bluuebunny/crossref_metadata_2025 huggingface dataset:
Do Substance Use and Delinquency have Differential Associations with Adolescents’ Peer Relations?
Authors: Maggs Jennifer L. , Hurrelmann Klaus
Published: July 2002

Despite their potentially serious short- and long-term risks, developmental theorists have argued that risk behaviours may play important constructive roles in adolescent development. The present study hypothesised that adolescent substance use, but not delinquency, would be associated with positive peer relations. Subjects were 491 German adolescents participating in the Bielefeld Longitudinal Youth Survey who completed questionnaires during grades 7, 8, 9, and 10. Measures were adolescents’ self-reported substance use (alcohol, tobacco), delinquency (antisocial acts), and peer relations (peer involvement frequency, perceived peer group closeness, perceived position in the peer group, having a boy- or girlfriend). Repeated measures analyses of variance examined gender and school type differences in risk behaviour and peer relations across four occasions. Within-time, peer relations were positively related to substance use and, in a few cases, positively related to delinquency. As hypothesised, substance use predicted increases in the frequency and intensity of peer relations, but delinquency did not do so consistently. The pattern of results did not differ by gender or school type. The discussion focuses on the meaning of risky behaviours and differences between substance use and delinquency, and argues that prevention efforts must take seriously the positive functions served by risk behaviours.
Social Problem Solving: Effect of Context and Parent Sex
Authors: Fagot Beverly I.
Published: July 2002

A group of 368 children, ages 3, 5, and 7, half boys and half girls, were seen with their mothers and fathers in two contexts. First, they were observed with all family members in their homes, with instructions to continue with normal activities. Then they were seen separately in the laboratory with each parent, who was asked to help them solve a problem involving a common social situation. In the laboratory situation, boys and girls were given equal amounts of help by their parents. Mothers and fathers behaved in similar fashion in the laboratory, and the only significant differences were with the children’s ages. In the home, mothers and fathers did differ, with the mother giving more instruction and providing more instruction to girls.
Response to Bartsch
Authors: Wimmer Heinz , Mayringer Heinz
Published: July 2002

Bartsch in her comment to our paper first presents a theoretical reason why false belief understanding should become evident earlier in children’s explanations than in their predictions and then disputes our evidence that this is not the case. We will go through the arguments in turn.
Memory Discrimination for Self-Performed and Imagined Acts: Bizarreness Effects in False Recognition
Authors: Worthen James B. , Wood Virginia V.
Published: July 2002

Two experiments investigated the susceptibility of common and unusual action events to memory distortion under incidental learning and delayed testing conditions. Experiment 1 tested the influence of imaginal and enactment instructions at testing on memory discrimination for self-performed and imagined acts. The influence of hypnotic procedures at testing on memory discrimination for action events was tested in Experiment 2. The results of both experiments suggest that the likelihood of confusing details associated with separate, previously experienced unusual action events is greater than the likelihood of confusing details associated with separate, previously experienced common action events. Based on the results of both experiments, it is concluded that bizarreness has both memory-facilitating and memory-inhibiting qualities.
Plausibility and subcategorization preference in children's processing of temporarily ambiguous sentences: Evidence from self-paced reading
Authors: Traxler Matthew J.
Published: July 2002

Three self-paced reading experiments investigated children's processing of temporarily ambiguous sentences. Across the three experiments, subcategorization preference of a verb in a subordinate clause and the semantic plausibility of the misanalysis were manipulated. Reading times in the temporarily ambiguous region and following syntactic disambiguation indicated that children in the age range tested (8 years, 11 months to 12 years, 11 months) routinely misanalyse sentences of the type tested, and their tendency to misanalyse the sentences does not depend on the subcategorization preferences of the initial verb. Additional correlational analyses suggested that subcategory information did affect the degree of difficulty that readers experienced processing the critical noun and matrix verb.
A Multispecificity Syntaxin Homologue, Vam3p, Essential for Autophagic and Biosynthetic Protein Transport to the Vacuole
Authors: Darsow Tamara , Rieder Stephanie E. , Emr Scott D.
Published: July 2002

Protein transport in eukaryotic cells requires the selective docking and fusion of transport intermediates with the appropriate target membrane. t-SNARE molecules that are associated with distinct intracellular compartments may serve as receptors for transport vesicle docking and membrane fusion through interactions with specific v-SNARE molecules on vesicle membranes, providing the inherent specificity of these reactions. VAM3 encodes a 283–amino acid protein that shares homology with the syntaxin family of t-SNARE molecules. Polyclonal antiserum raised against Vam3p recognized a 35-kD protein that was associated with vacuolar membranes by subcellular fractionation. Null mutants of vam3 exhibited defects in the maturation of multiple vacuolar proteins and contained numerous aberrant membrane-enclosed compartments. To study the primary function of Vam3p, a temperature-sensitive allele of vam3 was generated (vam3tsf). Upon shifting the vam3tsf mutant cells to nonpermissive temperature, an immediate block in protein transport through two distinct biosynthetic routes to the vacuole was observed: transport via both the carboxypeptidase Y pathway and the alkaline phosphatase pathway was inhibited. In addition, vam3tsf cells also exhibited defects in autophagy. Both the delivery of aminopeptidase I and the docking/ fusion of autophagosomes with the vacuole were defective at high temperature. Upon temperature shift, vam3tsf cells accumulated novel membrane compartments, including multivesicular bodies, which may represent blocked transport intermediates. Genetic interactions between VAM3 and a SEC1 family member, VPS33, suggest the two proteins may act together to direct the docking and/or fusion of multiple transport intermediates with the vacuole. Thus, Vam3p appears to function as a multispecificity receptor in heterotypic membrane docking and fusion reactions with the vacuole. Surprisingly, we also found that overexpression of the endosomal t-SNARE, Pep12p, suppressed vam3Δ mutant phenotypes and, likewise, overexpression of Vam3p suppressed the pep12Δ mutant phenotypes. This result indicated that SNAREs alone do not define the specificity of vesicle docking reactions.
Tyrosine Residue in Exon 14 of the Cytoplasmic Domain of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) Regulates Ligand Binding Specificity
Authors: Famiglietti Julie , Sun Jing , DeLisser Horace M. , Albelda Steven M.
Published: July 2002

Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19– amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell–cell aggregation while those variants missing exon 14 mediate homophilic cell–cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5–amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.
Targeted Deletion of the PEX2 Peroxisome Assembly Gene in Mice Provides a Model for Zellweger Syndrome, a Human Neuronal Migration Disorder
Authors: Faust Phyllis L. , Hatten Mary E.
Published: July 2002

Zellweger syndrome is a peroxisomal biogenesis disorder that results in abnormal neuronal migration in the central nervous system and severe neurologic dysfunction. The pathogenesis of the multiple severe anomalies associated with the disorders of peroxisome biogenesis remains unknown. To study the relationship between lack of peroxisomal function and organ dysfunction, the PEX2 peroxisome assembly gene (formerly peroxisome assembly factor-1) was disrupted by gene targeting.

Homozygous PEX2-deficient mice survive in utero but die several hours after birth. The mutant animals do not feed and are hypoactive and markedly hypotonic. The PEX2-deficient mice lack normal peroxisomes but do assemble empty peroxisome membrane ghosts. They display abnormal peroxisomal biochemical parameters, including accumulations of very long chain fatty acids in plasma and deficient erythrocyte plasmalogens. Abnormal lipid storage is evident in the adrenal cortex, with characteristic lamellar–lipid inclusions. In the central nervous system of newborn mutant mice there is disordered lamination in the cerebral cortex and an increased cell density in the underlying white matter, indicating an abnormality of neuronal migration. These findings demonstrate that mice with a PEX2 gene deletion have a peroxisomal disorder and provide an important model to study the role of peroxisomal function in the pathogenesis of this human disease.
The p75 Neurotrophin Receptor Mediates Neuronal Apoptosis and Is Essential for Naturally Occurring Sympathetic Neuron Death
Authors: Bamji Shernaz X. , Majdan Marta , Pozniak Christine D. , Belliveau Daniel J. , Aloyz Raquel , Kohn Judi , Causing Carrie G. , Miller Freda D.
Published: July 2002

Abstract. To determine whether the p75 neurotrophin receptor (p75NTR) plays a role in naturally occurring neuronal death, we examined neonatal sympathetic neurons that express both the TrkA tyrosine kinase receptor and p75NTR. When sympathetic neuron survival is maintained with low quantities of NGF or KCl, the neurotrophin brain-derived neurotrophic factor (BDNF), which does not activate Trk receptors on sympathetic neurons, causes neuronal apoptosis and increased phosphorylation of c-jun. Function-blocking antibody studies indicate that this apoptosis is due to BDNF-mediated activation of p75NTR. To determine the physiological relevance of these culture findings, we examined sympathetic neurons in BDNF−/− and p75NTR−/− mice. In BDNF−/− mice, sympathetic neuron number is increased relative to BDNF+/+ littermates, and in p75NTR−/− mice, the normal period of sympathetic neuron death does not occur, with neuronal attrition occurring later in life. This deficit in apoptosis is intrinsic to sympathetic neurons, since cultured p75NTR−/− neurons die more slowly than do their wild-type counterparts. Together, these data indicate that p75NTR can signal to mediate apoptosis, and that this mechanism is essential for naturally occurring sympathetic neuron death.
A Role for Tissue Factor in Cell Adhesion and Migration Mediated by Interaction with Actin-binding Protein 280
Authors: Ott Ilka , Fischer Edgar G. , Miyagi Yohei , Mueller Barbara M. , Ruf Wolfram
Published: July 2002

Tissue factor (TF), the protease receptor initiating the coagulation system, functions in vascular development, angiogenesis, and tumor cell metastasis by poorly defined molecular mechanisms. We demonstrate that immobilized ligands for TF specifically support cell adhesion, migration, spreading, and intracellular signaling, which are not inhibited by RGD peptides. Two-hybrid screening identified actin-binding protein 280 (ABP-280) as ligand for the TF cytoplasmic domain. Extracellular ligation of TF is necessary for ABP-280 binding. ABP-280 recruitment to TF adhesion contacts is associated with reorganization of actin filaments, but cytoskeletal adaptor molecules typically found in integrin-mediated focal contacts are not associated with TF. Chimeric molecules of the TF cytoplasmic domain and an unrelated extracellular domain support cell spreading and migration, demonstrating that the extracellular domain of TF is not involved in the recruitment of accessory molecules that influence adhesive functions. Replacement of TF's cytoplasmic Ser residues with Asp to mimic phosphorylation enhances the interaction with ABP-280, whereas Ala mutations abolish coprecipitation of ABP-280 with immobilized TF cytoplasmic domain, and severely reduce cell spreading. The specific interaction of the TF cytoplasmic domain with ABP-280 provides a molecular pathway by which TF supports tumor cell metastasis and vascular remodeling.
C-Nap1, a Novel Centrosomal Coiled-Coil Protein and Candidate Substrate of the Cell Cycle–regulated Protein Kinase Nek2
Authors: Fry Andrew M. , Mayor Thibault , Meraldi Patrick , Stierhof York-Dieter , Tanaka Kayoko , Nigg Erich A.
Published: July 2002

Nek2 (for NIMA-related kinase 2) is a mammalian cell cycle–regulated kinase structurally related to the mitotic regulator NIMA of Aspergillus nidulans. In human cells, Nek2 associates with centrosomes, and overexpression of active Nek2 has drastic consequences for centrosome structure. Here, we describe the molecular characterization of a novel human centrosomal protein, C-Nap1 (for centrosomal Nek2-associated protein 1), first identified as a Nek2-interacting protein in a yeast two-hybrid screen. Antibodies raised against recombinant C-Nap1 produced strong labeling of centrosomes by immunofluorescence, and immunoelectron microscopy revealed that C-Nap1 is associated specifically with the proximal ends of both mother and daughter centrioles. On Western blots, anti–C-Nap1 antibodies recognized a large protein (>250 kD) that was highly enriched in centrosome preparations. Sequencing of overlapping cDNAs showed that C-Nap1 has a calculated molecular mass of 281 kD and comprises extended domains of predicted coiled-coil structure. Whereas C-Nap1 was concentrated at centrosomes in all interphase cells, immunoreactivity at mitotic spindle poles was strongly diminished. Finally, the COOH-terminal domain of C-Nap1 could readily be phosphorylated by Nek2 in vitro, as well as after coexpression of the two proteins in vivo. Based on these findings, we propose a model implicating both Nek2 and C-Nap1 in the regulation of centriole–centriole cohesion during the cell cycle.
Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells
Authors: Hirose Masaya , Ishizaki Toshimasa , Watanabe Naoki , Uehata Masayoshi , Kranenburg Onno , Moolenaar Wouter H. , Matsumura Fumio , Maekawa Midori , Bito Haruhiko , Narumiya Shuh
Published: July 2002

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.
Mutational Analysis of the Structure and Localization of the Nucleolus in the Yeast Saccharomyces cerevisiae
Authors: Oakes M. , Aris J.P. , Brockenbrough J.S. , Wai H. , Vu L. , Nomura M.
Published: July 2002

The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I–defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.
Functional Differences between Keratins of Stratified and Simple Epithelia
Authors: Hutton Elizabeth , Paladini Rudolph D. , Yu Qian-Chun , Yen Mei , Coulombe Pierre A. , Fuchs Elaine
Published: July 2002

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.
Large-Scale Chromatin Unfolding and Remodeling Induced by VP16 Acidic Activation Domain
Authors: Tumbar Tudorita , Sudlow Gail , Belmont Andrew S.
Published: July 2002

Analysis of the relationship between transcriptional activators and chromatin organization has focused largely on lower levels of chromatin structure. Here we describe striking remodeling of large-scale chromatin structure induced by a strong transcriptional activator. A VP16-lac repressor fusion protein targeted the VP16 acidic activation domain to chromosome regions containing lac operator repeats. Targeting was accompanied by increased transcription, localized histone hyperacetylation, and recruitment of at least three different histone acetyltransferases. Observed effects on large-scale chromatin structure included unfolding of a 90-Mbp heterochromatic chromosome arm into an extended 25–40-μm chromonema fiber, remodeling of this fiber into a novel subnuclear domain, and propagation of large-scale chromatin unfolding over hundreds of kilobase pairs. These changes in large-scale chromatin structure occurred even with inhibition of ongoing transcription by α-amanitin. Our results suggest a functional link between recruitment of the transcriptional machinery and changes in large-scale chromatin structure. Based on the observed long-range propagation of changes in large-scale chromatin structure, we suggest a possible rationale for the observed clustering of housekeeping genes within Mbp-sized chromosome bands.
Exogenous Expression of N-Cadherin in Breast Cancer Cells Induces Cell Migration, Invasion, and Metastasis
Authors: Hazan Rachel B. , Phillips Greg R. , Qiao Rui Fang , Norton Larry , Aaronson Stuart A.
Published: July 2002

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell–cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin–mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin–expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin–expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin–expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin–expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin–expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin–expressing cells.
Determination of Cell Adhesion Sites of Neuropilin-1
Authors: Shimizu Masayuki , Murakami Yasunori , Suto Fumikazu , Fujisawa Hajime
Published: July 2002

Neuropilin-1 is a type 1 membrane protein with three distinct functions. First, it can mediate cell adhesion via a heterophilic molecular interaction. Second, in neuronal cells, neuropilin-1 binds the class 3 semaphorins, which are neuronal chemorepellents, and plays a role in the directional guidance of axons. Neuropilin-1 is expected to form complexes with the plexinA subfamily members and mediate the semaphorin-elicited inhibitory signals into neurons. Third, in endothelial cells, neuropilin-1 binds a potent endothelial cell mitogen, vascular endothelial growth factor (VEGF)165, and regulates vessel formation. Though the binding sites in neuropilin-1 for the class 3 semaphorins and VEGF165 have been analyzed, the sites involved in cell adhesion activity of the molecule have not been identified. In this study, we produced a variety of mutant neuropilin-1s and tested their cell adhesion activity. We showed that the b1 and b2 domains within the extracellular segment of neuropilin-1 were required for the cell adhesion activity, and peptides with an 18–amino acid stretch in the b1 and b2 domains were sufficient to induce the cell adhesion activity. In addition, we demonstrated that the cell adhesion ligands for neuropilin-1 were proteins and distributed in embryonic mesenchymal cells but distinct from the class 3 semaphorins, VEGF, or plexins.
The Dystrophin Complex Forms a Mechanically Strong Link between the Sarcolemma and Costameric Actin
Authors: Rybakova Inna N. , Patel Jitandrakumar R. , Ervasti James M.
Published: July 2002

The absence of dystrophin complex leads to disorganization of the force-transmitting costameric cytoskeleton and disruption of sarcolemmal membrane integrity in skeletal muscle. However, it has not been determined whether the dystrophin complex can form a mechanically strong bond with any costameric protein. We performed confocal immunofluorescence analysis of isolated sarcolemma that were mechanically peeled from skeletal fibers of mouse hindlimb muscle. A population of γ-actin filaments was stably associated with sarcolemma isolated from normal muscle and displayed a costameric pattern that precisely overlapped with dystrophin. However, costameric actin was absent from all sarcolemma isolated from dystrophin-deficient mdx mouse muscle even though it was localized to costameres in situ. Vinculin, α-actinin, β-dystroglycan and utrophin were all retained on mdx sarcolemma, indicating that the loss of costameric actin was not due to generalized membrane instability. Our data demonstrate that the dystrophin complex forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton through interaction with γ-actin filaments. Destabilization of costameric actin filaments may also be an important precursor to the costamere disarray observed in dystrophin-deficient muscle. Finally, these methods will be broadly useful in assessing the mechanical integrity of the membrane cytoskeleton in dystrophic animal models lacking other costameric proteins.
Dishevelled-1 Regulates Microtubule Stability
Authors: Krylova Olga , Messenger Marcus J. , Salinas Patricia C.
Published: July 2002

Dishevelled has been implicated in the regulation of cell fate decisions, cell polarity, and neuronal function. However, the mechanism of Dishevelled action remains poorly understood. Here we examine the cellular localization and function of the mouse Dishevelled protein, DVL-1. Endogenous DVL-1 colocalizes with axonal microtubules and sediments with brain microtubules. Expression of DVL-1 protects stable microtubules from depolymerization by nocodazole in both dividing cells and differentiated neuroblastoma cells. Deletion analyses reveal that the PDZ domain, but not the DEP domain, of DVL-1 is required for microtubule stabilization. The microtubule stabilizing function of DVL-1 is mimicked by lithium-mediated inhibition of glycogen synthase kinase-3β (GSK-3β) and blocked by expression of GSK-3β. These findings suggest that DVL-1, through GSK-3β, can regulate microtubule dynamics. This new function of DVL-1 in controlling microtubule stability may have important implications for Dishevelled proteins in regulating cell polarity.
Mechanism of Metabolic Control
Authors: Komeili Arash , Wedaman Karen P. , O'Shea Erin K. , Powers Ted
Published: July 2002

De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-β-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.
Dissection of Autophagosome Formation Using Apg5-Deficient Mouse Embryonic Stem Cells
Authors: Mizushima Noboru , Yamamoto Akitsugu , Hatano Masahiko , Kobayashi Yoshinori , Kabeya Yukiko , Suzuki Kuninori , Tokuhisa Takeshi , Ohsumi Yoshinori , Yoshimori Tamotsu
Published: July 2002

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein–tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.
Fibroblast Growth Factor Signaling and Basement Membrane Assembly Are Connected during Epithelial Morphogenesis of the Embryoid Body
Authors: Li Xiaofeng , Chen Yali , Schéele Susanne , Arman Esther , Haffner-Krausz Rebecca , Ekblom Peter , Lonai Peter
Published: July 2002

Fibroblast growth factors and receptors are intimately connected to the extracellular matrix by their affinity to heparan sulfate proteoglycans. They mediate multiple processes during embryonic development and adult life. In this study, embryonic stem cell–derived embryoid bodies were used to model fibroblast growth factor signaling during early epithelial morphogenesis. To avoid redundancy caused by multiple receptors, we employed a dominant negative mutation of Fgfr2. Mutant-derived embryoid bodies failed to form endoderm, ectoderm, and basement membrane and did not cavitate. However, in mixed cultures they displayed complete differentiation induced by extracellular products of the normal cell. Evidence will be presented here that at least one of these products is the basement membrane or factors connected to it. It will be shown that in the mutant, collagen IV and laminin-1 synthesis is coordinately suppressed. We will demonstrate that the basement membrane is required for embryoid body differentiation by rescuing columnar ectoderm differentiation and cavitation in the mutant by externally added basement membrane proteins. This treatment induced transcription of Eomesodermin, an early developmental gene, suggesting that purified basement membrane proteins can activate inherent developmental programs. Our results provide a new paradigm for the role of fibroblast growth factor signaling in basement membrane formation and epithelial differentiation.
Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1
Authors: Ito Koichi , Komazaki Shinji , Sasamoto Kazushige , Yoshida Morikatsu , Nishi Miyuki , Kitamura Kenji , Takeshima Hiroshi
Published: July 2002

In skeletal muscle excitation–contraction (E–C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E–C coupling in skeletal muscle.
NKG2A Complexed with CD94 Defines a Novel Inhibitory Natural Killer Cell Receptor
Authors: Brooks Andrew G. , Posch Phillip E. , Scorzelli Christopher J. , Borrego Francisco , Coligan John E.
Published: July 2002

CD94 is a C-type lectin expressed by natural killer (NK) cells and a subset of T cells. Blocking studies using anti-CD94 mAbs have suggested that it is a receptor for human leukocyte antigen class I molecules. CD94 has recently been shown to be a 26-kD protein covalently associated with an unidentified 43-kD protein(s). This report shows that NKG2A, a 43-kD protein, is covalently associated with CD94 on the surface of NK cells. Cell surface expression of NKG2A is dependent on the association with CD94 as glycosylation patterns characteristic of mature proteins are found only in NKG2A that is associated with CD94. Analysis of NK cell clones showed that NKG2A was expressed in all NK cell clones whose CD16-dependent killing was inhibited by cross-linking CD94. The induction of an inhibitory signal is consistent with the presence of two immunoreceptor tyrosine-based inhibitory motifs (V/LXYXXL) on the cytoplasmic domain of NKG2A. Similar motifs are found on Ly49 and killer cell inhibitory receptors, which also transmit negative signals to NK cells.
Interleukin 10 Secretion and Impaired Effector Function of Major Histocompatibility Complex Class II–restricted T Cells Anergized In Vivo
Authors: Buer Jan , Lanoue Astrid , Franzke Anke , Garcia Corinne , Boehmer Harald von , Sarukhan Adelaida
Published: July 2002

Continuous antigenic stimulation in vivo can result in the generation of so-called “anergic” CD4+ or CD8+ T cells that fail to proliferate upon antigenic stimulation and fail to develop cytolytic effector functions. Here we show that class II major histocompatibility complex–restricted T cells specific for influenza hemagglutinin (HA) that become anergic in mice expressing HA under control of the immunoglobulin κ promoter exhibit an impaired effector function in causing diabetes in vivo, as compared to their naive counterparts, when transferred into immunodeficient recipients expressing HA under the control of the insulin promoter. Furthermore, HA-specific T cells anergized in vivo contain higher levels of interleukin (IL)-4 messenger RNA (mRNA) than naive and recently activated T cells with the same specificity and more than a 100-fold higher levels of IL-10 mRNA. The higher expression of the IL-10 gene is also evident at the protein level. These findings raise the interesting possibility that T cells rendered anergic in vivo have in fact become regulatory T cells that may influence neighboring immune responses through the release of IL-10.
A T Cell Receptor–specific Blockade of Positive Selection
Authors: Baldwin Kristin K. , Reay Philip A. , Wu Lawren , Farr Andrew , Davis Mark M.
Published: July 2002

To investigate the influence of endogenous peptides on the developmental processes that occur during thymocyte selection, we have used monoclonal antibodies that preferentially recognize the major histocompatibility complex (MHC) molecule I-Ek when it is bound to the moth cytochrome c peptide (88–103). One of these antibodies (G35) specifically blocks the positive selection of transgenic thymocytes expressing a T cell receptor that is reactive to this peptide– MHC complex. Furthermore, G35 does not block the differentiation of transgenic T cells bearing receptors for a different I-Ek–peptide complex. This antibody recognizes a subset of endogenous I-Ek–peptide complexes found on a significant fraction of thymic antigen-presenting cells, including cortical and medullary epithelial cells. The sensitivity of G35 to minor alterations in peptide sequence suggests that the thymic peptide–MHC complexes that mediate the positive selection of a particular class II MHC–restricted thymocyte are structurally related to the complexes that can activate it in the periphery.
A Critical Role for the RelA Subunit of Nuclear Factor κB in Regulation of Multiple Immune-response Genes and in Fas-induced Cell Death
Authors: Ouaaz Fateh , Li Ming , Beg Amer A.
Published: July 2002

Binding sites for the nuclear factor (NF)-κB transcription factor have been identified within control regions of many genes involved in inflammatory and immune responses. Such κB sites are often found adjacent to those of interferon (IFN)-γ–inducible transcription factors, suggesting a requirement for multiple signaling pathways for gene regulation. Using fibroblasts from RelA (p65)-deficient mice generated by gene targeting, we have investigated the role of this subunit of NF-κB in gene activation by microbial lipopolysaccharide, tumor necrosis factor α, and in possible synergism with the IFN-γ–signaling pathway. Our results indicate not only that RelA is required for activation of key genes involved in adaptive (acquired) immune responses, including major histocompatibility complex class I, CD40, and the Fas death receptor, but also that both NF-κB–inducing signals and IFN-γ are necessary for maximal activation. In contrast, neutrophil-specific chemokine genes KC and MIP-2, which can function as nonspecific mediators in innate immune responses, were strongly induced by RelA in the absence of IFN-γ. Our results show that RelA plays a critical role in activation of immune system genes in response to nonspecific stimuli and demonstrate a novel proapoptotic function for this protein in Fas-induced cell death.
Fas Gene Mutation in the Progression of Adult T Cell Leukemia
Authors: Maeda Takahiro , Yamada Yasuaki , Moriuchi Ryozou , Sugahara Kazuyuki , Tsuruda Kazuto , Joh Tatsurou , Atogami Sunao , Tsukasaki Kunihiro , Tomonaga Masao , Kamihira Shimeru
Published: July 2002

Fas antigen (Apo-1/CD95) is an apoptosis-signaling cell surface receptor belonging to the tumor necrosis factor receptor superfamily. Adult T cell leukemia (ATL) cells express Fas antigen and show apoptosis after treatment with an anti-Fas monoclonal antibody. We established the ATL cell line KOB, which showed resistance to Fas-mediated apoptosis, and found that KOB expressed two forms of Fas mRNA, the normal form and a truncated form. The truncated transcript lacked 20 base pairs at exon 9, resulting in a frame shift and the generation of a premature stop codon at amino acid 239. The same mutation was detected in primary ascitic cells and peripheral blood cells. The mutation was not detected in lymph node cells, however, although all of the primary ATL cells were of the same clonal origin. A retroviral-mediated gene transfer of the truncated Fas to Jurkat cells rendered the cells resistant to Fas-mediated apoptosis, suggesting a dominant negative interference mechanism. These results indicate that an ATL subclone acquires a Fas mutation in the lymph nodes, enabling the subclone to escape from apoptosis mediated by the Fas/Fas ligand system and proliferate in the body. Mutation of the Fas gene may be one of the mechanisms underlying the progression of ATL.
Antigen Receptor–Induced Activation and Cytoskeletal Rearrangement Are Impaired in Wiskott-Aldrich Syndrome Protein–Deficient Lymphocytes
Authors: Zhang Jinyi , Shehabeldin Amro , da Cruz Luis A.G. , Butler Jeffrey , Somani Ally-Khan , McGavin Mary , Kozieradzki Ivona , dos Santos Antonio O. , Nagy Andras , Grinstein Sergio , Penninger Josef M. , Siminovitch Katherine A.
Published: July 2002

The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS null allele (WAS−/−). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44−CD25+ to the CD44−CD25− stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-ζ, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH2-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS−/− cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS−/− lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS−/− neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.
An Essential Role for Thymic Mesenchyme in Early T Cell Development
Authors: Suniara Ravinder K. , Jenkinson Eric J. , Owen John J.T.
Published: July 2002

We show that the mesenchymal cells that surround the 12-d mouse embryo thymus are necessary for T cell differentiation. Thus, epithelial lobes with attached mesenchyme generate all T cell populations in vitro, whereas lobes from which mesenchyme has been removed show poor lymphopoiesis with few cells progressing beyond the CD4−CD8− stage of development. Interestingly, thymic mesenchyme is derived from neural crest cells, and extirpation of the region of the neural crest involved results in impaired thymic development and craniofacial abnormalities similar to the group of clinical defects found in the DiGeorge syndrome.

Previous studies have suggested an inductive effect of mesenchyme on thymic epithelial morphogenesis. However, we have found that mesenchyme-derived fibroblasts are still required for early T cell development in the presence of mature epithelial cells, and hence mesenchyme might have a direct role in lymphopoiesis. We provide an anatomical basis for the role of mesenchyme by showing that mesenchymal cells migrate into the epithelial thymus to establish a network of fibroblasts and associated extracellular matrix. We propose that the latter might be important for T cell development through integrin and/or cytokine interactions with immature thymocytes.
Protein Kinase Cϵ Is Required for Macrophage Activation and Defense Against Bacterial Infection
Authors: Castrillo Antonio , Pennington Daniel J. , Otto Florian , Parker Peter J. , Owen Michael J. , Boscá Lisardo
Published: July 2002

To assess directly the role of protein kinase C (PKC)ϵ in the immune system, we generated mice that carried a homozygous disruption of the PKCϵ locus. PKCϵ−/− animals appeared normal and were generally healthy, although female mice frequently developed a bacterial infection of the uterus. Macrophages from PKCϵ−/− animals demonstrated a severely attenuated response to lipopolysaccharide (LPS) and interferon (IFN)γ, characterized by a dramatic reduction in the generation of NO, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. Further analysis revealed that LPS-stimulated macrophages from PKCϵ−/− mice were deficient in the induction of nitric oxide synthase (NOS)-2, demonstrating a decrease in the activation of IκB kinase, a reduction in IκB degradation, and a decrease in nuclear factor (NF)κB nuclear translocation. After intravenous administration of Gram-negative or Gram-positive bacteria, PKCϵ−/− mice demonstrated a significantly decreased period of survival. This study provides direct evidence that PKCϵ is critically involved at an early stage of LPS-mediated signaling in activated macrophages. Furthermore, we demonstrate that in the absence of PKCϵ, host defense against bacterial infection is severely compromised, resulting in an increased incidence of mortality.