bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of moisture content and prechill duration on dormancy breakage of Douglas fir seeds (Pseudotsuga menziesiivar.menziesii[Mirb.] Franco)
Authors: Gosling Peter G. , Samuel Yvonne , Peace Andrew
Published: August 2003

Seeds of Douglas fir (Pseudotsuga menziesiivar.menziesii[Mirb.] Franco) were initially germinated at six constant temperatures (10–35°C), following 0–48 weeks incubation under moist conditions at 4°C, i.e. prechill or stratification. The best, single germination temperature was 15°C for determining the efficacy of subsequent dormancy breakage treatments. Seeds from the same seedlot were then adjusted to 10, 15, 20, 25, 30, 35 and 40% moisture contents (mcfw, fresh weight basis), prechilled for 0, 2, 4, 8, 16, 32, 64 and 128 weeks and transferred to 15°C. A smoothed bivariate spline was used to model the results and showed that virtually all combinations of moisture content and prechill duration significantly stimulated germination capacity, but that the optimal germination percentage (≥93%) was stimulated only by various combinations of between 30 and 35% mcfw and 25 and 48 weeks prechill. At optimal moisture contents (30 and 35%) , extending the prechill duration beyond 48 weeks led to a decrease in germination capacity. This was not due to dormancy reintroduction, but was caused largely by seed death. Regression models using a weighting function, to account for differences in standard deviations, demonstrated significant increases in the mean moisture content of individual seeds at higher moisture contents (≥25%) and longer prechill durations (≥64 weeks) that were concomitant with significant decreases in dry weight. The most likely explanation for this was seed respiration. The combined results suggest that dormancy breakage in Douglas fir seeds requires a hydration level sufficient for respiration to take place, and that, after maximal dormancy release, seeds at the highest mc (35–40%) exhaust their food reserves and begin to deteriorate.
Ankyrin binding mediates L1CAM interactions with static components of the cytoskeleton and inhibits retrograde movement of L1CAM on the cell surface
Authors: Gil Orlando D. , Sakurai Takeshi , Bradley Ann E. , Fink Marc Y. , Cassella Melanie R. , Kuo James A. , Felsenfeld Dan P.
Published: August 2003

The function of adhesion receptors in both cell adhesion and migration depends critically on interactions with the cytoskeleton. During cell adhesion, cytoskeletal interactions stabilize receptors to strengthen adhesive contacts. In contrast, during cell migration, adhesion proteins are believed to interact with dynamic components of the cytoskeleton, permitting the transmission of traction forces through the receptor to the extracellular environment. The L1 cell adhesion molecule (L1CAM), a member of the Ig superfamily, plays a crucial role in both the migration of neuronal growth cones and the static adhesion between neighboring axons. To understand the basis of L1CAM function in adhesion and migration, we quantified directly the diffusion characteristics of L1CAM on the upper surface of ND-7 neuroblastoma hybrid cells as an indication of receptor–cytoskeleton interactions. We find that cell surface L1CAM engages in diffusion, retrograde movement, and stationary behavior, consistent with interactions between L1CAM and two populations of cytoskeleton proteins. We provide evidence that the cytoskeletal adaptor protein ankyrin mediates stationary behavior while inhibiting the actin-dependent retrograde movement of L1CAM. Moreover, inhibitors of L1CAM–ankyrin interactions promote L1CAM-mediated axon growth. Together, these results suggest that ankyrin binding plays a crucial role in the anti-coordinate regulation of L1CAM-mediated adhesion and migration.
Regulation of insulin-like growth factor–dependent myoblast differentiation by Foxo forkhead transcription factors
Authors: Hribal Marta L. , Nakae Jun , Kitamura Tadahiro , Shutter John R. , Accili Domenico
Published: August 2003

Insulin-like growth factors promote myoblast differentiation through phosphoinositol 3-kinase and Akt signaling. Akt substrates required for myogenic differentiation are unknown. Forkhead transcription factors of the forkhead box gene, group O (Foxo) subfamily are phosphorylated in an insulin-responsive manner by phosphatidylinositol 3-kinase–dependent kinases. Phosphorylation leads to nuclear exclusion and inactivation. We show that a constitutively active Foxo1 mutant inhibits differentiation of C2C12 cells and prevents myotube differentiation induced by constitutively active Akt. In contrast, a transcriptionally inactive mutant Foxo1 partially rescues inhibition of C2C12 differentiation mediated by wortmannin, but not by rapamycin, and is able to induce aggregation-independent myogenic conversion of teratocarcinoma cells. Inhibition of Foxo expression by siRNA resulted in more efficient differentiation, associated with increased myosin expression. These observations indicate that Foxo proteins are key effectors of Akt-dependent myogenesis.
ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis
Authors: Aikawa Yoshikatsu , Martin Thomas F.J.
Published: August 2003

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.
EphB1 recruits c-Src and p52Shc to activate MAPK/ERK and promote chemotaxis
Authors: Vindis Cécile , Cerretti Douglas Pat , Daniel Thomas O. , Huynh-Do Uyen
Published: August 2003

Eph receptors and their ligands (ephrins) play an important role in axonal guidance, topographic mapping, and angiogenesis. The signaling pathways mediating these activities are starting to emerge and are highly cell- and receptor-type specific. Here we demonstrate that activated EphB1 recruits the adaptor proteins Grb2 and p52Shc and promotes p52Shc and c-Src tyrosine phosphorylation as well as MAPK/extracellular signal–regulated kinase (ERK) activation. EphB1-mediated increase of cell migration was abrogated by the MEK inhibitor PD98059 and Src inhibitor PP2. In contrast, cell adhesion, which we previously showed to be c-jun NH2-terminal kinase (JNK) dependent, was unaffected by ERK1/2 and Src inhibition. Expression of dominant-negative c-Src significantly reduced EphB1-dependent ERK1/2 activation and chemotaxis. Site-directed mutagenesis experiments demonstrate that tyrosines 600 and 778 of EphB1 are required for its interaction with c-Src and p52Shc. Furthermore, phosphorylation of p52Shc by c-Src is essential for its recruitment to EphB1 signaling complexes through its phosphotyrosine binding domain. Together these findings highlight a new aspect of EphB1 signaling, whereby the concerted action of c-Src and p52Shc activates MAPK/ERK and regulates events involved in cell motility.
Spatial and temporal changes in Bax subcellular localization during anoikis
Authors: Valentijn Anthony J. , Metcalfe Anthony D. , Kott Jane , Streuli Charles H. , Gilmore Andrew P.
Published: August 2003

Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier.
Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss
Authors: Weaver Beth A.A. , Bonday Zahid Q. , Putkey Frances R. , Kops Geert J.P.L. , Silk Alain D. , Cleveland Don W.
Published: August 2003

Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in primary mouse fibroblasts in vitro and in 95% of regenerating hepatocytes in vivo. Without CENP-E, diminished levels of BubR1 are recruited to kinetochores and BubR1 kinase activity remains at basal levels. CENP-E binds to and directly stimulates the kinase activity of purified BubR1 in vitro. Thus, CENP-E is required for enhancing recruitment of its binding partner BubR1 to each unattached kinetochore and for stimulating BubR1 kinase activity, implicating it as an essential amplifier of a basal mitotic checkpoint signal.
Distinct roles of Gαi and Gβ13F subunits of the heterotrimeric G protein complex in the mediation of Drosophila neuroblast asymmetric divisions
Authors: Yu Fengwei , Cai Yu , Kaushik Rachna , Yang Xiaohang , Chia William
Published: August 2003

The asymmetric division of Drosophila neuroblasts involves the basal localization of cell fate determinants and the generation of an asymmetric, apicobasally oriented mitotic spindle that leads to the formation of two daughter cells of unequal size. These features are thought to be controlled by an apically localized protein complex comprising of two signaling pathways: Bazooka/Drosophila atypical PKC/Inscuteable/DmPar6 and Partner of inscuteable (Pins)/Gαi; in addition, Gβ13F is also required. However, the role of Gαi and the hierarchical relationship between the G protein subunits and apical components are not well defined. Here we describe the isolation of Gαi mutants and show that Gαi and Gβ13F play distinct roles. Gαi is required for Pins to localize to the cortex, and the effects of loss of Gαi or pins are highly similar, supporting the idea that Pins/Gαi act together to mediate various aspects of neuroblast asymmetric division. In contrast, Gβ13F appears to regulate the asymmetric localization/stability of all apical components, and Gβ13F loss of function exhibits phenotypes resembling those seen when both apical pathways have been compromised, suggesting that it acts upstream of the apical pathways. Importantly, our results have also revealed a novel aspect of apical complex function, that is, the two apical pathways act redundantly to suppress the formation of basal astral microtubules in neuroblasts.
Trafficking of prion proteins through a caveolae-mediated endosomal pathway
Authors: Peters Peter J. , Mironov Alexander , Peretz David , van Donselaar Elly , Leclerc Estelle , Erpel Susanne , DeArmond Stephen J. , Burton Dennis R. , Williamson R. Anthony , Vey Martin , Prusiner Stanley B.
Published: August 2003

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A–gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.
Impaired membrane resealing and autoimmune myositis in synaptotagmin VII–deficient mice
Authors: Chakrabarti Sabyasachi , Kobayashi Koichi S. , Flavell Richard A. , Marks Carolyn B. , Miyake Katsuya , Liston David R. , Fowler Kimberly T. , Gorelick Fred S. , Andrews Norma W.
Published: August 2003

Members of the synaptotagmin family have been proposed to function as Ca2+ sensors in membrane fusion. Syt VII is a ubiquitously expressed synaptotagmin previously implicated in plasma membrane repair and Trypanosoma cruzi invasion, events which are mediated by the Ca2+-regulated exocytosis of lysosomes. Here, we show that embryonic fibroblasts from Syt VII–deficient mice are less susceptible to trypanosome invasion, and defective in lysosomal exocytosis and resealing after wounding. Examination of mutant mouse tissues revealed extensive fibrosis in the skin and skeletal muscle. Inflammatory myopathy, with muscle fiber invasion by leukocytes and endomysial collagen deposition, was associated with elevated creatine kinase release and progressive muscle weakness. Interestingly, similar to what is observed in human polymyositis/dermatomyositis, the mice developed a strong antinuclear antibody response, characteristic of autoimmune disorders. Thus, defective plasma membrane repair in tissues under mechanical stress may favor the development of inflammatory autoimmune disease.
Arf6 and Phosphoinositol-4-Phosphate-5-Kinase Activities Permit Bypass of the Rac1 Requirement for β1 Integrin–mediated Bacterial Uptake
Authors: Wong Ka-Wing , Isberg Ralph R.
Published: August 2003

Efficient entry of the bacterium Yersinia pseudotuberculosis into mammalian cells requires the binding of the bacterial invasin protein to β1 integrin receptors and the activation of the small GTPase Rac1. We report here that this Rac1-dependent pathway involves recruitment of phosphoinositol-4-phosphate-5-kinase (PIP5K) to form phosphoinositol-4,5-bisphosphate (PIP2) at the phagocytic cup. Reducing the concentration of PIP2 in the target cell by using a membrane-targeted PIP2-specific phosphatase lowered bacterial uptake proportionately. PIP2 formation is regulated by Arf6. An Arf6 derivative defective for nucleotide binding (Arf6N122I) interfered with uptake and decreased the level of PIP2 around extracellular bacteria bound to host cells. This reduction in PIP2 occurred in spite of fact that PIP5K appeared to be recruited efficiently to the site of bacterial binding, indicating a role for Arf6 in activation of the kinase. The elimination of the Rac1-GTP–bound form from the cell by the introduction of the Y. pseudotuberculosis YopE RhoGAP protein could be bypassed by the overproduction of either PIP5K or Arf6, although the degree of bypass was greater for Arf6 transfectants. These results indicate that both Arf6 and PIP5K are involved in integrin-dependent uptake, and that Arf6 participates in both activation of PIP5K as well as in other events associated with bacterial uptake.
The B Cell Receptor Itself Can Activate Complement to Provide the Complement Receptor 1/2 Ligand Required to Enhance B Cell Immune Responses In Vivo
Authors: Rossbacher Joerg , Shlomchik Mark J.
Published: August 2003

B cells express complement receptors (CRs) that bind activated fragments of C3 and C4. Immunized CR knockout (KO) mice have lower antibody titers and smaller germinal centers (GCs), demonstrating the importance of CR signals for the humoral immune response. CR ligands were thought to be generated via complement fixation mediated by preexisting “natural” IgM or early Ab from inefficiently activated B cells. This concept was recently challenged by a transgenic (Tg) mouse model that lacks circulating antibody but still retains membrane IgM (mIgM) and mounts normal immune responses. To test whether CR ligands could be generated by the B cell receptor (BCR) itself, we generated similar mice carrying a mutated mIgM that was defective in C1q binding. We found that B cells from such mutant mice do not deposit C3 on B cells upon BCR ligation, in contrast to B cells from mIgM mice. This has implications for the immune response: the mutant mice have smaller GCs than mIgM mice, and they are particularly deficient in the maintenance of the GC response. These results demonstrate a new BCR-dependent pathway that is sufficient and perhaps necessary to provide a CR1/2 ligand that promotes efficient B cell activation.
Prostaglandin D2 Reinforces Th2 Type Inflammatory Responses of Airways to Low-dose Antigen through Bronchial Expression of Macrophage-derived Chemokine
Authors: Honda Kyoko , Arima Masafumi , Cheng Gang , Taki Shinsuke , Hirata Hirokuni , Eda Fukiko , Fukushima Fumiya , Yamaguchi Bunpei , Hatano Masahiko , Tokuhisa Takeshi , Fukuda Takeshi
Published: August 2003

PGD2, a lipid mediator released from mast cells, is known to participate in allergic reactions. However, the mechanism by which PGD2 contributes to such reactions remains unclear. We established a novel experimental model of asthma that permitted direct assessment of the role of PGD2 in airway inflammation. Antigen-sensitized mice were exposed to aerosolized prostaglandin D2 (PGD2) 1 d before challenge with low-dose aerosolized antigen. Not only the numbers of eosinophils, lymphocytes, and macrophages but also the levels of IL-4 and IL-5 in bronchoalveolar lavage fluid were higher in PGD2-pretreated mice than in control mice. The expression of macrophage-derived chemokine (MDC), a chemoattractant for Th2 cells, was greater in PGD2-pretreated mice than in control. Injection of anti-MDC antibody into PGD2-pretreated mice markedly inhibited inflammatory cell infiltration as well as Th2 cyto-kine production after antigen challenge. These results indicate that PGD2 accelerates Th2 type inflammation by induction of MDC. Our results suggest that this mechanism may play a key role in the development of human asthma and that MDC might be a target molecule for therapeutic intervention.
DAP12/TREM2 Deficiency Results in Impaired Osteoclast Differentiation and Osteoporotic Features
Authors: Paloneva Juha , Mandelin Jami , Kiialainen Anna , Böhling Tom , Prudlo Johannes , Hakola Panu , Haltia Matti , Konttinen Yrjö T. , Peltonen Leena
Published: August 2003

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), Nasu-Hakola disease, is a globally distributed recessively inherited disease. PLOSL is characterized by cystic bone lesions, osteoporotic features, and loss of white matter in the brain leading to spontaneous bone fractures and profound presenile dementia. We have earlier characterized the molecular genetic background of PLOSL by identifying mutations in two genes, DAP12 and TREM2. DAP12 is a transmembrane adaptor protein that associates with the cell surface receptor TREM2. The DAP12–TREM2 complex is involved in the maturation of dendritic cells. To test a hypothesis that osteoclasts would be the cell type responsible for the bone pathogenesis in PLOSL, we analyzed the differentiation of peripheral blood mononuclear cells isolated from DAP12- and TREM2-deficient PLOSL patients into osteoclasts. Here we show that loss of function mutations in DAP12 and TREM2 result in an inefficient and delayed differentiation of osteoclasts with a remarkably reduced bone resorption capability in vitro. These results indicate an important role for DAP12–TREM2 signaling complex in the differentiation and function of osteoclasts.
BrucellaEvades Macrophage Killing via VirB-dependent Sustained Interactions with the Endoplasmic Reticulum
Authors: Celli Jean , de Chastellier Chantal , Franchini Don-Marc , Pizarro-Cerda Javier , Moreno Edgardo , Gorvel Jean-Pierre
Published: August 2003

The intracellular pathogen Brucella is the causative agent of brucellosis, a worldwide zoonosis that affects mammals, including humans. Essential to Brucella virulence is its ability to survive and replicate inside host macrophages, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here we show in a model of Brucella abortus infection of murine bone marrow–derived macrophages that a fraction of the bacteria that survive an initial macrophage killing proceed to replicate in a compartment segregated from the endocytic pathway. The maturation of the Brucella-containing vacuole involves sustained interactions and fusion with the endoplasmic reticulum (ER), which creates a replicative compartment with ER-like properties. The acquisition of ER membranes by replicating Brucella is independent of ER-Golgi COPI-dependent vesicular transport. A mutant of the VirB type IV secretion system, which is necessary for intracellular survival, was unable to sustain interactions and fuse with the ER, and was killed via eventual fusion with lysosomes. Thus, we demonstrate that live intracellular Brucella evade macrophage killing through VirB-dependent sustained interactions with the ER. Moreover, we assign an intracellular function to the VirB system, as being required for late maturation events necessary for the biogenesis of an ER-derived replicative organelle.
Retinoids Regulate Survival and Antigen Presentation by Immature Dendritic Cells
Authors: Geissmann Frédéric , Revy Patrick , Brousse Nicole , Lepelletier Yves , Folli Claudia , Durandy Anne , Chambon Pierre , Dy Michel
Published: August 2003

Maturation of dendritic cells (DCs) is a critical step for the induction of an immune response. We have examined the role of retinoid nuclear receptor pathways in this process. Retinoids induce DC apoptosis, in the absence of inflammatory signals, through retinoic acid receptor (RAR)α/retinoic X receptor (RXR) heterodimers. In contrast, via a cross talk with inflammatory cytokines, retinoids increase DNA binding activity of nuclear factor κB in DCs, trigger membrane major histocompatibility complex class II and costimulatory molecule expression, induce the differentiation of immature DCs into mature DCs, and enhance antigen-specific T cell response. This maturation of DCs is mediated via a RXR-dependent/RAR-independent pathway and via an RARα/RXR pathway distinct from the one responsible for apoptosis. Apoptosis and activation, mediated through distinct nuclear retinoid receptor pathways, can be dissociated from each other with selective synthetic retinoids. We identify a novel cellular function for retinoids and suggest that selective retinoids might be of interest for controlling antigen presentation.
Regulation of Dendritic Cell Migration to the Draining Lymph Node
Authors: Martín-Fontecha Alfonso , Sebastiani Silvia , Höpken Uta E. , Uguccioni Mariagrazia , Lipp Martin , Lanzavecchia Antonio , Sallusto Federica
Published: August 2003

Antigen-pulsed dendritic cells (DCs) are used as natural adjuvants for vaccination, but the factors that influence the efficacy of this treatment are poorly understood. We investigated the parameters that affect the migration of subcutaneously injected mouse-mature DCs to the draining lymph node. We found that the efficiency of DC migration varied with the number of injected DCs and that CCR7+/+ DCs migrating to the draining lymph node, but not CCR7−/− DCs that failed to do so, efficiently induced a rapid increase in lymph node cellularity, which was observed before the onset of T cell proliferation. We also report that DC migration could be increased up to 10-fold by preinjection of inflammatory cytokines that increased the expression of the CCR7 ligand CCL21 in lymphatic endothelial cells. The magnitude and quality of CD4+ T cell response was proportional to the number of antigen-carrying DCs that reached the lymph node and could be boosted up to 40-fold by preinjection of tumor necrosis factor that conditioned the tissue for increased DC migration. These results indicate that DC number and tissue inflammation are critical parameters for DC-based vaccination.
Tuberculosis Toxin Blocking Phagosome Maturation Inhibits a Novel Ca2+/Calmodulin-PI3K hVPS34 Cascade
Authors: Vergne Isabelle , Chua Jennifer , Deretic Vojo
Published: August 2003

The capacity of Mycobacterium tuberculosis to infect latently over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome in infected macrophages. Here we describe how M. tuberculosis toxin lipoarabinomannan (LAM) causes phagosome maturation arrest, interfering with a new pathway connecting intracellular signaling and membrane trafficking. LAM from virulent M. tuberculosis, but not from avirulent mycobacteria, blocked cytosolic Ca2+ increase. Ca2+ and calmodulin were required for a newly uncovered Ca2+/calmodulin phosphatidylinositol (PI)3 kinase hVPS34 cascade, essential for production of PI 3 phosphate (PI3P) on liposomes in vitro and on phagosomes in vivo. The interference of the trafficking toxin LAM with the calmodulin-dependent production of PI3P described here ensures long-term M. tuberculosis residence in vacuoles sequestered away from the bactericidal and antigen-processing organelles in infected macrophages.
Tumor Regression and Autoimmunity after Reversal of a Functionally Tolerant State of Self-reactive CD8+ T Cells
Authors: Overwijk Willem W. , Theoret Marc R. , Finkelstein Steven E. , Surman Deborah R. , de Jong Laurina A. , Vyth-Dreese Florry A. , Dellemijn Trees A. , Antony Paul A. , Spiess Paul J. , Palmer Douglas C. , Heimann David M. , Klebanoff Christopher A. , Yu Zhiya , Hwang Leroy N. , Feigenbaum Lionel , Kruisbeek Ada M. , Rosenberg Steven A. , Restifo Nicholas P.
Published: August 2003

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I–restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.
Distinct Mutations in IRAK-4 Confer Hyporesponsiveness to Lipopolysaccharide and Interleukin-1 in a Patient with Recurrent Bacterial Infections
Authors: Medvedev Andrei E. , Lentschat Arnd , Kuhns Douglas B. , Blanco Jorge C.G. , Salkowski Cindy , Zhang Shuling , Arditi Moshe , Gallin John I. , Vogel Stefanie N.
Published: August 2003

We identified previously a patient with recurrent bacterial infections who failed to respond to gram-negative LPS in vivo, and whose leukocytes were profoundly hyporesponsive to LPS and IL-1 in vitro. We now demonstrate that this patient also exhibits deficient responses in a skin blister model of aseptic inflammation. A lack of IL-18 responsiveness, coupled with diminished LPS and/or IL-1–induced nuclear factor–κB and activator protein-1 translocation, p38 phosphorylation, gene expression, and dysregulated IL-1R–associated kinase (IRAK)–1 activity in vitro support the hypothesis that the defect lies within the signaling pathway common to toll-like receptor 4, IL-1R, and IL-18R. This patient expresses a “compound heterozygous” genotype, with a point mutation (C877T in cDNA) and a two-nucleotide, AC deletion (620–621del in cDNA) encoded by distinct alleles of the IRAK-4 gene (GenBank/EMBL/DDBJ accession nos. AF445802 and AY186092). Both mutations encode proteins with an intact death domain, but a truncated kinase domain, thereby precluding expression of full-length IRAK-4 (i.e., a recessive phenotype). When overexpressed in HEK293T cells, neither truncated form augmented endogenous IRAK-1 kinase activity, and both inhibited endogenous IRAK-1 activity modestly. Thus, IRAK-4 is pivotal in the development of a normal inflammatory response initiated by bacterial or nonbacterial insults.
129-derived Strains of Mice Are Deficient in DNA Polymerase ι and Have Normal Immunoglobulin Hypermutation
Authors: McDonald John P. , Frank Ekaterina G. , Plosky Brian S. , Rogozin Igor B. , Masutani Chikahide , Hanaoka Fumio , Woodgate Roger , Gearhart Patricia J.
Published: August 2003

Recent studies suggest that DNA polymerase η (polη) and DNA polymerase ι (polι) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of polι in generating mutations in an animal model, we first characterized the biochemical properties of murine polι. Like its human counterpart, murine polι is extremely error-prone when catalyzing synthesis on a variety of DNA templates in vitro. Interestingly, when filling in a 1 base-pair gap, DNA synthesis and subsequent strand displacement was greatest in the presence of both pols ι and η. Genomic sequence analysis of Poli led to the serendipitous discovery that 129-derived strains of mice have a nonsense codon mutation in exon 2 that abrogates production of polι. Analysis of hypermutation in variable genes from 129/SvJ (Poli−/−) and C57BL/6J (Poli+/+) mice revealed that the overall frequency and spectrum of mutation were normal in polι-deficient mice. Thus, either polι does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase.
ATM-related Tel1 associates with double-strand breaks through an Xrs2-dependent mechanism
Authors: Nakada Daisuke , Matsumoto Kunihiro , Sugimoto Katsunori
Published: August 2003

In budding yeast,TEL1encodes a protein closely related to ATM. Xrs2 is an Nbs1 homolog and forms a complex with Mre11 and Rad50. We show here that Tel1 associ ates with double-strand breaks (DSBs) through a mechanism dependent on the C terminus of Xrs2. Although Xrs2 is required for the DNA degradation at DSBs, the C-terminal Xrs2 truncation does not affect the degradation. Tel1 and the C terminus of Xrs2 are similarly involved in cell survival and Rad53 phosphorylation after DNA damage. Our findings suggest that the Tel1 association with DNA lesions is required for the activation of DNA damage responses.
Structure of HoxA9 and Pbx1 bound to DNA: Hox hexapeptide and DNA recognition anterior to posterior
Authors: LaRonde-LeBlanc Nicole A. , Wolberger Cynthia
Published: August 2003

The HOX/HOM superfamily of homeodomain proteins controls cell fate and segmental embryonic patterning by a mechanism that is conserved in all metazoans. The linear arrangement of the Hox genes on the chromosome correlates with the spatial distribution of HOX protein expression along the anterior–posterior axis of the embryo. Most HOX proteins bind DNA cooperatively with members of the PBC family of TALE-type homeodomain proteins, which includes human Pbx1. Cooperative DNA binding between HOX and PBC proteins requires a residue N-terminal to the HOX homeodomain termed the hexapeptide, which differs significantly in sequence between anterior- and posterior-regulating HOX proteins. We report here the 1.9-Å-resolution structure of a posterior HOX protein, HoxA9, complexed with Pbx1 and DNA, which reveals that the posterior Hox hexapeptide adopts an altered conformation as compared with that seen in previously determined anterior HOX/PBC structures. The additional nonspecific interactions and altered DNA conformation in this structure account for the stronger DNA-binding affinity and altered specificity observed for posterior HOX proteins when compared with anterior HOX proteins. DNA-binding studies of wild-type and mutant HoxA9 and HoxB1 show residues in the N-terminal arm of the homeodomains are critical for proper DNA sequence recognition despite lack of direct contact by these residues to the DNA bases. These results help shed light on the mechanism of transcriptional regulation by HOX proteins and show how DNA-binding proteins may use indirect contacts to determine sequence specificity.
Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation
Authors: Kim Sunhwa , Koga Takako , Isobe Miho , Kern Britt E. , Yokochi Taeko , Chin Y. Eugene , Karsenty Gerard , Taniguchi Tadatsugu , Takayanagi Hiroshi
Published: August 2003

Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, we demonstrate a novel function of the Stat1 transcription factor in the regulation of bone remodeling. In the bone of theStat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-β. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. Our study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling.
Genetic interaction between Wnt/β-catenin and BMP receptor signaling during formation of the AER and the dorsal–ventral axis in the limb
Authors: Soshnikova Natalia , Zechner Dietmar , Huelsken Joerg , Mishina Yuji , Behringer Richard R. , Taketo Makoto M. , Crenshaw E. Bryan , Birchmeier Walter
Published: August 2003

By conditional gene ablation in mice, we found that β-catenin, an essential downstream effector of canonical Wnt signaling, is a key regulator of formation of the apical ectodermal ridge (AER) and of the dorsal–ventral axis of the limbs. By generation of compound mutants, we also show that β-catenin acts downstream of the BMP receptor IA in AER induction, but upstream or parallel in dorsal–ventral patterning. Thus, AER formation and dorsal–ventral patterningof limbs are tightly controlled by an intricate interplay between Wnt/β-catenin and BMP receptor signaling.
DNA binding controls inactivation and nuclear accumulation of the transcription factor Stat1
Authors: Meyer Thomas , Marg Andreas , Lemke Petra , Wiesner Burkhard , Vinkemeier Uwe
Published: August 2003

Cytokine-dependent gene transcription greatly depends on the tyrosine phosphorylation (“activation”) of Stat proteins at the cell membrane. This rapidly leads to their accumulation in the nucleus by an unknown mechanism. We performed microinjections of recombinant Stat1 protein to show that nuclear accumulation of phosphorylated Stat1 can occur without cytokine stimulation of cells. Microinjection of Stat1 antibody and treatment of cells with kinase or phosphatase inhibitors revealed that nuclear accumulation is a highly dynamic process sustained by Stat1 nucleocytoplasmic cycling and continuous kinase activity. By characterizing nuclear accumulation mutants, it is demonstrated that nuclear import and nuclear retention are two separate steps leading up to nuclear accumulation, with nonspecific DNA binding of activated Stat1 being sufficient for nuclear retention. Critical for nuclear buildup of Stat1 and the subsequent nuclear export is the point of time of tyrosine dephosphorylation, because our data indicate that activated Stat1 is incapable of leaving the nucleus and requires dephosphorylation to do so. It is demonstrated that the inactivation of Stat1 is controlled by its exchange reaction with DNA, whereby DNA binding protects Stat1 from dephosphorylation in a sequence-specific manner. Thus, during nuclear accumulation, a surprisingly simple mechanism integrates central aspects of cytokine-dependent gene regulation, for example, receptor monitoring, promoter occupancy, and transcription factor inactivation.
Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers
Authors: Reményi Attila , Lins Katharina , Nissen L. Johan , Reinbold Rolland , Schöler Hans R. , Wilmanns Matthias
Published: August 2003

Members of the POU and SOX transcription factor families exemplify the partnerships established between various transcriptional regulators during early embryonic development. Although functional cooperativity between key regulator proteins is pivotal for milestone decisions in mammalian development, little is known about the underlying molecular mechanisms. In this study, we focus on two transcription factors, Oct4 and Sox2, as their combination on DNA is considered to direct the establishment of the first three lineages in the mammalian embryo. Using experimental high-resolution structure determination, followed by model building and experimental validation, we found that Oct4 and Sox2 were able to dimerize onto DNA in distinct conformational arrangements. We demonstrate that the DNA enhancer region of their target genes is responsible for the correct spatial alignment of glue-like interaction domains on their surface. Interestingly, these surfaces frequently have redundant functions and are instrumental in recruiting various interacting protein partners.
Laser cutting gains closed‐loop control
Authors: Kochan Anna
Published: September 2003

Eureka project E! 1784 Eurolaser Publics is creating a fully automated solution for the 2D and 3D laser cutting of metals up to 20 mm thick. The European consortium made up of Italian, French and Danish companies has developed a closed loop system incorporating innovative sensors and control algorithms.
Evolutionary perspectives on the capability accumulation process
Authors: Pandža Krsto , Polajnar Andrej , Buchmeister Borut , Thorpe Richard
Published: August 2003

There has been an increasing call from academics specialising in operations management to integrate different strategic management perspectives into operations strategy research. Recently some pieces of operations strategy research have used the resource‐based view. It is often suggested that the incorporation of resource‐based view ideas into the field of operations strategy is a search for a new paradigm, yet the ever‐increasing literature suffers from a lack of empirical research. Moreover, operations strategy research from the evolutionary perspective, using longitudinal field data, is almost completely neglected. This paper attempts to make two contributions. The first is to stimulate debate about the incorporation of resource‐based view and dynamic capabilities within operations strategy research. The second is to present a model based on in‐depth field research where the dynamics of the capability accumulation process is explored.
Implementing enterprise resource planning (ERP) systems in small and midsize manufacturing firms
Authors: Muscatello Joseph R. , Small Michael H. , Chen Injazz J.
Published: August 2003

Enterprise resource planning systems, if implemented successfully, can bestow impressive strategic, operational and information‐related benefits to adopting firms. A failed implementation can often spell financial doom. Currently, most of the information about the failures and successes are based on reports on implementations in large manufacturing and service organizations. But enterprise resource planning vendors are now steadily turning their marketing sights on small and medium‐sized manufacturers. The time is ripe for researchers to gather, analyze and disseminate information that will help these firms to implement their projects successfully. This research adopts a multiple case study approach to investigate the implementation process in small and midsize manufacturing firms in the US. The research focuses on implementation activities that foster successful installations and are developed using information gleaned from our field studies of four projects. Avenues for future research are also suggested.
The implications of fit between planning environments and manufacturing planning and control methods
Authors: Jonsson Patrik , Mattsson Stig‐Arne
Published: August 2003

The applicability of manufacturing planning and control methods differs between environments. This paper explains the fit between the planning environment and material and capacity planning on the detailed material planning and shop‐floor planning levels. The study is based on a conceptual discussion and a survey of 84 Swedish manufacturing companies. Results show the use of planning methods and their levels of user satisfaction in complex customer order production, configure to order production, batch production of standardized products and repetitive mass production, respectively.
Service process design flexibility and customer waiting time
Authors: Sheu Chwen , McHaney Roger , Babbar Sunil
Published: August 2003

Customer waiting is regarded as one of the most critical aspects of service quality. Research has suggested various approaches to reduce the negative impact of waiting. This article investigates the waiting time performance of alternative service process designs that consist of two operations, order taking and order preparation. The research premise is that no single service process design is the best in all operating conditions. Managers should build flexibility into service process design by using alternative designs in combination. Several break‐even models are developed to examine the contingent nature of the performance of alternative designs. The results point to the need for building flexibility into service process designs by demonstrating that waiting time performance can only be optimized if design strategies are altered in response to ongoing changes in service system input parameters.
Advancing theory development in total quality management
Authors: Rungtusanatham Manus , Ogden Jeffrey A. , Wu Bin
Published: August 2003

The Deming management method, which encompasses the breadth of W. Edwards Deming's approach to quality management, has had significant impact on the practice and scholarship of quality management. In this paper we present a scholarly introspection and discussion on scientific research related to the Deming management method, the contributions of such research to theory development in quality management, and future research directions to pursue in order to continue to advance scientific knowledge in quality management. In doing so, we traced the historical development of the Deming management method. We present and discuss two streams of research related to the Deming management method, namely the concept of profound knowledge and the Deming‐based theory of TQM. We also highlight several opportunities for future research that would advance theory development in the TQM discipline – opportunities anchored by the imperative for confirmatory research that requires proper operationalization and primary data, and the plausibility of theory refinement and extension through the specification of new relationships and the inclusion of moderators and mediators.
Strategic planning and firms' competencies
Authors: De Toni Alberto , Tonchia Stefano
Published: August 2003

The traditional strategic model of industrial organization is criticized by the upholders of the theories that regard the resources and competencies of a firm as the source of its competitive advantage. This article presents these new strategic dictates in a critical form, examining the reasons that justify the striking interest being taken by researchers and managers, but also the limits of those principles, some intrinsic others that can be overcome by an integration with tradition. As a consequence, the article presents a framework which attempts to relate the constituent elements that distinguish the two main strategic theories and shows that both must be considered for strategic planning and performance management.
Competence and resource architectures
Authors: Mills John , Platts Ken , Bourne Mike
Published: August 2003

This paper aims to produce a justified, generic, pictorial architecture of the relationships between resources and competences within firms. It begins by providing definitions of the nature, scope and relationships between resources, capabilities and competences from the resource and competence literatures. In so doing, theory is refined and a linked resource and competence architecture is developed. The architecture distinguishes between high‐level competences that customers recognize, for example fast product delivery, and competences that support high‐level competences but are less visible customers, like competences in rapid knowledge acquisition and deployment. An empirical example is then used to illustrate how the architecture enables the construction of structured pictures of connected competences and co‐ordinated resources within a manufacturing business. Finally, the architecture is critiqued and its value for managers in structuring competence performance improvement activities is discussed.