bluuebunny/crossref_metadata_2025 huggingface dataset:
Frontal EEG asymmetry in response to emotional vignettes in preschool age children
Authors: Pickens Jeffrey , Field Tiffany , Nawrocki Thomas
Published: July 2002

EEG recordings were conducted with preschool children during presentations of videotaped vignettes depicting a fictional young child experiencing happy, sad, angry, and fearful events. Significant EEG asymmetry in the frontal region (left frontal activation) occurred during all types of emotional vignettes, but not during baseline periods (a neutral star-field image presented before and after each vignette). This pattern of left frontal cortical activation during each emotional vignette may represent a generally positive and low intensity emotional “approach” response even in the case of the negative emotion vignettes. These children displayed mostly neutral facial expressions during the vignettes, with some evidence of lip movements during the negative episodes (suggesting that the stories were eliciting some mild emotional responses or empathy). There was no evidence of more intense emotional responses that have been associated with right frontal cortical activation. These EEG patterns may reflect cortical mechanisms underlying mild emotional responses and affective displays in preschool children, as well as their developing ability to regulate their affective systems.
Efficacy of an intensive home-based educational intervention programme for 4- to 6-year-old ethnic minority children in the Netherlands
Authors: van Tuijl Cathy , Leseman Paul P.M. , Rispens Jan
Published: July 2002

This paper reports the results of an intensive home-based educational intervention programme for 4- to 6-year-old children at risk of educational failure. The programme, Opstap Opnieuw (“Step-up Anew”), was developed in the Netherlands as an alternative to the well-known HIPPY-programme, of which a Dutch version was carried out in the early 1990s for ethnic minority groups, without apparent success. Building on the basic intervention strategy of HIPPY (i.e., involving mothers and paraprofessional aides), a new curriculum was developed based on recent theoretical insights in cognitive and language development, and emergent literacy and numeracy. The programme was carried out with Turkish and Moroccan immigrant families. For the Turkish group, the results were partly positive: There were modest effects of the programme on cognitive development and emergent numeracy, small effects on Turkish language development, but no effects on Dutch language development. In contrast, for the Moroccan group the effects were disappointing. The results are evaluated with respect to recent insights into effective strategies and essential ingredients of early educational intervention programmes.
Sociometric status of young adults: Behavioural correlates, and cognitive-motivational antecedents and consequences
Authors: Eronen Sanna , Nurmi Jari-Erik
Published: July 2002

A cross-lagged longitudinal study was carried out to investigate whether social reaction styles and loneliness serve as antecedents and consequences of sociometric status among young adults. Behavioural correlates of sociometric status were also studied. Questionnaires measuring sociometric ratings, social reaction styles, loneliness, and group atmosphere were ”lled in by 154 students one week after starting at a new school, then half a year later, and ”nally, one year later. In Measurement 2, the participants’ social behaviour was rated by their classmates. The results indicated that social reaction styles, feelings of loneliness, and satisfaction with the group atmosphere prospectively predicted sociometric status: Popularity, for example, was predicted by a high level of approach orientation. Sociometric status also predicted changes in individuals’ reaction styles and feelings of loneliness. For instance, popularity increased optimism and decreased loneliness. Finally, high sociometric status was related to prosocial behaviours, whereas low sociometric status was associated with behavioural deficiencies.
A comparison of four types of cognitive conflict and their effect on cognitive development
Authors: Druyan Sara
Published: July 2002

The study examines the differential influence of four types of conflict—two physical conflicts and two social conflicts—on the improvement of the ability to solve balance problems. A total of 395 subjects, consisting of preschoolers, 3rd, and 5th graders, participated in three sessions: the pre-test, the intervention in which visual, kinesthetic, peer, or child-adult conflicts were presented, and the post-test. In each of the sessions the subjects were required to solve balance problems based on prototype problems employed by Siegler (1976). The ” ndings indicate that the kinesthetic conflict is the most effective in promoting the consideration of the distance dimension at the preschool age and that child-adult conflict is the most effective in promoting the coordination of weight and distance dimensions at school age. In addition, peer conflict was found to be significantly effective but only in 5th grade. In conclusion, the results suggest that the effectiveness of cognitive conflicts depends on both the demands of the task and the developmental stage of the child.
Words versus faces in evoking preschool children’s knowledge of the causes of emotions
Authors: Russell James A. , Widen Sherri C.
Published: July 2002

Children ( N = 160), aged 3 to 4 years, generated stories describing the causes of six different emotions: happiness, surprise, fear, anger, disgust, and sadness. The emotion was specified to the child either by a word (such as scared or disgusted) or by a photograph of a facial expression said to be a universal, biologically based signal for that emotion. For no emotion did the face produce significantly better performance than did the word. For fear and disgust, the word produced significantly better performance than did the face.
A Behavioural Genetic Perspective on Close Relationships
Authors: Pike Alison , Plomin Robert
Published: July 2002

This paper examines how genetic, shared environment, and nonshared environment influence individual differences in close relationships. First, a brief explanation of behavioural genetic methods is given. Next, we review behavioural genetic studies of the close relationships in which people are typically involved (parent-child, sibling, friend, and spouse). Many of the studies reviewed indicate a modest to moderate role for genetic factors. This suggests that genetic make-up can affect the nature of close relationships. It also implies that genetic factors may be involved in the influence of close relationships on individual adaptation. Behavioural genetic research has begun to show that this is the case. Close relationships may be especially important sources of nonshared environment. Behavioural genetic research consistently shows that environmental factors that have a functional effect on psychological traits work to make siblings in the same family different from one another rather than similar. Attempts to pinpoint specific aspects of the nonshared environment are under way. Parent-child relationships have been systematically explored, and found not to play an important role in nonshared environment. This leaves open the possibility that extrafamilial relationships, in particular friendships, may be important contributors to the nonshared environment.
Glutamate‐dopamine interactions mediate the effects of psychostimulant drugs
Authors: WANG JOHN Q. , McGINTY JACQUELINE F.
Published: July 2002

The striatum, a major central nervous system structure modulating movement, is enriched with glutamatergic and dopaminergic innervation. By altering activities of both glutamatergic and dopaminergic transmissions the psychostimulants, amphetamine and cocaine, induce behavioral changes in experimental animals. Activation of the two systems is also essential in the mediation of drug‐stimulated gene expression in striatal neurons, which is considered to be an important component of the neuroplasticity underlying long‐term profiles of stimulant use. Interactions between the two systems occur at multiple levels that determine the final outcome of drug stimulation. Emerging studies on the detailed transsynaptic and intracellular mechanisms of glutamatedopamine interactions in response to stimulant exposure are providing cellular and molecular insight into the pathophysiology of stimulant abuse.
A Multinational Comparison Of Aphasia Management Practices
Authors: Katz Richard C. , Hallowell Brooke , Code Chris , Armstrong Elizabeth , Roberts Patricia , Pound Carole , Katz Lynn
Published: July 2002

The effect of restructuring of healthcare on the quality, quantity, and nature of aphasia management is largely unknown. The current study is the first to examine access, diagnostic, treatment, and discharge patterns of patients with aphasia in Australia, Canada, the UK, the US private sector (US‐Private), and the US Veterans Health Administration in the Department of Veterans Affairs (US‐VA). The authors developed a 37‐item survey to be completed by clinicians working with aphasic patients. The survey focused on eight areas: access to care, evaluation procedures, group treatment, number and duration of treatment sessions, limitations of the number of sessions, termination of treatment, follow‐up practices, and resumption of treatment. 394 surveys were distributed and 175 were returned completed (44% return rate). Respondents represented a range of ages, work experiences, and work settings. There was considerable consistency among respondents from our five healthcare systems. Results suggest that patients may be routinely denied treatment in direct contradiction to the research literature. Just as we carefully monitor the progress of patients receiving our treatment, we are obliged to monitor the effects of managed care on our patients, fellow clinicians, and our profession.
Thinking out loud: an exploration of problem‐solving language in preschoolers with and without language impairment
Authors: Sturn Arlene , Johnston Judith
Published: July 2002

This study investigates the use of language while engaged in solving a complex spatial problem. Children with specific language impairment (SLI) have been reported to lag behind age peers in certain cognitive tasks, despite normal range non‐verbal intelligence test scores. It was hypothesized that these delays could reflect some failure to employ language to direct and facilitate thought. Dyads of preschool children were taped during a three‐dimensional construction task. Task‐relevant utterances were then coded for function and addressee. The speech of six children with SLI, aged 4;10, was compared with that of two control groups, one matched by age, the other by language level. The SLI group used less problem‐solving speech and fewer modal expressions than their age peers. Not all differences were statistically significant, but the pattern of findings indicated that what differences did occur were due to overall reductions in the amount of speech rather than any specific failure to use language as a tool of thought. Correlations between the use of problem‐solving speech and a measure of cognitive style indicated clear group differences. For children in the control groups, greater use of private speech was associated with greater cognitive efficiency; for children in the SLI group the relationship was in the opposite direction, greater use of private speech being associated with less cognitive efficiency. In all three groups, problem‐solving speech could be addressed either to partner or to self, although some individual preferences were observed. The findings, while preliminary, suggest new directions for studies of cognitive deficits in children with SLI. They also indicate the importance of including socially directed utterances, and controls for language proficiency, in future research on private speech
Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis
Authors: Spann Timothy P. , Moir Robert D. , Goldman Anne E. , Stick Reimer , Goldman Robert D.
Published: July 2002

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.
Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud
Authors: Shaw Sidney L. , Yeh Elaine , Maddox Paul , Salmon E.D. , Bloom Kerry
Published: July 2002

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at ≅0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.
Cyclin-dependent Kinases Participate in Death of Neurons Evoked by DNA-damaging Agents
Authors: Park David S. , Morris Erick J. , Padmanabhan Jaya , Shelanski Michael L. , Geller Herbert M. , Greene Lloyd A.
Published: July 2002

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus–induced expression of CKIs p16ink4, p21waf/cip1, and p27kip1, as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation– and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1–associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.
Sphingosine Kinase Expression Increases Intracellular Sphingosine-1-Phosphate and Promotes Cell Growth and Survival
Authors: Olivera Ana , Kohama Takafumi , Edsall Lisa , Nava Victor , Cuvillier Olivier , Poulton Samantha , Spiegel Sarah
Published: July 2002

Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein–coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229–240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G1/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.
In Situ Biochemical Demonstration That P-Glycoprotein Is a Drug Efflux Pump with Broad Specificity
Authors: Chen Yu , Simon Sanford M.
Published: July 2002

While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.
A Phosphorylation Site Regulates Sorting of the Vesicular Acetylcholine Transporter to Dense Core Vesicles
Authors: Krantz David E. , Waites Clarissa , Oorschot Viola , Liu Yongjian , Wilson Rachel I. , Tan Philip K. , Klumperman Judith , Edwards Robert H.
Published: July 2002

Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.
Activated ras Prevents Downregulation of Bcl-XL Triggered by Detachment from the Extracellular Matrix
Authors: Rosen Kirill , Rak Janusz , Leung Thomas , Dean Nicholas M. , Kerbel Robert S. , Filmus Jorge
Published: July 2002

Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptotic member of the Bcl-2 family. Since exogenous Bak could only partially restore susceptibility to anoikis in the ras-transformed cells, the existence of at least another component of the apoptotic machinery mediating the effect of activated ras on anoikis was suggested. Indeed, here we show that, in nonmalignant rat and human intestinal epithelial cells, detachment from the ECM or disruption of the cytoskeleton results in a significant downregulation of the antiapoptotic effector Bcl-XL, and that activated H- or K-ras oncogenes completely abrogate this downregulation. In addition, we found that enforced downregulation of Bcl-XL in the ras-transformed cells promotes anoikis and significantly inhibits tumorigenicity, indicating that disruption of the adhesion-dependent regulation of Bcl-XL is an essential part of the molecular changes associated with transformation by ras. While the ras-induced downregulation of Bak could be reversed by pharmacological inhibition of phosphatidylinositol 3 kinase (PI 3-kinase), the effect of ras on Bcl-XL was PI 3-kinase– and mitogen-activated protein kinase (MAP kinase)–independent. We conclude that ras-induced resistance to anoikis in intestinal epithelial cells is mediated by at least two distinct mechanisms: one that triggers downregulation of Bak and another that stabilizes Bcl-XL expression in the absence of the ECM.
Distinct Membrane Domains on Endosomes in the Recycling Pathway Visualized by Multicolor Imaging of Rab4, Rab5, and Rab11
Authors: Sönnichsen Birte , De Renzis Stefano , Nielsen Erik , Rietdorf Jens , Zerial Marino
Published: July 2002

Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.
Reduced Mobility of the Alternate Splicing Factor (Asf) through the Nucleoplasm and Steady State Speckle Compartments
Authors: Kruhlak Michael J. , Lever Melody A. , Fischle Wolfgang , Verdin Eric , Bazett-Jones David P. , Hendzel Michael J.
Published: July 2002

Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF–GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF–GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.
Gradient of Increasing Affinity of Importin β for Nucleoporins along the Pathway of Nuclear Import
Authors: Ben-Efraim Iris , Gerace Larry
Published: July 2002

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin β cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin β during import. Our results indicate that the importin β transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin β moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.
The Plasminogen Activator Inhibitor PAI-1 Controls in Vivo Tumor Vascularization by Interaction with Proteases, Not Vitronectin
Authors: Bajou Khalid , Masson Véronique , Gerard Robert D. , Schmitt Petra M. , Albert Valérie , Praus Michael , Lund Leif R. , Frandsen Thomas L. , Brunner Nils , Dano Keld , Fusenig Norbert E. , Weidle Ulrich , Carmeliet Geert , Loskutoff David , Collen Desiré , Carmeliet Peter , Foidart Jean Michel , Noël Agnès
Published: July 2002

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.
The Short Arm of the Laminin γ2 Chain Plays a Pivotal Role in the Incorporation of Laminin 5 into the Extracellular Matrix and in Cell Adhesion
Authors: Gagnoux-Palacios Laurent , Allegra Maryline , Spirito Flavia , Pommeret Olivier , Romero Christine , Ortonne Jean-paul , Meneguzzi Guerrino
Published: July 2002

Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the γ2 chain that removes the NH2-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the γ2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the γ2 chain short arm, we expressed mutated γ2 cDNAs into immortalized γ2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the γ2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the γ2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.
9-O-Acetylation of Sialomucins: A Novel Marker of Murine CD4 T Cells that Is Regulated during Maturation and Activation
Authors: Krishna Murli , Varki Ajit
Published: July 2002

Terminal sialic acids on cell surface glycoconjugates can carry 9-O-acetyl esters. For technical reasons, it has previously been difficult to determine their precise distribution on different cell types. Using a recombinant soluble form of the Influenza C virus hemagglutinin-esterase as a probe for 9-O-acetylated sialic acids, we demonstrate here their preferential expression on the CD4 T cell lineage in normal B10.A mouse lymphoid organs. Of total thymocytes, 8–10% carry 9-O-acetylation; the great majority of these are the more mature PNA−, HSA−, and TCRhi medullary cells. While low levels of 9-O-acetylation are seen on some CD4/CD8 double positive (DP) and CD8 single positive (SP) cells, high levels are present primarily on 80– 85% of CD4 SP cells. Correlation with CD4 and CD8 levels suggests that 9-O-acetylation appears as an early differentiation marker as cells mature from the DP to the CD4 SP phenotype. This high degree of 9-O-acetylation is also present on 90–95% of peripheral spleen and lymph node CD4 T cells. In contrast, only a small minority of CD8 T cells and B cells show such levels of 9-O-acetylation. Among mature peripheral CD4 T lymphocytes, the highly O-acetylated cells are Mel 14hi, CD44lo, and CD45R(exon B)hi, features typical of naive cells. Digestions with trypsin and O-sialoglycoprotease (OSGPase) and ELISA studies of lipid extracts indicate that the 9-O-acetylated sialic acids on peripheral CD4 T cells are predominantly on O-linked mucintype glycoproteins and to a lesser degree, on sialylated glycolipids (gangliosides). In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells. The 9-O-acetylated gangliosides on mouse T cells are not bound by CD60 antibodies, which recognize O-acetylated gangliosides in human T cells. Tethering 9-O-acetylated mucins with the Influenza C probe with or without secondary cross-linking did not cause activation of CD4 T cells. However, activation by other stimuli including TCR ligation is associated with a substantial decrease in surface 9-O-acetylation, primarily in the mucin glycoprotein component. Thus, 9-O-acetylation of sialic acids on cell surface mucins is a novel marker on CD4 T cells that appears on maturation and is modulated downwards upon activation.
Membrane Fas Ligand Kills Human Peripheral Blood T Lymphocytes, and Soluble Fas Ligand Blocks the Killing
Authors: Suda Takashi , Hashimoto Hideo , Tanaka Masato , Ochi Takahiro , Nagata Shigekazu
Published: July 2002

It has been believed that the Fas expressed on human peripheral blood T cells (PBT) is nonfunctional, because these cells are insensitive to agonistic anti-Fas/Apo-1 mAbs that efficiently kill in vitro–activated T cells and many Fas-expressing cell lines. Here, we demonstrate that membrane-bound Fas ligand (FasL) kills both fresh and in vitro–activated PBT, indicating that the Fas expressed on fresh PBT is functional. In contrast, soluble FasL kills only the latter. Naive T cells in umbilical cord blood do not express Fas, but can be induced to express Fas by IFN-γ or by a combination of IL-2 and anti-CD28 mAb, after which they acquire sensitivity to membrane but not to soluble FasL. Soluble FasL inhibited the killing of fresh PBT by membrane FasL. These results indicate that the shedding of FasL from the membrane is a mechanism for downregulating at least part of its killing activity.
Disruption of Fas Receptor Signaling by Nitric Oxide in Eosinophils
Authors: Hebestreit Holger , Dibbert Birgit , Balatti Ivo , Braun Doris , Schapowal Andreas , Blaser Kurt , Simon Hans-Uwe
Published: July 2002

It has been suggested that Fas ligand–Fas receptor interactions are involved in the regulation of eosinophil apoptosis and that dysfunctions in this system could contribute to the accumulation of these cells in allergic and asthmatic diseases. Here, we demonstrate that nitric oxide (NO) specifically prevents Fas receptor–mediated apoptosis in freshly isolated human eosinophils. In contrast, rapid acceleration of eosinophil apoptosis by activation of the Fas receptor occurs in the presence of eosinophil hematopoietins. Analysis of the intracellular mechanisms revealed that NO disrupts Fas receptor–mediated signaling events at the level of, or proximal to, Jun kinase (JNK), but distal to sphingomyelinase (SMase) activation and ceramide generation. In addition, activation of SMase occurs downstream of an interleukin 1 converting enzyme–like (ICE-like) protease(s) that is not blocked by NO. However, NO prevents activation of a protease that targets lamin B1. These findings suggest a role for an additional NO-sensitive apoptotic signaling pathway that amplifies the proteolytic cascade initialized by activation of the Fas receptor. Therefore, NO concentrations within allergic inflammatory sites may be important in determining whether an eosinophil survives or undergoes apoptosis upon Fas ligand stimulation.
Inhibition of Constitutive Signaling of Kaposi's Sarcoma–associated Herpesvirus G Protein–Coupled Receptor by Protein Kinases in Mammalian Cells in Culture
Authors: Geras-Raaka Elizabeth , Arvanitakis Leandros , Bais Carlos , Cesarman Ethel , Mesri Enrique A. , Gershengorn Marvin C.
Published: July 2002

Kaposi's sarcoma–associated herpesvirus (KSHV)/human herpesvirus 8, which is consistently present in tissues of patients with Kaposi's sarcoma and primary effusion lymphomas, contains a gene that encodes a G protein–coupled receptor (KSHV-GPCR). We recently showed that KSHV-GPCR exhibits constitutive signaling via activation of phosphoinositide-specific phospholipase C and stimulates cell proliferation and transformation. In this study, we determined whether normal cellular mechanisms could inhibit constitutive signaling by KSHV-GPCR and thereby KSHV-GPCR–stimulated proliferation. We show that coexpression of GPCR-specific kinases (GRKs) and activation of protein kinase C inhibit constitutive signaling by KSHV-GPCR in COS-1 monkey kidney cells and in mouse NIH 3T3 cells. Moreover, GRK-5 but not GRK-2 inhibits KSHV-GPCR–stimulated proliferation of rodent fibroblasts. These data provide evidence that cell regulatory pathways of receptor desensitization may be therapeutic targets in human diseases involving constitutively active receptors.
Transgene Expression of bcl-xL Permits Anti-immunoglobulin (Ig)–induced Proliferation in xid B Cells
Authors: Solvason Nanette , Wu Wei Wei , Kabra Nisha , Lund-Johansen Fridjtof , Roncarolo Maria Grazia , Behrens Timothy W. , Grillot Didier A.M. , Nunez Gabriel , Lees Emma , Howard Maureen
Published: July 2002

Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.
T Helper 1 (Th1) and Th2 Characteristics Start to Develop During T Cell Priming and Are Associated with an Immediate Ability to Induce Immunoglobulin Class Switching
Authors: Toellner Kai-Michael , Luther Sanjiv A. , Sze Daniel M.-Y. , Choy Richard K.-W. , Taylor Dale R. , MacLennan Ian C.M. , Acha-Orbea Hans
Published: July 2002

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cγ2a and Cγ1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon γ in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone.

The addition of killed Bordetella pertussis to the hapten–protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten–protein can proceed in a node where there is substantial Th1 activity.
Cerebral Ischemia Enhances Polyamine Oxidation: Identification of Enzymatically Formed 3-Aminopropanal as an Endogenous Mediator of Neuronal and Glial Cell Death
Authors: Ivanova Svetlana , Botchkina Galina I. , Al-Abed Yousef , Meistrell Malcolm , Batliwalla Franak , Dubinsky Janet M. , Iadecola Constantino , Wang Haichao , Gregersen Peter K. , Eaton John W. , Tracey Kevin J.
Published: July 2002

To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after the onset of ischemia in brain homogenates (15.8 ± 0.9 nmol/h/mg protein) as compared with homogenates prepared from the normally perfused contralateral side (7.4 ± 0.5 nmol/h/mg protein) (P <0.05). The major catabolic products of polyamine oxidase are putrescine and 3-aminopropanal. Although 3-aminopropanal is a potent cytotoxin, essential information was previously lacking on whether 3-aminopropanal is produced during cerebral ischemia. We now report that 3-aminopropanal accumulates in the ischemic brain within 2 h after permanent forebrain ischemia in rats. Cytotoxic levels of 3-aminopropanal are achieved before the onset of significant cerebral cell damage, and increase in a time-dependent manner with spreading neuronal and glial cell death. Glial cell cultures exposed to 3-aminopropanal undergo apoptosis (LD50 = 160 μM), whereas neurons are killed by necrotic mechanisms (LD50 = 90 μM). The tetrapeptide caspase 1 inhibitor (Ac-YVAD-CMK) prevents 3-aminopropanal–mediated apoptosis in glial cells. Finally, treatment of rats with two structurally distinct inhibitors of polyamine oxidase (aminoguanidine and chloroquine) attenuates brain polyamine oxidase activity, prevents the production of 3-aminopropanal, and significantly protects against the development of ischemic brain damage in vivo. Considered together, these results indicate that polyamine oxidase–derived 3-aminopropanal is a mediator of the brain damaging sequelae of cerebral ischemia, which can be therapeutically modulated.
An Essential Role for Nuclear Factor κB in Promoting Double Positive Thymocyte Apoptosis
Authors: Hettmann Thore , DiDonato Joseph , Karin Michael , Leiden Jeffrey M.
Published: July 2002

To examine the role of nuclear factor (NF)-κB in T cell development and activation in vivo, we produced transgenic mice that express a superinhibitory mutant form of inhibitor κB-α (IκB-αA32/36) under the control of the T cell–specific CD2 promoter and enhancer (mutant [m]IκB-α mice). Thymocyte development proceeded normally in the mIκB-α mice. However, the numbers of peripheral CD8+ T cells were significantly reduced in these animals. The mIκB-α thymocytes displayed a marked proliferative defect and significant reductions in interleukin (IL)-2, IL-3, and granulocyte/macrophage colony-stimulating factor production after cross-linking of the T cell antigen receptor. Perhaps more unexpectedly, double positive (CD4+CD8+; DP) thymocytes from the mIκB-α mice were resistant to α-CD3–mediated apoptosis in vivo. In contrast, they remained sensitive to apoptosis induced by γ-irradiation. Apoptosis of wild-type DP thymocytes after in vivo administration of α-CD3 mAb was preceded by a significant reduction in the level of expression of the antiapoptotic gene, bcl-xL. In contrast, the DP mIκB-α thymocytes maintained high level expression of bcl-xL after α-CD3 treatment. Taken together, these results demonstrated important roles for NF-κB in both inducible cytokine expression and T cell proliferation after TCR engagement. In addition, NF-κB is required for the α-CD3–mediated apoptosis of DP thymocytes through a pathway that involves the regulation of the antiapoptotic gene, bcl-xL.
Receptor Avidity and Costimulation Specify the Intracellular Ca2+ Signaling Pattern in Cd4+Cd8+ Thymocytes
Authors: Freedman Bruce D. , Liu Qing-Hua , Somersan Selin , Kotlikoff Michael I. , Punt Jennifer A.
Published: July 2002

Thymocyte maturation is governed by antigen–T cell receptor (TCR) affinity and the extent of TCR aggregation. Signals provided by coactivating molecules such as CD4 and CD28 also influence the fate of immature thymocytes. The mechanism by which differences in antigen–TCR avidity encode unique maturational responses of lymphocytes and the influence of coactivating molecules on these signaling processes is not fully understood. To better understand the role of a key second messenger, calcium, in governing thymocyte maturation, we measured the intracellular free calcium concentration ([Ca2+]i) response to changes in TCR avidity and costimulation. We found that TCR stimulation initiates either amplitude- or frequency-encoded [Ca2+]i changes depending on (a) the maturation state of stimulated thymocytes, (b) the avidity of TCR interactions, and (c) the participation of specific coactivating molecules. Calcium signaling within immature but not mature thymocytes could be modulated by the avidity of CD3/CD4 engagement. Low avidity interactions induced biphasic calcium responses, whereas high avidity engagement initiated oscillatory calcium changes. Notably, CD28 participation converted the calcium response to low avidity receptor engagement from a biphasic to oscillatory pattern. These data suggest that calcium plays a central role in encoding the nature of the TCR signal received by thymocytes and, consequently, a role in thymic selection.