bluuebunny/crossref_metadata_2025 huggingface dataset:
Singapore's Little India: A Tourist Attraction as a Contested Landscape
Authors: Chang T.C.
Published: July 2002

This paper explores Singapore's Little India historic district as an example of a contested urban landscape. Specifically, it argues that Little India is a site of struggle between 'insider' and 'outsider' groups. Using primarily Relph's notion of 'insideness' and 'outsideness', and other concepts dealing with spatial resistance and domination, different groups of people with differing degrees of attachment to Little India are identified. The insider-outsider cleavage is interrogated from three perspectives: the relationship between tourists and locals; ethnic tensions between Indian and Chinese communities; and, interaction between planners and users of the urban landscape. In exploring the myriad insider-outsider dynamics, it is contended that who represents an 'insider' and who is considered an 'outsider' is open to negotiation. This is because different people possess differing conceptions of 'inside ness', in turn giving rise to varying senses of attachment and belonging to place. This paper critiques existing tourism writings which focus predominantly on the relationship between tourists and locals, and it argues that in any tourist destination the tourist-local conflict is only one aspect of a much larger struggle over place. For this reason, urban tourism studies must focus on the wider arena in which the tourist-local interaction is set.
Parental influences on Chinese literacy development: A comparison of preschoolers in Beijing, Hong Kong, and Singapore
Authors: Li Hui , Rao Nirmala
Published: July 2002

Parents of 480 Chinese preschoolers in Beijing, Hong Kong, and Singapore reported on their involvement in literacy teaching, the home literacy environment, and their beliefs about language learning. The preschoolers, ranging in age from 2 to 6 years, completed the Preschool and Primary Chinese Literacy Scale (PPCLS), in individual sessions. Results indicated significant age and societal differences on the total PPCLS score and also on the following subscales: Character Identification, Visual and Auditory Discrimination, and Word Recognition. In all three societies, older children outperformed younger children on these subscales. Preschoolers from Hong Kong and Singapore did significantly better than those from Beijing. Despite sociocultural variations (e.g., status of the Chinese language, government directives regarding the age at which to start literacy teaching, documented goals of the preschool curriculum), which contributed to societal differences, home literacy education significantly contributed to the prediction of Chinese literacy attainment in Beijing, Hong Kong, and Singapore.
Attachment security and adjustment to school in middle childhood
Authors: Granot David , Mayseless Ofra
Published: July 2002

The concurrent association between security of attachment and adaptive functioning at school in middle childhood was examined. A sample of 113 children of 4th and 5th grade ” lled out a self-report measure of attachment security (Kerns, Klepac, & Cole, 1996) and were administered the Doll Story Completion task (Bretherton, Ridgeway, & Cassidy, 1990b) modi” ed for use with children in middle childhood to assess the quality and the security of attachment-related representations of the relationship with the mother. According to the latter measure children were classi” ed as secure, avoidant, ambivalent, or disorganised with regard to attachment. Their teachers completed several questionnaires assessing each child’s academic achievement, emotional and social adjustment, and frequency of behavioural problems. In addition, each participating class underwent a sociometric procedure. Findings based on correlations and comparisons of attachment groups indicated that secure children showed better adjustment to school as reflected in teachers’ reports of scholastic, emotional, social, and behavioural adjustment, as well as in peer-rated social status. Avoidant and disorganised children showed the poorest adjustment. Findings indicated the usefulness of attachment theory in understanding adjustment to the school environment in middle childhood.
Optimism and pessimism in children: A study of parenting correlates
Authors: Hasan Naheed , Power Thomas G.
Published: July 2002

The present study investigated the relation between mothers’ generalised expectancies, mothers’ self-reported parenting practices, and their children’s optimism, pessimism, and depressive symptoms. A community sample of 81 children, 8–12 years old, and their mothers participated. Questionnaires were used to assess four parenting dimensions (control, structure, support, and autonomy granting), as well as mothers’ and children’s optimism, pessimism, and depressive symptoms. Results showed that maternal pessimism correlated with child pessimism, and that maternal depressive symptoms correlated negatively with child optimism. Multiple regressions indicated that mothers who were moderately controlling had children who showed the most optimism, whereas those who allowed their children little autonomy in problem solving had children with the highest level of depressive symptoms.
Residential Treatment for Dually Diagnosed Homeless Veterans: A Comparison of Program Types
Authors: Kasprow Wesley J. , Rosenheck Robert , Frisman Linda , DiLella Diane
Published: July 2002

This study compared two types of residential programs that treat dually diagnosed homeless veterans. Programs specializing in the treatment of substance abuse disorders (SA) and those programs addressing both psychiatric disorders and substance abuse problems within the same setting (DDX) were compared on (1) program characteristics, (2) clients' perceived environment, and (3) outcomes of treatment. The study was based on surveys and discharge reports from residential treatment facilities that were under contract to the Department of Veterans Affairs Health Care for Homeless Veterans program, a national outreach and case management program operating at 71 sites across the nation. Program characteristics surveys were completed by program administrators, perceived environment surveys were completed by veterans in treatment, and discharge reports were completed by VA case managers. DDX programs were characterized by lower expectations for functioning, more acceptance of problem behavior, and more accommodation for choice and privacy, relative to SA programs after adjusting for baseline differences. Dually diagnosed veterans in DDX programs perceived these programs as less controlling than SA programs, but also as having lower involvement and less practical and personal problem orientations. At discharge, a lower percentage of veterans from DDX than SA programs left without staff consultation. A higher percentage of veterans from DDX than SA programs were discharged to community housing rather than to further institutional treatment. Program effects were not different for psychotic and non‐psychotic veterans. Although differences were modest, integration of substance abuse and psychiatric treatment may promote a faster return to community living for dually diagnosed homeless veterans. Such integration did not differentially benefit dually diagnosed veterans whose psychiatric problems included a psychotic disorder.
The Dose Determines the Stimulation (and Poison): Development of A Chemical Hormesis Database
Authors: Calabrese Edward J. , Baldwin Linda A.
Published: July 2002

A comprehensive effort was undertaken to identify articles demonstrating chemical hormesis. Nearly 4000 potentially relevant articles were retrieved from preliminary computer searches utilizing various keyword descriptors and extensive cross-referencing. A priori evaluation criteria were established including study design features (e.g., number of doses, dose range), statistical analysis, and reproducibility of results. Evidence of chemical hormesis was judged to have occurred in approximately 350 of the 4000 studies evaluated. Chemical hormesis was observed in a wide range of taxonomic groups and involved agents representing highly diverse chemical classes, many of potential environmental relevance. Numerous biologic endpoints were assessed, with growth responses the most prevalent, followed by metabolic effects, longevity, reproductive responses, and survival. Hormetic responses were generally observed to be of limited magnitude with the average low-dose maximum stimulation approximately 50% greater than controls. The hormetic dose-response range was generally limited to about one order of magnitude with the upper end of the hormetic curve approaching the estimated no-observed-effect level (NOEL) for the particular endpoint. Based on the evaluation criteria, high to moderate evidence of hormesis was observed in studies comprised of ≥ doses with <3 doses in the hormetic zone. The present analysis suggests that chem ical hormesis is a reproducible and generalizable biologic phenomenon. Over the last decade advances have been made providing mechanistic insight helpful in explaining the phenomenon of chemical hormesis in multiple biologic systems with various endpoints. The reason for the uncertainty surrounding the existence of hormesis as a “real phenomenon” is believed to be the result of its relatively infrequent observation in the literature due to experimental design considerations, especially with respect to the number of doses, range of doses, and endpoint selection.
A dependency syndrome related to areca nut use: some medical and psychological aspects among areca nut users in the Gujarat community in the UK
Authors: Winstock A. R. , Trivedy C. R. , Warnakulasuriya K. A. A. S. , Peters T. J.
Published: July 2002

Use of betel nut (areca nut) and its products is widespread, particularly in the Indo‐Chinese continents, being the fourth most widely used substance after tobacco, alcohol and caffeine, affecting approximately 20% of the world's population. Betel nut, with or without admixed tobacco, is widely used among UK Indo‐Asian immigrants, particularly Gujurate speakers. To date most research has concentrated on oral submucous fibrosis and malignancy. This paper reports detailed socio‐demographic, clinical, laboratory and psychological studies in 11 current and former heavy betel nut users, referred by an Oral Medicine Unit in NW London. The patients, nine males, two females, had a high incidence of cardiovascular disease and truncal obesity. Laboratory investigations showed a high incidence of reduced serum B12 levels (4/9) and raised urinary cotinine levels (6/11), although none were current cigarette smokers. These findings are consistent with heavy usage of tobacco‐areca combinations by this group. Routine biochemical and haematological investigations and clinical examination revealed no consistent abnormalities. Subjects had used areca for an average of 35 years with the mean age of first use being 13 years. Most subjects reported beneficial psychosocial effects. Ten subjects reported cessation withdrawal effects with the mean Severity of Dependence Score of 7.3. These findings are consistent with the existence of a dependency syndrome among those who use areca nut products. Further research is required to delineate the relative contributions of areca nut and tobacco to this clinical picture. Use of the areca nut, especially with tobacco, represents an area of health prevention among the UK minority populations that has, to date, been overlooked.
GOS.SP.ASS.'98: an assessment for speech disorders associated with cleft palate and/or velopharyngeal dysfunction (revised)
Authors: Sell Debbie , Harding Anne , Grunwell Pamela
Published: July 2002

In 1994 the present authors proposed a speech assessment protocol for speech disorders associated with cleft palate and/or velopharyngeal dysfunction known as GOS.SP.ASS. (Great Ormond Street Speech Assessment). In a recent survey undertaken to review the different speech assessment protocols used in six cleft palate centres in the UK, GOS.SP.ASS. was selected from six protocols as the optimal procedure for clinical and research purposes. The process of identifying an optimal procedure involved analysis of completed forms for each assessment. Analysis of the completed GOS.SP.ASS. forms revealed significant ambiguities in the protocol which led to differences in form completion. This paper describes important revisions to the original GOS.SP.ASS. protocol in order to ensure comparable data from different clinicians. This detailed speech assessment is now complemented by the Cleft Audit Protocol for Speech (CAPS), a tool recommended for clinical audit. As a result of close collaboration in their preparation, the results are directly comparable. In addition, the speech elicitation sentences and the phonetic diagram have been modified.
The Autophagic and Endocytic Pathways Converge at the Nascent Autophagic Vacuoles
Authors: Liou Willisa , Geuze Hans J. , Geelen Math. J.H. , Slot Jan W.
Published: July 2002

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV—initial (AVi), intermediate (AVi/d), and degradative (AVd)—were defined by morphological criteria and immunogold labeling characteristics of marker enzymes.

The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV+) or fusion profiles (FP+) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV+ exhibited an exponential increase with time. FP+ between MVE or VE and all three types of AV were observed. Among the 112 FP+ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15–45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.
Interphase Nuclei of Many Mammalian Cell Types Contain Deep, Dynamic, Tubular Membrane-bound Invaginations of the Nuclear Envelope
Authors: Fricker Mark , Hollinshead Michael , White Nick , Vaux David
Published: July 2002

The nuclear envelope consists of a doublemembraned extension of the rough endoplasmic reticulum. In this report we describe long, dynamic tubular channels, derived from the nuclear envelope, that extend deep into the nucleoplasm. These channels show cell-type specific morphologies ranging from single short stubs to multiple, complex, branched structures. Some channels transect the nucleus entirely, opening at two separate points on the nuclear surface, while others terminate at or close to nucleoli. These channels are distinct from other topological features of the nuclear envelope, such as lobes or folds.

The channel wall consists of two membranes continuous with the nuclear envelope, studded with features indistinguishable from nuclear pore complexes, and decorated on the nucleoplasmic surface with lamins. The enclosed core is continuous with the cytoplasm, and the lumenal space between the membranes contains soluble ER-resident proteins (protein disulphide isomerase and glucose-6-phosphatase).

Nuclear channels are also found in live cells labeled with the lipophilic dye DiOC6. Time-lapse imaging of DiOC6-labeled cells shows that the channels undergo changes in morphology and spatial distribution within the interphase nucleus on a timescale of minutes.

The presence of a cytoplasmic core and nuclear pore complexes in the channel walls suggests a possible role for these structures in nucleo–cytoplasmic transport. The clear association of a subset of these structures with nucleoli would also be consistent with such a transport role.
Bee1, a Yeast Protein with Homology to Wiscott-Aldrich Syndrome Protein, Is Critical for the Assembly of Cortical Actin Cytoskeleton
Authors: Li Rong
Published: July 2002

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.
Centrosomes Isolated from Spisula solidissima Oocytes Contain Rings and an Unusual Stoichiometric Ratio of α/β Tubulin
Authors: Vogel Jacalyn M. , Stearns Tim , Rieder Conly L. , Palazzo Robert E.
Published: July 2002

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures ∼25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase–specific phosphoepitopes (MPM-2), (b) α-, β-, and γ-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. γ-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little α- or β-tubulin copurified with centrosomes, and the ratio of α- to β-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain β-tubulin that is not dimerized with α-tubulin.
Telomeres Cluster De Novo before the Initiation of Synapsis: A Three-dimensional Spatial Analysis of Telomere Positions before and during Meiotic Prophase
Authors: Bass Hank W. , Marshall Wallace F. , Sedat John W. , Agard David A. , Cande W. Zacheus
Published: July 2002

We have analyzed the progressive changes in the spatial distribution of telomeres during meiosis using three-dimensional, high resolution fluorescence microscopy. Fixed meiotic cells of maize (Zea mays L.) were subjected to in situ hybridization under conditions that preserved chromosome structure, allowing identification of stage-dependent changes in telomere arrangements. We found that nuclei at the last somatic prophase before meiosis exhibit a nonrandom, polarized chromosome organization resulting in a loose grouping of telomeres. Quantitative measurements on the spatial arrangements of telomeres revealed that, as cells passed through premeiotic interphase and into leptotene, there was an increase in the frequency of large telomere-to-telomere distances and a decrease in the bias toward peripheral localization of telomeres. By leptotene, there was no obvious evidence of telomere grouping, and the large, singular nucleolus was internally located, nearly concentric with the nucleus. At the end of leptotene, telomeres clustered de novo at the nuclear periphery, coincident with a displacement of the nucleolus to one side. The telomere cluster persisted throughout zygotene and into early pachytene. The nucleolus was adjacent to the cluster at zygotene. At the pachytene stage, telomeres rearranged again by dispersing throughout the nuclear periphery. The stagedependent changes in telomere arrangements are suggestive of specific, active telomere-associated motility processes with meiotic functions. Thus, the formation of the cluster itself is an early event in the nuclear reorganizations associated with meiosis and may reflect a control point in the initiation of synapsis or crossing over.
High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A
Authors: Ayscough Kathryn R. , Stryker Joel , Pokala Navin , Sanders Miranda , Crews Phil , Drubin David G.
Published: July 2002

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2–5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site.

LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actindependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.
Chlamydomonas Inner-Arm Dynein Mutant, ida5, Has a Mutation in an Actin-encoding Gene
Authors: Kato-Minoura Takako , Hirono Masafumi , Kamiya Ritsu
Published: July 2002

Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5′ end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = ∼5.3) but had two novel spots with pIs of ∼5.6 and ∼5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional actin gene, it is likely that the novel spots in ida5 and ida5-t originated from another gene(s) that codes for a novel actin-like protein(s) (NAP), which has hitherto been undetected in wildtype cells. These mutants retain the two inner-arm subspecies b and g, in addition to f, possibly because NAP can functionally substitute for the actin in these subspecies while they cannot in other subspecies. The net growth rate of ida5 and ida5-t cells did not differ from that of wild type, but the mating efficiency was greatly reduced. This defect was apparently caused by deficient growth of the fertilization tubule. These results suggest that NAP can carry out some, but not all, functions performed by conventional actin in the cytoplasm and raise the possibility that Chlamydomonas can live without ordinary actin.
Sec2p Mediates Nucleotide Exchange on Sec4p and Is Involved in Polarized Delivery of Post-Golgi Vesicles
Authors: Walch-Solimena Christiane , Collins Ruth N. , Novick Peter J.
Published: July 2002

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.
Dynamics of β-Catenin Interactions with APC Protein Regulate Epithelial Tubulogenesis
Authors: Pollack Anne L. , Barth Angela I.M. , Altschuler Yoram , Nelson W. James , Mostov Keith E.
Published: July 2002

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, β-catenin, with several molecular partners have been shown to be important for cell signaling and cell–cell adhesion. To examine if β-catenin has a role in tubulogenesis, we tested the effect of expressing NH2-terminal deleted β-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH2-terminal deleted β-catenins inhibited formation of these cell extensions. Both ΔN90 β-catenin, which binds to α-catenin, and ΔN131 β-catenin, which does not bind to α-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of β-catenin distinct from its role in cadherin-mediated cell–cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH2-terminal deleted β-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH2-terminal deleted β-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for β-catenin in cell migration and indicate that dynamic β-catenin–APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.
A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization
Authors: Arkowitz Robert A. , Lowe Nick
Published: July 2002

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.
Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase Cγ
Authors: Carroll David J. , Ramarao Chodavarapu S. , Mehlmann Lisa M. , Roche Serge , Terasaki Mark , Jaffe Laurinda A.
Published: July 2002

Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.
Actomyosin-based Retrograde Flow of Microtubules in the Lamella of Migrating Epithelial Cells Influences Microtubule Dynamic Instability and Turnover and Is Associated with Microtubule Breakage and Treadmilling
Authors: Waterman-Storer Clare M. , Salmon E.D.
Published: July 2002

We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally “pioneering” MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 μm/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione–2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that ∼80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.
Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction
Authors: Mandai Kenji , Nakanishi Hiroyuki , Satoh Ayako , Obaishi Hiroshi , Wada Manabu , Nishioka Hideo , Itoh Masahiko , Mizoguchi Akira , Aoki Takeo , Fujimoto Toyoshi , Matsuda Yoichi , Tsukita Shoichiro , Takai Yoshimi
Published: July 2002

A novel actin filament (F-actin)–binding protein with a molecular mass of ∼205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.
Targeting of NH2-terminal–processed Microsomal Protein to Mitochondria: A Novel Pathway for the Biogenesis of Hepatic Mitochondrial P450MT2
Authors: Addya Sankar , Anandatheerthavarada Hindupur K. , Biswas Gopa , Bhagwat Shripad V. , Mullick Jayati , Avadhani Narayan G.
Published: July 2002

Cytochrome P4501A1 is a hepatic, microsomal membrane–bound enzyme that is highly induced by various xenobiotic agents. Two NH2-terminal truncated forms of this P450, termed P450MT2a and MT2b, are also found localized in mitochondria from β-naphthoflavone–induced livers. In this paper, we demonstrate that P4501A1 has a chimeric NH2-terminal signal that facilitates the targeting of the protein to both the ER and mitochondria. The NH2-terminal 30–amino acid stretch of P4501A1 is thought to provide signals for ER membrane insertion and also stop transfer. The present study provides evidence that a sequence motif immediately COOH-terminal (residues 33–44) to the transmembrane domain functions as a mitochondrial targeting signal under both in vivo and in vitro conditions, and that the positively charged residues at positions 34 and 39 are critical for mitochondrial targeting. Results suggest that 25% of P4501A1 nascent chains, which escape ER membrane insertion, are processed by a liver cytosolic endoprotease. We postulate that the NH2-terminal proteolytic cleavage activates a cryptic mitochondrial targeting signal. Immunofluorescence microscopy showed that a portion of transiently expressed P4501A1 is colocalized with the mitochondrial-specific marker protein cytochrome oxidase subunit I. The mitochondrial-associated MT2a and MT2b are localized within the inner membrane compartment, as tested by resistance to limited proteolysis in both intact mitochondria and mitoplasts. Our results therefore describe a novel mechanism whereby proteins with chimeric signal sequence are targeted to the ER as well as to the mitochondria.
Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton
Authors: Vaduva Gabriela , Martin Nancy C. , Hopper Anita K.
Published: July 2002

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.
Periplakin, a Novel Component of Cornified Envelopes and Desmosomes That Belongs to the Plakin Family and Forms Complexes with Envoplakin
Authors: Ruhrberg Christiana , Hajibagheri M.A. Nasser , Parry David A.D. , Watt Fiona M.
Published: July 2002

The cornified envelope is a layer of transglutaminase cross-linked protein that is assembled under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We have determined the cDNA sequence of one of the proteins that becomes incorporated into the cornified envelope of cultured epidermal keratinocytes, a protein with an apparent molecular mass of 195 kD that is encoded by a mRNA with an estimated size of 6.3 kb. The protein is expressed in keratinizing and nonkeratinizing stratified squamous epithelia and in a number of other epithelia. Expression of the protein is upregulated during the terminal differentiation of epidermal keratinocytes in vivo and in culture. Immunogold electron microscopy was used to demonstrate an association of the 195-kD protein with the desmosomal plaque and with keratin filaments in the differentiated layers of the epidermis. Sequence analysis showed that the 195-kD protein is a member of the plakin family of proteins, to which envoplakin, desmoplakin, bullous pemphigoid antigen 1, and plectin belong. Envoplakin and the 195-kD protein coimmunoprecipitate. Analysis of their rod domain sequences suggests that the formation of both homodimers and heterodimers would be energetically favorable. Confocal immunofluorescent microscopy of cultured epidermal keratinocytes revealed that envoplakin and the 195-kD protein form a network radiating from desmosomes, and we speculate that the two proteins may provide a scaffolding onto which the cornified envelope is assembled. We propose to name the 195-kD protein periplakin.
Kar9p Is a Novel Cortical Protein Required for Cytoplasmic Microtubule Orientation in Yeast
Authors: Miller Rita K. , Rose Mark D.
Published: July 2002

kar9 was originally identified as a bilateral karyogamy mutant, in which the two zygotic nuclei remained widely separated and the cytoplasmic microtubules were misoriented (Kurihara, L.J., C.T. Beh, M. Latterich, R. Schekman, and M.D. Rose. 1994. J. Cell Biol. 126:911–923.). We now report a general defect in nuclear migration and microtubule orientation in kar9 mutants. KAR9 encodes a novel 74-kD protein that is not essential for life. The kar9 mitotic defect was similar to mutations in dhc1/dyn1 (dynein heavy chain gene), jnm1, and act5. kar9Δ dhc1Δ, kar9Δ jnm1Δ, and kar9Δ act5Δ double mutants were synthetically lethal, suggesting that these genes function in partially redundant pathways to carry out nuclear migration. A functional GFP-Kar9p fusion protein localized to a single dot at the tip of the shmoo projection. In mitotic cells, GFP-Kar9p localized to a cortical dot with both mother–daughter asymmetry and cell cycle dependence. In small-budded cells through anaphase, GFP-Kar9p was found at the tip of the growing bud. In telophase and G1 unbudded cells, no localization was observed. By indirect immunofluorescence, cytoplasmic microtubules intersected the GFP-Kar9p dot. Nocodazole experiments demonstrated that Kar9p's cortical localization was microtubule independent. We propose that Kar9p is a component of a cortical adaptor complex that orients cytoplasmic microtubules.
NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival
Authors: Scatena Marta , Almeida Manuela , Chaisson Michelle L. , Fausto Nelson , Nicosia Roberto F. , Giachelli Cecilia M.
Published: July 2002

The αvβ3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β3 integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β1-integrin ligand) did not induce NF-κB activity. The αvβ3 integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β3 integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in αvβ3 integrin-mediated endothelial cell survival.
The Src Homology Domain 3 (SH3) of a Yeast Type I Myosin, Myo5p, Binds to Verprolin and Is Required for Targeting to Sites of Actin Polarization
Authors: Anderson Blake L. , Boldogh Istvan , Evangelista Marie , Boone Charles , Greene Lloyd A. , Pon Liza A.
Published: July 2002

The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Δ myo5Δ cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A–induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37°C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Δ myo5Δ cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Δ myo5Δ cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.