Taken together, our results demonstrate that the keratin filament system is not homogenous but is organized into temporally and spatially distinct subdomains. Furthermore, the cortical localization of the regulatory cues for keratin filament turnover provides an ideal way to adjust the epithelial cytoskeleton to dynamic cellular processes.
Identification and Characterization of Two Novel Proteins Affecting Fission Yeast γ-tubulin Complex Function
Authors: Venkatram Srinivas , Tasto Joseph J. , Feoktistova Anna , Jennings Jennifer L. , Link Andrew J. , Gould Kathleen L.
Published: March 2004

The γ-tubulin complex, via its ability to organize microtubules, is critical for accurate chromosome segregation and cytokinesis in the fission yeast, Schizosaccharomyces pombe. To better understand its roles, we have purified the S. pombe γ-tubulin complex. Mass spectrometric analyses of the purified complex revealed known components and identified two novel proteins (i.e., Mbo1p and Gfh1p) with homology to γ-tubulin–associated proteins from other organisms. We show that both Mbo1p and Gfh1p localize to microtubule organizing centers. Although cells deleted for either mbo1+or gfh1+are viable, they exhibit a number of defects associated with altered microtubule function such as defects in cell polarity, nuclear positioning, spindle orientation, and cleavage site specification. In addition, mbo1Δ and gfh1Δ cells exhibit defects in astral microtubule formation and anchoring, suggesting that these proteins have specific roles in astral microtubule function. This study expands the known roles of γ-tubulin complex components in organizing different types of microtubule structures in S. pombe.
Listeria monocytogenesActin-based Motility Varies Depending on Subcellular Location: A Kinematic Probe for Cytoarchitecture
Authors: Lacayo Catherine I. , Theriot Julie A.
Published: March 2004

Intracellular Listeria monocytogenes actin-based motility is characterized by significant individual variability, which can be influenced by cytoarchitecture. L. monocytogenes was used as a probe to transmit information about structural variation among subcellular domains defined by mitochondrial density. By analyzing the movement of a large population of L. monocytogenes in PtK2 cells, we found that mean speed and trajectory curvature were significantly larger for bacteria moving in mitochondria-containing domains (generally perinuclear) than for bacteria moving in mitochondria-free domains (generally peripheral). Analysis of bacteria that traversed both mitochondria-containing and mitochondria-free domains revealed that these motile differences were not intrinsic to bacteria themselves. Disruption of mitochondrial respiration did not affect bacterial mean speed, speed persistence, or trajectory curvature. In contrast, microtubule depolymerization lead to decreased mean speed per bacterium and increased mean speed persistence of L. monocytogenes moving in mitochondria-free domains compared with untreated cells. L. monocytogenes were also observed to physically collide with mitochondria and push them away from the bacterial path of motion, causing bacteria to slow down before rapidly resuming their speed. Our results show that subcellular domains along with microtubule depolymerization may influence the actin cytoskeleton to affect L. monocytogenes speed, speed persistence, and trajectory curvature.
Rab14 Is Involved in Membrane Trafficking between the Golgi Complex and Endosomes
Authors: Junutula Jagath R. , De Maziére Ann M. , Peden Andrew A. , Ervin Karen E. , Advani Raj J. , van Dijk Suzanne M. , Klumperman Judith , Scheller Richard H.
Published: March 2004

Rab GTPases are localized to various intracellular compartments and are known to play important regulatory roles in membrane trafficking. Here, we report the subcellular distribution and function of Rab14. By immunofluorescence and immunoelectron microscopy, both endogenous as well as overexpressed Rab14 were localized to biosynthetic (rough endoplasmic reticulum, Golgi, and trans-Golgi network) and endosomal compartments (early endosomal vacuoles and associated vesicles). Notably overexpression of Rab14Q70L shifted the distribution toward the early endosome associated vesicles, whereas the S25N and N124I mutants induced a shift toward the Golgi region. A similar, although less pronounced, redistribution of the transferrin receptor was also observed in cells overexpressing Rab14 mutants. Impairment of Rab14 function did not however affect transferrin uptake or recycling kinetics. Together, these findings suggest that Rab14 is involved in the biosynthetic/recycling pathway between the Golgi and endosomal compartments.
Diverse and Specific Gene Expression Responses to Stresses in Cultured Human Cells
Authors: Murray John Isaac , Whitfield Michael L. , Trinklein Nathan D. , Myers Richard M. , Brown Patrick O. , Botstein David
Published: March 2004

We used cDNA microarrays in a systematic study of the gene expression responses of HeLa cells and primary human lung fibroblasts to heat shock, endoplasmic reticulum stress, oxidative stress, and crowding. Hierarchical clustering of the data revealed groups of genes with coherent biological themes, including genes that responded to specific stresses and others that responded to multiple types of stress. Fewer genes increased in expression after multiple stresses than in free-living yeasts, which have a large general stress response program. Most of the genes induced by multiple diverse stresses are involved in cell-cell communication and other processes specific to higher organisms. We found substantial differences between the stress responses of HeLa cells and primary fibroblasts. For example, many genes were induced by oxidative stress and dithiothreitol in fibroblasts but not HeLa cells; conversely, a group of transcription factors, including c-fos and c-jun, were induced by heat shock in HeLa cells but not in fibroblasts. The dataset is freely available for search and download at http://microarray-pubs.stanford.edu/human_stress/Home.shtml .
Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor
Authors: Freedman Neal D. , Yamamoto Keith R.
Published: March 2004

The vertebrate glucocorticoid receptor (GR) is cytoplasmic without hormone and localizes to the nucleus after hormone binding. GR has two nuclear localization signals (NLS): NL1 is similar in sequence to the SV40 NLS; NL2 is poorly defined, residing in the ligand-binding domain. We found that GR displayed similar hormone-regulated compartmentalization in Saccharomyces cerevisiae and required the Sxm1 nuclear import receptor for NL2-mediated import. Two metazoan homologues of Sxm1, importin 7 and importin 8, bound both NL1 and NL2, whereas importin α selectively bound NL1. In an in vitro nuclear import assay, both importin 7 and the importin α-importin β heterodimer could import a GR NL1 fragment. Under these conditions, full-length GR localized to nuclei in the presence but not absence of an unidentified component in cell extracts. Interestingly, importin 7, importin 8, and importin α bound GR even in the absence of hormone; thus, hormonal control of localization is exerted at a step downstream of import receptor binding.
Dynamic Regulation of a GPCR-Tetraspanin-G Protein Complex on Intact Cells: Central Role of CD81 in Facilitating GPR56-Gαq/11Association
Authors: Little Kevin D. , Hemler Martin E. , Stipp Christopher S.
Published: March 2004

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein–coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Gαq, Gα11, and Gβ, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Gαq/11complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Gαq/11complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.
Desmin Aggregate Formation by R120G αB-Crystallin Is Caused by Altered Filament Interactions and Is Dependent upon Network Status in Cells
Authors: Der Perng Ming , Wen Shu Fang , van den IJssel Paul , Prescott Alan R. , Quinlan Roy A.
Published: March 2004

The R120G mutation in αB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of αB-crystallin for desmin filaments. The apparent dissociation constant of R120G αB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G αB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type αB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G αB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in αB-crystallin.
Deoxycholic Acid Activates β-Catenin Signaling Pathway and Increases Colon Cell Cancer Growth and Invasiveness
Authors: Pai Rama , Tarnawski Andrzej S. , Tran Teresa
Published: March 2004

Colorectal cancer is often lethal when invasion and/or metastasis occur. Tumor progression to the metastatic phenotype is mainly dependent on tumor cell invasiveness. Secondary bile acids, particularly deoxycholic acid (DCA), are implicated in promoting colon cancer growth and progression. Whether DCA modulates β-catenin and promotes colon cancer cell growth and invasiveness remains unknown. Because β-catenin and its target genes urokinase-type plasminogen activator receptor (uPAR) and cyclin D1 are overexpressed in colon cancers, and are linked to cancer growth, invasion, and metastasis, we investigated whether DCA activates β-catenin signaling and promotes colon cancer cell growth and invasiveness. Our results show that low concentrations of DCA (5 and 50 μM) significantly increase tyrosine phosphorylation of β-catenin, induce urokinase-type plasminogen activator, uPAR, and cyclin D1 expression and enhance colon cancer cell proliferation and invasiveness. These events are associated with a substantial loss of E-cadherin binding to β-catenin. Inhibition of β-catenin with small interfering RNA significantly reduced DCA-induced uPAR and cyclin D1 expression. Blocking uPAR with a neutralizing antibody significantly suppressed DCA-induced colon cancer cell proliferation and invasiveness. These findings provide evidence for a novel mechanism underlying the oncogenic effects of secondary bile acids.
Biophysical Parameters Influence Actin-based Movement, Trajectory, and Initiation in a Cell-free System
Authors: Cameron Lisa A. , Robbins Jennifer R. , Footer Matthew J. , Theriot Julie A.
Published: March 2004

Using a biochemically complex cytoplasmic extract to reconstitute actin-based motility of Listeria monocytogenes and polystyrene beads coated with the bacterial protein ActA, we have systematically varied a series of biophysical parameters and examined their effects on initiation of motility, particle speed, speed variability, and path trajectory. Bead size had a profound effect on all aspects of motility, with increasing size causing slower, straighter movement and inhibiting symmetry-breaking. Speed also was reduced by extract dilution, by addition of methylcellulose, and paradoxically by addition of excess skeletal muscle actin, but it was enhanced by addition of nonmuscle (platelet) actin. Large, persistent individual variations in speed were observed for all conditions and their relative magnitude increased with extract dilution, indicating that persistent alterations in particle surface properties may be responsible for intrinsic speed variations. Trajectory curvature was increased for smaller beads and also for particles moving in the presence of methylcellulose or excess skeletal muscle actin. Symmetry breaking and movement initiation occurred by two distinct modes: either stochastic amplification of local variation for small beads in concentrated extracts, or gradual accumulation of strain in the actin gel for large beads in dilute extracts. Neither mode was sufficient to enable spherical particles to break symmetry in the cytoplasm of living cells.
Uncoated Endocytic Vesicles Require the Unconventional Myosin, Myo6, for Rapid Transport through Actin Barriers
Authors: Aschenbrenner Laura , Naccache Samia N. , Hasson Tama
Published: March 2004

After clathrin-mediated endocytosis, clathrin removal yields an uncoated vesicle population primed for fusion with the early endosome. Here we present the first characterization of uncoated vesicles and show that myo6, an unconventional myosin, functions to move these vesicles out of actin-rich regions found in epithelial cells. Time-lapse microscopy revealed that myo6-associated uncoated vesicles were motile and exhibited fusion and stretching events before endosome delivery, processes that were dependent on myo6 motor activity. In the absence of myo6 motor activity, uncoated vesicles remained trapped in the actin mesh, where they exhibited Brownian-like motion. Exit from the actin mesh occurred by a slow diffusion-based mechanism, delaying transferrin trafficking to the early endosome. Expression of a myo6 mutant that bound tightly to F-actin produced immobilized vesicles and blocked trafficking. Depolymerization of the actin cytoskeleton rescued this block and specifically accelerated transferrin delivery to the early endosome without affecting earlier steps in endocytosis. Therefore actin is a physical barrier impeding uncoated vesicle trafficking, and myo6 is recruited to move the vesicles through this barrier for fusion with the early endosome.
An Assembly-incompetent Mutant Establishes a Requirement for Dynamin Self-assembly in Clathrin-mediated Endocytosis In Vivo
Authors: Song Byeong Doo , Yarar Defne , Schmid Sandra L.
Published: March 2004

Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, I690, K694, and I697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-I690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.
Cyclase-associated Protein 1 (CAP1) Promotes Cofilin-induced Actin Dynamics in Mammalian Nonmuscle Cells
Authors: Bertling Enni , Hotulainen Pirta , Mattila Pieta K. , Matilainen Tanja , Salminen Marjo , Lappalainen Pekka
Published: March 2004

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, the mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. Here, we show that CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects to those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals.
CisML: an XML-based format for sequence motif detection software
Authors: Haverty Peter M. , Weng Zhiping
Published: March 2004

Summary: CisML is an XML-based format for sequence motif detection software. This proposed standard is applicable to many types of sequence motif detection programs. It is intended to facilitate the integration of data and the comparison of results from different software packages, and to simplify the development of downstream tools. XSL stylesheets are provided for easy generation of text, html and graphical reports from CisML-formatted data.

Availability:  http://zlab.bu.edu/CisML/

Supplementary information: Example CisML-formatted data and XSL stylesheets for report generation are available along with the sample output.
Efficient sampling algorithm for estimating subgraph concentrations and detecting network motifs
Authors: Kashtan N. , Itzkovitz S. , Milo R. , Alon U.
Published: March 2004

Summary: Biological and engineered networks have recently been shown to display network motifs: a small set of characteristic patterns that occur much more frequently than in randomized networks with the same degree sequence. Network motifs were demonstrated to play key information processing roles in biological regulation networks. Existing algorithms for detecting network motifs act by exhaustively enumerating all subgraphs with a given number of nodes in the network. The runtime of such algorithms increases strongly with network size. Here, we present a novel algorithm that allows estimation of subgraph concentrations and detection of network motifs at a runtime that is asymptotically independent of the network size. This algorithm is based on random sampling of subgraphs. Network motifs are detected with a surprisingly small number of samples in a wide variety of networks. Our method can be applied to estimate the concentrations of larger subgraphs in larger networks than was previously possible with exhaustive enumeration algorithms. We present results for high-order motifs in several biological networks and discuss their possible functions.

Availability: A software tool for estimating subgraph concentrations and detecting network motifs (mfinder 1.1) and further information is available at http://www.weizmann.ac.il/mcb/UriAlon/
A Web-based design center for vector-based siRNA and siRNA cassette
Authors: Wang Luquan , Mu Forest Y.
Published: March 2004

Summary: To facilitate the designing process for vector-based siRNA and siRNA cassette, a tool set has been developed consisting of a siRNA target finder, a siRNA construct builder and a siRNA sequence scrambler. The siRNA target finder is used to identify candidate siRNA target sites. The program automates homology filtering, minimizes non-specific cross-reaction, filters target sites based on RNA duplex internal stability and siRNA sense/anti-sense strand secondary structure. The siRNA construct builder is used to create a siRNA hairpin construct as a vector insert or a cassette insert. The siRNA sequence scrambler is used to generate a negative control sequence for siRNA experiments. Together, these programs provide a comprehensive utility set to address the specific bioinformatics need of DNA-based siRNA design.

Availability: The Web-based design tools are available at GenScript Website (siRNA design center). http://www.genscript.com or http://www.genscript.com/rnai.html#design or http://www.genscript.com/sirna_ca.html#design
Building a skills portfolio for the information professional
Authors: Brine Alan , Feather John
Published: March 2004

This paper reports on research in progress that reviews the skills used by library and information science (LIS) professionals and how to record them in a manner that encourages continuing professional and personal development. A portfolio was developed by the learning and teaching support network for information and computer sciences (LTSN‐ICS) in conjunction with the Chartered Institute for Library and Information Professionals (CILIP) and academic departments in the United Kingdom (UK). The portfolio was evaluated at pilot sites in UK LIS schools by surveying the progress of those students who took part. Evaluation of the participants indicates that the portfolio is a useful tool in the personal and professional development of LIS students. The general consensus among students themselves is that the portfolio provides a tool to help them plan their future professional development. Further evaluation is planned in 2003/2004 with LIS professionals to enable both the professional body and the LTSN‐ICS to determine the appropriateness of the portfolio as a tool for continuing personal and professional development.
Stone buildings, cyberspace, and the library user
Authors: Agee Jim , Antrim Patricia
Published: March 2004

Although libraries provide quiet and well‐equipped places for students to seek and use information, the trend librarians are seeing is a decline in the number of people coming to the library. In contrast, they are seeing a dramatic increase in the use of remotely accessed research databases. From these two trends emerges the concept of disintermediation, where library users seek and retrieve information without the assistance of the librarian, and the realization that librarians are no longer present when users need help in developing successful search strategies and evaluating the information they find. Librarians are not present at that teachable moment. To overcome the effects of disintermediation, librarians need to evaluate their services and recreate their instructional strategies in innovative ways so that they are available to the users of information wherever those users are.
Funding down under: entrepreneurial approaches to generating income at the University of Queensland Cybrary
Authors: Schmidt Janine , Peachey Lucy
Published: March 2004

The government, from both national and regional sources, is the primary source of funds for universities and their libraries in Australia. Student numbers and financial allocations are set by the federal government. Government policy changes in relation to higher education funding have impacted considerably in the last few years and the percentage of total income emanating from the federal government has steadily deteriorated. Student fees, on both a partial and full basis, have been introduced as part of the funding mix. Most university libraries in Australia are funded by their parent organizations, usually through a fixed percentage of overall university income. Cost pressures have been experienced by university libraries through reductions in government income, and excessive increases in the costs of library materials, but also through a steadily falling value of the Australian currency against other major world currencies. This paper reviews the environment of Australian university libraries and focuses on the University of Queensland Cybrary as a case study, reviewing initiatives that have been adopted to increase income.
The 2002 female FTSE index and women directors
Authors: Singh Val , Vinnicombe Susan
Published: March 2004

The stagnation in the position of female directorships in the UK’s FTSE 100 companies appears to be very slowly changing. After a review of previous research on women directors, this paper reports the statistics on women directors in the top 100 listed companies. The paper comments on the findings regarding companies with women directors, female directorships and the women holding those directorships. It reviews the backgrounds (demographic profiles including age, education, marital status and children; corporate experience, international experience, etc.) of the top women executive directors. The paper also examines the minority of top companies with women executive directors, to see how their particular characteristics and contingencies (e.g. sector, chairmen, CEO and board demographics) may have influenced the environment as incubators for these successful women. The paper considers the findings through several theoretical lenses for explanations of the results, and conclude by commenting on the progress being made in other European countries.
A longitudinal study of women directors in Australia
Authors: Burgess Zena , Fallon Barry
Published: March 2004

Using social identity theory as a framework, the present study empirically tests the idea that women can maintain positions on corporate boards over a number of years through becoming part of the board’s ingroup. A sample of 32 women directors who were part of a study of corporate directors in 1995 participated in the six‐year follow‐up. A series of hypotheses are tested using nonparametric statistical techniques to test differences in women directors’ personal and board characteristics over the two time periods. Implications of the results are drawn for women seeking to maintain or gain board positions and for the applicability of social identity theory to the research area.
Girl power: double jeopardy or diversity in action behind boardroom doors in New Zealand?
Authors: McGregor Judy
Published: March 2004

A dramatic acceleration in “girl power” predicted for the boardrooms of major New Zealand business is examined against the notion that age is a traditional demographic variable influencing board selection. For example, the average age of directors in Canada and the USA is 59 years, with 56 per cent of Canadian directors over 60 years. The paper examines whether there is a generational divide between younger women with higher educational qualifications who have “fast‐tracked” onto boards and older female directors with substantial business experience and expertise and the seniority to dedicate the time to board membership. Six interviews were undertaken with women under 40 years who are directors, and with older women, aged 45 years and over, on the same three boards. Similarities and differences in selection and perceptions of the role are analysed. Whether being young and female is a form of double jeopardy or an expression of boardroom diversity in action is explored.
The experience of women corporate inside directors on the boards of Fortune 1,000 firms
Authors: Dahlen Zelechowski Deborah , Bilimoria Diana
Published: March 2004

Inside directors are executives who hold the dual roles of officers of the firm and corporate board members. Six women inside directors from Fortune 1,000 corporations were interviewed for this exploratory study. Through systematic coding of the interviews, two independent dimensions of influence and inclusion emerged as the critical factors that enhance or restrict the performance and contributions of women at the top of corporations. Three sub‐themes characterized women inside directors’ influence: their role in the board’s decision making, the bases of influence, and influence strategies used. Three sub‐themes characterized the inclusion dynamics experienced by women inside directors: support and acceptance, exclusion, and the nature of the advice they received. Conclusions are drawn regarding the convergence of the influence and inclusion dynamics for women to function most effectively at the top of corporations.
African‐American policewomen: An exploration of professional relationships
Authors: Dodge Mary , Pogrebin Mark
Published: March 2004

Explores the professional, job‐related relationships of African‐American policewomen. The difficulties black women police officers face often are exacerbated by the intersection of race and gender that influence their self‐perceptions and the perceptions of those with whom they interact, including other officers and members of the community. Additionally, job‐related issues and interactions appear to impact community relations, particularly in circumstances involving minorities. The experiences of the women suggest that marginalization continues to be problematic for some minorities working in law enforcement. The research is based on qualitative data obtained from black female police officers in a large urban city.
The Missouri State Highway Patrol as a representative model
Authors: Falcone David N.
Published: March 2004

Using a socio‐historical analytical approach, explains the emergence and evolutionary iterations of the Missouri State Highway Patrol (MSHP) as it developed into a virtual state police organization. The legal history of the MSHP is chronicled, and the cultural opposition to a state police department in Missouri is explained. Despite the show‐me states’ long‐standing opposition to state‐level policing authority, the evolution of the MSHP into a state‐police‐like organization is developed as a model that is representative of numerous other state‐level police organizations across the nation. The evolutionary patterns in the development of the MSHP are foreshadowed by other state‐level police organizations, like the Illinois State Police.
Reading about “community (oriented) policing” and police models
Authors: Ponsaers Paul
Published: March 2004

Discusses the actual conceptions about policing used by social scientists. Police models are central entities of thoughts and ideas on policing, which include an observable internal coherence. Stresses that there are in fact only four central police models: the military‐bureaucratic model; the lawful policing model; community‐oriented policing (COP); and public‐private divide policing. Precisely in articulating COP against its negative references, the essence becomes clearer. Concludes that each concrete police apparatus can be considered as a combination of police models. The democratization process can be endangered by the growing dominance of a public‐private (divide) police model. That is the main reason why it is important to encourage the search for a more profound theoretical basis for policing the community.