bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular‐level pursuit of yeast mitosis by time‐ and space‐resolved Raman spectroscopy
Authors: Huang Yu‐San , Karashima Takeshi , Yamamoto Masayuki , Hamaguchi Hiro‐o
Published: January 2003

The process of eukaryotic cell division has been studied at the molecular level by Raman spectroscopy. Time‐ and space‐resolved Raman spectra of a living fission yeast cell were successfully recorded with a 100 s time resolution and a 250 nm spatial resolution by using a confocal multi‐channel Raman microspectrometer. Raman spectra characteristic of the nucleus, mitochondrion and septum were identified and time‐dependent changes of major molecular components during the cell cycle were elucidated. Copyright © 2002 John Wiley & Sons, Ltd.
Comparative essential oil composition of various parts of the turpentine tree (Pistacia terebinthus L) growing wild in Turkey
Authors: Couladis M , Özcan M , Tzakou O , Akgül A
Published: January 2003

The essential oil contents (dry weight basis) of young shoots, flowers, unripe and ripe fruits of the turpentine tree (Pistacia terebinthus L) were determined as 0.74, 0.70, 0.54 and 0.73% respectively. The main components identified by GC/MS analysis (the identified components represents 90.7, 96.1, 98.0 and 99.0% of the respective oils) were limonene (3.0, 9.4, 34.2 and 32.8%), α‐pinene (5.3, 12.4, 15.6 and 5.3%), β‐pinene (1.4, 8.0, 11.5 and 22.5%) and germacrene D (trace, 19.9, 3.5 and 4.6%). Minor qualitative and major quantitative variations in some compounds of the analysed oils were determined with respect to the different parts of the turpentine tree.

© 2002 Society of Chemical Industry
Effect of acetylation on the functional properties of lentil (Lens culinaris) globulin
Authors: Bora Pushkar Singh
Published: January 2003

The functional properties of native and acetylated (62.5, 84.1, 90.1 and 93.0%) globulins were evaluated. Acetylation caused a shift in isoelectric pH from 4.5 to 3.5. The solubility of acetylated globulins improved considerably above pH 4.0 but deteriorated below this pH. Increasing acetylation enhanced water absorption (from 1.08 to 1.67, 1.96, 2.29 and 2.33 ml g−1) but reduced oil absorption capacity (from 2.61 to 2.17, 2.15, 2.05 and 1.76 ml g−1). In comparison with native globulin the relative viscosity of acetylated globulins also increased, but with an increase in acetylation level there was a decrease in relative viscosity (2.13, 1.81, 1.65 and 1.62). The emulsifying activity of 62.5% acetylated globulin increased from 54.2% for native globulin to 56.0%, but above this level of acetylation the activity decreased. Emulsion stability followed a similar behaviour. The foaming capacity of native globulin was improved by 62.5% acetylation but reduced at higher acetylation levels. Maximum foam stability was observed for 90.1% acetylated globulin.

© 2002 Society of Chemical Industry
Adaptive QoS parameters approach to modeling Internet performance
Authors: Yang Shin‐Jer , Chou Hung‐Cheng
Published: January 2003

Due to the recent advances in Internet technologies and applications, the issue of Quality of Service (QoS) is more essential to Internet performance. In this paper, we address and discuss the influence factors and also finalize the QoS parameters for Internet performance. Then we present the simulation procedure for monitoring the performance evaluation and propose the algorithm for tuning the performance value. Based on simulation results and performance analysis, we can tune and adjust possible parameters' values and then refine the influence level of each QoS parameter. Finally, Internet performance can be enhanced and maintained through using the proposed approach over several sub‐networks. Hence, it increases the adaptability for implementing the QoS Internet. Copyright © 2002 John Wiley & Sons, Ltd.
The structure of self‐reported problem behaviors in Chinese children
Authors: Xin Tao , Zhou Zheng , Bray Melissa A. , Kehle Thomas J.
Published: January 2003

This study examined the structure of self‐reported problem behaviors in Mainland Chinese children. Third grade (n = 3011) and fifth grade (n = 3708) students completed a 20‐item Problem Behavior Frequency Checklist (PBFC). Findings suggested that among the four factors studied (physical aggression, substance use, delinquency, and self‐centered behaviors), self‐centered behaviors and physical aggression were reported most frequently by the Chinese elementary school children. Furthermore, boys had higher prevalence rates of problem behaviors than girls. Developmental differences were also present. Confirmatory factor analyses supported a model that included specific factors related to substance use, physical aggression, delinquency, and self‐centered behaviors. Therefore, the single factor conceptualization in which different types of problem behaviors are hypothesized to reflect a single underlying common factor may not hold for this population. © 2003 Wiley Periodicals, Inc. Psychol Schs 40: 19–33, 2003.
Chinese children's self‐concepts in the domains of learning and social relations
Authors: Wang Qi , Li Jin
Published: January 2003

In both academia and pop psychology, the Chinese self is often characterized as collectivist, putting group interest above self‐interest. This account of self misconstrues the complex nature of human psychosocial functioning in cultural contexts. This account further overlooks the dynamic process in which the self is formed and sustained. In this article, we propose, from a domain‐specific perspective, to reexamine the construct of self and its development in Chinese children. By presenting others' and our own research, we analyze Chinese children's self‐concept in two distinct domains—the domain of learning and the domain of social relations. We demonstrate that Chinese children's self‐concept has a strong autonomous component in the context of learning and achievement while displaying a social orientation in the context of relationships and family. We show how these tendencies of Chinese self relate to Confucian ethics and how they are shaped by early socialization. We then discuss implications of these findings for theories of the self. © 2003 Wiley Periodicals, Inc. Psychol Schs 40: 85–101, 2003.
PTPα regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration
Authors: Zeng Li , Si Xiaoning , Yu Wei-Ping , Le Hoa Thi , Ng Kwok Peng , Teng Raymond M.H. , Ryan Kenneth , Wang Dennis Z.-M. , Ponniah Sathivel , Pallen Catherine J.
Published: January 2003

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) α in integrin signaling using immortalized fibroblasts derived from wild-type and PTPα-deficient mouse embryos. Defects in PTPα−/− migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTPα−/− cells was increased by expression of active (but not inactive) PTPα. Integrin-mediated formation of src–FAK and fyn–FAK complexes was reduced or abolished in PTPα−/− cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTPα restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTPα−/− cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTPα−/− fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTPα functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTPα is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.
Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1
Authors: Innocenti Metello , Frittoli Emanuela , Ponzanelli Isabella , Falck John R. , Brachmann Saskia M. , Di Fiore Pier Paolo , Scita Giorgio
Published: January 2003

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.
A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy
Authors: Monani Umrao R. , Pastore Matthew T. , Gavrilina Tatiana O. , Jablonka Sibylle , Le Thanh T. , Andreassi Catia , DiCocco Jennifer M. , Lorson Christian , Androphy Elliot J. , Sendtner Michael , Podell Michael , Burghes Arthur H.M.
Published: January 2003

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.
Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis
Authors: Ricci Jean-Ehrland , Gottlieb Roberta A. , Green Douglas R.
Published: January 2003

During apoptosis, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which induces caspase activation to orchestrate the death of the cell. Mitochondria rapidly lose their transmembrane potential (ΔΨm) and generate reactive oxygen species (ROS), both of which are likely to contribute to the dismantling of the cell. Here we show that both the rapid loss of ΔΨm and the generation of ROS are due to the effects of activated caspases on mitochondrial electron transport complexes I and II. Caspase-3 disrupts oxygen consumption induced by complex I and II substrates but not that induced by electron transfer to complex IV. Similarly, ΔΨm generated in the presence of complex I or II substrates is disrupted by caspase-3, and ROS are produced. Complex III activity measured by cytochrome c reduction remains intact after caspase-3 treatment. In apoptotic cells, electron transport and oxygen consumption that depends on complex I or II was disrupted in a caspase-dependent manner. Our results indicate that after cytochrome c release the activation of caspases feeds back on the permeabilized mitochondria to damage mitochondrial function (loss of ΔΨm) and generate ROS through effects of caspases on complex I and II in the electron transport chain.
Imaging kinase–AKAP79–phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy
Authors: Oliveria Seth F. , Gomez Lisa L. , Dell'Acqua Mark L.
Published: January 2003

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A–kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton–localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 Å of each other, thus providing the first direct evidence of a ternary kinase–scaffold–phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.
Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway
Authors: Goshima Gohta , Kiyomitsu Tomomi , Yoda Kinya , Yanagida Mitsuhiro
Published: January 2003

Kinetochores are the chromosomal sites for spindle interaction and play a vital role for chromosome segregation. The composition of kinetochore proteins and their cellular roles are, however, poorly understood in higher eukaryotes. We identified a novel kinetochore protein family conserved from yeast to human that is essential for equal chromosome segregation. The human homologue hMis12 of yeast spMis12/scMtw1 retains conserved sequence features and locates at the kinetochore region indistinguishable from CENP-A, a centromeric histone variant. RNA interference (RNAi) analysis of HeLa cells shows that the reduced hMis12 results in misaligned metaphase chromosomes, lagging anaphase chromosomes, and interphase micronuclei without mitotic delay, while CENP-A is located at kinetochores. Further, the metaphase spindle length is abnormally extended. Spindle checkpoint protein hMad2 temporally localizes at kinetochores at early mitotic stages after RNAi. The RNAi deficiency of CENP-A leads to a similar mitotic phenotype, but the kinetochore signals of other kinetochore proteins, hMis6 and CENP-C, are greatly diminished. RNAi for hMis6, like that of a kinetochore kinesin CENP-E, induces mitotic arrest. Kinetochore localization of hMis12 is unaffected by CENP-A RNAi, demonstrating an independent pathway of CENP-A in human kinetochores.
Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane
Authors: Kaufmann Thomas , Schlipf Sarah , Sanz Javier , Neubert Karin , Stein Reuven , Borner Christoph
Published: January 2003

It is assumed that the survival factors Bcl-2 and Bcl-xL are mainly functional on mitochondria and therefore must contain mitochondrial targeting sequences. Here we show, however, that only Bcl-xL is specifically targeted to the mitochondrial outer membrane (MOM) whereas Bcl-2 distributes on several intracellular membranes. Mitochondrial targeting of Bcl-xL requires the COOH-terminal transmembrane (TM) domain flanked at both ends by at least two basic amino acids. This sequence is a bona fide targeting signal for the MOM as it confers specific mitochondrial localization to soluble EGFP. The signal is present in numerous proteins known to be directed to the MOM. Bcl-2 lacks the signal and therefore localizes to several intracellular membranes. The COOH-terminal region of Bcl-2 can be converted into a targeting signal for the MOM by increasing the basicity surrounding its TM. These data define a new targeting sequence for the MOM and propose that Bcl-2 acts on several intracellular membranes whereas Bcl-xL specifically functions on the MOM.
Critical Role of the Carboxyl Terminus of Proline-rich Tyrosine Kinase (Pyk2) in the Activation of Human Neutrophils by Tumor Necrosis Factor
Authors: Han Hyunsil , Fuortes Michele , Nathan Carl
Published: January 2003

Transduction of Tat-tagged fusion proteins confirmed a hypothesis based on pharmacologic inhibitors (Fuortes, M., M. Melchior, H. Han, G.J. Lyon, and C. Nathan. 1999. J. Clin. Invest. 104:327–335) that proline-rich tyrosine kinase (Pyk2) plays a critical role in the activation of adherent human neutrophils, and allowed an analysis of individual Pyk2 domains not possible with chemical inhibitors. Acting as a dominant negative, the COOH terminus of Pyk2 fused to a Tat peptide (Tat-CT), but not other regions of Pyk2, specifically inhibited the respiratory burst of cells responding to tumor necrosis factor (TNF), Salmonella, or Listeria, while sparing responses induced by phorbol ester. Tat-CT suppressed TNF-triggered cell spreading and the phosphorylation of endogenous Pyk2 and the associated tyrosine kinase Syk without blocking the ability of neutrophils to degranulate and kill bacteria. Thus, separate signals control the respiratory burst and degranulation, and a normal rate of killing of some bacteria can be sustained by granule products in conjunction with a minimal residual respiratory burst. Inhibition of select inflammatory functions without impairment of antibacterial activity may commend the Pyk2 pathway as a potential target for antiinflammatory therapy.
Negative Regulation of T Cell Receptor–Lipid Raft Interaction by Cytotoxic T Lymphocyte–associated Antigen 4
Authors: Chikuma Shunsuke , Imboden John B. , Bluestone Jeffrey A.
Published: January 2003

Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is an essential negative regulator of T cell activation. Recent evidence suggests that CTLA-4 association with the immunological synapse during contact with antigen-presenting cells is important for its inhibitory function. In the present study, we observed a direct interaction of CTLA-4 with the phosphorylated form of T cell receptor (TCR)ζ within the glycolipid-enriched microdomains associated with the T cell signaling complex. In this setting, CTLA-4 regulated the accumulation/retention of TCRζ in the signaling complex, as the lipid raft fractions from CTLA-4KO T cells contained significantly higher amounts of the TCR components when compared with wild-type littermates. In contrast, coligation of CTLA-4 with the TCR during T cell activation selectively decreased the amount of TCRζ that accumulated in the rafts. These results suggest that CTLA-4 functions to regulate T cell signaling by controlling TCR accumulation and/or retention within this a critical component of the immunological synapse.
Flexibility of Mouse Classical and Plasmacytoid-derived Dendritic Cells in Directing T Helper Type 1 and 2 Cell Development
Authors: Boonstra André , Asselin-Paturel Carine , Gilliet Michel , Crain Chad , Trinchieri Giorgio , Liu Yong-Jun , O'Garra Anne
Published: January 2003

Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the development of both Th1 and Th2 effector cells depending on the dose of antigen. In general, high antigen doses induced Th1 cell development whereas low antigen doses induced Th2 cell development. Both cultured and ex vivo splenic plasmacytoid-derived DCs enhanced CD4+ T cell proliferation and induced strong Th1 cell development when activated with the Toll-like receptor (TLR)9 ligand CpG, and not with the TLR4 ligand lipopolysaccharide (LPS). The responsiveness of plasmacytoid pDCs to CpG correlated with high TLR9 expression similarly to human plasmacytoid pDCs. Conversely, myeloid DCs generated with granulocyte/macrophage colony-stimulating factor enhanced Th1 cell development when stimulated with LPS as a result of their high level of TLR4 expression. Polarized Th1 responses resulting from high antigen dose were not additionally enhanced by stimulation of DCs by TLR ligands. Thus, the net effect of antigen dose, the state of maturation of the DCs together with the stimulation of DCs by pathogen-derived products, will determine whether a Th1 or Th2 response develops.
NOISE-INDUCED ORDER OUT OF CHAOS BY BAILOUT EMBEDDING
Authors: CARTWRIGHT JULYAN H. E. , MAGNASCO MARCELO O. , PIRO ORESTE , TUVAL IDAN
Published: January 2003

We review the concept of bailout embedding; a general process for obtaining order from chaotic dynamics by embedding the system within another larger one. Such an embedding can target islands of order and hence control chaos. Moreover, a small amount of noise enhances this process.
CELLULAR DYNAMICS IN NOISY ENVIRONMENT
Authors: SUGUNA C. , SINHA SOMDATTA
Published: January 2003

Cells are continuously subjected to noisy internal and external environments. Though cellular functions are carried on quite reliably under such noise, they can also result in fluctuations of parameters of the various biochemical processes and cause the dynamics of the reaction pathway to change. To show the varied effects of noisy environment on cellular dynamics, we consider a model cell containing a minimal biochemical pathway that exhibits a wide array of dynamical behaviors – from equilibrium, simple limit cycles and higher periodics, birhythmicity, complex oscillations and chaos - that are observed in many cellular functions. We show that, even under constant parameters, small fluctuations in the variables (i.e., substrate concentrations) can facilitate switching of the dynamics between oscillatory states with very different amplitudes and frequencies. The final temporal behavior of the pathway is unpredictable for the range of parameters where birhythmicity is observed. Parametric noise can mask the original dynamics of the pathway in the birhythmic state, though considerable robustness in dynamics is seen in the presence of noise for other values of parameters. Thus we show that the cellular dynamics can exhibit both robust and non-robust behavior in noisy environment for different parameters in the same pathway.
CAPITAL REDISTRIBUTION BRINGS WEALTH BY PARRONDO'S PARADOX
Authors: TORAL RAÚL
Published: January 2003

We present new versions of the Parrondo's paradox by which a losing game can be turned into winning by including a mechanism that allows redistribution of the capital amongst an ensemble of players. This shows that, for this particular class of games, redistribution of the capital is beneficial for everybody. The same conclusion arises when the redistribution goes from the richer players to the poorer.
Scientific Time and Temporal Experience: Pragmatism's Contribution to the Search for a Synthesis
Authors: Rosenthal Sandra
Published: January 2003

The problem of the relation between lived temporal experience and scientific time is an ongoing philosophical issue which has led to numerous and well entrenched radical solutions that in one way or another sever human temporal experience from the time of the universe. This presentation will offer a bird's eye view of the roots of the philosophical problem both historically and as it manifests itself today in major positions or movements that contour the contemporary philosophical landscape. It will then sketch the path to a possible solution from the perspective of classical American pragmatism, the philosophical movement encompassing the writings of Charles Peirce, William James, John Dewey, C.I. Lewis, and G.H. Mead. For pragmatism the roots of the problem are ultimately located in the understanding of time as discrete and confusions among the mathematical time of physical science, the time of the universe, and lived temporal experience.
Functional ablation of the mouseLdb1gene results in severe patterning defects during gastrulation
Authors: Mukhopadhyay Mahua , Teufel Andreas , Yamashita Tsuyoshi , Agulnick Alan D. , Chen Lan , Downs Karen M. , Schindler Alice , Grinberg Alexander , Huang Sing-Ping , Dorward David , Westphal Heiner
Published: January 2003

The LIM domain-binding protein 1 (Ldb1) is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA and Otx transcription factors. These proteins exert key functions during embryogenesis. Here we show that targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype. There is no heart anlage and head structures are truncated anterior to the hindbrain. In about 40% of the mutants, posterior axis duplication is observed. There are also severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells and the amnion. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in Ldb1 mutant embryos. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning regulated by Ldb1.
Evidence for a direct functional antagonism of the selector genesproboscipediaandeyelessinDrosophilahead development
Authors: Benassayag Corinne , Plaza Serge , Callaerts Patrick , Clements Jason , Romeo Yves , Gehring Walter J. , Cribbs David L.
Published: January 2003

Diversification of Drosophila segmental and cellular identities both require the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selectorproboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that we used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated EY proteins. Apart from eye loss, these new eyeless alleles lead to defects in the adult olfactory appendages: the maxillary palps and antennae. In support of these observations, both ey and pb are expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either PB or EY proteins. A combination of in vivo and in vitro approaches indicates that PB suppresses EY transactivation activity via protein-protein contacts of the PB homeodomain and EY Paired domain. The direct functional antagonism between PB and EY proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis.
The promoter targeting sequence facilitates and restricts a distant enhancer to a single promoter in theDrosophilaembryo
Authors: Lin Qing , Wu Di , Zhou Jumin
Published: January 2003

Transcriptional enhancers in large gene complexes activate promoters over huge distances, yet little is known about the mechanism of these long-range interactions. We report that the promoter targeting sequence (PTS) from theAbdominal-B locus of the Drosophila bithorax complex facilitates the activity of a distantly located enhancer in transgenic embryos and that it restricts the enhancer to a single promoter. These functions are heritable in all successive generations. We also show that the PTS functions only when itself and an insulator are located between the enhancer and the promoter. These findings suggest that the PTS may facilitate long-range enhancer-promoter interactions in the endogenous Abdominal-B locus. We propose that the PTS establishes a stable chromatin structure between an enhancer and a promoter, which facilitates yet restricts an enhancer to a single promoter.
Pax1 and Pax9 activateBapx1to induce chondrogenic differentiation in the sclerotome
Authors: Rodrigo Isabel , Hill Robert E. , Balling Rudi , Münsterberg Andrea , Imai Kenji
Published: January 2003

We have previously shown that the paired-box transcription factors Pax1 and Pax9 synergistically act in the proper formation of the vertebral column. Nevertheless, downstream events of the Pax1/Pax9 action and their target genes remain to be elucidated. We show, by analyzing Pax1;Pax9 double mutant mice, that expression of Bapx1 in the sclerotome requires the presence of Pax1 and Pax9, in a gene dose-dependent manner. By using a retroviral system to overexpress Pax1 in chick presomitic mesoderm explants, we show that Pax1 can substitute for Shh in inducing Bapx1expression and in initiating chondrogenic differentiation. Furthermore, we demonstrate that Pax1 and Pax9 can transactivate regulatory sequences in theBapx1 promoter and that they physically interact with theBapx1 promoter region. These results strongly suggest thatBapx1 is a direct target of Pax1 and Pax9. Together, we conclude that Pax1 and Pax9 are required and sufficient for the chondrogenic differentiation of sclerotomal cells.
The chickoligozeugodactyly(ozd) mutant lacks sonic hedgehog function in the limb
Authors: Ros Maria A. , Dahn Randall D. , Fernandez-Teran Marian , Rashka Kay , Caruccio Nicholas C. , Hasso Sean M. , Bitgood J. James , Lancman Joseph J. , Fallon John F.
Published: January 2003

We have analyzed a new limb mutant in the chicken that we nameoligozeugodactyly (ozd). The limbs of this mutant have a longitudinal postaxial defect, lacking the posterior element in the zeugopod(ulna/fibula) and all digits except digit 1 in the leg. Classical recombination experiments show that the limb mesoderm is the defective tissue layer in ozd limb buds. Molecular analysis revealed that theozd limbs develop in the absence of Shh expression, while all other organs express Shh and develop normally. NeitherPtc1 nor Gli1 are detectable in mutant limb buds. However,Bmp2 and dHAND are expressed in the posterior wing and leg bud mesoderm, although at lower levels than in normal embryos. Activation ofHoxd11-13 occurs normally in ozd limbs but progressively declines with time. Phase III of expression is more affected than phase II,and expression is more severely affected in the more 5′ genes. Interestingly, re-expression of Hoxd13 occurs at late stages in the distal mesoderm of ozd leg buds, correlating with formation of digit 1. Fgf8 and Fgf4 expression are initiated normally in the mutant AER but their expression is progressively downregulated in the anterior AER. Recombinant Shh protein or ZPA grafts restore normal pattern toozd limbs; however, retinoic acid fails to induce Shh in ozdlimb mesoderm. We conclude that Shh function is required for limb development distal to the elbow/knee joints, similar to the Shh-/-mouse. Accordingly we classify the limb skeletal elements as Shh dependent or independent, with the ulna/fibula and digits other than digit 1 in the leg being Shh dependent. Finally we propose that the ozd mutation is most likely a defect in a regulatory element that controls limb-specific expression of Shh.
Transcriptional activation inDrosophilaspermatogenesis involves the mutually dependent function ofalyand a novel meiotic arrest genecookie monster
Authors: Jiang Jianqiao , White-Cooper Helen
Published: January 2003

In Drosophila spermatogenesis, meiotic cell cycle progression and cellular differentiation are linked by the function of the meiotic arrest genes. The meiotic arrest genes control differentiation by regulating the transcriptional activation of many differentiation-specific genes. The meiotic arrest genes have been subdivided into aly and can classes,based on the mechanism by which they control cell cycle progression.aly has previously been shown to encode a chromatin-associated protein. We present the identification, cloning and characterisation of a novel Drosophila meiotic arrest gene, cookie monster(comr), that has a mutant phenotype indistinguishable from that ofaly. A null mutant allele of comr is viable but male sterile. Mutant primary spermatocytes fail to initiate transcription of a large number of genes, and arrest before entry into the meiotic divisions. In adult males, expression of comr is testis specific, low levels of transcripts are detected at other stages of development. comr encodes a novel acidic protein, which is nuclear and primarily localised to regions of chromatin in primary spermatocytes. The nuclear localisation of Aly and Comr proteins are mutually dependent. Finally, we show that active RNA polymerase II is found in distinct domains in the nucleus that constitute a subset of the total Comr stained chromatin.
Mbt, aDrosophilaPAK protein, combines with Cdc42 to regulate photoreceptor cell morphogenesis
Authors: Schneeberger Daniela , Raabe Thomas
Published: January 2003

The Drosophila gene mushroom bodies tiny (mbt)encodes a putative p21-activated kinase (PAK), a family of proteins that has been implicated in a multitude of cellular processes including regulation of the cytoskeleton, cell polarisation, control of MAPK signalling cascades and apoptosis. The mutant phenotype of mbt is characterised by fewer neurones in the brain and the eye, indicating a role of the protein in cell proliferation, differentiation or survival. We show that mutations inmbt interfere with photoreceptor cell morphogenesis. Mbt specifically localises at adherens junctions of the developing photoreceptor cells. A structure-function analysis of the Mbt protein in vitro and in vivo revealed that the Mbt kinase domain and the GTPase binding domain, which specifically interacts with GTP-loaded Cdc42, are important for Mbt function. Besides regulation of kinase activity, another important function of Cdc42 is to recruit Mbt to adherens junctions. We propose a role for Mbt as a downstream effector of Cdc42 in photoreceptor cell morphogenesis.
Genetic rescue of cell number in a mouse model of microphthalmia:interactions between Chx10 and G1-phase cell cycle regulators
Authors: Green Eric S. , Stubbs Jennifer L. , Levine Edward M.
Published: January 2003

Insufficient cell number is a primary cause of failed retinal development in the Chx10 mutant mouse. To determine if Chx10 regulates cell number by antagonizing p27Kip1 activity, we generated Chx10,p27Kip1 double null mice. The severe hypocellular defect in Chx10 single null mice is alleviated in the double null, and whileChx10-null retinas lack lamination, double null retinas have near normal lamination. Bipolar cells are absent in the double null retina, a defect that is attributable to a requirement for Chx10 that is independent of p27Kip1. We find that p27Kip1 is abnormally present in progenitors of Chx10-null retinas, and that its ectopic localization is responsible for a significant amount of the proliferation defect in this microphthalmia model system. mRNA and protein expression patterns in these mice and in cyclin D1-null mice suggest that Chx10 influences p27Kip1 at a post-transcriptional level, through a mechanism that is largely dependent on cyclin D1. This is the first report of rescue of retinal proliferation in a microphthalmia model by deletion of a cell cycle regulatory gene.
A re-evaluation of the contributions of Apterous and Notch to the dorsoventral lineage restriction boundary in theDrosophilawing
Authors: Milán Marco , Cohen Stephen M.
Published: January 2003

The Drosophila limb primordia are subdivided into compartments:cell populations that do not mix during development. The wing is subdivided into dorsal (D) and ventral (V) compartments by the activity of the selector gene apterous in D cells. Apterous causes segregation of D and V cell populations by at least two distinct mechanisms. The LRR transmembrane proteins Capricious and Tartan are transiently expressed in D cells and contribute to initial segregation of D and V cells. Signaling between D and V cells mediated by Notch and Fringe contributes to the maintenance of the DV affinity boundary. Given that Notch is activated symmetrically, in D and V cells adjacent to the boundary, its role in boundary formation remains somewhat unclear. We re-examine the roles of Apterous and Fringe activities in DV boundary formation and present evidence that Fringe cannot, by itself,generate an affinity difference between D and V cells. Although not sufficient, Fringe is required via Notch activation for expression of an Apterous-dependent affinity difference. We propose that Apterous controls expression of surface proteins that confer an affinity difference in conjunction with activated Notch. Thus, we view Apterous as instructive and Notch activity as essential, but permissive.
Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis
Authors: Frank Mary J. , Cartwright Heather N. , Smith Laurie G.
Published: January 2003

We have taken a genetic approach to investigating cytoskeleton-dependent mechanisms governing cell morphogenesis in the maize leaf epidermis. Previously, we showed that the Brick1 (Brk1) gene is required for the formation of epidermal cell lobes as well as for properly polarized divisions of stomatal subsidiary mother cells, and encodes an 8 kDa protein highly conserved in plants and animals. Here, we show that two additional Brick genes, Brk2 and Brk3, are involved in the same aspects of epidermal cell morphogenesis and division. As shown previously for Brk1, analysis of the cytoskeleton shows that Brk2 andBrk3 are required for the formation of local F-actin enrichments associated with lobe outgrowth in wild-type cells. Analysis of brk1;brk2,brk1;brk3 and brk2;brk3 double mutants shows that their phenotypes are the same as those of brk single mutants. Mosaic analysis shows that Brk1 acts non cell-autonomously over a short distance. By contrast, Brk2 and Brk3 act cell-autonomously to promote pavement cell lobe formation, but Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote polarized subsidiary mother cell divisions. Together, these observations indicate that all three Brk genes act in a common pathway in which each Brk gene has a distinct function. Recent work demonstrating a function for the mammalian homolog of BRK1 (HSPC300) in activation of Arp2/3-dependent actin polymerization implicates theBrk pathway in local regulation of actin polymerization in plant cells.
Discovery of genes with highly restricted expression patterns in theDrosophilawing disc using DNA oligonucleotide microarrays
Authors: Butler Miranda J. , Jacobsen Thomas L. , Cain Donna M. , Jarman Michael G. , Hubank Michael , Whittle J. Robert S. , Phillips Roger , Simcox Amanda
Published: January 2003

The Drosophila wing disc is divided along the proximaldistal axis into regions giving rise to the body wall (proximal), wing hinge(central) and wing blade (distal). We applied DNA microarray analysis to discover genes with potential roles in the development of these regions. We identified a set of 94 transcripts enriched (two fold or greater) in the body wall and 56 transcripts enriched in the wing/hinge region. Transcripts that are known to have highly restricted expression patterns, such aspannier, twist and Bar-H1 (body wall) and knot,nubbin and Distal-less (wing/hinge), showed strong differential expression on the arrays. In situ hybridization for 50 previously uncharacterized genes similarly revealed that transcript enrichment identified by the array analysis was consistent with the observed spatial expression. There was a broad spectrum of patterns, in some cases suggesting that the genes could be targets of known signaling pathways. We show that three of these genes respond to wingless signaling. We also discovered genes likely to play specific roles in tracheal and myoblast cell types, as these cells are part of the body wall fragment. In summary, the identification of genes with restricted expression patterns using whole genome profiling suggests that many genes with potential roles in wing disc development remain to be characterized.
Invariant Sema5A inhibition serves an ensheathing function during optic nerve development
Authors: Oster Stephen F. , Bodeker MacDara O. , He Fengling , Sretavan David W.
Published: January 2003

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, we found that transmembrane Semaphorin5A mRNA and protein was specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, we examined whether retinal axon responses to Sema5A were modulated by other guidance signals axons encountered along the retinal pathway. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggered an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibited retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations showed that blocking of Sema5A function led to retinal axons straying out of the optic nerve bundle, indicating that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules.
The actin-binding protein UNC-115 is an effector of Rac signaling during axon pathfinding in C. elegans
Authors: Struckhoff Eric C. , Lundquist Erik A.
Published: January 2003

Rac GTPases control cell shape by regulating downstream effectors that influence the actin cytoskeleton. UNC-115, a putative actin-binding protein similar to human abLIM/limatin, has previously been implicated in axon pathfinding. We have discovered the role of UNC-115 as a downstream cytoskeletal effector of Rac signaling in axon pathfinding. We show thatunc-115 double mutants with ced-10 Rac, mig-2 Rac orunc-73 GEF but not with rac-2/3 Rac displayed synthetic axon pathfinding defects, and that loss of unc-115 function suppressed the formation of ectopic plasma membrane extensions induced by constitutively-active rac-2 in neurons. Furthermore, we show that UNC-115 can bind to actin filaments. Thus, UNC-115 is an actin-binding protein that acts downstream of Rac signaling in axon pathfinding.
The Dictyostelium prestalk cell inducer DIF regulates nuclear accumulation of a STAT protein by controlling its rate of export from the nucleus
Authors: Fukuzawa Masashi , Abe Tomoaki , Williams Jeffrey G.
Published: January 2003

Dd-STATc becomes tyrosine phosphorylated, dimerises and accumulates in the nuclei of Dictyostelium cells exposed to DIF, the chlorinated hexaphenone that directs prestalk cell differentiation. By performing cytoplasmic photobleaching of living cells, we show that DIF inhibits the nuclear export of Dd-STATc. Within Dd-STATc there is a 50 amino acid region containing several consensus CRM1 (exportin 1)-dependent nuclear export signals (NESs). Deletion of this region causes Dd-STATc to accumulate in the nucleus constitutively and, when coupled to GFP, the same region directs nuclear export. We show that the N-terminal-proximal 46 amino acids are necessary for nuclear accumulation of Dd-STATc and sufficient to direct constitutive nuclear accumulation when fused to GFP. Combining the photobleaching and molecular analyses, we suggest that DIF-induced dimerisation of Dd-STATc functionally masks the NES-containing region and that this leads to nett nuclear accumulation, directed by the N-terminal-proximal import signals. These results show that the regulated nuclear accumulation of a STAT protein can be controlled at the level of nuclear export and they also provide a better understanding of the mechanism whereby DIF directs cell type divergence.
The tumor-suppressor and cell adhesion molecule Fat controls planar polarity via physical interactions with Atrophin, a transcriptional co-repressor
Authors: Fanto Manolis , Clayton Lesley , Meredith Jamie , Hardiman Kirsten , Charroux Bernard , Kerridge Stephen , McNeill Helen
Published: January 2003

Fat is an atypical cadherin that controls both cell growth and planar polarity. Atrophin is a nuclear co-repressor that is also essential for planar polarity; however, it is not known what genes Atrophin controls in planar polarity, or how Atrophin activity is regulated during the establishment of planar polarity. We show that Atrophin binds to the cytoplasmic domain of Fat and that Atrophin mutants show strong genetic interactions with fat. We find that both Atrophin and fat clones in the eye have non-autonomous disruptions in planar polarity that are restricted to the polar border of clones and that there is rescue of planar polarity defects on the equatorial border of these clones. Both fat and Atrophin are required to control four-jointed expression. In addition our mosaic analysis demonstrates an enhanced requirement for Atrophin in the R3 photoreceptor. These data lead us to a model in which fat andAtrophin act twice in the determination of planar polarity in the eye: first in setting up positional information through the production of a planar polarity diffusible signal, and later in R3 fate determination.