|
| ||
|
|
||
Blocking
Terms modified by Blocking Selected AbstractsBlocking Dishevelled signaling in the noncanonical Wnt pathway in sea urchins disrupts endoderm formation and spiculogenesis, but not secondary mesoderm formationDEVELOPMENTAL DYNAMICS, Issue 7 2009Christine A. Byrum Abstract Dishevelled (Dsh) is a phosphoprotein key to beta-catenin dependent (canonical) and beta-catenin independent (noncanonical) Wnt signaling. Whereas canonical Wnt signaling has been intensively studied in sea urchin development, little is known about other Wnt pathways. To examine roles of these beta-catenin independent pathways in embryogenesis, we used Dsh-DEP, a deletion construct blocking planar cell polarity (PCP) and Wnt/Ca2+ signaling. Embryos overexpressing Dsh-DEP failed to gastrulate or undergo skeletogenesis, but produced pigment cells. Although early mesodermal gene expression was largely unperturbed, embryos exhibited reduced expression of genes regulating endoderm specification and differentiation. Overexpressing activated beta-catenin failed to rescue Dsh-DEP embryos, indicating that Dsh-DEP blocks endoderm formation downstream of initial canonical Wnt signaling. Because Dsh-DEP-like constructs block PCP signaling in other metazoans, and disrupting RhoA or Fz 5/8 in echinoids blocks subsets of the Dsh-DEP phenotypes, our data suggest that noncanonical Wnt signaling is crucial for sea urchin endoderm formation and skeletogenesis. Developmental Dynamics 238:1649,1665, 2009. © 2009 Wiley-Liss, Inc. [source] Priming of CD8+ T cell responses by pathogens typically depends on CD70-mediated interactions with dendritic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2007Anita Schildknecht Abstract The CD27/CD70-interaction has been shown to provide a costimulatory and survival signal for T cells in vitro and in vivo. Recently, CD70 expression by DC was found to be important for the priming of CD8+ T cells. We show here that blocking CD70 interactions has a significant impact on priming of CD8+ T cell responses by vaccinia virus (VV), Listeria monocytogenes and vesicular stomatitis virus (VSV) in mice. However, the priming of specific CD8+ T cells upon infection with lymphocytic choriomeningitis virus (LCMV) was only marginally reduced by CD70-blockade. Blocking of CD70 prevented CD8+ T cell priming in DIETER mice, a model in which presentation of LCMV-derived epitopes can be induced selectively in dendritic cells (DC). In contrast, CD70-CD27 interactions were not important for the priming of VSV-specific CD4+ T cells or class switch of neutralizing antibodies. As we show that priming of CD8+ T cells by the pathogens used here is dependent on antigen presentation by DC and that infection results in up-regulation of CD70 on DC, we conclude that CD70 expression on DC plays an important role in the priming of CD8+ T cells by pathogens. Moreover, the lack of CD70 cannot be completely compensated for by other costimulatory molecules. [source] Multifunctional Crosslinkable Iridium Complexes as Hole Transporting/Electron Blocking and Emitting Materials for Solution-Processed Multilayer Organic Light-Emitting DiodesADVANCED FUNCTIONAL MATERIALS, Issue 7 2009Biwu Ma Abstract Here, a new series of crosslinkable heteroleptic iridium (III) complexes for use in solution processed phosphorescent organic light emitting diodes (OLEDs) is reported. These iridium compounds have the general formula of (PPZ-VB)2Ir(C,N), where PPZ-VB is phenylpyrazole (PPZ) vinyl benzyl (VB) ether; and the C,N ligands represent a family of four different cyclometallating ligands including 1-phenylpyrazolyl (PPZ) (1), 2-(4,6-difluorophenyl)pyridyl (DFPPY) (2), 2-(p-tolyl)pyridyl (TPY) (3), and 2-phenylquinolyl (PQ) (4). With the incorporation of two crosslinkable VB ether groups, these compounds can be fully crosslinked after heating at 180,°C for 30,min. The crosslinked films exhibit excellent solvent resistance and film smoothness which enables fabrication of high-performance multilayer OLEDs by sequential solution processing of multiple layers. Furthermore, the photophysical properties of these compounds can be easily controlled by simply changing the cyclometallating C,N ligand in order to tune the triplet energy within the range of 3.0,2.2,eV. This diversity makes these materials not only suitable for use in hole transporting and electron blocking but also as emissive layers of several colors. Therefore, these compounds are applied as effective materials for all-solution processed OLEDs with (PPZ-VB)2IrPPZ (1) acting as hole transporting and electron blocking layer and host material, as well as three other compounds, (PPZ-VB)2IrDFPPY (2), (PPZ-VB)2IrTPY(3), and (PPZ-VB)2IrPQ(4), used as crosslinkable phosphorescent emitters. [source] Novel mechanism of antibodies to hepatitis B virus in blocking viral particle release from cells,HEPATOLOGY, Issue 3 2010Avidan U. Neumann Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti-HBs (HepeX-B) antibodies in patients with chronic hepatitis B. The antibody HBV-17 recognizes a conformational epitope, whereas antibody HBV-19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX-B treatment of HBsAg-producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX-B was removed from the cell culture supernatants. Conclusion: These results identify a novel antiviral mechanism of antibodies to HBsAg (anti-HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections. (HEPATOLOGY 2010;) [source] A New Method for Post-Synthesis Coating of Zirconia on the Mesopore Walls of SBA-15 Without Pore Blocking,ADVANCED MATERIALS, Issue 11 2008Cheralathan Kanakkampalayan Krishnan Zirconia coating of the mesopore walls of the mesoporous silica material SBA-15 is achieved by internal hydrolysis of a zirconia precursor, which is loaded inside the mesopores, using NH3/H2O vapor at elevated temperature and subsequent calcination (see figure). High loadings of zirconia, more than 30 wt%, can be coated on the mesopore walls without any pore blocking. [source] p38 MAPK inhibition modulates rabbit nucleus pulposus cell response to IL-1,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 7 2008Rebecca K. Studer Abstract Analysis of disc gene expression implicated IL-1 in the development of intervertebral disc degeneration (IDD) in a rabbit stab model. The purpose of these studies is to determine the role of p38 Mitogen Activated Protein Kinase (p38 MAPK) signaling in nucleus pulposus cell response to IL-1, and to compare rabbit nucleus pulposus (rNP) cell responses to IL-1 activation with those in a stab model of disc degeneration. NP cells maintained in alginate bead culture were exposed to IL-1, with or without p38 MAPK inhibition. RNA was isolated for reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression, conditioned media analyzed for accumulation of nitric oxide (NO) and prostaglandin E-2 (PGE-2), and proteoglycan synthesis measured after 10 days. IL-1 upregulation of mRNA for cycloxygenase-2 (COX-2), matrix metalloproteinase-3 (MMP-3), IL-1, and IL-6, was blunted by p38 inhibition while downregulation of matrix proteins (collagen I, collagen II, aggrecan) and insulin-like-growth-factor I (IFG-1) was also reversed. mRNA for tissue inhibitor of matrixmetalloproteinase-1 (TIMP-1) was modestly increased by IL-1, while those for Transforming Growth Factor-, (TGF-,) SOX-9, and versican remained unchanged. Blocking p38 MAPK reduced IL-1 induced NO and PGE-2 accumulation and partially restored proteoglycan synthesis. p38 MAPK inhibition in control cells increased mRNA for matrix proteins (aggrecan, collagen II, versican, collagen I) and anabolic factors (IGF-1, TGF, and SOX-9) from 50% to 120%, decreased basal PGE-2 accumulation, but had no effect on message for TIMP-1, MMP-3, or COX-2. Inhibition of p38 MAPK in cytokine-activated disc cells blunts gene expression and production of factors associated with inflammation, pain, and disc matrix catabolism while reversing IL-1 downregulation of matrix protein gene expression and proteoglycan synthesis. The results support the hypothesis that IL-1 could be responsible for many of the mRNA changes seen in rabbit NP in the stab model of disc degeneration, and uphold the concept that development of molecular techniques to block p38 MAPK could provide a therapeutic approach to slow the course of intervertebral disc degeneration. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:991,998, 2008 [source] The allergy-protective properties of Acinetobacter lwoffii F78 are imparted by its lipopolysaccharideALLERGY, Issue 6 2010J. Debarry To cite this article: Debarry J, Hanuszkiewicz A, Stein K, Holst O, Heine H. The allergy-protective properties of Acinetobacter lwoffii F78 are imparted by its lipopolysaccharide. Allergy 2010; 65: 690,697. Abstract Background:, An increasing number of epidemiological studies show that exposure to farming environment during early childhood strongly influences the development of allergic reactions later in life (,hygiene hypothesis'). Also, it had been shown that certain bacteria from this environment may have allergy-protective properties. In the present study, we further characterized one of these bacteria, namely Acinetobacter lwoffii F78, with regard to the bacteria-induced signaling and possible mechanisms of allergy protection. Methods:, The impact of A. lwoffii F78 on human monocyte-derived dendritic cells especially with respect to their THelper cell polarization capacity was investigated by ELISA and real-time PCR experiments as well as confocal microscopy. The responsible molecule for these effects was further characterized and identified using blocking experiments. Results:, It was shown that A. lwoffii F78 induced a TH1-polarizing program in human dendritic cells which led to TH1 differentiation. In addition, a positive influence on the TBet/GATA3 level could be detected. Blocking experiments revealed that the lipopolysaccharide (LPS) of A. lwoffii F78 was the responsible molecule promoting these effects. Conclusion:, We found evidence that the allergy-protecting effects of A. lwoffii F78 are because of the activation of a TH1-polarizing program in human dendritic cells, and that the LPS of A. lwoffii F78 is responsible for these beneficial effects. [source] Actinobacillus actinomycetemcomitans induces apoptosis of T lymphocytes by the Fas and Fas ligand pathwayMOLECULAR ORAL MICROBIOLOGY, Issue 5 2002A. Nalbant Actinobacillus actinomycetemcomitans expresses a number of toxins capable of inducing apoptotic cell death of T lymphocytes. However, the exact mechanism(s) has not been elucidated. The present study investigated the involvement of the Fas (CD95)-mediated apoptotic pathway in A. actinomycetemcomitans -induced T-cell apoptosis. To that end, peripheral blood mononuclear cells (PBMC) were cultured with or without A. actinomycetemcomitans cell-free culture supernatant (CFCS) for 0,96 h. The cells were then labeled with specific monoclonal antibodies and flow cytometry was performed. Results demonstrated up-regulation of Fas and activation of caspase-3 in T cells in response to A. actinomycetemcomitans CFCS. Monocytes were the only cells analyzed to express Fas ligand (FasL) constitutively, and this was further up-regulated in response to A. actinomycetemcomitans CFCS, while T cells expressed FasL only after this stimulation. Depletion of monocytes prior to stimulation with A. actinomycetemcomitans CFCS led to a marked decline in apoptosis. Blocking of Fas,FasL interactions with anti-Fas monoclonal antibody or Fas:Fc fusion protein lead to a significant decline, but not abolition, of T-cell apoptosis. Nearly all T cells expressed Bcl-2 at the outset of culture, and Bcl-2 expression declined in T cells stimulated with A. actinomycetemcomitans CFCS. Collectively, these data provide evidence for the induction of T-cell apoptosis by A. actinomycetemcomitans via the Fas-mediated pathway, involving caspase-3 and Bcl- 2. Moreover, this apoptotic response was dependent on the presence of monocytes. [source] Role for CTLA-4 but not CD25+ T cells during Schistosoma mansoni infection of micePARASITE IMMUNOLOGY, Issue 6 2007C. M. WALSH SUMMARY Schistosoma mansoni infection of mice increases the frequency of cells that are CD4+CD25+ in the acute (4 and 8 weeks) and chronic (16 week) stages of infection. Depletion of > 85% of CD25+ cells in the acute or chronic stages of schistosome infection caused no overt changes in morbidity or immunological responses. The absence of effect in mice with CD25+ cells depleted may be due to the preferential expression of IL-4 and IL-10, two cytokines that are protective in schistosome infection, on CD25, CD4+ cells. We also assessed infection-induced changes of other regulatory markers, GITR, CD103 and CTLA-4 on CD4+ cells. We identified a marked expansion of CTLA-4+ population on CD25, CD4+ cells in acute and chronic infection. Blocking of CTLA-4 during acute, but not chronic infection, caused significant weight loss and altered the type 2 cytokine response of mice, with increased IL-4 and IL-5 production associated with significantly more Th2 cells and eosinophils in the liver granuloma. This study illustrates the complexity of regulation of T cells in schistosome infection and highlights a specific role for CTLA-4+, but not CD25+ cells, in the regulation of Th2 responses in helminth infection. [source] Blocking Two-level Factorial ExperimentsQUALITY AND RELIABILITY ENGINEERING INTERNATIONAL, Issue 3 2007Murat Kulahci Abstract Blocking is commonly used in experimental design to eliminate unwanted variation by creating more homogeneous conditions for experimental treatments within each block. While it has been a standard practice in experimental design, blocking fractional factorials still presents many challenges due to differences between treatment and blocking variables. Lately, new design criteria such as the total number of clear effects and fractional resolution have been proposed to design blocked two-level fractional factorial experiments. This article presents a flexible matrix representation for two-level fractional factorials that will allow experimenters and software developers to block such experiments based on any design criterion that is suitable with the experimental conditions. Copyright © 2006 John Wiley & Sons, Ltd. [source] Sulindac Inhibits Canonical Wnt Signaling by Blocking the PDZ Domain of the Protein Dishevelled,ANGEWANDTE CHEMIE, Issue 35 2009Ho-Jin Lee Dr. Neuer Nutzen: Der nichtsteroidale Entzündungshemmer Sulindac wechselwirkt direkt und spezifisch mit der PDZ-Domäne des Proteins Dishevelled (Dvl), einer zentralen intrazellulären Komponente des Wnt-Signalwegs. Sulindac bindet an die konventionelle Peptidbindetasche der Domäne (siehe Bild) und könnte durch ihr Blockieren das kanonische Wnt-Signal inhibieren und so einen chemischen Schutz vor Krebs bieten. [source] The impact of anterior cruciate ligament injury on lubricin metabolism and the effect of inhibiting tumor necrosis factor , on chondroprotection in an animal modelARTHRITIS & RHEUMATISM, Issue 10 2009K. A. Elsaid Objective To examine the effects of anterior cruciate ligament transection (ACLT) in a rat model on lubricin metabolism and its relationship to markers of inflammation and cartilage damage, and to determine whether blocking the metabolic effects of tumor necrosis factor , (TNF,) by etanercept increases the chondroprotection provided by lubricin. Methods Unilateral ACLT was performed in Lewis rats. Levels of lubricin, TNF,, interleukin-1, (IL-1,), and sulfated glycosaminoglycans (sGAG) in synovial fluid (SF) lavage specimens and synovial tissue lubricin gene expression were evaluated at 1 week and 4 weeks following ACLT. Histologic evaluation of articular cartilage included staining with lubricin-specific monoclonal antibody 9G3 and Safranin O. The percentage of lubricin staining on the surface of articular cartilage in weight-bearing areas was estimated by digital imaging. Blocking of TNF, was performed using etanercept, which was administered subcutaneously at a dose of 0.5 mg/kg around the ACL-transected joints, using different dosing strategies. The ACL-transected and contralateral joints of these rats were harvested 4 weeks following surgery. Results Four weeks following ACLT, SF lubricin concentrations and the percentage of cartilage surface lubricin staining were significantly lower in the injured joints compared with the contralateral joints. A significant decrease in synovial tissue lubricin gene expression was associated with elevated TNF, and IL-1, concentrations in SF lavage samples. With all of the etanercept treatment strategies, blocking of TNF, significantly increased the amount of lubricin bound to cartilage, coupled with a significant decrease in sGAG release. However, changes in the concentrations of lubricin in SF were variable. Conclusion Blocking TNF, resulted in a chondroprotective effect, exemplified by increased lubricin deposition on articular cartilage and a decrease in sGAG release from articular cartilage in an animal model of posttraumatic arthritis. [source] Nephritogenic Anti-DNA antibodies regulate gene expression in MRL/lpr mouse glomerular mesangial cellsARTHRITIS & RHEUMATISM, Issue 7 2006Xiaoping Qing Objective Lupus-associated IgG anti,double-stranded DNA antibodies are thought to be pathogenic in the kidney due to cross-reaction with glomerular antigens, leading subsequently to immune complex formation in situ and complement activation. We undertook this study to determine if pathogenic anti-DNA antibodies may also contribute to renal damage by directly influencing mesangial gene expression. Methods Complementary DNA microarray gene profiling was performed in primary mesangial cells (derived from lupus-prone MRL/lpr mice) treated with pathogenic, noncomplexed anti-DNA antibodies. Significant gene up-regulation induced by anti-DNA antibodies as determined by microarray analysis was further investigated by real-time polymerase chain reaction and methods to detect the relevant proteins. Induction of proinflammatory genes by pathogenic antibodies was confirmed by comparing gene expression in glomeruli of old versus young MRL/lpr mice, and by antibody injection in vivo. Results Pathogenic, but not nonpathogenic, antibodies significantly induced a number of transcripts, including CXCL1/KC, LCN2, iNOS, CX3CL1/fractalkine, SERPINA3G, and I,B, ("marker genes"). Blocking of Fc, receptors or using Fc, chain,knockout mesangial cells had no effect on the gene regulation effect of the pathogenic antibody R4A, indicating a non,Fc-dependent mechanism. The glomerular expression of these marker genes increased over time with the development of glomerular antibody deposition and active nephritis in MRL/lpr mice. Moreover, injection of R4A into SCID mice in vivo significantly up-regulated glomerular marker gene expression. Conclusion These findings indicate that the renal pathogenicity of anti-DNA antibodies may be attributed in part to their ability to directly modulate gene expression in kidney mesangial cells through both Fc-dependent and non,Fc-dependent mechanisms. [source] Attenuated endothelin-1 mRNA expression with endothelin-1 receptor blockade during hypoxaemia and reoxygenation in newborn pigletsACTA PAEDIATRICA, Issue 6 2000S Medbø We investigated the cause of decreased plasma endothelin-1 (ET-1) during hypoxaemia and reoxygenation in newborn piglets subjected to simultaneous blocking of the ET-1 receptors. Changes in plasma ET-1 and prepro-ET-1 mRNA expression in the main pulmonary artery and the left lower lobe in the lung were studied in 1-2-d-old piglets. Ten minutes prior to hypoxaemia, the hypoxaemia group (n = 10) was given saline, two groups (both n = 9) were given 1 and 5 mg/kg i.v. SB 217242 (an ET-1 receptor antagonist). Two groups served as normoxic controls, with and without SB 217242 5 mg/kg i.v. Hypoxaemia was induced by ventilating with 8% O2 until base excess was 20mmol/l or mean arterial blood pressure was < 20mmHg. Reoxygenation was performed for 2h with room air. During hypoxaemia, plasma ET-1 decreased in the hypoxaemia group, remained unchanged in the 1-mg group and increased in the 5-mg group. At the end of reoxygenation, plasma ET-1 was above baseline in the 1-mg and 5-mg groups. In the pulmonary artery, the hypoxaemia group showed 2- to 5-fold higher prepro-ET-1 mRNA expression compared to all the other groups (p < 0.05). There were trends for higher prepro-ET-1 mRNA expression in pulmonary tissue in the hypoxaemia group compared to the two receptor-blocking groups (p < 0.07). Conclusions: We conclude that hypoxaemia and reoxygenation increase prepro-ET-1 mRNA expression in the pulmonary artery in newborn piglets. These observations suggest that the half-life of ET-1 is decreased during hypoxaemia and reoxygenation in newborn piglets. [source] Deadlock detection in MPI programsCONCURRENCY AND COMPUTATION: PRACTICE & EXPERIENCE, Issue 11 2002Glenn R. Luecke Abstract The Message-Passing Interface (MPI) is commonly used to write parallel programs for distributed memory parallel computers. MPI-CHECK is a tool developed to aid in the debugging of MPI programs that are written in free or fixed format Fortran 90 and Fortran 77. This paper presents the methods used in MPI-CHECK 2.0 to detect many situations where actual and potential deadlocks occur when using blocking and non-blocking point-to-point routines as well as when using collective routines. Copyright © 2002 John Wiley & Sons, Ltd. [source] Staying alive: Dalmatian mediated blocking of apoptosis is essential for tissue maintenanceDEVELOPMENTAL DYNAMICS, Issue 6 2010Bilal E. Kerman Abstract In an EMS screen for mutations disrupting tracheal development, we identified new alleles of the dalmation (dmt) gene, which had previously been shown to affect peripheral nervous system (PNS) development. Here, we demonstrate that dmt loss results in programmed cell death, disrupting PNS patterning and leading to large gaps in the salivary duct and trachea. Dmt loss results in increased expression of the proapoptotic regulator genes head involution defective (hid) and reaper (rpr), and deletion of these genes or tissue-specific expression of the baculoviral apoptotic inhibitor P35 rescues the dmt defects. dmt is also required to protect cells from irradiation induced expression of hid and rpr during the irradiation resistant stage, which begins as cells become irreversibly committed to their final fates. Thus, we propose that Dmt keeps cells alive by blocking activation of hid and rpr as cells become irreversibly committed. Developmental Dynamics 239:1609,1621, 2010. © 2010 Wiley-Liss, Inc. [source] Iris as a recipient tissue for pigment cells: Organized in vivo differentiation of melanocytes and pigmented epithelium derived from embryonic stem cells in vitroDEVELOPMENTAL DYNAMICS, Issue 9 2008Hitomi Aoki Abstract Regenerative transplantation of embryonic stem (ES) cell-derived melanocytes into adult tissues, especially skin that includes hair follicles or the hair follicle itself, generally not possible, whereas that of ES cell-derived pigmented epithelium was reported previously. We investigated the in vivo differentiation of these two pigment cell types derived from ES cells after their transfer into the iris. Melanocytes derived from ES cells efficiently integrated into the iris and expanded to fill the stromal layer of the iris, like those prepared from neonatal skin. Transplanted pigmented epithelium from either ES cells or the neonatal eye was also found to be integrated into the iris. Both types of these regenerated pigment cells showed the correct morphology. Regenerated pigment epithelium expressed its functional marker. Functional blocking of signals required for melanocyte development abolished the differentiation of transplanted melanocytes. These results indicate successful in vivo regenerative transfer of pigment cells induced from ES cells in vitro. Developmental Dynamics 237:2394,2404, 2008. © 2008 Wiley-Liss, Inc. [source] Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro,DEVELOPMENTAL DYNAMICS, Issue 2 2002Anthony M. Delise Abstract During embryonic limb development, cartilage formation is presaged by a crucial mesenchymal cell condensation phase. N-Cadherin, a Ca2+ -dependent cell,cell adhesion molecule, is expressed in embryonic chick limb buds in a spatiotemporal pattern suggestive of its involvement during cellular condensation; functional blocking of N-cadherin homotypic binding, by using a neutralizing monoclonal antibody, results in perturbed chondrogenesis in vitro and in vivo. In high-density micromass cultures of embryonic limb mesenchymal cells, N-cadherin expression level is high during days 1 and 2, coincident with active cellular condensation, and decreases upon overt chondrogenic differentiation from day 3 on. In this study, we have used a transfection approach to evaluate the effects of gain- and loss-of-function expression of N-cadherin constructs on mesenchymal condensation and chondrogenesis in vitro. Chick limb mesenchymal cells were transfected by electroporation with recombinant expression plasmids encoding wild-type or two mutant extracellular/cytoplasmic deletion forms of N-cadherin. Expression of the transfected N-cadherin forms showed a transient profile, being high on days 1,2 of culture, and decreasing by day 3, fortuitously coincident with the temporal profile of endogenous N-cadherin gene expression. Examined by means of peanut agglutinin (PNA) staining for condensing precartilage mesenchymal cells, cultures overexpressing wild-type N-cadherin showed enhanced cellular condensation on culture days 2 and 3, whereas expression of the deletion mutant forms (extracellular/cytoplasmic) of N-cadherin resulted in a decrease in PNA staining, suggesting that a complete N-cadherin protein is required for normal cellular condensation to occur. Subsequent chondrogenesis was also affected. Cultures overexpressing the wild-type N-cadherin protein showed enhanced chondrogenesis, indicated by increased production of cartilage matrix (sulfated proteoglycans, collagen type II, and cartilage proteoglycan link protein), as well as increased cartilage nodule number and size of individual nodules, compared with control cultures and cultures transfected with either of the two mutant N-cadherin constructs. These results demonstrate that complete N-cadherin function, at the levels of both extracellular homotypic binding and cytoplasmic linkage to the cytoskeleton by means of the catenin complex, is required for chondrogenesis by mediating functional mesenchymal cell condensation. © 2002 Wiley-Liss, Inc. [source] Arachidonic acid as a retrograde signal controlling growth and dynamics of retinotectal arborsDEVELOPMENTAL NEUROBIOLOGY, Issue 1 2008B.H. Leu Abstract In the developing visual system, correlated presynaptic activity between neighboring retinal ganglion cells (RGC) stabilizes retinotopic synapses via a postsynaptic NMDAR (N -methyl- D -aspartate receptor)-dependent mechanism. Blocking NMDARs makes individual axonal arbors larger, which underlies an unsharpened map, and also increases branch turnover, as if a stabilizing factor from the postsynaptic partner is no longer released. Arachidonic acid (AA), a candidate retrograde stabilizing factor, is released by cytoplasmic phospholipase A2 (cPLA2) after Ca2+ entry through activated NMDARs, and can activate presynaptic protein kinase C to phosphorylate various substrates such as GAP43 to regulate cytoskeletal dynamics. To test the role of cPLA2 in the retinotectal system of developing zebrafish, we first used PED6, a fluorescent reporter of cPLA2 activity, to show that 1,3 min of strobe flashes activated tectal cPLA2 by an NMDAR-dependent mechanism. Second, we imaged the dynamic growth of retinal arbors during both local inhibition of tectal cPLA2 by a pharmacological inhibitor, arachidonic tri-fluoromethylketone, and its suppression by antisense oligonucleotides (both injected intraventricularly). Both methods produced larger arbors and faster branch dynamics as occurs with blocking NMDARs. In contrast, intraocular suppression of retinal cPLA2 with large doses of antisense oligos produced none of the effects of tectal cPLA2 inhibition. Finally, if AA is the retrograde messenger, the application of exogenous AA to the tectum should reverse the increased branch turnover caused by blocking either NMDARs or cPLA2. In both cases, intraventricular injection of AA stabilized the overall branch dynamics, bringing rates down below the normal values. The results suggest that AA generated postsynaptically by cPLA2 downstream of Ca2+ entry through NMDARs acts as a retrograde signal to regulate the dynamic growth of retinal arbors. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2008. [source] Oxidative stress, nitric oxide, and the mechanisms of cell death in Lurcher Purkinje cellsDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2007Rebecca McFarland Abstract Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (+/Lc). Lurcher is a gain of function mutation in the ,2 glutamate receptor (GluR,2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of +/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all +/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in +/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and +/Lc Purkinje cells at P10, P15, and P25. The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in +/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for +/Lc Purkinje cell death, +/Lc mutants were crossed with an ,-nNOS knockout mutant (nNOS,,/,) to reduce the production of NO. Analysis of the double mutants showed that blocking ,-nNOS expression does not rescue +/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluR,2+/Lc:nNOS,/, double mutant Purkinje cells, which suggests that the failure to rescue GluR,2+/Lc:nNOS,/, Purkinje cells may be explained by the induction of alternative nNOS isoforms. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Differential roles of two major brain structures, mushroom bodies and central complex, for Drosophila male courtship behaviorDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2006Takaomi Sakai Abstract Drosophila male courtship is a complex and robust behavior, the potential for which is genetically built into specific neural circuits in the central nervous system. Previous studies using male-female mosaics and the flies with defects in particular brain structures implicated the critical central regions involved in male courtship behavior. However, their acute physiological roles in courtship regulation still largely remain unknown. Using the temperature-sensitive Dynamin mutation, shibirets1, here we demonstrate the significance of two major brain structures, the mushroom bodies and the central complex, in experience-independent aspects of male courtship. We show that blocking of synaptic transmission in the mushroom body intrinsic neurons significantly delays courtship initiation and reduces the courtship activity by shortening the courtship bout length when virgin females are used as a sexual target. Interestingly, however, the same treatment affects neither initiation nor maintenance of courtship toward young males that release courtship-stimulating pheromones different from those of virgin females. In contrast, blocking of synaptic transmission in a central complex substructure, the fan-shaped body, slightly but significantly reduces courtship activity toward both virgin females and young males with little effect on courtship initiation. Taken together, our results indicate that the neuronal activity in the mushroom bodies plays an important role in responding to female-specific sex pheromones that stimulate initiation and maintenance of male courtship behavior, whereas the fan-shaped body neurons are involved in maintenance of male courtship regardless of the nature of courtship-stimulating cues. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] A developmental bottleneck in dispersing larvae: implications for spatial population dynamicsECOLOGY LETTERS, Issue 4 2003Daniel W. Schneider Abstract We found evidence for a critical population bottleneck at a developmental-stage transition in larvae of the zebra mussel Dreissena polymorpha Pallas from field estimates of mortality. Identification of this critical period in the field was made possible by closely tracking cohorts of larvae over 5 days of development as they dispersed 128 km in a river system. The presence of a survival bottleneck during development was confirmed in laboratory studies of zebra mussel larvae. Development-specific mortality has important implications for spatial population dynamics of the zebra mussel in particular, and all species with indirect development in general. Marine reserves that do not take development-specific mortality into account may dramatically underestimate reserve size needed to protect rare and/or exploited marine populations. Conversely, for the zebra mussel, the lower contribution of dispersing individuals to population growth downstream of reserves can lead to more feasible control through the blocking of dispersal. [source] DO GOVERNMENTS SUPPRESS GROWTH?ECONOMICS & POLITICS, Issue 1 2007AND INNOVATION BLOCKING IN A MODEL OF SCHUMPETERIAN GROWTH, INSTITUTIONS, RENT-SEEKING This paper argues that some governments adopt growth-reducing policies due to the rational self-interest of the political elites. The model takes a rent-seeking government that can block innovation and incorporates it into a Schumpeterian growth model. The quality of a country's institutions is reflected in the cost of innovation blocking. An increase in the level of innovation-blocking activity will reduce the rate of innovation and therefore reduce growth. The government also faces the possibility of losing power whenever an innovation occurs. We examine the conditions under which a government will choose to block innovation and suppress growth. [source] Natural killer cells become tolerogenic after interaction with apoptotic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2010Wai Po Chong Abstract NK cells are effectors in innate immunity and also participate in immunoregulation through the release of TGF-,1 and lysis of activated/autoreactive T cells. Apoptotic cells (AC) have been shown to induce tolerogenic properties in innate immune cells, including macrophages and dendritic cells, but not NK cells. In this study, we demonstrated that after interaction with AC, NK cells released TGF-,1, which in turn suppressed the production of IFN-, by NK cells upon IL-12 and IgG activation. We further identified phosphatidylserine as a potential target on AC for the NK cells, as phosphatidylserine could stimulate NK cells to release TGF-,1, which in turn suppressed CD4+ T-cell proliferation and activation. Moreover, AC-treated NK cells displayed cytotoxicity against autologous-activated CD4+ T cells by upregulating NKp46. This lysis occurred in part through the NKp46-vimentin pathway, as activated CD4+ T cells expressed vimentin on the cell surface and blocking of vimentin or NKp46, but not other NK-cell receptors, significantly suppressed the NK-cell cytotoxicity. We report here a novel interaction between NK cells and AC, resulting in the tolerogenic properties of NK cells required for immune contraction. [source] In vivo tumor cell rejection induced by NK cell inhibitory receptor blockade: Maintained tolerance to normal cells even in the presence of IL-2EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2010Gustaf Vahlne Abstract Missing-self-reactivity can be mimicked by blocking self-specific inhibitory receptors on NK cells, leading to increased rejection of syngeneic tumor cells. Using a mouse model, we investigated whether Ab-mediated blocking of inhibitory receptors, to a degree where NK cells rejected syngeneic tumor cells, would still allow self-tolerance toward normal syngeneic cells. Ly49C/I inhibitory receptors on C57BL/6 (H-2b) NK cells were blocked with F(ab')2 fragments of the mAb 5E6. Inhibitory receptor blockade in vivo caused rejection of i.v. inoculated fluorescence-labeled syngeneic lymphoma line cells but not of syngeneic spleen cells, BM cells or lymphoblasts. The selective rejection of tumor cells was NK cell-dependent and specifically induced by Ly49C/I blockade. Moreover, selective tumor rejection was maintained after treatment with 5E6 F(ab')2 for 9 wk, arguing against the induction of NK cell anergy or autoreactivity during this time. Combination therapy using 5E6 F(ab')2 together with high dose IL-2 treatment further increased lymphoma cell rejection. In addition, combination therapy reduced growth of melanoma cell line tumors established by s.c. inoculation 3 days before start of treatment. Our results demonstrate that inhibitory receptor blockade does not result in attack on normal cells, despite potent reactivity against MHC class I-expressing tumors. [source] NK cells of human secondary lymphoid tissues enhance T cell polarization via IFN-, secretionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2006Barbara Morandi Abstract Human secondary lymphoid tissues harbor NK cells that predominantly secrete cytokines in response to activation. Here, we demonstrate that these immunoregulatory NK cells assist in the Th1 polarization of primary immune responses, induced by dendritic cells. Tonsilar, but not peripheral blood NK cells enhanced the expansion of IFN-,-producing CD4+ T cells via their superior ability to produce IFN-,. Addition of IFN-, increased Th1 polarization while antibody blocking of this cytokine abolished NK cell-dependent Th1 polarization. Our data suggest that NK cells in secondary lymphoid organs assist priming of Th1 cells via cytokine secretion and this effect should be harnessed during vaccination against viruses and tumors. [source] AQP4 expression in striatal primary cultures is regulated by dopamine , implications for proliferation of astrocytesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2008Eva Küppers Abstract Proliferation of astrocytes plays an essential role during ontogeny and in the adult brain, where it occurs following trauma and in inflammation and neurodegenerative diseases as well as in normal, healthy mammals. The cellular mechanisms underlying glial proliferation remain poorly understood. As dopamine is known to modulate proliferation in different cell populations, we investigated the effects of dopamine on the proliferation of striatal astrocytes in vitro. We found that dopamine reduced proliferation. As proliferation involves, among other things, a change in cell volume, which normally comes with water movement across the membrane, water channels might represent a molecular target of the dopamine effect. Therefore we studied the effect of dopamine on aquaporin 4 (AQP4) expression, the main aquaporin subtype expressed in glial cells, and observed a down-regulation of the AQP4-M23 isoform. This down-regulation was the cause of the dopamine-induced decrease in proliferation as knockdown of AQP4 using siRNA techniques mimicked the effects of dopamine on proliferation. Furthermore, stimulation of glial proliferation by basic fibroblast growth factor was also abolished by knocking down AQP4. In addition, blocking of AQP4 with 10 ,m tetraethylammonium inhibited osmotically induced cell swelling and stimulation of glial cell proliferation by basic fibroblast growth factor. These results demonstrate a clear-cut involvement of AQP4 in the regulation of proliferation and implicate that modulation of AQP4 could be used therapeutically in the treatment of neurodegenerative diseases as well as in the regulation of reactive astrogliosis by preventing or reducing the glia scar formation, thus improving regeneration following ischemia or other trauma. [source] In vivo optical recordings of synaptic transmission and intracellular Ca2+ and Cl, in the superior colliculus of fetal ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2006Yoshiyuki Sakata Abstract Although the N -methyl- d -aspartate (NMDA) receptor is known to play a crucial role in activity-dependent remodeling of synaptic connections in the fetal superior colliculus (SC), its contribution to the electrical activity of fetal SC neurons has not been determined. Furthermore, whether ,-aminobutyric acid (GABA)-mediated inhibition occurs either as early as prenatal periods or only after eye opening has been controversial. We therefore performed optical recordings using voltage-, Ca2+ - and Cl, -sensitive fluorescent dyes to analyse synaptic transmission and changes in intracellular Ca2+ and Cl, in the SC of fetal rats that were still connected with the dams by the umbilical cord. Excitatory and inhibitory responses were evoked by focal SC stimulation. The excitatory synaptic responses are composed of early and late components. The early component was mediated by both non-NMDA and NMDA receptors, whereas the late component occurred mainly via NMDA receptors. Train pulse stimulation at higher currents was required for induction of the inhibition, which was antagonized by bicuculline, and blocking of the GABA-mediated inhibition by bicuculline uncovered masked excitatory synaptic responses. Focal SC stimulation induced increases in [Cl,]i and [Ca2+]i that were mediated by GABA-A receptors and mainly by NMDA receptors, respectively. GABA antagonists augmented SC-induced increases in [Ca2+]i. These results indicate that, in the fetal SC, excitatory and inhibitory synaptic transmissions occur before birth, that the NMDA receptor is a major contributor to excitatory synaptic transmission and increased [Ca2+]i, and that the GABA-A receptor is already functioning to inhibit excitatory neurotransmission. [source] Bi-directional regulation of postsynaptic cortactin distribution by BDNF and NMDA receptor activityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005Junko Iki Abstract Cortactin is an F-actin-associated protein which interacts with the postsynaptic scaffolding protein Shank at the SH3 domain and is localized within the dendritic spine in the mouse neuron. Green fluorescent protein (GFP)-based time-lapse imaging revealed cortactin redistribution from dendritic cytoplasm to postsynaptic sites by application of brain-derived neurotrophic factor (BDNF). This response was mediated by mitogen-activated protein (MAP) kinase activation and was dependent on the C-terminal SH3 domain. In contrast, activation of N -methyl- d -aspartate (NMDA) receptors induced loss of cortactin from postsynaptic sites. This NMDA-dependent redistribution was blocked by an Src family kinase inhibitor. Conversely, increasing Src family kinase activity induced cortactin phosphorylation and loss of cortactin from the postsynaptic sites. Finally, blocking of endogenous BDNF reduced the amount of cortactin at the postsynaptic sites and an NMDA receptor antagonist prevented this reduction. These results indicate the importance of counterbalance between BDNF and NMDA receptor-mediated signalling in the reorganization of the postsynaptic actin cytoskeleton during neuronal development. [source] Spontaneous electrical activity and dendritic spine size in mature cerebellar Purkinje cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2005Robin J. Harvey Abstract Previous experiments have shown that in the mature cerebellum both blocking of spontaneous electrical activity and destruction of the climbing fibres by a lesion of the inferior olive have a similar profound effect on the spine distribution on the proximal dendrites of the Purkinje cells. Many new spines develop that are largely innervated by parallel fibers. Here we show that blocking electrical activity leads to a significant decrease in size of the spines on the branchlets. We have also compared the size of the spines of the proximal dendritic domain that appear during activity block and after an inferior olive lesion. In this region also, the spines in the absence of activity are significantly smaller. In the proximal dendritic domain, the new spines that develop in the absence of activity are innervated by parallel fibers and are not significantly different in size from those of the branchlets, although they are shorter. Thus, the spontaneous activity of the cerebellar cortex is necessary not only to maintain the physiological spine distribution profile in the Purkinje cell dendritic tree, but also acts as a signal that prevents spines from shrinking. [source] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||