bluuebunny/crossref_metadata_2025 huggingface dataset:
Historicizing Slavery in West Indian Feminisms
Authors: Beckles Hilary McD.
Published: July 2002

This paper traces the evolution of a coherent feminist genre in written historical texts during and after slavery, and in relation to contemporary feminist writing in the West Indies. The paper problematizes the category ‘woman’ during slavery, arguing that femininity was itself deeply differentiated by class and race, thus leading to historical disunity in the notion of feminine identity during slavery. This gender neutrality has not been sufficiently appreciated in contemporary feminist thought leading to liberal feminist politics in the region. This has proved counter productive in the attempts of Caribbean feminist theorizing to provide alternative understandings of the construction of the nation-state as it emerged out of slavery and the role of women themselves in the shaping of modern Caribbean society.
Women's Organizations and Movements in the Commonwealth Caribbean: The Response to Global Economic Crisis in the 1980s
Authors: Reddock Rhoda
Published: July 2002

In this paper I explore the emergence of women's organizations and feminist consciousness in the twentieth century in the English-speaking (Commonwealth) Caribbean. The global ideas concerning women's equality from the 1960s onwards clearly informed the initiatives taken by both women and states of the Caribbean. None the less, the paper illustrates, by use of examples, the interlocked nature of women's struggles with the economic, social and political issues which preoccupy the region's population. I examine in greater detail two case studies of women's activism and mobilization around the impact of structural adjustment policies in the two territories of Jamaica and Trinidad and Tobago. By tracing the connections between and among the organizations and initiatives of women in the region, the paper situates the feminist movement in the English-speaking Caribbean as a continuously evolving one, fusing episodic struggles in different territories, engaging women of different classes and groups, and continuously building on past experience.
Understanding Women in Scotland
Authors: Breitenbach Esther , Brown Alice , Myers Fiona
Published: July 2002

This article explores obstacles to understanding the history and contemporary experiences of women in Scotland, and to the development of feminist research in Scotland. It is argued that explanations which invoke Scottish male chauvinism and misogyny alone are insufficient, and that the marginalization of women in Scotland is produced both by male domination within Scotland, and by English cultural and political hegemony within the UK. The article comments on the relationship of the concept of ‘Britishness’ to that of ‘Scottishness’ (and other identities within the UK) and illustrates how the frequent confusion of ‘British’ with ‘English’ serves to obscure Scottish experience. It is also argued that the place of Scotland within the British state has led to the creation of an institutional framework that disadvantages women, and a system of government that excludes women. This implies that feminist debates on the state in Britain require a specific focus on the form of the British state, and in the context of constitutional change in particular this is important for the development of future strategies. It is argued that the double marginalization of women in Scotland is not just a problem in relation to the development of feminist research, but is also a political problem in that it contributes to a degree of alienation from feminism in England. The article concludes by arguing for the necessity of recognition of difference, but also for dialogue, as the basis for feminist alliances in different parts of the UK.
Kindergarten Disruptive Behaviour, Family Adversity, Gender, and Elementary School Failure
Authors: Mâsse Louise C. , Tremblay Richard E.
Published: July 2002

Objective: To investigate the developmental pathway of school failure from age 8 to age 11 by gender when the main effects and interactions of family adversity and the level of kindergarten disruptive behaviour were accounted for. Method: A population sample of boys ( n = 862) and girls ( n = 985) were followed from kindergarten up to age 11. Family adversity and disruptive behaviour were measured in kindergarten and school failure was assessed from ages 8 to 11. Results: A logit-linear analysis on the patterns of school failure revealed that there was no significant difference in the proportion of boys and girls transferred from an age-appropriate regular class by the end of elementary school. However, the age at which this transfer occurred differed by gender: Girls were found to experience failure later than boys do. The results also indicated that both family adversity and kindergarten disruptive behaviour had an effect on the proportion and age at which children were transferred from an age-appropriate regular class. Conclusion: Findings underline that the academic performance and delinquency relationship may be developmentally different by gender. Further research focused on understanding the impact of school failure by gender is needed to clarify these issues.
Predicting the Implementation Effort of Erp Projects: Empirical Evidence on SAP/R3
Authors: Francalanci Chiara
Published: July 2002

This paper investigates the impact of the technical size and organizational complexity of SAP/R3 projects on implementation effort. Traditional models for predicting software implementation effort tie measures of code size and programming complexity to development time. ERP projects shift a significant proportion of the implementation effort from code development to the parameterization of a pre-existing software package. At the same time, they move complexity from technical to organizational factors since they force companies to adapt to predefined work processes embedded in the software. This paper redefines the concepts of size and complexity for ERP projects and empirically verifies their impact on implementation effort. Specifically, project size is measured in terms of the number of SAP modules and submodules that are implemented, while complexity is defined as the organizational scope of the project in terms of users involved and overall company size. Hypotheses are tested on 43 SAP/R3 projects conducted in a cross-section of manufacturing companies. The findings show that both the technical size and organizational complexity of projects are relevant drivers of implementation effort. The results indicate that implementation effort not only grows with the number of modules and submodules that are selected for implementation, but that SAP is found to require increasing resources to be implemented in larger companies and for a higher number of users, thus indicating that, while there is a technical component of effort that is independent of the organizational breadth of the project, each user adds an organizational component of costs.
A novel anti‐inflammatory activity of lysozyme: modulation of serum complement activation
Authors: Ogundele M. O.
Published: July 2002

Lysozyme is an ubiquitous enzyme found in most biological secretions and leukocytes. This study was aimed at investigating its interaction with other inflammatory mediators on mucosa surfaces, particularly the complement system. Lysozyme has been shown in our present study, to inhibit the haemolytic activity of serum complement in a dose‐dependent fashion, when tested within the levels present in normal and inflamed breast‐milk samples, and other mucosal secretions. This represents a new antiinflammatory action of lysozym e in relation to the serum complement, and the exact mode of the interaction need further studies.
A Modern Post-Mortem on Böhm's Capital Theory: Its Vital Normative Flaw Shared by Presraffian Mainstream Capital Theory
Authors: Samuelson Paul A.
Published: July 2002

The Nobel Prize of Piero Sraffa and Joan Robinson that Stockholm never awarded might have pleased at least one of them. Its citation would have included: “Their investigations uncovered a fatal normative flaw in Böhm-Bawerkian and modern mainstream capital theory.”

Just prior to Alfred Marshall's 1890 ascendancy as leading world economist, Eugen von Böhm-Bawerk (1851–1914) perhaps wore that crown thanks to his three-volume treatise on the history and fundamentals of interest theories. Böhm (1884, 1889, 1909, 1912) somewhat independently followed in the footsteps of Stanley Jevons (1871) and himself strongly stimulated Knut Wicksell (1893), Irving Fisher (1906, 1907, 1930), and Friedrich Hayek (1931, 1941). Pugnacious and somewhat incoherent, Böhm and his disciples battled cogently the competing school of John Bates Clark (1899) and Frank Knight (1934, 1935a, 1935b), which idealized a permanentscalarcapital alleged to be virtually permanent and with a marginal productivity determining its interest rate in much the same way that primary labor's marginal productivity determines its real wage rate and primary land's marginal productivity determines its real rent rate(s). The Clark-Knight paradigm—and, for that matter, Frank Ramsey's 1928 mathematical clone—shares the Böhm-Hayek vital normative flaw.
Establishment of the Dorso-ventral Axis in Xenopus Embryos Is Presaged by Early Asymmetries in β-Catenin That Are Modulated by the Wnt Signaling Pathway
Authors: Larabell Carolyn A. , Torres Monica , Rowning Brian A. , Yost Cynthia , Miller Jeffrey R. , Wu Mike , Kimelman David , Moon Randall T.
Published: July 2002

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on β-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that β-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with β-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous β-catenin. Steadystate levels and nuclear accumulation of β-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased β-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of β-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in β-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in β-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.
Transmembrane Domain Sequence Requirements for Activation of the p185c-neu Receptor Tyrosine Kinase
Authors: Chen Lucinda I. , Webster Melanie K. , Meyer April N. , Donoghue Daniel J.
Published: July 2002

The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664→ Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.
Flexibility within Myosin Heads Revealed by Negative Stain and Single-Particle Analysis
Authors: Burgess S.A. , Walker M.L. , White H.D. , Trinick J.
Published: July 2002

Electron microscopy of negatively stained myosin has previously revealed three discrete regions within the heads of the molecule. However, despite a probable resolution of ∼2 nm, it is difficult to discern directly consistent details within these regions. This is due to variability in both head conformation and in staining. In this study, we applied single-particle image processing and classified heads into homogeneous groups. The improved signal-to-noise ratio after averaging these groups reveals substantially improved detail. The image averages were compared to a model simulating negative staining of the atomic structure of subfragment-1 (S1). This shows that the three head regions correspond to the motor domain and the essential and regulatory light chains. The image averages were very similar to particular views of the S1 model. They also revealed considerable flexibility between the motor and regulatory domains, despite the molecules having been prepared in the absence of nucleotide. This flexibility probably results from rotation of the regulatory domain about the motor domain, where the relative movement of the regulatory light chain is up to 12 nm, and is most clearly illustrated in animated sequences (available at http://www.leeds.ac.uk/chb/muscle/ myosinhead.html). The sharply curved conformation of the atomic model of S1 is seen only rarely in our data, with straighter heads being more typical.
Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins
Authors: Barlowe Charles
Published: July 2002

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-α-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-α-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.
Ripped Pocket and Pickpocket, Novel Drosophila DEG/ENaC Subunits Expressed in Early Development and in Mechanosensory Neurons
Authors: Adams Christopher M. , Anderson Michael G. , Motto David G. , Price Margaret P. , Johnson Wayne A. , Welsh Michael J.
Published: July 2002

Drosophila melanogaster has proven to be a good model for understanding the physiology of ion channels. We identified two novel Drosophila DEG/ ENaC proteins, Pickpocket (PPK) and Ripped Pocket (RPK). Both appear to be ion channel subunits. Expression of RPK generated multimeric Na+ channels that were dominantly activated by a mutation associated with neurodegeneration. Amiloride and gadolinium, which block mechanosensation in vivo, inhibited RPK channels. Although PPK did not form channels on its own, it associated with and reduced the current generated by a related human brain Na+ channel. RPK transcripts were abundant in early stage embryos, suggesting a role in development. In contrast, PPK was found in sensory dendrites of a subset of peripheral neurons in late stage embryos and early larvae. In insects, such multiple dendritic neurons play key roles in touch sensation and proprioception and their morphology resembles human mechanosensory free nerve endings. These results suggest that PPK may be a channel subunit involved in mechanosensation.
Differential Stimulation of the Na+/H+ Exchanger Determines Chloroquine Uptake in Plasmodium falciparum
Authors: Wünsch Stefan , Sanchez Cecilia P. , Gekle Michael , Große-Wortmann Lars , Wiesner Jochen , Lanzer Michael
Published: July 2002

Here we describe the identification and characterization of a physiological marker that is associated with the chloroquine-resistant (CQR) phenotype in the human malarial parasite Plasmodium falciparum. Single cell in vivo pH measurements revealed that CQR parasites consistently have an elevated cytoplasmic pH compared to that of chloroquine-sensitive (CQS) parasites because of a constitutively activated Na+/H+ exchanger (NHE). Together, biochemical and physiological data suggest that chloroquine activates the plasmodial NHE of CQS parasites, resulting in a transitory phase of rapid sodium/hydrogen ion exchange during which chloroquine is taken up by this protein. The constitutively stimulated NHE of CQR parasites are capable of little or no further activation by chloroquine. We propose that the inability of chloroquine to stimulate its own uptake through the constitutively activated NHE of resistant parasites constitutes a minimal and necessary event in the generation of the chloroquine-resistant phenotype.
ARF6 Targets Recycling Vesicles to the Plasma Membrane: Insights from an Ultrastructural Investigation
Authors: D'Souza-Schorey Crislyn , van Donselaar Elly , Hsu Victor W. , Yang Chunzhi , Stahl Philip D. , Peters Peter J.
Published: July 2002

We have shown previously that the ADP- ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.
The Cohesion Protein MEI-S332 Localizes to Condensed Meiotic and Mitotic Centromeres until Sister Chromatids Separate
Authors: Moore Daniel P. , Page Andrea W. , Tang Tracy Tzu-Ling , Kerrebrock Anne W. , Orr-Weaver Terry L.
Published: July 2002

The Drosophila MEI-S332 protein has been shown to be required for the maintenance of sister-chromatid cohesion in male and female meiosis. The protein localizes to the centromeres during male meiosis when the sister chromatids are attached, and it is no longer detectable after they separate. Drosophila melanogaster male meiosis is atypical in several respects, making it important to define MEI-S332 behavior during female meiosis, which better typifies meiosis in eukaryotes. We find that MEI-S332 localizes to the centromeres of prometaphase I chromosomes in oocytes, remaining there until it is delocalized at anaphase II. By using oocytes we were able to obtain sufficient material to investigate the fate of MEI-S332 after the metaphase II–anaphase II transition. The levels of MEI-S332 protein are unchanged after the completion of meiosis, even when translation is blocked, suggesting that the protein dissociates from the centromeres but is not degraded at the onset of anaphase II. Unexpectedly, MEI-S332 is present during embryogenesis, localizes onto the centromeres of mitotic chromosomes, and is delocalized from anaphase chromosomes. Thus, MEI-S332 associates with the centromeres of both meiotic and mitotic chromosomes and dissociates from them at anaphase.
Association of the Myosin-binding Subunit of Myosin Phosphatase and Moesin: Dual Regulation of Moesin Phosphorylation by Rho-associated Kinase and Myosin Phosphatase
Authors: Fukata Yuko , Kimura Kazushi , Oshiro Noriko , Saya Hideyuki , Matsuura Yoshiharu , Kaibuchi Kozo
Published: July 2002

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho- kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at the free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and inactivates Rho. MBS was colocalized with moesin at the cell–cell contact sites in MDCK cells. We also found that moesin was coimmunoprecipitated with MBS from MDCK cells. Recombinant MBS interacted with the amino-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moesin, which was phosphorylated by Rho-kinase. The phosphatase activity was inhibited when MBS was phosphorylated by Rho-kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho.
G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton
Authors: Peracino Barbara , Borleis Jane , Jin Tian , Westphal Monika , Schwartz Jean-Marc , Wu Lijun , Bracco Enrico , Gerisch Günther , Devreotes Peter , Bozzaro Salvatore
Published: July 2002

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.
Mechanical Fluctuations of the Membrane–Skeleton Are Dependent on F-Actin ATPase in Human Erythrocytes
Authors: Tuvia Shmuel , Levin Shlomo , Bitler Arkady , Korenstein Rafi
Published: July 2002

Cell membrane fluctuations (CMF) of human erythrocytes, measured by point dark field microscopy, were shown to depend, to a large extent, on intracellular MgATP (Levin, S.V., and R. Korenstein. 1991. Biophys. J. 60:733–737). The present study extends that investigation and associates CMF with F-actin's ATPase activity. MgATP was found to reconstitute CMF in red blood cell (RBC) ghosts and RBC skeletons to their levels in intact RBCs, with an apparent Kd of 0.29 mM. However, neither non-hydrolyzable ATP analogues (AMP-PNP, ATPγS) nor hydrolyzable ones (ITP, GTP), were able to elevate CMF levels. The inhibition of ATPase activity associated with the RBC's skeleton, carried out either by the omission of the MgATP substrate or by the use of several inhibitors (vanadate, phalloidin, and DNase I), resulted in a strong decrease of CMF. We suggest that the actin's ATPase, located at the pointed end of the short actin filament, is responsible for the MgATP stimulation of CMF in RBCs.
Spindle Checkpoint Protein Xmad1 Recruits Xmad2 to Unattached Kinetochores
Authors: Chen Rey-Huei , Shevchenko Andrej , Mann Matthias , Murray Andrew W.
Published: July 2002

The spindle checkpoint prevents the metaphase to anaphase transition in cells containing defects in the mitotic spindle or in chromosome attachment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentration fails to restore the checkpoint, suggesting that other checkpoint component(s) were depleted from the extract through their association with Xmad2. Mass spectrometry provided peptide sequences from an 85-kD protein that coimmunoprecipitates with Xmad2 from egg extracts. This information was used to clone XMAD1, which encodes a homologue of the budding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint in egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and to those kinetochores that are not bound to spindle microtubules during mitosis. Adding an anti-Xmad1 antibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xmad2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to recruit Xmad2 to kinetochores that have not bound microtubules.
An Unconventional Role for Cytoplasmic Disulfide Bonds in Vaccinia Virus Proteins
Authors: Locker Jacomine Krijnse , Griffiths Gareth
Published: July 2002

Previous data have shown that reducing agents disrupt the structure of vaccinia virus (vv). Here, we have analyzed the disulfide bonding of vv proteins in detail. In vv-infected cells cytoplasmically synthesized vv core proteins became disulfide bonded in the newly assembled intracellular mature viruses (IMVs). vv membrane proteins also assembled disulfide bonds, but independent of IMV formation and to a large extent on their cytoplasmic domains. If disulfide bonding was prevented, virus assembly was only partially impaired as shown by electron microscopy as well as a biochemical assay of IMV formation. Under these conditions, however, the membranes around the isolated particles appeared less stable and detached from the underlying core. During the viral infection process the membrane proteins remained disulfide bonded, whereas the core proteins were reduced, concomitant with delivery of the cores into the cytoplasm. Our data show that vv has evolved an unique system for the assembly of cytoplasmic disulfide bonds that are localized both on the exterior and interior parts of the IMV.
Localization of Core Spindle Pole Body (SPB) Components during SPB Duplication in Saccharomyces cerevisiae
Authors: Adams Ian R. , Kilmartin John V.
Published: July 2002

We have examined the process of spindle pole body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear envelope. We analyzed the role of the main SPB components in the formation of these structures by identifying them from an SPB core fraction by mass spectrometry. Temperature-sensitive mutants for two of the components, Spc29p and Nud1p, were prepared to partly define their function. The composition of two of the intermediates in SPB duplication, the satellite and the duplication plaque, was examined by immunoelectron microscopy. Both contain cytoplasmic SPB components showing that duplication has already been partly achieved by the end of the preceding cell cycle when the satellite is formed. We show that by overexpression of SPB components the structure of the satellite can be changed and SPB duplication inhibited by disrupting the attachment of the plaque-like intermediate to the half-bridge. We present a model for SPB duplication where binding of SPB components to either end of the bridge structure ensures two separate SPBs.
Cdc25b and Cdc25c Differ Markedly in Their Properties as Initiators of Mitosis
Authors: Karlsson Christina , Katich Stephanie , Hagting Anja , Hoffmann Ingrid , Pines Jonathon
Published: July 2002

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.
Hcp-1, a Protein Involved in Chromosome Segregation, Is Localized to the Centromere of Mitotic Chromosomes in Caenorhabditis elegans
Authors: Moore Landon L. , Morrison Mike , Roth Mark B.
Published: July 2002

To learn more about holocentric chromosome structure and function, we generated a monoclonal antibody (mAb), 6C4, that recognizes the poleward face of mitotic chromosomes in Caenorhabditis elegans. Early in mitosis, mAb 6C4 stains dots throughout the nucleoplasm. Later in prophase, mAb 6C4 stains structures on opposing faces of chromosomes which orient towards the centrosomes at metaphase. Colocalization with an antibody against a centromeric histone H3–like protein and the MPM-2 antibody, which identifies a kinetochore-associated phosphoepitope present in a variety of organisms, shows that the mAb 6C4 staining is present adjacent to the centromere.

Expression screening using mAb 6C4 identified a protein in C. elegans that we named HCP-1 (for holocentric protein 1). We also identified a second protein from the C. elegans genome sequence database, HCP-2, that is 54% similar to HCP-1. When expression of HCP-1 is reduced by RNA interference (RNAi), staining with mAb 6C4 is eliminated, indicating that hcp-1 encodes the major mAb 6C4 antigen. RNAi with hcp-1 and hcp-2 together results in aberrant anaphases and embryonic arrest at ∼100 cells with different amounts of DNA in individual nuclei. These results suggest that HCP-1 is a centromere-associated protein that is involved in the fidelity of chromosome segregation.
Cingulin Contains Globular and Coiled-Coil Domains and Interacts with Zo-1, Zo-2, Zo-3, and Myosin
Authors: Cordenonsi Michelangelo , D'Atri Fabio , Hammar Eva , Parry David A.D. , Kendrick-Jones John , Shore David , Citi Sandra
Published: July 2002

We characterized the sequence and protein interactions of cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (Kd ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.
Regulation of Actin Filament Cross-linking and Bundle Shape in Drosophila Bristles
Authors: Tilney Lewis G. , Connelly Patricia S. , Vranich Kelly A. , Shaw Michael K. , Guild Gregory M.
Published: July 2002

Previous studies demonstrate that in developing Drosophila bristles, two cross-linking proteins are required sequentially to bundle the actin filaments that support elongating bristle cells. The forked protein initiates the process and facilitates subsequent cross-linking by fascin. Using cross-linker–specific antibodies, mutants, and drugs we show that fascin and actin are present in excessive amounts throughout bundle elongation. In contrast, the forked cross-linker is limited throughout bundle formation, and accordingly, regulates bundle size and shape. We also show that regulation of cross-linking by phosphorylation can affect bundle size. Specifically, inhibition of phosphorylation by staurosporine results in a failure to form large bundles if added during bundle formation, and leads to a loss of cross-linking by fascin if added after the bundles form. Interestingly, inhibition of dephosphorylation by okadaic acid results in the separation of the actin bundles from the plasma membrane. We further show by thin section electron microscopy analysis of mutant and wild-type bristles that the amount of material that connects the actin bundles to the plasma membrane is also limited throughout bristle elongation. Therefore, overall bundle shape is determined by the number of actin filaments assembled onto the limited area provided by the connector material. We conclude that assembly of actin bundles in Drosophila bristles is controlled in part by the controlled availability of a single cross-linking protein, forked, and in part by controlled phosphorylation of cross-links and membrane actin connector proteins.
A Direct Interaction of Axonin-1 with Ngcam-Related Cell Adhesion Molecule (Nrcam) Results in Guidance, but Not Growth of Commissural Axons
Authors: Fitzli Dora , Stoeckli Esther T. , Kunz Stefan , Siribour Kingsley , Rader Christoph , Kunz Beat , Kozlov Serguei V. , Buchstaller Andrea , Lane Robert P. , Suter Daniel M. , Dreyer William J. , Sonderegger Peter
Published: July 2002

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti–axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.
The Unfolded Protein Response Regulates Multiple Aspects of Secretory and Membrane Protein Biogenesis and Endoplasmic Reticulum Quality Control
Authors: Ng Davis T.W. , Spear Eric D. , Walter Peter
Published: July 2002

The unfolded protein response (UPR) is an intracellular signaling pathway that relays signals from the lumen of the ER to activate target genes in the nucleus. We devised a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutants that are dependent on activation of the pathway for viability. Using this strategy, we isolated mutants affecting various aspects of ER function, including protein translocation, folding, glycosylation, glycosylphosphatidylinositol modification, and ER-associated protein degradation (ERAD). Extending results gleaned from the genetic studies, we demonstrate that the UPR regulates trafficking of proteins at the translocon to balance the needs of biosynthesis and ERAD. The approach also revealed connections of the UPR to other regulatory pathways. In particular, we identified SON1/RPN4, a recently described transcriptional regulator for genes encoding subunits of the proteasome. Our genetic strategy, therefore, offers a powerful means to provide insight into the physiology of the UPR and to identify novel genes with roles in many aspects of secretory and membrane protein biogenesis.
Tenascin-C Suppresses Rho Activation
Authors: Wenk Melissa B. , Midwood Kim S. , Schwarzbauer Jean E.
Published: July 2002

Cell binding to extracellular matrix (ECM) components changes cytoskeletal organization by the activation of Rho family GTPases. Tenascin-C, a developmentally regulated matrix protein, modulates cellular responses to other matrix proteins, such as fibronectin (FN). Here, we report that tenascin-C markedly altered cell phenotype on a three-dimensional fibrin matrix containing FN, resulting in suppression of actin stress fibers and induction of actin-rich filopodia. This distinct morphology was associated with complete suppression of the activation of RhoA, a small GTPase that induces actin stress fiber formation. Enforced activation of RhoA circumvented the effects of tenascin. Effects of active Rho were reversed by a Rho inhibitor C3 transferase. Suppression of GTPase activation allows tenascin-C expression to act as a regulatory switch to reverse the effects of adhesive proteins on Rho function. This represents a novel paradigm for the regulation of cytoskeletal organization by ECM.
Dral Is a P53-Responsive Gene Whose Four and a Half Lim Domain Protein Product Induces Apoptosis
Authors: Scholl Florence A. , McLoughlin Patricia , Ehler Elisabeth , de Giovanni Carla , Schäfer Beat W.
Published: July 2002

DRAL is a four and a half LIM domain protein identified because of its differential expression between normal human myoblasts and the malignant counterparts, rhabdomyosarcoma cells. In the current study, we demonstrate that transcription of the DRAL gene can be stimulated by p53, since transient expression of functional p53 in rhabdomyosarcoma cells as well as stimulation of endogenous p53 by ionizing radiation in wild-type cells enhances DRAL mRNA levels. In support of these observations, five potential p53 target sites could be identified in the promoter region of the human DRAL gene. To obtain insight into the possible functions of DRAL, ectopic expression experiments were performed. Interestingly, DRAL expression efficiently triggered apoptosis in three cell lines of different origin to the extent that no cells could be generated that stably overexpressed this protein. However, transient transfection experiments as well as immunofluorescence staining of the endogenous protein allowed for the localization of DRAL in different cellular compartments, namely cytoplasm, nucleus, focal contacts, as well as Z-discs and to a lesser extent the M-bands in cardiac myofibrils. These data suggest that downregulation of DRAL might be involved in tumor development. Furthermore, DRAL expression might be important for heart function.
Engineering the Processive Run Length of the Kinesin Motor
Authors: Thorn Kurt S. , Ubersax Jeffrey A. , Vale Ronald D.
Published: July 2002

Conventional kinesin is a highly processive molecular motor that takes several hundred steps per encounter with a microtubule. Processive motility is believed to result from the coordinated, hand-over-hand motion of the two heads of the kinesin dimer, but the specific factors that determine kinesin's run length (distance traveled per microtubule encounter) are not known. Here, we show that the neck coiled-coil, a structure adjacent to the motor domain, plays an important role in governing the run length. By adding positive charge to the neck coiled-coil, we have created ultra-processive kinesin mutants that have fourfold longer run lengths than the wild-type motor, but that have normal ATPase activity and motor velocity. Conversely, adding negative charge on the neck coiled-coil decreases the run length. The gain in processivity can be suppressed by either proteolytic cleavage of tubulin's negatively charged COOH terminus or by high salt concentrations. Therefore, modulation of processivity by the neck coiled-coil appears to involve an electrostatic tethering interaction with the COOH terminus of tubulin. The ability to readily increase kinesin processivity by mutation, taken together with the strong sequence conservation of the neck coiled-coil, suggests that evolutionary pressures may limit kinesin's run length to optimize its in vivo function.
In Vitro Assembly and Structure of Trichocyte Keratin Intermediate Filaments
Authors: Wang He , Parry David A.D. , Jones Leslie N. , Idler William W. , Marekov Lyuben N. , Steinert Peter M.
Published: July 2002

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from “microfibrils.” These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.
Metaphase to Anaphase (mat) Transition–Defective Mutants inCaenorhabditis elegans
Authors: Golden Andy , Sadler Penny L. , Wallenfang Matthew R. , Schumacher Jill M. , Hamill Danielle R. , Bates Gayle , Bowerman Bruce , Seydoux Geraldine , Shakes Diane C.
Published: July 2002

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants “mat” for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.