bluuebunny/crossref_metadata_2025 huggingface dataset:
Grouping in short-term verbal memory: Is position coded temporally?
Authors: Ng Honey L.H. , Maybery Murray T.
Published: July 2002

The nature of the mechanisms that code item position in serial short-term verbal recall was investigated with reference to temporal grouping phenomena—effects that arise when additional pauses are inserted in a presented list to form groups of items. Several recent models attempt to explain these phenomena by assuming that positional information is retained by associating items with contextual information. According to two of the models—the Phonological Loop model (Hitch, Burgess, Towse, & Culpin, 1996) and the OSCAR model (Brown, Preece, & Hulme, 2000)—contextual information depends critically on the timing of item presentation with reference to group onset. By contrast, according to the Start-End model (Henson, 1998) and a development from it, which we label the Oscillator-Revised Start-End model (Henson & Burgess, 1997), contextual information is independent of time from group onset. Three experiments examined whether coding of position is time dependent. The critical manipulation was to vary stimulus-onset asynchrony from one group to the next in the same list. Lists of consonants were presented visually, but with vocalization in Experiment 1, auditorily in Experiment 2, and auditorily with articulatory suppression in Experiment 3. The pattern of order errors consistently favoured the predictions of the time-independent models over those of the time-dependent models in that across-group transpositions reflected within-group serial position rather than time from group onset. Errors involving intrusions from previous lists also reflected within-group serial position, thereby extending support for the time-independent models.
Task-induced fatigue states and simulated driving performance
Authors: Matthews Gerald , Desmond Paula A.
Published: July 2002

States of fatigue are implicated in driver impairment and motor vehicle accidents. This article reports two studies investigating two possible mechanisms for performance impairment: (1) loss of attentional resources; and (2) active regulation of matching effort to task demands. The first hypothesis predicts that fatigue effects will be accentuated by high task demands, but the second hypothesis predicts that fatigue effects will be strongest in “underload” conditions. In two studies, drivers performed a stimulated driving task, in which task demands were manipulated by varying road curvature. In a “fatigue induction” condition, the early part of the drive was occupied by performance of a demanding secondary task concurrently with driving, after which the concurrent task ceased. Post-induction driving performance was compared with a control condition in which drivers were not exposed to the induction. In both studies, the fatigue induction elicited various subjective fatigue and stress symptoms, and also raised reported workload. Fatigue effects on vehicle control and signal detection were assessed during and after the fatigue induction. The fatigue induction increased heading error, reduced steering activity, and, in the second study, reduced perceptual sensitivity on a secondary detection task. These effects were confined to driving on straight rather than on curved road sections, consistent with the effort regulation hypothesis. The second study showed that fatigue effects were moderated by a motivational manipulation. Results are interpreted within a control model, such that task-induced fatigue may reduce awareness of performance impairment, rather than reluctance or inability to mobilize compensatory effort following detection of impairment.
Age of Acquisition Norms for a Large Set of Object Names and Their Relation to Adult Estimates and Other Variables
Authors: Morrison Catriona M. , Chappell Tameron D. , Ellis Andrew W.
Published: July 2002

Studies of lexical processing have relied heavily on adult ratings of word learning age or age of acquisition, which have been shown to be strongly predictive of processing speed. This study reports a set of objective norms derived in a large-scale study of British children's naming of 297 pictured objects (including 232 from the Snodgrass & Vanderwart, 1980, set). In addition, data were obtained on measures of rated age of acquisition, rated frequency, imageability, object familiarity, picture-name agreement, and name agreement. We discuss the relationship between the objective measure and adult ratings of word learning age. Objective measures should be used when available, but where not, our data suggest that adult ratings provide a reliable and valid measure of real word learning age.
Investigating the Relation between Imagery and Perception: Evidence from Face Priming
Authors: Cabeza Roberto , Burton A. Mike , Kelly Stephen W. , Shigeru Akamatsu
Published: July 2002

The relation between imagery and perception was investigated in face priming. Two experiments are reported in which subjects either saw or imagined the faces of celebrities. They were later given a speeded perceptual test (familiarity judgement to pictures of celebrities) or a speeded imagery test (in which they were told the names of celebrities and asked to make a decision about their appearance). Seeing faces primed the perceptual test, and imaging faces primed the imagery test; however, there was no priming between seeing and imaging faces. These results show that perception and imagery can be dissociated in normal subjects. In two further experiments, we examined the effects of imaging faces on a subsequent face-naming task and on a task requiring familiarity judgements to partial faces. Both these tasks were facilitated by prior imaging of faces. These results are discussed in relation to those of McDermott & Roediger (1994), who found that imagery promoted object priming in a perceptual test involving naming partial line drawings. The implications for models of face recognition are also discussed.
Summation: Further Assessment of a Configural Theory
Authors: Pearce John M. , George David N. , Aydin Aydan
Published: July 2002

Rats received Pavlovian conditioning in which food was signalled by a visual stimulus, A+, an auditory stimulus, B+, and a compound composed of different visual and auditory stimuli, CD+. Test trials were then given with the compound AB. Experiments 1 and 2A revealed stronger responding during AB than during CD. In Experiment 2B, there was no evidence of a summation of responding during AB when A+ B+ training was conducted in the absence of CD+ trials. A further failure to observe abnormally strong responding during ABwas found in Experiment 3 for which the training trials with A+ B+ CD+ were accompanied by trials in which C and D were separately paired with food. The results are explained in terms of a configural theory of conditioning, which assumes that responding during a compound is determined by generalization from its components, as well as from other compounds to which it is similar.
An overview of diversion strategies for Australian drug‐related offenders
Authors: SPOONER CATHERINE , HALL WAYNE , MATTICK RICHARD P.
Published: July 2002

This paper provides a descriptive overview of options for diversion of drug‐related offenders from the criminal justice system. Drug‐related offences include drug offences (for example, possession of a prohibited substance); offences that are directly linked to intoxication (for example, drink‐driving or assault); and offences committed to support drug use (for example, theft). After an offence has been detected by police, multiple opportunities for diversion occur throughout the criminal justice process. (a) Pre‐arrest: when an offence is first detected, prior to a charge being laid. This is known as police diversion and includes fines, warnings and cautions, sometimes with educational information or referral to assessment and treatment. (b) Pre‐trial: when a charge is made but before the matter is heard at court. Examples are treatment as a condition of bail, conferencing and prosecutor discretion. (c) Pre‐sentence: a delay of sentence while assessment and treatment are sought. (d) Post‐sentence: as part of sentencing, for example suspended sentences, drug courts, noncustodial sentences and circle sentencing. (e) Pre‐release: prior to release from a sentence, on parole. Issues for diversion programmes include net widening, the ethics of coercion to treatment, the needs of families and intersectoral collaboration. A framework for diversion is presented in which increasingly treatment‐focused and coercive diversion strategies are used as offenders' criminal careers and drug problems increase.
Serum levels of interleukin‐6 type cytokines and soluble interleukin‐6 receptor in patients with rheumatoid arthritis
Authors: Robak T. , Gladalska A. , Stepień H. , Robak E.
Published: July 2002

We investigated the serum concentrations of interleukin‐6 (IL‐6) and two IL‐6 family of cytokines (leukaemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) as well as IL‐6 soluble receptor (sIL‐6R) using an enzyme‐linked immunosorbent assay (ELISA) in 66 patients with rheumatoid arthritis (RA) and 24 healthy controls. We examined a possible association between the serum levels of these peptides and RA activity according to the Mallya and Mace scoring system and Ritchie′s index. We also evaluated the correlation between the serum levels of IL‐6, LIF, CNTF and sIL‐6R and duration of the disease and calculated sIL‐6R/IL‐6 ratio in RA patients and in the control group. IL‐6 and sIL‐6R were detectable in all 66 patients with RA and 24 normal individuals. LIF was also found in the serum of all patients with RA and in 16 (66.7%) normal individuals. In contrast CNTF was measurable only in 15 (22.7%) patients with RA and 24 (33.3%) normal individuals. The highest IL‐6 and sIL‐6R levels were found in the patients with Stages 3 and 4 of RA activity and the lowest in the control group. In contrast there were no statistically significant diferences between the LIF and CNTF levels in RA patients and normal individuals. We found positive correlation between IL‐6 and sIL‐6R concentrations and Ritchie′s index and a lack of such correlation with LIF and CNTF. IL‐6 serum level correlated positively with the disease duration, but sIL‐6R, LIF and CNTF did not. Serum sIL‐6R/IL‐6 ratio was significantly lower in RA patients than in healthy controls. In conclusion, an increase in the serum levels of IL‐6 and sIL‐6R, but not LIF and CNTF concentrations, may be useful markers for RA activity.
Hyperresponsiveness in the Human Nasal Airway: New Targets for the Treatment of Allergic Airway Disease
Authors: Turner P. J. , Foreman J. C.
Published: July 2002

Allergic rhinitis is a condition which affects over 15% of the population in the United Kingdom. The pathological process involves two stages: nasal inflammation, and the development of nasal airway hyperresponsiveness (AHR) to allergen and a number of other stimuli. This results in the amplification of any subsequent allergic reaction, contributing to the chronic allergic state. A number of different hypotheses have been proposed to explain the underlying mechanism of AHR, including a role for eosinophil‐derived proteins, free radicals and neuropeptides. While there may be a number of independent pathways which can result in AHR, evidence obtained from both animal models and in vivo experiments in humans indicate that some mediators may interact with one another, resulting in AHR. Further research into these interactions may open new avenues for the pharmacological treatment of chronic allergic rhinitis, and possibly other allergic airway diseases.
Inflammatory Markers in Cystic Fibrosis Patients with Lung Pseudomonas aeruginosa Infection
Authors: Pukhalsky A. L. , Kapranov N. I. , Kalashnikova E. A. , Shmarina G. V. , Shabalova L. A. , Kokarovtseva S. N. , Pukhalskaya D. A. , Kashirskaja N. J. , Simonova O. I.
Published: July 2002

Chronic endobronchial inflammation and bacterial infection are the main causes of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive genetic disorder associated with improper function of chloride channels. Inflammation in CF lung is greatly amplified after Pseudomonas aeruginosa infection. In this study the relationship between P. aeruginosa status and inflammatory markers has been investigated. Seventeen CF children in acute lung exacerbation were examined. CF patients without P. aeruginosa infection were characterized by elevated activity of sputum elastase, reduced response of peripheral blood lymphocytes to PHA and significant resistance to the antiproliferative action of glucocorticoids. These parameters were normalized after antibiotic treatment. The patients with prolonged P. aeruginosa infection demonstrated extremely high levels of elastase activity and elevated amounts of sputum IL‐8 and TNF‐α. Although antibiotic treatment resulted in clinical improvement, it failed to suppress excessive immune response in the lung. The data indicate that CF patients with prolonged P. aeruginosa need the modified treatment, which should include immunomodulating drugs and protease inhibitors as well as antibacterial therapy.
A Role for the M9 Transport Signal of hnRNP A1 in mRNA Nuclear Export
Authors: Izaurralde Elisa , Jarmolowski Artur , Beisel Christina , Mattaj Iain W. , Dreyfuss Gideon , Fischer Utz
Published: July 2002

Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.
The Lumenal Domain of Sec63p Stimulates the ATPase Activity of BiP and Mediates BiP Recruitment to the Translocon in Saccharomyces cerevisiae
Authors: Corsi Ann K. , Schekman Randy
Published: July 2002

We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S–transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.
In Vivo Analysis of the Major Exocytosis-sensitive Phosphoprotein in Tetrahymena
Authors: Chilcoat N. Doane , Turkewitz Aaron P.
Published: July 2002

Phosphoglucomutase (PGM) is a ubiquitous highly conserved enzyme involved in carbohydrate metabolism. A number of recently discovered PGM-like proteins in a variety of organisms have been proposed to function in processes other than metabolism. In addition, sequence analysis suggests that several of these may lack PGM enzymatic activity. The best studied PGM-like protein is parafusin, a major phosphoprotein in the ciliate Paramecium tetraurelia that undergoes rapid and massive dephosphorylation when cells undergo synchronous exocytosis of their dense-core secretory granules. Indirect genetic and biochemical evidence also supports a role in regulated exocytotic membrane fusion. To examine this matter directly, we have identified and cloned the parafusin homologue in Tetrahymena thermophila, a ciliate in which protein function can be studied in vivo. The unique T. thermophila gene, called PGM1, encodes a protein that is closely related to parafusin by sequence and by characteristic post-translational modifications. Comparison of deduced protein sequences, taking advantage of the known atomic structure of rabbit muscle PGM, suggests that both ciliate enzymes and all other PGM-like proteins have PGM activity. We evaluated the activity and function of PGM1 through gene disruption. Surprisingly, ΔPGM1 cells displayed no detectable defect in exocytosis, but showed a dramatic decrease in PGM activity. Both our results, and reinterpretation of previous data, suggest that any potential role for PGM-like proteins in regulated exocytosis is unlikely to precede membrane fusion.
Assembly of the Yeast Vacuolar H+-ATPase Occurs in the Endoplasmic Reticulum and Requires a Vma12p/Vma22p Assembly Complex
Authors: Graham Laurie A. , Hill Kathryn J. , Stevens Tom H.
Published: July 2002

Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex. Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex. Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of ∼5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane. Analysis of these protein–protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked. Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway.
Clb5-associated Kinase Activity is Required Early in the Spindle Pathway for Correct Preanaphase Nuclear Positioning in Saccharomyces cerevisiae
Authors: Segal Marisa , Clarke Duncan J. , Reed Steven I.
Published: July 2002

In Saccharomyces cerevisiae, a single cyclin-dependent kinase, Cdc28, regulates both G1/S and G2/M phase transitions by associating with stage-specific cyclins. During progression through S phase and G2/M, Cdc28 is activated by the B-type cyclins Clb1–6. Because of functional redundancy, specific roles for individual Clbs have been difficult to assign. To help genetically define such roles, strains carrying a cdc28ts allele, combined with single CLB deletions were studied. We assumed that by limiting the activity of the kinase, these strains would be rendered more sensitive to loss of individual Clbs.

By this approach, a novel phenotype associated with CLB5 mutation was observed. Homozygous cdc28-4ts clb5 diploids were inviable at room temperature. Cells were defective in spindle positioning, leading to migration of undivided nuclei into the bud. Occasionally, misplaced spindles were observed in cdc28-4 clb5 haploids; additional deletion of CLB6 caused full penetrance. Thus, CLB5 effects proper preanaphase spindle positioning, yet the requirement differs in haploids and diploids. The execution point for the defect corresponded to the time of Clb5-dependent kinase activation. Nevertheless, lethality of cdc28-4 clb5 diploids was not rescued by CLB2 or CLB4 overexpression, indicating a specificity of Clb5 function beyond temporality of expression.
Drosophila Polo Kinase Is Required for Cytokinesis
Authors: Carmena Mar , Riparbelli Maria Giovanna , Minestrini Gianluca , Tavares Álvaro M. , Adams Richard , Callaini Giuliano , Glover David M.
Published: July 2002

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.
Paralemmin, a Prenyl-Palmitoyl–anchored Phosphoprotein Abundant in Neurons and Implicated in Plasma Membrane Dynamics and Cell Process Formation
Authors: Kutzleb Christian , Sanders Gabriele , Yamamoto Raina , Wang Xiaolu , Lichte Beate , Petrasch-Parwez Elisabeth , Kilimann Manfred W.
Published: July 2002

We report the identification and initial characterization of paralemmin, a putative new morphoregulatory protein associated with the plasma membrane. Paralemmin is highly expressed in the brain but also less abundantly in many other tissues and cell types. cDNAs from chicken, human, and mouse predict acidic proteins of 42 kD that display a pattern of sequence cassettes with high inter-species conservation separated by poorly conserved linker sequences. Prenylation and palmitoylation of a COOH-terminal cluster of three cysteine residues confers hydrophobicity and membrane association to paralemmin. Paralemmin is also phosphorylated, and its mRNA is differentially spliced in a tissue-specific and developmentally regulated manner. Differential splicing, lipidation, and phosphorylation contribute to electrophoretic heterogeneity that results in an array of multiple bands on Western blots, most notably in brain. Paralemmin is associated with the cytoplasmic face of the plasma membranes of postsynaptic specializations, axonal and dendritic processes and perikarya, and also appears to be associated with an intracellular vesicle pool. It does not line the neuronal plasmalemma continuously but in clusters and patches. Its molecular and morphological properties are reminiscent of GAP-43, CAP-23, and MARCKS, proteins implicated in plasma membrane dynamics. Overexpression in several cell lines shows that paralemmin concentrates at sites of plasma membrane activity such as filopodia and microspikes, and induces cell expansion and process formation. The lipidation motif is essential for this morphogenic activity. We propose a function for paralemmin in the control of cell shape, e.g., through an involvement in membrane flow or in membrane–cytoskeleton interaction.
Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B
Authors: Arregui Carlos O. , Balsamo Janne , Lilien Jack
Published: July 2002

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.
The Cell Adhesion Molecule L1 Is Developmentally Regulated in the Renal Epithelium and Is Involved in Kidney Branching Morphogenesis
Authors: Debiec Hanna , Christensen Erik Ilsø , Ronco Pierre Marie
Published: July 2002

We immunopurified a surface antigen specific for the collecting duct (CD) epithelium. Microsequencing of three polypeptides identified the antigen as the neuronal cell adhesion molecule L1, a member of the immunoglobulin superfamily. The kidney isoform showed a deletion of exon 3. L1 was expressed in the mesonephric duct and the metanephros throughout CD development. In the adult CD examined by electron microscopy, L1 was not expressed on intercalated cells but was restricted to CD principal cells and to the papilla tall cells. By contrast, L1 appeared late in the distal portion of the elongating nephron in the mesenchymally derived epithelium and decreased during postnatal development. Immunoblot analysis showed that expression, proteolytic cleavage, and the glycosylation pattern of L1 protein were regulated during renal development. L1 was not detected in epithelia of other organs developing by branching morphogenesis. Addition of anti-L1 antibody to kidney or lung organotypic cultures induced dysmorphogenesis of the ureteric bud epithelium but not of the lung. These results suggest a functional role for L1 in CD development in vitro. We further postulate that L1 may be involved in the guidance of developing distal tubule and in generation and maintenance of specialized cell phenotypes in CD.
Ponsin/SH3P12: An l-Afadin– and Vinculin-binding Protein Localized at Cell–Cell and Cell–Matrix Adherens Junctions
Authors: Mandai Kenji , Nakanishi Hiroyuki , Satoh Ayako , Takahashi Kenichi , Satoh Keiko , Nishioka Hideo , Mizoguchi Akira , Takai Yoshimi
Published: July 2002

We recently isolated a novel actin filament (F-actin)–binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell–cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin–binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin–binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell–cell AJ in nonepithelial cells, we attempted here to identify an l-afadin–binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin–binding protein localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin– and vinculin-binding protein localized at ZA in epithelial cells, at cell–cell AJ in nonepithelial cells, and at cell–matrix AJ in both types of cells.
p120ctn Acts as an Inhibitory Regulator of Cadherin Function in Colon Carcinoma Cells
Authors: Aono Shinya , Nakagawa Shinichi , Reynolds Albert B. , Takeichi Masatoshi
Published: July 2002

p120ctn binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin–dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120ctn to a faster position. To investigate whether p120ctn plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120ctn deletion constructs. Notably, expression of NH2-terminally deleted p120ctn induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120ctn-binding site was introduced into Colo 205 cells, this molecule also induced cell aggregation, indicating that cadherins can function normally if they do not bind to p120ctn. These findings suggest that in Colo 205 cells, a signaling mechanism exists to modify a biochemical state of p120ctn and the modified p120ctn blocks the cadherin system. The NH2 terminus–deleted p120ctn appears to compete with the endogenous p120ctn to abolish the adhesion-blocking action.
Intracellular Distribution of Mammalian Protein Kinase a Catalytic Subunit Altered by Conserved Asn2 Deamidation
Authors: Pepperkok Rainer , Hotz-Wagenblatt Agnes , König Norbert , Girod Andreas , Bossemeyer Dirk , Kinzel Volker
Published: July 2002

The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH2-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. König, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457–469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Cα and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH2-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.
PNG1, a Yeast Gene Encoding a Highly Conserved Peptide:N-Glycanase
Authors: Suzuki Tadashi , Park Hangil , Hollingsworth Nancy M. , Sternglanz Rolf , Lennarz William J.
Published: July 2002

It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.
Molecular Basis for Rab Prenylation
Authors: Alory Christelle , Balch William E.
Published: July 2002

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.
Pivotal role of VASP in Arp2/3 complex–mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility
Authors: Skoble Justin , Auerbuch Victoria , Goley Erin D. , Welch Matthew D. , Portnoy Daniel A.
Published: July 2002

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.
Specific tetraspanin functions
Authors: Hemler Martin E.
Published: July 2002

Relatively little attention has been given to the large family of abundantly expressed transmembrane proteins known as tetraspanins. Now, the importance of tetraspanins is strongly supported by emerging genetic evidence, coupled with new insights into the biochemistry and functions of tetraspanin protein complexes.
Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate
Authors: Lefebvre Christophe , Terret M. Emilie , Djiane Alexandre , Rassinier Pascale , Maro Bernard , Verlhac Marie-Hélène
Published: July 2002

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/…/MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/…/MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase–interacting and spindle-stabilizing protein).
Downregulation of CD1 Marks Acquisition of Functional Maturation of Human Thymocytes and Defines a Control Point in Late Stages of Human T Cell Development
Authors: Res Pieter , Blom Bianca , Hori Toshiyuki , Weijer Kees , Spits Hergen
Published: July 2002

We have investigated whether in the human thymus transition of CD4+CD8+ double positive (DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both SP and DP thymocytes correlates with downregulation of CD1a. CD1a− cells with a persistent DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of mature DP cells can emigrate from the thymus. The requirements for generating functional T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II– positive, but not MHC class II–negative, mouse thymic microenvironments support differentiation of human progenitors into TCRαβ+CD4+ SP cells, indicating that mouse MHC class II can positively select TCRαβ+CD4+ SP human cells. Strikingly, these SP are arrested in the CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes differentiate to expandable CD1a− cells upon cocultivation with human thymic stromal cells. Taken together these data indicate that when CD1a+ DP TCRαβlow cells mature, these cells interact with MHC, but that an additional, apparently species-specific, signal is required for downregulation of CD1a to generate functional mature TCRαβ+ cells.
Tyrosine Phosphorylation of Pyk2 Is Selectively Regulated by Fyn During TCR Signaling
Authors: Qian Dapeng , Lev Sima , Oers Nicolai S.C. van , Dikic Ivan , Schlessinger Joseph , Weiss Arthur
Published: July 2002

The Src family protein tyrosine kinases (PTKs), Lck and Fyn, are coexpressed in T cells and perform crucial functions involved in the initiation of T cell antigen receptor (TCR) signal transduction. However, the mechanisms by which Lck and Fyn regulate TCR signaling are still not completely understood. One important question is whether Lck and Fyn have specific targets or only provide functional redundancy during TCR signaling. We have previously shown that Lck plays a major role in the tyrosine phosphorylation of the TCR-ζ chain and the ZAP-70 PTK. In an effort to identify the targets that are specifically regulated by Fyn, we have studied the tyrosine phosphorylation of Pyk2, a recently discovered new member of the focal adhesion kinase family PTK. We demonstrated that Pyk2 was rapidly tyrosine phosphorylated following TCR stimulation. TCR-induced tyrosine phosphorylation of Pyk2 was selectively dependent on Fyn but not Lck. Moreover, in heterologous COS-7 cells, coexpression of Pyk2 with Fyn but not Lck resulted in substantial increases in Pyk2 tyrosine phosphorylation. The selective regulation of Pyk2 tyrosine phosphorylation by Fyn in vivo correlated with the preferential phosphorylation of Pyk2 by Fyn in vitro. Our results demonstrate that Pyk2 is a specific target regulated by Fyn during TCR signaling.
Activity and Phenotype of Natural Killer Cells in Peptide Transporter (TAP)-deficient Patients (Type I Bare Lymphocyte Syndrome)
Authors: Zimmer Jacques , Donato Lionel , Hanau Daniel , Cazenave Jean-Pierre , Tongio Marie-Marthe , Moretta Alessandro , Salle Henri de la
Published: July 2002

In this paper we describe the function and phenotype of natural killer (NK) lymphocytes from HLA class I–deficient patients. These cells are, as has been previously reported, unable to lyse HLA class I− K562 cells, but are able to perform antibody-dependent cellular cytotoxicity (ADCC), although with lower efficiency as compared to NK cells from normal individuals. Transporter associated to antigen processing (TAP)− NK cells proliferate when cultured in the presence of lymphoblastoid B cells (B-LCs) and interleukin 2 and develop a spectrum of cytotoxicity similar to that of activated normal NK cells. Importantly, activation of the TAP− NK cells induces strong cytotoxicity to autologous B-LCs. Analysis of the phenotype of circulating TAP− NK lymphocytes showed them to display a normal diverse repertoire of HLA class I–specific NK receptors. These receptors were expressed at normal levels, apart from the CD94–NKG2A complex, which appeared to be overexpressed. This latter finding could reflect an adaptation to the low expression of HLA class I molecules. Finally, functional analyses indicated that the inhibitory receptors in TAP− individuals can transduce inhibitory signals. Our results suggest that in vivo, the NK cells of TAP− patients could participate in immune defense, at least through ADCC, but upon activation, may be involved in autoimmune processes.
B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement
Authors: Bleul Conrad C. , Schultze Joachim L. , Springer Timothy A.
Published: July 2002

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.
Regulation of Anti-DNA B Cells in Recombination-activating Gene–deficient Mice
Authors: Xu Hui , Li Hui , Suri-Payer Elisabeth , Hardy Richard R. , Weigert Martin
Published: July 2002

Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti–double-stranded (ds)DNA and anti–single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B cells seem to be functionally silenced. Here we have investigated how anti-DNA B cells are regulated in recombination- activating gene (RAG)-2−/− mice. In this setting, anti-dsDNA B cells are eliminated by apoptosis in the bone marrow and anti-ssDNA B cells are partially activated.