Science.gov

Sample records for l1 regulates transendothelial

  1. Localized signals that regulate transendothelial migration.

    PubMed

    Muller, William A

    2016-02-01

    Transendothelial migration (TEM) of leukocytes is the step in leukocyte emigration in which the leukocyte actually leaves the blood vessel to carry out its role in the inflammatory response. It is therefore, arguably the most critical step in emigration. This review focuses on two of the many aspects of this process that have seen important recent developments. The adhesion molecules, PECAM (CD31) and CD99 that regulate two major steps in TEM, do so by regulating specific signals. PECAM initiates the signaling pathway responsible for the calcium flux that is required for TEM. Calcium enters through the cation channel TRPC6 and recruits the first wave of trafficking of membrane from the lateral border recycling compartment (LBRC). CD99 signals through soluble adenylate cyclase to activate protein kinase A to recruit a second wave of LBRC trafficking. Another process that is critical for TEM is transient removal of VE-cadherin from the site of TEM. However, the local signaling pathways that are responsible for this appear to be different from those that open the junctions to increase vascular permeability.

  2. The regulation of transendothelial migration: new knowledge and new questions.

    PubMed

    Muller, William A

    2015-08-01

    Leucocyte transendothelial migration (TEM) involves a co-operative series of interactions between surface molecules on the leucocyte and cognate counter-ligands on the endothelial cell. These interactions set up a cascade of signalling events inside the endothelial cell that both allow for the junctions to loosen and for membrane to be recruited from the lateral border recycling compartment (LBRC). The LBRC is thought to provide an increased surface area and unligated receptors to the leucocyte to continue the process. The relative importance of the individual adhesion/signalling molecules that promote transmigration may vary depending on the type of leucocyte, the vascular bed, the inflammatory stimulus, and the stage of the inflammatory response. However, the molecular interactions between leucocyte and endothelial cell activate signalling pathways that disengage the adherens and tight junctions and recruit the LBRC to the site of transmigration. With the exception of disengaging the junctions, similar molecules and mechanisms promote transcellular migration as paracellular migration of leucocytes. This review will discuss the molecular interactions and signalling pathways that regulate transmigration, and the common themes that emerge from studying TEM of different leucocyte subsets under different inflammatory conditions. We will also raise some unanswered questions in need of future research.

  3. Epithin/PRSS14 proteolytically regulates angiopoietin receptor Tie2 during transendothelial migration.

    PubMed

    Kim, Chungho; Lee, Hyo Seon; Lee, Deokjae; Lee, Sang Don; Cho, Eun-Gyung; Yang, Soo Jung; Kim, Sang Bum; Park, Dongeun; Kim, Moon Gyo

    2011-01-27

    Epithin/PRSS14, a type II transmembrane serine protease, is involved in normal epithelial development and tumor progression. Here we report, as an interacting substrate of epithin, a receptor tyrosine kinase Tie2 that is well known for important roles in the vessel stability. Epithin interacts with and degrades the Tie2 extracellular portion that contains the ligand-binding domain. Epithin is located in the neighbor of Tie2-expressing vessels in normal tissue. Furthermore, epithin can cleave and degrade Tie2 not only in the same cell but also from neighboring cells nearby, resulting in the degradation of the Tie2 ectodomain. The remaining Tie2 fragment was highly phosphorylated and was able to recruit a downstream effector, phosphatidylinositol 3-kinase. Knocking down epithin expression using short hairpin RNA in thymoma cell severely impaired the migration through endothelial cells that show the actin rearrangement during the process. The diminution of epithin protein expression in 4T1 breast cancer cells caused the significant decrease in the number of transendothelial migrating cells in vitro as well as in those of metastasizing tumor nodules in vivo, Therefore, we propose that epithin, which regulates endothelial Tie2 functions, plays a critical role in the fine tuning of transendothelial migration for normal and cancer cells.

  4. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration

    PubMed Central

    Watson, Richard L.; Buck, Jochen; Levin, Lonny R.; Winger, Ryan C.; Wang, Jing; Arase, Hisashi

    2015-01-01

    CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM. PMID:26101266

  5. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

    PubMed

    Watson, Richard L; Buck, Jochen; Levin, Lonny R; Winger, Ryan C; Wang, Jing; Arase, Hisashi; Muller, William A

    2015-06-29

    CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM.

  6. The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo.

    PubMed

    Woodfin, Abigail; Voisin, Mathieu-Benoit; Beyrau, Martina; Colom, Bartomeu; Caille, Dorothée; Diapouli, Frantzeska-Maria; Nash, Gerard B; Chavakis, Triantafyllos; Albelda, Steven M; Rainger, G Ed; Meda, Paolo; Imhof, Beat A; Nourshargh, Sussan

    2011-06-26

    The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

  7. Poliovirus receptor (CD155) regulates a step in transendothelial migration between PECAM and CD99.

    PubMed

    Sullivan, David P; Seidman, Michael A; Muller, William A

    2013-03-01

    The movement of leukocytes across endothelium [referred to as diapedesis or transendothelial migration (TEM)] is a critical step in the inflammatory process. Recently, it was demonstrated that treatment of endothelial cells and monocytes with antibodies against poliovirus receptor (PVR; CD155) and DNAX-associated molecule-1 (DNAM-1; CD226) arrested monocytes over endothelial junctions and prevented TEM, suggesting that these molecules are involved in diapedesis. However, nothing was known about the mechanism by which PVR and DNAM-1 work in TEM. Herein, we show that, similar to endothelial PECAM interacting with leukocyte PECAM, activation of endothelial PVR with anti-PVR antibodies or interaction with its ligand, DNAM-1, results in recruitment of the tyrosine phosphatase Shp-2, and this process is dependent on Src kinases. Furthermore, differential and sequential treatment with blocking antibodies directed against PVR, DNAM-1, PECAM, and CD99 showed that endothelial PVR and monocyte DNAM-1 interact at and regulate a step between those regulated by PECAM and CD99. Further studies demonstrate that PVR resides in the recently identified lateral border recycling compartment, similar to PECAM and CD99. These findings suggest that the localization of adhesion/signaling molecules to the lateral border recycling compartment and the recruitment of Shp-2 may be common mechanisms for the regulation of TEM by endothelial cells.

  8. TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response

    PubMed Central

    Weber, Evan W.; Han, Fei; Tauseef, Mohammad; Birnbaumer, Lutz; Mehta, Dolly

    2015-01-01

    Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca2+]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca2+]i regulates TEM and the channels mediating this ↑[Ca2+]i are unknown. Buffering ↑[Ca2+]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca2+]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca2+ channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca2+]i required for TEM at a step downstream of PECAM homophilic interactions. PMID:26392222

  9. Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration.

    PubMed

    Estin, Miriam L; Thompson, Scott B; Traxinger, Brianna; Fisher, Marlie H; Friedman, Rachel S; Jacobelli, Jordan

    2017-04-04

    Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.

  10. MIP-1α enhances Jurkat cell transendothelial migration by up-regulating endothelial adhesion molecules VCAM-1 and ICAM-1.

    PubMed

    Ma, Yi-Ran; Ma, Ying-Huan

    2014-11-01

    The aim of this study is to evaluate the expression of macrophage inflammatory protein-1α (MIP-1α) in Jurkat cells and its effect on transendothelial migration. In the present study, human acute lymphoblastic leukemia Jurkat cells (Jurkat cells) were used as a model of T cells in human T-cell acute lymphoblastic leukemia (T-ALL), which demonstrated significantly higher MIP-1α expression compared with that in normal T-cell controls. The ability of Jurkat cells to cross a human brain microvascular endothelial cell (HBMEC) monolayer was almost completely abrogated by MIP-1α siRNA. In addition, the overexpression of MIP-1α resulted in the up-regulated expression of endothelial adhesion molecules, which enhanced the migration of Jurkat cells through a monolayer of HBMEC. MIP-1α levels in Jurkat cells appeared to be an important factor for its transendothelial migration, which may provide the theoretical basis to understand the mechanisms of brain metastases of T-ALL at cellular and molecular levels.

  11. Stanniocalcin-1 regulates endothelial gene expression and modulates trans-endothelial migration of leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) inhibits monocyte chemotactic protein-1- and stromal-derived factor-1alpha (SDF-1alpha)-mediated chemotaxis and diminishes chemokinesis in macrophage-like RAW264.7 and U937 cells in a manner that may involve atte...

  12. Transcriptional profiling of human monocytes identifies the inhibitory receptor CD300a as regulator of transendothelial migration.

    PubMed

    Ghavampour, Sharang; Lange, Carsten; Bottino, Cristina; Gerke, Volker

    2013-01-01

    Local inflammatory responses are characterized by the recruitment of circulating leukocytes from the blood to sites of inflammation, a process requiring the directed migration of leukocytes across the vessel wall and hence a penetration of the endothelial lining. To identify underlying signalling events and novel factors involved in these processes we screened for genes differentially expressed in human monocytes following their adhesion to and passage through an endothelial monolayer. Functional annotation clustering of the genes identified revealed an overrepresentation of those associated with inflammation/immune response, in particular early monocyte to macrophage differentiation. Among the gene products so far not implicated in monocyte transendothelial migration was the inhibitory immune receptor CD300a. CD300a mRNA and protein levels were upregulated following transmigration and engagement of the receptor by anti-CD300a antibodies markedly reduced monocyte transendothelial migration. In contrast, siRNA mediated downregulation of CD300a in human monocytes increased their rate of migration. CD300a colocalized and cosedimented with actin filaments and, when activated, caused F-actin cytoskeleton alterations. Thus, monocyte transendothelial migration is accompanied by an elevation of CD300a which serves an inhibitory function possibly required for termination of the actual transmigration.

  13. L1 adhesion molecule on mouse leukocytes: regulation and involvement in endothelial cell binding.

    PubMed

    Hubbe, M; Kowitz, A; Schirrmacher, V; Schachner, M; Altevogt, P

    1993-11-01

    L1 is a cell surface glycoprotein of the immunoglobulin superfamily which was initially shown to mediate adhesion between neural cells. Recently we have reported that L1 is expressed by bone marrow cells and the majority of mature lymphocytes (Kowitz et al., Eur. J. Immunol. 1992. 22: 1199-1205). To analyze the function of L1 on leukocytes we studied its regulation following cell activation. In vitro activation of B lymphocytes with lipopolysaccharide or T lymphocytes with phorbol 12-myristate 13-acetate/Ca2+ ionophore, concanavalin A or anti-CD3 monoclonal antibody as well as in vivo activation of V beta 8+ T cells with staphylococcal enterotoxin B (SEB) revealed a down-regulation of L1 within 48 h. A rapid loss of L1 expression was seen when mouse neutrophils were activated with PMA alone. This rapid loss paralleled the shedding of L-selectin. We also studied a possible role of L1 in the binding of leukocytes to endothelial cells. ESb-MP lymphoma cells with a high expression of L1 (L1hi) could bind to bend3 endothelioma cells without prior activation with inflammatory cytokines. The interaction was inhibited by anti-L1 antibodies. In contrast, ESb-MP cells with low L1 expression (L1lo) were only marginally bound. Latex beads coated with affinity-isolated L1 antigen were also able to bind to the endothelioma cells in a specific fashion. The binding of ESb-MP lymphoma cells required Ca2+ and Mg2+ ions and was sensitive to cold temperature. Since the endothelioma cells did not express L1 the binding mechanism studied here is distinct from the established L1-L1 homotypic interaction. It is possible that the novel L1-mediated adhesion pathway involves an unidentified ligand and could play a role in leukocyte migration.

  14. Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: implications for transendothelial chemotaxis

    PubMed Central

    1996-01-01

    Leukocyte emigration possibly requires dynamic regulation of integrin adhesiveness for endothelial and extracellular matrix ligands. Adhesion assays on purified vascular cell adhension molecule (VCAM)-1, fibronectin, and fibronectin fragments revealed distinct kinetic patterns for the regulation of very late antigen (VLA)-4 (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) avidity by the CC chemokines monocyte inflammatory protein (MIP)-1 alpha, RANTES (regulated on activation, normal T expressed and secreted), or monocyte chemoattractant protein (MCP)-1 in monocytes. CC chemokines induced early activation and subsequent deactivation of VLA-4, whereas upregulation of VLA-5 avidity occurred later and persisted. Controlled detachment assays in shear flow suggested that adhesive strength of VLA-4 for VCAM-1 or the 40-kD fragment of fibronectin (FN40) is more rapidly increased and subsequently reduced by MCP-1 than by MIP-1 alpha, and confirmed late and sustained activation of the adhesive strength of VLA-5 for the 120- kD fragment of fibronectin (FN120). Mn2+ or the stimulating beta 1 mAb TS2/16 strongly and stably enhanced monocyte binding to VCAM-1 or fibronectin, and locked beta 1 integrins in a high avidity state, which was not further modulated by CC chemokines. Mn2+ and mAb TS2/16 inhibited CC chemokine-induced transendothelial migration, particularly chemotaxis across stimulated endothelium that involved VLA-4 and VCAM- 1. VLA-4 on Jurkat cells is of constitutively high avidity and interfered with migration across barriers expressing VCAM-1. Low but not high site densities of VCAM-1 or FN40 promoted, while FN120 impaired, beta 1 integrin-dependent monocyte chemotaxis to MCP-1 across filters coated with these substrates. Thus, we show that CC chemokines can differentially and selectively regulate avidity of integrins sharing common beta subunits. Transient activation and deactivation of VLA-4 may serve to facilitate transendothelial diapedesis, whereas late and

  15. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  16. Mechanisms of Leukocyte Transendothelial Migration

    PubMed Central

    Muller, William A.

    2013-01-01

    Neither the innate nor adaptive immune system “responds” unless leukocytes cross blood vessels. This process occurs through diapedesis, in which the leukocyte moves in an ameboid fashion through tightly apposed endothelial borders and, in some cases, through the endothelial cell itself. This review focuses on the active role of the endothelial cell in diapedesis. Several mechanisms play a critical role in transendothelial migration, including signals derived from clustering of apically disposed intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, disruption or loosening of adherens junctions, and targeted recycling of platelet/endothelial cell adhesion molecule and other molecules from the recently described lateral border recycling compartment. Surprisingly, many of the same molecules and mechanisms that regulate paracellular migration also control transcellular migration. A hypothesis that integrates the various known mechanisms of transmigration is proposed. PMID:21073340

  17. Mechanisms of leukocyte transendothelial migration.

    PubMed

    Muller, William A

    2011-01-01

    Neither the innate nor adaptive immune system "responds" unless leukocytes cross blood vessels. This process occurs through diapedesis, in which the leukocyte moves in an ameboid fashion through tightly apposed endothelial borders and, in some cases, through the endothelial cell itself. This review focuses on the active role of the endothelial cell in diapedesis. Several mechanisms play a critical role in transendothelial migration, including signals derived from clustering of apically disposed intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, disruption or loosening of adherens junctions, and targeted recycling of platelet/endothelial cell adhesion molecule and other molecules from the recently described lateral border recycling compartment. Surprisingly, many of the same molecules and mechanisms that regulate paracellular migration also control transcellular migration. A hypothesis that integrates the various known mechanisms of transmigration is proposed.

  18. Rho GTPases and cancer cell transendothelial migration.

    PubMed

    Reymond, Nicolas; Riou, Philippe; Ridley, Anne J

    2012-01-01

    Small Rho GTPases are major regulators of actin cytoskeleton dynamics and influence cell shape and migration. The expression of several Rho GTPases is often up-regulated in tumors and this frequently correlates with a poor prognosis for patients. Migration of cancer cells through endothelial cells that line the blood vessels, called transendothelial migration or extravasation, is a critical step during the metastasis process. The use of siRNA technology to target specifically each Rho family member coupled with imaging techniques allows the roles of individual Rho GTPases to be investigated. In this chapter we describe methods to assess how Rho GTPases affect the different steps of cancer cell transendothelial cell migration in vitro.

  19. Reversible monoubiquitination regulates the Parkinson disease-associated ubiquitin hydrolase UCH-L1.

    PubMed

    Meray, Robin K; Lansbury, Peter T

    2007-04-06

    Deubiquitinating enzymes (DUBs) are negative regulators of protein ubiquitination and play an important role in ubiquitin-dependent processes. Recent studies have found that diverse cellular mechanisms are employed to control the activity of DUBs. Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a highly expressed neuronal DUB linked to Parkinson disease; however, little is known about its specific functions or modes of regulation. Here, we demonstrate that UCH-L1 is post-translationally modified by monoubiquitin in cells, at lysine residues near the active site. This modification restricts enzyme activity by preventing binding to ubiquitinated targets, and permanent monoubiquitination, as mimicked by a ubiquitin-UCH-L1 fusion, inhibits UCH-L1 in its capacity to increase free ubiquitin levels in cells. Interestingly, UCH-L1 catalyzes its own deubiquitination in an intramolecular manner, thereby regulating the lifetime of this modification. Our results illustrate monoubiquitination as a reversible regulatory mechanism for DUB activity involving auto-deubiquitination.

  20. Reactivation of L1 retrotransposon by benzo(a)pyrene involves complex genetic and epigenetic regulation.

    PubMed

    Teneng, Ivo; Montoya-Durango, Diego E; Quertermous, James L; Lacy, Mary E; Ramos, Kenneth S

    2011-03-01

    Benzo(a)pyrene (BaP), is an environmental pollutant present in tobacco smoke and a byproduct of fossil fuel combustion which likely contributes to the tumorigenic processes in human cancers including lung and esophageal. Long Interspersed Nuclear Element-1 (LINE-1) or L1 is a mobile element within the mammalian genome that propagates via a "copy-and-paste" mechanism using reverse transcriptase and RNA intermediates. L1 is strongly expressed during early embryogenesis and then silenced as cells initiate differentiation programming. Although the complex transcriptional control mechanisms of L1 are not well understood, L1 reactivation has been described in several human cancers and following exposure of mouse or human cells to BaP. In this study we investigated the molecular mechanisms and epigenetic events that regulate L1 reactivation following BaP exposure. We show that challenge of HeLa cells with BaP induces early enrichment of the transcriptionally-active chromatin markers histone H3 trimethylated at lysine 4 (H3K4Me3) and histone H3 acetylated at lysine 9 (H3K9Ac), and reduces association of DNA methyltransferase-1 (DNMT1) with the L1 promoter. These changes are followed by proteasome-dependent decreases in cellular DNMT1 expression and sustained reduction of cytosine methylation within the L1 promoter CpG island. Pharmacological inhibition of the proteasome signaling pathway with the inhibitor MG132 blocks degradation of DNMT1 and alters BaP-mediated histone epigenetic modifications. We conclude that genetic reactivation of L1 by BaP involves an ordered cascade of epigenetic events that begin with nucleosomal histone modifications and is completed with alterations in DNMT1 recruitment to the L1 promoter and reduced DNA methylation of CpG islands.

  1. Association between PD-1/PD-L1 and T regulate cells in early recurrent miscarriage.

    PubMed

    Li, Guiyu; Lu, Caixia; Gao, Jing; Wang, Xietong; Wu, Huanling; Lee, Chao; Xing, Baoxiang; Zhang, Qi

    2015-01-01

    In this study, we try to testify the relationship between the programmed cell death receptor-1 (PD-1)/programmed cell death ligand 1 (PD-L1) passway and Treg cells in maternal-fetal immune regulation through PD-1 blockade on lymphocytes of normal early pregnancy in vitro and investigation of the PD-1 and PD-L1 changes in early recurrent miscarriage patients. CD4+ CD25+ Treg cells and PD-1 (CD279) positive cell were detected in deciduas in early recurrent miscarriage patients by flow cytometry. And the normal early pregnant women were as controls. Meanwhile the mRNA level of PD-1 and molecular expression of PD-L1 in deciduas of early recurrent miscarriage patients were detected by real time RT-PCR test and Immunohistochemical staining respectively. Also through antibody blocking assay to block PD-1 on lymphocytes of normal early pregnancy in vitro further testify the relationship between PD-1/PD-L1 and Treg cells, the results were analyzed by flow cytometry. CD4+ CD25+ Treg cells decreased both in deciduas in RM (P < 0.05), and for all almost 100% Treg cells (CD4+ CD25+) expressed PD-1, but there was no difference between the PD-1 positive cells in decidual lymphocytes in RM and that in normal pregnancy women (P > 0.05). PD-L1 mRNA in deciduas decreased in RM (P < 0.001), but PD-1 mRNA no difference (P > 0.1). After PD-1 blockade there was no change in CD4+ CD25+ Treg cells percentage, while the CD4+ T cell percentage increased (P < 0.01), as well as the level of IFN-gamma in cells supernatant (P < 0.01). PD-1 blockade has a little influence on the number of Treg cells, and may lead to impaired Treg cells function, the decrease of PD-L1 may closely relates to the occurrence of early recurrent miscarriage and implies that Treg cells may through PD-1/PD-L1 pathway play a role of immunosuppression regulation, and the impairment of Treg cells function in recurrent early abortion cases may be due to PD-L1 decrease in deciduas or trophoblast cells rather than PD-1 change.

  2. Differential L1 regulation in pluripotent stem cells of humans and apes.

    PubMed

    Marchetto, Maria C N; Narvaiza, Iñigo; Denli, Ahmet M; Benner, Christopher; Lazzarini, Thomas A; Nathanson, Jason L; Paquola, Apuã C M; Desai, Keval N; Herai, Roberto H; Weitzman, Matthew D; Yeo, Gene W; Muotri, Alysson R; Gage, Fred H

    2013-11-28

    Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have

  3. Atypical calcium regulation of the PKD2-L1 polycystin ion channel

    PubMed Central

    DeCaen, Paul G; Liu, Xiaowen; Abiria, Sunday; Clapham, David E

    2016-01-01

    Native PKD2-L1 channel subunits are present in primary cilia and other restricted cellular spaces. Here we investigate the mechanism for the channel's unusual regulation by external calcium, and rationalize this behavior to its specialized function. We report that the human PKD2-L1 selectivity filter is partially selective to calcium ions (Ca2+) moving into the cell, but blocked by high internal Ca2+concentrations, a unique feature of this transient receptor potential (TRP) channel family member. Surprisingly, we find that the C-terminal EF-hands and coiled-coil domains do not contribute to PKD2-L1 Ca2+-induced potentiation and inactivation. We propose a model in which prolonged channel activity results in calcium accumulation, triggering outward-moving Ca2+ ions to block PKD2-L1 in a high-affinity interaction with the innermost acidic residue (D523) of the selectivity filter and subsequent long-term channel inactivation. This response rectifies Ca2+ flow, enabling Ca2+ to enter but not leave small compartments such as the cilium. DOI: http://dx.doi.org/10.7554/eLife.13413.001 PMID:27348301

  4. The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells.

    PubMed

    Yu, Bangning; Cook, Carla; Santanam, Nalini

    2009-10-01

    Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease.

  5. The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 Cells

    PubMed Central

    Yu, Bangning; Cook, Carla

    2009-01-01

    Abstract Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor (PPAR)-γ, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H2O2) (100 μM) or tumor necrosis factor-α (TNFα) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5–100 μM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARγ, and C/EBPα to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H2O2 or TNFα and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5–25 μM) having a larger inductive effect compared to higher concentrations (50–100 μM). Boldine treatment alone in the absence of H2O2 or TNFα was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  6. Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

    SciTech Connect

    Li, Ying; Huang, Xiaohua; An, Yue; Ren, Feng; Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei; He, Xiaowen; Schachner, Melitta; Xiao, Zhicheng; Ma, Keli; Li, Yali

    2013-10-25

    Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

  7. The RNA stability regulator HuR regulates L1 protein expression in vivo in differentiating cervical epithelial cells

    SciTech Connect

    Cumming, S.A.; Chuen-Im, T.; Zhang, J.; Graham, S.V.

    2009-01-05

    Human papillomavirus (HPV) L1 and L2 capsid protein expression is restricted to the granular layer of infected, stratified epithelia and is regulated at least partly at post-transcriptional levels. For HPV16, a 79 nt late regulatory element (LRE) is involved in this control. Using W12 cells as a model for HPV16-infected differentiating cervical epithelial cells we show that HuR, a key cellular protein that controls mRNA stability, binds the LRE most efficiently in nuclear and cytoplasmic extracts of differentiated cells. Further, HuR binds the 3' U-rich portion of the LRE directly in vitro. Overexpression of HuR in undifferentiated W12 cells results in an increase in L1 mRNA and protein levels while siRNA knock-down of HuR in differentiated W12 cells depletes L1 expression. In differentiated cervical epithelial cells HuR may bind and stabilise L1 mRNAs aiding translation of L1 protein.

  8. Epigenetic regulation of L1CAM in endometrial carcinoma: comparison to cancer–testis (CT-X) antigens

    PubMed Central

    2013-01-01

    Background L1CAM was originally identified as an adhesion molecule involved in neural development. In many human carcinomas L1CAM is over-expressed and is associated with a bad prognosis. We previously reported that L1CAM was absent in the vast majority of endometrioid endometrial carcinomas (ECs) (type 1) but was strongly expressed in the more aggressive serous and clear-cell ECs (termed type 2). The differential regulation of L1CAM in ECs is not well understood. Recent evidence suggests that it can be regulated by epigenetic mechanisms. Here we investigated the role of DNA-methylation of the L1CAM promoter for expression. We also studied the relationship to cancer testis (CT-X) antigens that co-localize with L1CAM on chromosome Xq28, a region that is often activated in human tumors. Methods We used EC cell lines and primary tumor tissues for our analysis. For expression analysis we employed RT-PCR and Western blotting. DNA-Methylation of the L1CAM promoter was determined after bisulfite conversation and DNA sequencing. Tumor tissues were examined by immunohistochemical (IHC) staining. Results We demonstrate that the treatment of L1CAM low/negative expressing EC cell lines with 5′-Azacytidine (5-AzaC) or knock-down of DNMT1 (DNA methyltransferase 1) as well as the HDAC (histone deacetylase) inhibitor Trichostatin A (TSA) up-regulated L1CAM at the mRNA and protein level. The L1CAM gene has two promoter regions with two distinct CpG islands. We observed that the expression of L1CAM correlated with hypermethylation in promoter 1 and 5-AzaC treatment affected the DNA-methylation pattern in this region. The CT-X antigens NY-ESO-1, MAGE-A3 and MAGE-A4 were also strongly up-regulated by 5-AzaC or knock-down of DNMT1 but did not respond to treatment with TSA. Primary EC tumor tissues showed a variable methylation pattern of the L1CAM promoter. No striking differences in promoter methylation were observed between tumor areas with L1CAM expression and those without

  9. miR-21-3p is a positive regulator of L1CAM in several human carcinomas.

    PubMed

    Doberstein, Kai; Bretz, Niko P; Schirmer, Uwe; Fiegl, Heidi; Blaheta, Roman; Breunig, Christian; Müller-Holzner, Elisabeth; Reimer, Dan; Zeimet, Alain G; Altevogt, Peter

    2014-11-28

    Expression of L1 cell adhesion molecule (L1CAM) occurs frequently in human cancers and is associated with poor prognosis in cancers such as ovarian, endometrial, breast, renal cell carcinoma and pancreatic ductal adenocarcinoma. L1CAM promotes cell motility, invasion, chemoresistance and metastasis formation. Elucidating genetic processes involved in the expression of L1CAM in cancers is of considerable importance. Transcription factors such as SLUG, β-catenin/TCF-LEF, PAX8 and VHL have been implicated in the re-activation of L1CAM in various types of cancers. There is increasing evidence that micro-RNAs can also have strong effects on gene expression. Here we have identified miR-21-3p as a positive regulator of L1CAM expression. Over-expression of miR-21-3p (miR-21*) but not the complementary sequence miR-21-5p (miR-21) could strongly augment L1CAM expression in renal, endometrial and ovarian carcinoma derived cell lines by an unknown mechanism involving transcriptional activation of the L1CAM gene. In patient cohorts from renal, endometrial and ovarian cancers we observed a strong positive correlation of L1CAM and miR-21-3p expressions. Although L1CAM alone was a reliable marker for overall and disease free survival, the combination of L1CAM and miR-21-3p expressions strongly enhanced the predictive power. Our findings shed new light on the complex regulation of L1CAM in cancers and advocate the use of L1CAM/miR-21-3p for diagnostic application.

  10. Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation.

    PubMed

    Henderson, Mark J; Vij, Neeraj; Zeitlin, Pamela L

    2010-04-09

    DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.

  11. The L1TD1 protein interactome reveals the importance of post-transcriptional regulation in human pluripotency.

    PubMed

    Emani, Maheswara Reddy; Närvä, Elisa; Stubb, Aki; Chakroborty, Deepankar; Viitala, Miro; Rokka, Anne; Rahkonen, Nelly; Moulder, Robert; Denessiouk, Konstantin; Trokovic, Ras; Lund, Riikka; Elo, Laura L; Lahesmaa, Riitta

    2015-03-10

    The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  12. KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

    PubMed Central

    Gupta, Namrata; Thakker, Suhani; Verma, Subhash C.

    2016-01-01

    The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA. PMID:27599637

  13. Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    PubMed

    Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

    2009-06-17

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

  14. Hormone and pharmaceutical regulation of ASP production in 3T3-L1 adipocytes.

    PubMed

    Gao, Ying; Gauvreau, Danny; Cianflone, Katherine

    2010-04-01

    Several studies have demonstrated increases in acylation stimulating protein (ASP), and precursor protein C3 in obesity, diabetes and dyslipidemia, however the nature of the regulation is unknown. To evaluate chronic hormonal and pharmaceutical mediated changes in ASP and potential mechanisms, 3T3-L1 adipocytes were treated with physiological concentrations of relevant hormones and drugs currently used in treatment of metabolic diseases for 48 h. Medium ASP production and C3 secretion were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride (TG) mass, non-esterified fatty acid (NEFA) release and real-time FA uptake). Chylomicrons increased ASP production (up to 411 +/- 133% P < 0.05), while leptin, triiodothyronine, and beta-blockers atenolol and propranolol had no effect. Dexamethasone, lovastatin, rosiglitazone and rimonabant decreased ASP production (-53 to -85%, P < 0.05), associated with a decrease in the precursor protein C3 (-37% to -65%, P < 0.01). By contrast, epinephrine, progesterone, testosterone, angiotensin II and metformin also decreased ASP (-54% to -100%, P < 0.05), but without change in precursor protein C3, suggesting a direct effect on convertase activity, possibly mediated by interference (except metformin) due to marked increases in NEFA (5.6-31-fold, increased P < 0.05). Both lovastatin and metformin induced decreases in ASP were also associated with decreased TG mass (maximal -60%, P < 0.05) and real-time FA uptake (maximum -75%, P < 0.05), suggesting a change in adipocyte differentiation status. These in vitro results are consistent with in vivo ASP profiles in subjects, and suggest that ASP may be regulated through precursor C3 availability, convertase activity and differentiation status.

  15. The Wheat GT Factor TaGT2L1D Negatively Regulates Drought Tolerance and Plant Development

    PubMed Central

    Zheng, Xin; Liu, Haipei; Ji, Hongtao; Wang, Youning; Dong, Baodi; Qiao, Yunzhou; Liu, Mengyu; Li, Xia

    2016-01-01

    GT factors are trihelix transcription factors that specifically regulate plant development and stress responses. Recently, several GT factors have been characterized in different plant species; however, little is known about the role of GT factors in wheat. Here, we show that TaGT2L1A, TaGT2L1B, and TaGT2L1D are highly homologous in hexaploid wheat, and are localized to wheat chromosomes 2A, 2B, and 2D, respectively. These TaGT2L1 genes encode proteins containing two SANT domains and one central helix. All three homologs were ubiquitously expressed during wheat development and were responsive to osmotic stress. Functional analyses demonstrated that TaGT2L1D acts as a transcriptional repressor; it was able to suppress the expression of AtSDD1 in Arabidopsis by binding directly to the GT3 box in its promoter that negatively regulates drought tolerance. TaGT2L1D overexpression markedly increased the number of stomata and reduced drought tolerance in gtl1-3 plants. Notably, ectopic expression of TaGT2L1D also affected floral organ development and overall plant growth. These results demonstrate that TaGT2L1 is an ortholog of AtGTL1, and that it plays an evolutionarily conserved role in drought resistance by fine tuning stomatal density in wheat. Our data also highlight the role of TaGT2L1 in plant growth and development. PMID:27245096

  16. Regulation of PD-L1 expression in a high-grade invasive human oral squamous cell carcinoma microenvironment.

    PubMed

    Hirai, Mariko; Kitahara, Hiroko; Kobayashi, Yutaka; Kato, Koroku; Bou-Gharios, George; Nakamura, Hiroyuki; Kawashiri, Shuichi

    2017-01-01

    Blockade of the programmed-death 1 receptor (PD-1)/programmed-death ligand (PD-L1) pathway efficiently reduces tumour growth and improves survival. Durable tumour regression with blockade of the PD-1/PD-L1 checkpoint has been demonstrated in recent clinical studies. Oral squamous cell carcinoma (OSCC) is highly immunosuppressive, and PD-L1 expression has been proposed as a potential mechanism responsible for this phenotype. Despite the fact that anti-PD-1 treatment can produce durable responses, such therapy appears to benefit only a subset of patients. Thus, it is important to understand the mechanisms underlying regulation of PD-L1 expression in the OSCC microenvironment. In this study, we showed that PD-L1 expression in high-grade invasive OSCC cell lines was lower than that in a low-grade invasive OSCC line and found a close correlation between PD-L1 expression and the epithelial-mesenchymal transition (EMT). PD-L1 expression was upregulated in macrophages and dendritic cells (DCs) in high-grade invasive human OSCC tissues or co-cultured with mesenchymal-phenotype OSCC cells in vitro. TLR4-inhibitory peptide successfully suppressed PD-L1 upregulation on macrophages and DCs co-cultured with mesenchymal-phenotype OSCC cells, suggesting that some EMT-induced tumour antigen is critical for PD-L1 induction on tumour-associated macrophages and DCs. Further studies are necessary to explore the impact of EMT on the tumour immune microenvironment and to identify potential biomarkers for selecting patients who might preferentially benefit from PD-1/PD-L1 blockade or immunotherapies more broadly.

  17. Regulation of PD-L1 expression in a high-grade invasive human oral squamous cell carcinoma microenvironment

    PubMed Central

    Hirai, Mariko; Kitahara, Hiroko; Kobayashi, Yutaka; Kato, Koroku; Bou-Gharios, George; Nakamura, Hiroyuki; Kawashiri, Shuichi

    2017-01-01

    Blockade of the programmed-death 1 receptor (PD-1)/programmed-death ligand (PD-L1) pathway efficiently reduces tumour growth and improves survival. Durable tumour regression with blockade of the PD-1/PD-L1 checkpoint has been demonstrated in recent clinical studies. Oral squamous cell carcinoma (OSCC) is highly immunosuppressive, and PD-L1 expression has been proposed as a potential mechanism responsible for this phenotype. Despite the fact that anti-PD-1 treatment can produce durable responses, such therapy appears to benefit only a subset of patients. Thus, it is important to understand the mechanisms underlying regulation of PD-L1 expression in the OSCC microenvironment. In this study, we showed that PD-L1 expression in high-grade invasive OSCC cell lines was lower than that in a low-grade invasive OSCC line and found a close correlation between PD-L1 expression and the epithelial-mesenchymal transition (EMT). PD-L1 expression was upregulated in macrophages and dendritic cells (DCs) in high-grade invasive human OSCC tissues or co-cultured with mesenchymal-phenotype OSCC cells in vitro. TLR4-inhibitory peptide successfully suppressed PD-L1 upregulation on macrophages and DCs co-cultured with mesenchymal-phenotype OSCC cells, suggesting that some EMT-induced tumour antigen is critical for PD-L1 induction on tumour-associated macrophages and DCs. Further studies are necessary to explore the impact of EMT on the tumour immune microenvironment and to identify potential biomarkers for selecting patients who might preferentially benefit from PD-1/PD-L1 blockade or immunotherapies more broadly. PMID:27922697

  18. Myeloid ZFP36L1 Does Not Regulate Inflammation or Host Defense in Mouse Models of Acute Bacterial Infection

    PubMed Central

    Hyatt, Lynnae D.; Wasserman, Gregory A.; Rah, Yoon J.; Matsuura, Kori Y.; Coleman, Fadie T.; Hilliard, Kristie L.; Pepper-Cunningham, Zachary Ash; Ieong, Michael; Stumpo, Deborah J.; Blackshear, Perry J.; Quinton, Lee J.; Mizgerd, Joseph P.; Jones, Matthew R.

    2014-01-01

    Zinc finger protein 36, C3H type-like 1 (ZFP36L1) is one of several Zinc Finger Protein 36 (Zfp36) family members, which bind AU rich elements within 3′ untranslated regions (UTRs) to negatively regulate the post-transcriptional expression of targeted mRNAs. The prototypical member of the family, Tristetraprolin (TTP or ZFP36), has been well-studied in the context of inflammation and plays an important role in repressing pro-inflammatory transcripts such as TNF-α. Much less is known about the other family members, and none have been studied in the context of infection. Using macrophage cell lines and primary alveolar macrophages we demonstrated that, like ZFP36, ZFP36L1 is prominently induced by infection. To test our hypothesis that macrophage production of ZFP36L1 is necessary for regulation of the inflammatory response of the lung during pneumonia, we generated mice with a myeloid-specific deficiency of ZFP36L1. Surprisingly, we found that myeloid deficiency of ZFP36L1 did not result in alteration of lung cytokine production after infection, altered clearance of bacteria, or increased inflammatory lung injury. Although alveolar macrophages are critical components of the innate defense against respiratory pathogens, we concluded that myeloid ZFP36L1 is not essential for appropriate responses to bacteria in the lungs. Based on studies conducted with myeloid-deficient ZFP36 mice, our data indicate that, of the Zfp36 family, ZFP36 is the predominant negative regulator of cytokine expression in macrophages. In conclusion, these results imply that myeloid ZFP36 may fully compensate for loss of ZFP36L1 or that Zfp36l1-dependent mRNA expression does not play an integral role in the host defense against bacterial pneumonia. PMID:25299049

  19. Microgravity Effects on Transendothelial Transport

    NASA Technical Reports Server (NTRS)

    Tarbell, John M.

    1996-01-01

    The Endothelial Cell (EC) layer which lines blood vessels from the aorta to the capillaries provides the principal barrier to transport of water and solutes between blood and underlying tissue. Endothelial cells are continuously exposed to the mechanical shearing force (shear stress) and normal force (pressure) imposed by flowing blood on their surface, and they are adapted to this mechanical environment. When the cardiovascular system is exposed to microgravity, the mechanical environmental of endothelial cells is perturbed drastically and the transport properties of EC layers are altered in response. We have shown recently that step changes in shear stress have an acute effect on transport properties of EC layers in a cell culture model, and several recent studies in different vessels of live animals have confirmed the shear-dependent transport properties of the endothelium. We hypothesize that alterations in mechanical forces induced by microgravity and their resultant influence on transendothelial transport of water and solutes are, in large measure, responsible for the characteristic cephalad fluid shift observed in humans experiencing microgravity. To study the effects of altered mechanical forces on transendothelial transport and to test pharmacologic agents as counter measures to microgravity induced fluid shifts we have proposed ground-based studies using well defined cell culture models.

  20. TLR3-Induced Maturation of Murine Dendritic Cells Regulates CTL Responses by Modulating PD-L1 Trafficking

    PubMed Central

    Varthaman, Aditi; Moreau, Hélène D.; Maurin, Mathieu; Benaroch, Philippe

    2016-01-01

    Targeting TLR3 through formulations of polyI:C is widely studied as an adjuvant in cancer immunotherapy. The efficacy of such targeting has been shown to increase in combination with anti-PD-L1 treatment. Nevertheless, the mechanistic details of the effect of polyI:C on DC maturation and the impact on T-DC interactions upon PD-L1 blockade is largely unknown. Here we found that although DC treatment with polyI:C induced a potent inflammatory response including the production of type I interferon, polyI:C treatment of DCs impaired activation of peptide specific CD8+ T cells mainly due to PD-L1. Interestingly, we found that PD-L1 trafficking to the cell surface is regulated in two waves in polyI:C-treated DCs. One induced upon overnight treatment and a second more rapid one, specific to polyI:C treatment, was induced upon CD40 signaling leading to a further increase in surface PD-L1 in DCs. The polyI:C-induced cell surface PD-L1 reduced the times of contact between DCs and T cells, potentially accounting for limited T cell activation. Our results reveal a novel CD40-dependent regulation of PD-L1 trafficking induced upon TLR3 signaling that dictates its inhibitory activity. These results provide a mechanistic framework to understand the efficacy of anti-PD-L1 cancer immunotherapy combined with TLR agonists. PMID:27911948

  1. TLR3 triggering regulates PD-L1 (CD274) expression in human neuroblastoma cells.

    PubMed

    Boes, Marianne; Meyer-Wentrup, Friederike

    2015-05-28

    Neuroblastoma is the most common extracranial solid tumor in children, causing 12% of all pediatric cancer mortality. Neuroblastoma specific T-cells have been detected in patients, but usually fail to attack and eradicate the tumors. Tumor immune evasion may thus play an important role in neuroblastoma pathogenicity. Recent research in adult cancer patients shows that targeting T-cell check-point molecules PD-1/PD-L1 (or CD279/CD274) may bolster immune reactivity against solid tumors. Also, infections can be associated with spontaneous neuroblastoma regression. In our current study, we therefore investigated if antibody targeting of PD-L1 and triggering of selective pathogen-receptor Toll-like receptors (TLRs) potentiates immunogenicity of neuroblastoma cells. We find this to be the case. TLR3 triggering induced strong upregulation of both MHC class I and PD-L1 on neuroblastoma cells. At the same time TGF-β levels decreased and IL-8 secretion was induced. The combined neuroblastoma cell treatment using PD-L1 blockade and TLR3 triggering using virus analog poly(I:C) moreover induced CD4(+) and CD8(+) T-cell activation. Thus, we propose combined treatment using PD-L1 blockade with synthetic TLR ligands as an avenue toward new immunotherapy against human neuroblastoma.

  2. EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

    PubMed

    Nakajo, Atsuhiro; Yoshimura, Shin-ichiro; Togawa, Hiroko; Kunii, Masataka; Iwano, Tomohiko; Izumi, Ayaka; Noguchi, Yuria; Watanabe, Ayako; Goto, Ayako; Sato, Toshiro; Harada, Akihiro

    2016-02-01

    The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

  3. EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

    PubMed Central

    Nakajo, Atsuhiro; Togawa, Hiroko; Kunii, Masataka; Iwano, Tomohiko; Izumi, Ayaka; Noguchi, Yuria; Watanabe, Ayako; Goto, Ayako; Sato, Toshiro

    2016-01-01

    The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain–binding protein 1–like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1–dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport. PMID:26833786

  4. Sustained Induction of Collagen Synthesis by TGF-β Requires Regulated Intramembrane Proteolysis of CREB3L1

    PubMed Central

    Chen, Qiuyue; Lee, Ching-En; Denard, Bray; Ye, Jin

    2014-01-01

    CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor and activated through Regulated Intramembrane Proteolysis (RIP), is essential for collagen production by osteoblasts during bone development. Here, we show that TGF-β (transforming growth factor-β), a cytokine known to stimulate production of collagen during wound healing and fibrotic diseases, induces proteolytic activation of CREB3L1 in human A549 cells. This activation results from inhibition of expression of TM4SF20 (transmembrane 4 L6 family member 20), which normally inhibits RIP of CREB3L1. Cleavage of CREB3L1 releases its NH2-terminal domain from membranes, allowing it to enter the nucleus where it binds to Smad4 to activate transcription of genes encoding proteins required for assembly of collagen-containing extracellular matrix. Our findings raise the possibility that inhibition of RIP of CREB3L1 could prevent excess deposition of collagen in certain fibrotic diseases. PMID:25310401

  5. LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts

    PubMed Central

    Kuwahara, Tomoki; Inoue, Keiichi; D’Agati, Vivette D.; Fujimoto, Tetta; Eguchi, Tomoya; Saha, Shamol; Wolozin, Benjamin; Iwatsubo, Takeshi; Abeliovich, Asa

    2016-01-01

    Leucine-rich repeat kinase 2 (LRRK2) has been linked to several clinical disorders including Parkinson’s disease (PD), Crohn’s disease, and leprosy. Furthermore in rodents, LRRK2 deficiency or inhibition leads to lysosomal pathology in kidney and lung. Here we provide evidence that LRRK2 functions together with a second PD-associated gene, RAB7L1, within an evolutionarily conserved genetic module in diverse cellular contexts. In C. elegans neurons, orthologues of LRRK2 and RAB7L1 act coordinately in an ordered genetic pathway to regulate axonal elongation. Further genetic studies implicated the AP-3 complex, which is a known regulator of axonal morphology as well as of intracellular protein trafficking to the lysosome compartment, as a physiological downstream effector of LRRK2 and RAB7L1. Additional cell-based studies implicated LRRK2 in the AP-3 complex-related intracellular trafficking of lysosomal membrane proteins. In mice, deficiency of either RAB7L1 or LRRK2 leads to prominent age-associated lysosomal defects in kidney proximal tubule cells, in the absence of frank CNS pathology. We hypothesize that defects in this evolutionarily conserved genetic pathway underlie the diverse pathologies associated with LRRK2 in humans and in animal models. PMID:27424887

  6. Transcription Factor CREB3L1 Regulates Endoplasmic Reticulum Stress Response Genes in the Osmotically Challenged Rat Hypothalamus

    PubMed Central

    Greenwood, Mingkwan; Greenwood, Michael Paul; Paton, Julian F. R.; Murphy, David

    2015-01-01

    Arginine vasopressin (AVP) is synthesised in magnocellular neurons (MCNs) of supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. In response to the hyperosmotic stressors of dehydration (complete fluid deprivation, DH) or salt loading (drinking 2% salt solution, SL), AVP synthesis increases in MCNs, which over-burdens the protein folding machinery in the endoplasmic reticulum (ER). ER stress and the unfolded protein response (UPR) are signaling pathways that improve ER function in response to the accumulation of misfold/unfold protein. We asked whether an ER stress response was activated in the SON and PVN of DH and SL rats. We observed increased mRNA expression for the immunoglobulin heavy chain binding protein (BiP), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), and cAMP responsive element binding protein 3 like 1 (Creb3l1) in both SON and PVN of DH and SL rats. Although we found no changes in the splicing pattern of X box-binding protein 1 (Xbp1), an increase in the level of the unspliced form of Xbp1 (Xbp1U) was observed in DH and SL rats. CREB3L1, a novel ER stress inducer, has been shown to be activated by ER stress to regulate the expression of target genes. We have previously shown that CREB3L1 is a transcriptional regulator of the AVP gene; however, a role for CREB3L1 in the response to ER stress has yet to be investigated in MCNs. Here, we used lentiviral vectors to introduce a dominant negative form of CREB3L1 (CREB3L1DN) in the rat SON. Expression of CREB3L1DN in the SON decreased Chop and Xbp1U mRNA levels, but not BiP and Atf4 transcript expression. CREB3L1 is thus implicated as a transcriptional mediator of the ER stress response in the osmotically stimulated SON. PMID:25915053

  7. The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

    PubMed Central

    2013-01-01

    Background In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. Results The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. Conclusions The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic

  8. [Techniques to study transendothelial migration in vitro].

    PubMed

    Knopfová, L; Bouchal, P; Smarda, J

    2014-01-01

    The most dangerous aspect of cancer is the metastatic spread to other parts of the body. Cancer cells frequently use circulation to spread to secondary locations. By entering the blood-stream (in a process called intravasation) and by crossing the vessel walls at the metastatic sites (extravasation) tumor cells disseminate to distal organs and eventually form life  threatening metastases. Crossing the vessel walls (transendothelial migration) is a vital step of metastatic cascade and the elucidation of mechanisms involved in transendothelial migration might inspire new strategies of targeted antimetastatic therapy. There are several methods to study transendothelial migration in living models (in vivo). Although they offer complex physiological microenvironment, they are expensive and technically demanding, therefore not widely used. As an alternative, sophisticated techniques to investigate transendothelial migration in vitro have been developed. They are generally more available and feasible, but there is still considerable variability in the difficulty of performance, the requirements for specialized devices, accuracy of in vivo simulation and relevance for oncological applications. The classification, various modifications, pros and cons of in vitro techniques for studying transendothelial migration are summarized in this review.

  9. Ubiquitin Ligase NEDD4 Regulates PPARγ Stability and Adipocyte Differentiation in 3T3-L1 Cells

    PubMed Central

    Li, Jing Jing; Wang, Ruishan; Lama, Rati; Wang, Xinjiang; Floyd, Z. Elizabeth; Park, Edwards A.; Liao, Francesca-Fang

    2016-01-01

    Peroxisome proliferator–activated receptor-γ (PPARγ) is a ligand-activated nuclear receptor which controls lipid and glucose metabolism. It is also the master regulator of adipogenesis. In adipocytes, ligand-dependent PPARγ activation is associated with proteasomal degradation; therefore, regulation of PPARγ degradation may modulate its transcriptional activity. Here, we show that neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4), an E3 ubiquitin ligase, interacts with the hinge and ligand binding domains of PPARγ and is a bona fide E3 ligase for PPARγ. NEDD4 increases PPARγ stability through the inhibition of its proteasomal degradation. Knockdown of NEDD4 in 3T3-L1 adipocytes reduces PPARγ protein levels and suppresses adipocyte conversion. PPARγ correlates positively with NEDD4 in obese adipose tissue. Together, these findings support NEDD4 as a novel regulator of adipogenesis by modulating the stability of PPARγ. PMID:27917940

  10. TNF-α and TGF-β Counter-Regulate PD-L1 Expression on Monocytes in Systemic Lupus Erythematosus

    PubMed Central

    Ou, Jing-Ni; Wiedeman, Alice E.; Stevens, Anne M.

    2012-01-01

    Monocytes in patients with systemic lupus erythematosus (SLE) are hyperstimulatory for T lymphocytes. We previously found that the normal program for expression of a negative costimulatory molecule programmed death ligand-1 (PD-L1) is defective in SLE patients with active disease. Here, we investigated the mechanism for PD-L1 dysregulation on lupus monocytes. We found that PD-L1 expression on cultured SLE monocytes correlated with TNF-α expression. Exogenous TNF-α restored PD-L1 expression on lupus monocytes. Conversely, TGF-β inversely correlated with PD-L1 in SLE and suppressed expression of PD-L1 on healthy monocytes. Therefore, PD-L1 expression in monocytes is regulated by opposing actions of TNF-α and TGF-β. As PD-L1 functions to fine tune lymphocyte activation, dysregulation of cytokines resulting in reduced expression could lead to loss of peripheral T cell tolerance. PMID:22389764

  11. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    SciTech Connect

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  12. Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L. 1

    PubMed Central

    Farago, S.; Brunold, C.

    1990-01-01

    Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of 35SO42−, and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using 35SO42−, roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5′-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5′-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5′-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved. PMID

  13. β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase, UCH-L1*

    PubMed Central

    Poon, Wayne W.; Carlos, Anthony J.; Aguilar, Brittany L.; Berchtold, Nicole C.; Kawano, Crystal K.; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W.

    2013-01-01

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival. PMID:23599427

  14. β-Amyloid (Aβ) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1.

    PubMed

    Poon, Wayne W; Carlos, Anthony J; Aguilar, Brittany L; Berchtold, Nicole C; Kawano, Crystal K; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W

    2013-06-07

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.

  15. Increased soluble and membrane-bound PD-L1 contributes to immune regulation and disease progression in patients with tuberculous pleural effusion

    PubMed Central

    Pan, Xue; Zhong, Anyuan; Xing, Yufei; Shi, Minhua; Qian, Bin; Zhou, Tong; Chen, Yongjing; Zhang, Xueguang

    2016-01-01

    Soluble and membrane-bound programmed death ligand-1 (sPD-L1 and mPD-L1, respectively) have been demonstrated to participate in the immune suppression of non-small cell lung cancer. However, the contribution of sPD-L1 and mPD-L1 to immune regulation and disease progression in patients with pleural effusions remains unknown. The present study evaluated the levels of sPD-L1 and membrane-bound PD-1/PD-L1 in the peripheral blood and pleural effusions of patients with tuberculous pleural effusion (TPE), malignant pleural effusion (MPE) and non-tuberculous non-malignant pleural effusion (n-TB n-M). Furthermore, selected T lymphocytes and cluster of differentiation (CD)14+ monocytes were co-cultured to investigate the potential effect of the PD-1/PD-L1 pathway in TPE. Levels of sPD-L1 and PD-L1 on CD14+ monocytes were increased in the TPE group, as compared with the MPE and n-TB n-M groups. Furthermore, sPD-L1 levels and the expression levels of PD-L1 on CD14+ monocytes were demonstrated to be positively correlated with interferon (IFN)-γ concentration in pleural effusions. Therefore, IFN-γ may increase the expression of PD-L1 on CD14+ monocytes in vitro. Cell counting kit-8 analysis demonstrated that anti-PD-L1 antibody was able to partially reverse the proliferation of T lymphocytes in the co-culture system. The results of the present study indicated that sPD-L1 or mPD-L1 are associated with the immune regulation and disease progression of TPE, and may serve as possible biomarkers of TPE. Furthermore, sPD-L1 and the PD-1/PD-L1 pathway of TPE may be associated with the Th1 immune response; therefore, an anti-PD-1/PD-L1 pathway suggests a potential immune therapy strategy for the treatment of TPE. PMID:27698705

  16. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  17. Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night.

    PubMed

    deHaro, Dawn; Kines, Kristine J; Sokolowski, Mark; Dauchy, Robert T; Streva, Vincent A; Hill, Steven M; Hanifin, John P; Brainard, George C; Blask, David E; Belancio, Victoria P

    2014-07-01

    Expression of long interspersed element-1 (L1) is upregulated in many human malignancies. L1 can introduce genomic instability via insertional mutagenesis and DNA double-strand breaks, both of which may promote cancer. Light exposure at night, a recently recognized carcinogen, is associated with an increased risk of cancer in shift workers. We report that melatonin receptor 1 inhibits mobilization of L1 in cultured cells through downregulation of L1 mRNA and ORF1 protein. The addition of melatonin receptor antagonists abolishes the MT1 effect on retrotransposition in a dose-dependent manner. Furthermore, melatonin-rich, but not melatonin-poor, human blood collected at different times during the circadian cycle suppresses endogenous L1 mRNA during in situ perfusion of tissue-isolated xenografts of human cancer. Supplementation of human blood with exogenous melatonin or melatonin receptor antagonist during the in situ perfusion establishes a receptor-mediated action of melatonin on L1 expression. Combined tissue culture and in vivo data support that environmental light exposure of the host regulates expression of L1 elements in tumors. Our data imply that light-induced suppression of melatonin production in shift workers may increase L1-induced genomic instability in their genomes and suggest a possible connection between L1 activity and increased incidence of cancer associated with circadian disruption.

  18. AKT-STAT3 Pathway as a Downstream Target of EGFR Signaling to Regulate PD-L1 Expression on NSCLC cells.

    PubMed

    Abdelhamed, Sherif; Ogura, Keisuke; Yokoyama, Satoru; Saiki, Ikuo; Hayakawa, Yoshihiro

    2016-01-01

    While cancer development and progression can be controlled by cytotoxic T cells, it is also known that tumor-specific CD8(+)T cells become functionally impaired by acquiring a group of inhibitory receptors known as immune checkpoints. Amongst those, programmed death-1 (PD-1) is one of the most recognized negative regulators of T cell function. In non-small lung cancers (NSCLCs), the aberrant activation of epidermal growth factor receptor (EGFR) is known to induce PD-L1 expression and further the treatment with gefitinib, a tyrosine kinase inhibitor (TKI) for EGFR, decrease the expression of PD-L1 on NSCLC. Given the acquired resistance to gefitinib treatment frequently observed by developing secondary-site mutations limiting its efficacy, it is important to understand the downstream mechanism of activated-EGFR signaling for regulating PD-L1 in NSCLC. In this study, we demonstrated that AKT-STAT3 pathway could be a potential target for regulating the surface expression of PD-L1 on NSCLCs with aberrant EGFR activity and, further, the inhibition of AKT or STAT3 activity could down-regulate the expression of PD-L1 even in gefitinib-resistant NSCLCs. These results highlight an importance of AKT-STAT3 pathway as a promising target for potentiating anti-tumor immune responses by regulating PD-L1 expression on cancer cells with aberrant EGFR activity.

  19. WHSC1L1 drives cell cycle progression through transcriptional regulation of CDC6 and CDK2 in squamous cell carcinoma of the head and neck

    PubMed Central

    Saloura, Vassiliki; Vougiouklakis, Theodore; Zewde, Makda; Kiyotani, Kazuma; Park, Jae-Hyun; Gao, Guimin; Karrison, Theodore; Lingen, Mark; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-01-01

    Wolf-Hisrchhorn Syndrome Candidate 1-Like 1 (WHSC1L1) is a protein lysine methyltransferase that is recurrently amplified (8p11.23) in patients with squamous cell carcinoma of the head and neck (SCCHN). In this study, we investigated the oncogenic role of WHSC1L1 in SCCHN. Using immunohistochemistry on tissue microarrays of patients with locoregionally advanced SCCHN, we found that WHSC1L1 is significantly overexpressed in patients with SCCHN, and is associated with poor grade and heavy smoking history. Knockdown of WHSC1L1 expression resulted in significant growth suppression and reduction of H3K36 dimethylation (H3K36me2) in SCCHN cells. Chromatin immunoprecipitation analysis showed that WHSC1L1 and H3K36me2 are enriched in the gene bodies of the cell cycle-related genes CDC6 and CDK2, implying that WHSC1L1 directly regulates the transcription of these genes. According to the importance of CDC6 and CDK2 for G1 to S transition, WHSC1L1 knockdown induced strong G0/G1 arrest which was rescued by introduction of wild-type WHSC1L1 but not by that of enzyme-inactive WHSC1L1. Our results imply that WHSC1L1 and its product H3K36me2 are essential for the transition from G1 to S phase in SCCHN cells and that WHSC1L1 could serve as a rational target for anticancer drug development for patients with head and neck cancer. PMID:27285764

  20. Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

    PubMed

    Locard-Paulet, Marie; Lim, Lindsay; Veluscek, Giulia; McMahon, Kelly; Sinclair, John; van Weverwijk, Antoinette; Worboys, Jonathan D; Yuan, Yinyin; Isacke, Clare M; Jørgensen, Claus

    2016-02-09

    The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.

  1. Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells.

    PubMed

    Ji, Shuhan; Doumit, Matthew E; Hill, Rodney A

    2015-01-01

    The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)2D3) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100 nM) of 1, 25-(OH)2D3 were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)2D3 were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)2D3. In response to 1, 25-(OH)2D3 (1, 10, and 100 nM), lipid accumulation and the expression of PPARγ, C/EBPα, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)2D3 tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBPα promoter activity response to 1, 25-(OH)2D3 was also tested, with no effect detected. These results indicate that 1, 25-(OH)2D3 inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPARγ activation or immediately up-stream thereof. Gene expression down-stream of PPARγ especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)2D3 in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity.

  2. Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/MenaINV-initiated invadopodium formation

    PubMed Central

    Pignatelli, Jeanine; Bravo-Cordero, Jose Javier; Roh-Johnson, Minna; Gandhi, Saumil J.; Wang, Yarong; Chen, Xiaoming; Eddy, Robert J.; Xue, Alice; Singer, Robert H.; Hodgson, Louis; Oktay, Maja H.; Condeelis, John S.

    2016-01-01

    The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/MenaINV signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell – macrophage interaction results in the upregulation of MenaINV through the activation of MENA transcription. Notch1 and MenaINV expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of MenaINV expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis. PMID:27901093

  3. Novel roles of c-Met in the survival of renal cancer cells through the regulation of HO-1 and PD-L1 expression.

    PubMed

    Balan, Murugabaskar; Mier y Teran, Eduardo; Waaga-Gasser, Ana Maria; Gasser, Martin; Choueiri, Toni K; Freeman, Gordon; Pal, Soumitro

    2015-03-27

    The receptor tyrosine kinase c-Met is overexpressed in renal cancer cells and can play major role in the growth and survival of tumor. We investigated how the c-Met-mediated signaling through binding to its ligand hepatocyte growth factor (HGF) can modulate the apoptosis and immune escape mechanism(s) of renal cancer cells by the regulations of novel molecules heme oxygenase-1 (HO-1) and programmed death-1 ligand 1 (PD-L1). We found that HGF/c-Met-mediated signaling activated the Ras/Raf pathway and down-regulated cancer cell apoptosis; and it was associated with the overexpression of cytoprotective HO-1 and anti-apoptotic Bcl-2/Bcl-xL. c-Met-induced HO-1 overexpression was regulated at the transcriptional level. Next, we observed that c-Met induction markedly up-regulated the expression of the negative co-stimulatory molecule PD-L1, and this can be prevented following treatment of the cells with pharmacological inhibitors of c-Met. Interestingly, HGF/c-Met-mediated signaling could not induce PD-L1 at the optimum level when either Ras or HO-1 was knocked down. To study the functional significance of c-Met-induced PD-L1 expression, we performed a co-culture assay using mouse splenocytes (expressing PD-L1 receptor PD-1) and murine renal cancer cells (RENCA, expressing high PD-L1). We observed that the splenocyte-mediated apoptosis of cancer cells during co-culture was markedly increased in the presence of either c-Met inhibitor or PD-L1 neutralizing antibody. Finally, we found that both c-Met and PD-L1 are significantly up-regulated and co-localized in human renal cancer tissues. Together, our study suggests a novel mechanism(s) by which c-Met can promote increased survival of renal cancer cells through the regulation of HO-1 and PD-L1.

  4. Novel Roles of c-Met in the Survival of Renal Cancer Cells through the Regulation of HO-1 and PD-L1 Expression*

    PubMed Central

    Balan, Murugabaskar; Mier y Teran, Eduardo; Waaga-Gasser, Ana Maria; Gasser, Martin; Choueiri, Toni K.; Freeman, Gordon; Pal, Soumitro

    2015-01-01

    The receptor tyrosine kinase c-Met is overexpressed in renal cancer cells and can play major role in the growth and survival of tumor. We investigated how the c-Met-mediated signaling through binding to its ligand hepatocyte growth factor (HGF) can modulate the apoptosis and immune escape mechanism(s) of renal cancer cells by the regulations of novel molecules heme oxygenase-1 (HO-1) and programmed death-1 ligand 1 (PD-L1). We found that HGF/c-Met-mediated signaling activated the Ras/Raf pathway and down-regulated cancer cell apoptosis; and it was associated with the overexpression of cytoprotective HO-1 and anti-apoptotic Bcl-2/Bcl-xL. c-Met-induced HO-1 overexpression was regulated at the transcriptional level. Next, we observed that c-Met induction markedly up-regulated the expression of the negative co-stimulatory molecule PD-L1, and this can be prevented following treatment of the cells with pharmacological inhibitors of c-Met. Interestingly, HGF/c-Met-mediated signaling could not induce PD-L1 at the optimum level when either Ras or HO-1 was knocked down. To study the functional significance of c-Met-induced PD-L1 expression, we performed a co-culture assay using mouse splenocytes (expressing PD-L1 receptor PD-1) and murine renal cancer cells (RENCA, expressing high PD-L1). We observed that the splenocyte-mediated apoptosis of cancer cells during co-culture was markedly increased in the presence of either c-Met inhibitor or PD-L1 neutralizing antibody. Finally, we found that both c-Met and PD-L1 are significantly up-regulated and co-localized in human renal cancer tissues. Together, our study suggests a novel mechanism(s) by which c-Met can promote increased survival of renal cancer cells through the regulation of HO-1 and PD-L1. PMID:25645920

  5. COX2/mPGES1/PGE2 pathway regulates PD-L1 expression in tumor-associated macrophages and myeloid-derived suppressor cells.

    PubMed

    Prima, Victor; Kaliberova, Lyudmila N; Kaliberov, Sergey; Curiel, David T; Kusmartsev, Sergei

    2017-01-31

    In recent years, it has been established that programmed cell death protein ligand 1 (PD-L1)-mediated inhibition of activated PD-1(+) T lymphocytes plays a major role in tumor escape from immune system during cancer progression. Lately, the anti-PD-L1 and -PD-1 immune therapies have become an important tool for treatment of advanced human cancers, including bladder cancer. However, the underlying mechanisms of PD-L1 expression in cancer are not fully understood. We found that coculture of murine bone marrow cells with bladder tumor cells promoted strong expression of PD-L1 in bone marrow-derived myeloid cells. Tumor-induced expression of PD-L1 was limited to F4/80(+) macrophages and Ly-6C(+) myeloid-derived suppressor cells. These PD-L1-expressing cells were immunosuppressive and were capable of eliminating CD8 T cells in vitro. Tumor-infiltrating PD-L1(+) cells isolated from tumor-bearing mice also exerted morphology of tumor-associated macrophages and expressed high levels of prostaglandin E2 (PGE2)-forming enzymes microsomal PGE2 synthase 1 (mPGES1) and COX2. Inhibition of PGE2 formation, using pharmacologic mPGES1 and COX2 inhibitors or genetic overexpression of PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH), resulted in reduced PD-L1 expression. Together, our study demonstrates that the COX2/mPGES1/PGE2 pathway involved in the regulation of PD-L1 expression in tumor-infiltrating myeloid cells and, therefore, reprogramming of PGE2 metabolism in tumor microenvironment provides an opportunity to reduce immune suppression in tumor host.

  6. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    SciTech Connect

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui; Zhang, Jun; Xia, Ning-Shao; Miao, Ji; Zhao, Qinjian

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  7. Annexin A2 Limits Neutrophil Transendothelial Migration by Organizing the Spatial Distribution of ICAM-1.

    PubMed

    Heemskerk, Niels; Asimuddin, Mohammed; Oort, Chantal; van Rijssel, Jos; van Buul, Jaap D

    2016-03-15

    ICAM-1 is required for firm adhesion of leukocytes to the endothelium. However, how the spatial organization of endothelial ICAM-1 regulates leukocyte adhesion is not well understood. In this study, we identified the calcium-effector protein annexin A2 as a novel binding partner for ICAM-1. ICAM-1 clustering promotes the ICAM-1-annexin A2 interaction and induces translocation of ICAM-1 into caveolin-1-rich membrane domains. Depletion of endothelial annexin A2 using RNA interference enhances ICAM-1 membrane mobility and prevents the translocation of ICAM-1 into caveolin-1-rich membrane domains. Surprisingly, this results in increased neutrophil adhesion and transendothelial migration under flow conditions and reduced crawling time, velocity, and lateral migration distance of neutrophils on the endothelium. In conclusion, our data show that annexin A2 limits neutrophil transendothelial migration by organizing the spatial distribution of ICAM-1.

  8. Modulation of integrin α4β1 by ADAM28 promotes lymphocyte adhesion and transendothelial migration.

    PubMed

    McGinn, Owen J; English, William R; Roberts, Stephanie; Ager, Ann; Newham, Peter; Murphy, Gillian

    2011-10-01

    ADAMs (a disintegrin and metalloproteinase) are a family of type I transmembrane glycoproteins related to snake venom metalloproteases and disintegrins. They are regulatory proteins that modulate intercellular adhesion and the bioavailability of growth factors, and have been implicated in many disease states, including cancer, immunity and inflammation. One member of the ADAM family, ADAM28, has been reported to bind to the integrin α4β1 in humans; however, the distribution of ADAM28 and the biological consequences of ADAM28-α4β1 interactions are yet to be fully elucidated. The expression of ADAM28 in human and murine tissues was examined by multiple Affymetrix microarray analyses, real-time RT-PCR (reverse transcription-PCR) and immunohistochemical staining. We found that ADAM28 has a relatively restricted expression pattern in mouse and human and is highly expressed in the B-lymphocyte lineage, including chronic lymphocytic leukaemic B-cells. The murine B-lymphoma line L1-2 and recombinant soluble murine ADAM28 were used to investigate ADAM28-α4β1 interactions. Our data reveal that ADAM28 binding to α4β1 is typical of integrin-ligand interactions, since it is attenuated by anti-functional integrin antibodies, and is enhanced by Mn2+ and the integrin mAb (monoclonal antibody) 9EG7. However, a key finding was that soluble ADAM28 unexpectedly enhanced α4β1-dependent cell adhesion to VCAM-1 (vascular cell adhesion molecule-1). In so doing ADAM28 was able to influence lymphocyte adhesion to, and migration through, endothelial monolayers, suggesting a physiological role for ADAM28 in regulating the specific spatial and temporal transendothelial migration of lymphocytes.

  9. NPC1L1 is a key regulator of intestinal vitamin K absorption and a modulator of warfarin therapy.

    PubMed

    Takada, Tappei; Yamanashi, Yoshihide; Konishi, Kentaro; Yamamoto, Takehito; Toyoda, Yu; Masuo, Yusuke; Yamamoto, Hideaki; Suzuki, Hiroshi

    2015-02-18

    Vitamin K (VK) is a micronutrient that facilitates blood coagulation. VK antagonists, such as warfarin, are used in the clinic to prevent thromboembolism. Because VK is not synthesized in the body, its intestinal absorption is crucial for maintaining whole-body VK levels. However, the molecular mechanism of this absorption is unclear. We demonstrate that Niemann-Pick C1-like 1 (NPC1L1) protein, a cholesterol transporter, plays a central role in intestinal VK uptake and modulates the anticoagulant effect of warfarin. In vitro studies using NPC1L1-overexpressing intestinal cells and in vivo studies with Npc1l1-knockout mice revealed that intestinal VK absorption is NPC1L1-dependent and inhibited by ezetimibe, an NPC1L1-selective inhibitor clinically used for dyslipidemia. In addition, in vivo pharmacological studies demonstrated that the coadministration of ezetimibe and warfarin caused a reduction in hepatic VK levels and enhanced the pharmacological effect of warfarin. Adverse events caused by the coadministration of ezetimibe and warfarin were rescued by oral VK supplementation, suggesting that the drug-drug interaction effects observed were the consequence of ezetimibe-mediated VK malabsorption. This mechanism was supported by a retrospective evaluation of clinical data showing that, in more than 85% of warfarin-treated patients, the anticoagulant activity was enhanced by cotreatment with ezetimibe. Our findings provide insight into the molecular mechanism of VK absorption. This new drug-drug interaction mechanism between ezetimibe (a cholesterol transport inhibitor) and warfarin (a VK antagonist and anticoagulant) could inform clinical care of patients on these medications, such as by altering the kinetics of essential, fat-soluble vitamins.

  10. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with spec...

  11. The autophagy gene Atg16l1 differentially regulates Treg and TH2 cells to control intestinal inflammation

    PubMed Central

    Kabat, Agnieszka M; Moghaddam, Amin E; Pearson, Claire F; Laing, Adam; Abeler-Dörner, Lucie; Forman, Simon P; Grencis, Richard K; Sattentau, Quentin; Simon, Anna Katharina; Pott, Johanna; Maloy, Kevin J

    2016-01-01

    A polymorphism in the autophagy gene Atg16l1 is associated with susceptibility to inflammatory bowel disease (IBD); however, it remains unclear how autophagy contributes to intestinal immune homeostasis. Here, we demonstrate that autophagy is essential for maintenance of balanced CD4+ T cell responses in the intestine. Selective deletion of Atg16l1 in T cells in mice resulted in spontaneous intestinal inflammation that was characterized by aberrant type 2 responses to dietary and microbiota antigens, and by a loss of Foxp3+ Treg cells. Specific ablation of Atg16l1 in Foxp3+ Treg cells in mice demonstrated that autophagy directly promotes their survival and metabolic adaptation in the intestine. Moreover, we also identify an unexpected role for autophagy in directly limiting mucosal TH2 cell expansion. These findings provide new insights into the reciprocal control of distinct intestinal TH cell responses by autophagy, with important implications for understanding and treatment of chronic inflammatory disorders. DOI: http://dx.doi.org/10.7554/eLife.12444.001 PMID:26910010

  12. Galangin induces apoptosis in gastric cancer cells via regulation of ubiquitin carboxy-terminal hydrolase isozyme L1 and glutathione S-transferase P.

    PubMed

    Kim, Deuk Ae; Jeon, Young Keul; Nam, Myeong Jin

    2012-03-01

    Galangin has been shown to have anti-cancer property against several types of cancer cells. Many studies have described the anti-oxidant and apoptotic effects of galangin. However, the mechanism of galangin-induced apoptosis has not yet been studied for human gastric cancer cells. We investigated galangin-induced apoptosis of human gastric cancer SNU-484 cells. Galangin inhibited proliferation of SNU-484 cells in a dose- and time-dependent manner. The results showed that galangin significantly decreased the viability of SNU-484 cells at 50-200 μM for 24 h and 48 h. Galangin-induced cell death was characterized with the changes in cell morphology, DNA fragmentation, cell cycle, activation of caspase-3/-9, poly (ADP-ribose) polymerase (PARP) cleavage, and expression of MAP kinase such as ERK1/2 and JNK. For identification of proteins potentially involved in apoptosis, a two-dimensional electrophoresis was employed. Proteomic analysis showed that several proteins were associated with anti-cancer properties of galangin. Of particular interest, these proteins included ubiquitin carboxy-terminal hydrolase isozyme L1 (Uch-L1) and glutathione S-transferase P (GSTP), which are involved in apoptosis of SNU-484 cells. Western blot analysis confirmed up-regulation of Uch-L1 and down-regulation of GSTP following galangin treatment. Our results suggest that Uch-L1 and GSTP be involved in galangin-induced apoptosis in human gastric cancer SNU-484 cells.

  13. Transendothelial Transport and Its Role in Therapeutics

    PubMed Central

    Upadhyay, Ravi Kant

    2014-01-01

    Present review paper highlights role of BBB in endothelial transport of various substances into the brain. More specifically, permeability functions of BBB in transendothelial transport of various substances such as metabolic fuels, ethanol, amino acids, proteins, peptides, lipids, vitamins, neurotransmitters, monocarbxylic acids, gases, water, and minerals in the peripheral circulation and into the brain have been widely explained. In addition, roles of various receptors, ATP powered pumps, channels, and transporters in transport of vital molecules in maintenance of homeostasis and normal body functions have been described in detail. Major role of integral membrane proteins, carriers, or transporters in drug transport is highlighted. Both diffusion and carrier mediated transport mechanisms which facilitate molecular trafficking through transcellular route to maintain influx and outflux of important nutrients and metabolic substances are elucidated. Present review paper aims to emphasize role of important transport systems with their recent advancements in CNS protection mainly for providing a rapid clinical aid to patients. This review also suggests requirement of new well-designed therapeutic strategies mainly potential techniques, appropriate drug formulations, and new transport systems for quick, easy, and safe delivery of drugs across blood brain barrier to save the life of tumor and virus infected patients. PMID:27355037

  14. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation

    SciTech Connect

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  15. Mesenchymal stem cells increase skin graft survival time and up-regulate PD-L1 expression in splenocytes of mice.

    PubMed

    Moravej, Ali; Geramizadeh, Bita; Azarpira, Negar; Zarnani, Amir-Hassan; Yaghobi, Ramin; Kalani, Mehdi; Khosravi, Maryam; Kouhpayeh, Amin; Karimi, Mohammad-Hossein

    2017-02-01

    Recently, mesenchymal stem cells (MSCs) have gained considerable interests as hopeful therapeutic cells in transplantation due to their immunoregulatory functions. But exact mechanisms underlying MSCs immunoregulatory function is not fully understood. Herein, in addition to investigate the ability of MSCs to prolong graft survival time, the effects of them on the expression of PD-L1 and IDO immunomodulatory molecules in splenocytes of skin graft recipient mice was clarified. To achieve this goal, full-thickness skins were transplanted from C57BL/6 to BALB/c mice. MSCs were isolated from bone marrow of BALB/c mice and injected to the recipient mice. Skin graft survival was monitored daily to determine graft rejection time. On days 2, 5 and 10 post skin transplantation, serum cytokine levels and expression of PD-L1 and IDO mRNA and protein in the splenocytes of recipient mice were evaluated. The results showed that administration of MSCs prolonged skin graft survival time from 11 to 14 days. On days 2 and 5 post transplantation, splenocytes PD-L1 expression and IL-10 serum level in MSCs treated mice were higher than those in the controls, while IL-2 and IFN-γ levels were lower. Rejection in MSCs treated mice was accompanied by an increase in IL-2 and IFN-γ, and decrease in PD-L1 expression and IL-10 level. No difference in the expression of IDO between MSCs treated mice and controls was observed. In conclusion, we found that one of the mechanisms underlying MSCs immunomodulatory function could be up-regulating PD-L1 expression.

  16. A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes.

    PubMed

    Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-10-14

    Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity.

  17. A novel IRS-1-associated protein, DGKζ regulates GLUT4 translocation in 3T3-L1 adipocytes

    PubMed Central

    Liu, TingYu; Yu, BuChin; Kakino, Mamoru; Fujimoto, Hitoshi; Ando, Yasutoshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Insulin receptor substrates (IRSs) are major targets of insulin receptor tyrosine kinases. Here we identified diacylglycerol kinase zeta (DGKζ) as an IRS-1-associated protein, and examined roles of DGKζ in glucose transporter 4 (GLUT4) translocation to the plasma membrane. When DGKζ was knocked-down in 3T3-L1 adipocytes, insulin-induced GLUT4 translocation was inhibited without affecting other mediators of insulin-dependent signaling. Similarly, knockdown of phosphatidylinositol 4-phosphate 5-kinase 1α (PIP5K1α), which had been reported to interact with DGKζ, also inhibited insulin-induced GLUT4 translocation. Moreover, DGKζ interacted with IRS-1 without insulin stimulation, but insulin stimulation decreased this interaction. Over-expression of sDGKζ (short-form DGKζ), which competed out DGKζ from IRS-1, enhanced GLUT4 translocation without insulin stimulation. Taking these results together with the data showing that cellular PIP5K activity was correlated with GLUT4 translocation ability, we concluded that IRS-1-associated DGKζ prevents GLUT4 translocation in the absence of insulin and that the DGKζ dissociated from IRS-1 by insulin stimulation enhances GLUT4 translocation through PIP5K1α activity. PMID:27739494

  18. L1/Laminin modulation of growth cone response to EphB triggers growth pauses and regulates the microtubule destabilizing protein SCG10.

    PubMed

    Suh, Leejee H; Oster, Stephen F; Soehrman, Sophia S; Grenningloh, Gabriele; Sretavan, David W

    2004-02-25

    During development, EphB proteins serve as axon guidance molecules for retinal ganglion cell axon pathfinding toward the optic nerve head and in midbrain targets. To better understand the mechanisms by which EphB proteins influence retinal growth cone behavior, we investigated how axon responses to EphB were modulated by laminin and L1, two guidance molecules that retinal axons encounter during in vivo pathfinding. Unlike EphB stimulation in the presence of laminin, which triggers typical growth cone collapse, growth cones co-stimulated by L1 did not respond to EphB. Moreover, EphB exposure in the presence of both laminin and L1 resulted in a novel growth cone inhibition manifested as a pause in axon elongation with maintenance of normal growth cone morphology and filopodial activity. Pauses were not associated with loss of growth cone actin but were accompanied by a redistribution of the microtubule cytoskeleton with increased numbers of microtubules extending into filopodia and to the peripheral edge of the growth cone. This phenomenon was accompanied by reduced levels of the growth cone microtubule destabilizing protein SCG10. Antibody blockade of SCG10 function in growth cones resulted in both changes in microtubule distribution and pause responses mirroring those elicited by EphB in the presence of laminin and L1. These results demonstrate that retinal growth cone responsiveness to EphB is regulated by co-impinging signals from other axon guidance molecules. Furthermore, the results are consistent with EphB-mediated axon guidance mechanisms that involve the SCG10-mediated regulation of the growth cone microtubule cytoskeleton.

  19. CD169+ macrophages regulate PD-L1 expression via type I interferon and thereby prevent severe immunopathology after LCMV infection

    PubMed Central

    Shaabani, Namir; Duhan, Vikas; Khairnar, Vishal; Gassa, Asmae; Ferrer-Tur, Rita; Häussinger, Dieter; Recher, Mike; Zelinskyy, Gennadiy; Liu, Jia; Dittmer, Ulf; Trilling, Mirko; Scheu, Stefanie; Hardt, Cornelia; Lang, Philipp A; Honke, Nadine; Lang, Karl S

    2016-01-01

    Upon infection with persistence-prone virus, type I interferon (IFN-I) mediates antiviral activity and also upregulates the expression of programmed death ligand 1 (PD-L1), and this upregulation can lead to CD8+ T-cell exhaustion. How these very diverse functions are regulated remains unknown. This study, using the lymphocytic choriomeningitis virus, showed that a subset of CD169+ macrophages in murine spleen and lymph nodes produced high amounts of IFN-I upon infection. Absence of CD169+ macrophages led to insufficient production of IFN-I, lower antiviral activity and persistence of virus. Lack of CD169+ macrophages also limited the IFN-I-dependent expression of PD-L1. Enhanced viral replication in the absence of PD-L1 led to persistence of virus and prevented CD8+ T-cell exhaustion. As a consequence, mice exhibited severe immunopathology and died quickly after infection. Therefore, CD169+ macrophages are important contributors to the IFN-I response and thereby influence antiviral activity, CD8+ T-cell exhaustion and immunopathology. PMID:27809306

  20. Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

    PubMed

    Zhang, Song-An; Niyazi, Hu-Er-Xi-Dan; Hong, Wen; Tuluwengjiang, Gu-Li-Xian; Zhang, Lei; Zhang, Yang; Su, Wei-Peng; Bao, Yong-Xing

    2017-03-01

    This study aimed to investigate the effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 signaling pathway. A total of 43 adult female Wistar rats were selected and injected with HeLa cells in the caudal vein to construct a rat model of cervical cancer. All model rats were randomly divided into the radiotherapy group ( n = 31) and the control group ( n = 12). The immunophenotype of Treg cells was detected by the flow cytometry. The protein expressions of EBI3, PD-1, and PD-L1 in cervical cancer tissues were tested by the streptavidin-peroxidase method. HeLa cells in the logarithmic growth phase were divided into four groups: the blank, the negative control group, the EBI3 mimics group, and the EBI3 inhibitors group. Western blotting was used to detect PD-1 and PD-L1 protein expressions. MTT assay was performed to measure the proliferation of Treg cells. Flow cytometry was used to detect cell cycle and apoptosis, and CD4(+)/CD8(+) T cell ratio in each group. Compared with before and 1 week after radiotherapy, the percentages of CD4(+)T cells and CD8(+)T cells were significantly decreased in the radiotherapy group at 1 month after radiotherapy. Furthermore, down-regulation of EBI3 and up-regulation of PD-1 and PD-L1 were observed in cervical cancer tissues at 1 month after radiotherapy. In comparison to the blank and negative control groups, increased expression of EBI3 and decreased expressions of PD-1 and PD-L1 were found in the EBI3 mimics group. However, the EBI3 inhibitors group had a lower expression of EBI3 and higher expressions of PD-1 and PD-L1 than those in the blank and negative control groups. The EBI3 mimics group showed an increase in the optical density value (0.43 ± 0.05), while a decrease in the optical density value (0.31 ± 0.02) was found in the EBI3 inhibitors group. Moreover, compared with the blank and negative control groups, the apoptosis rates

  1. Regulation of myosin IIA and filamentous actin during insulin-stimulated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Stall, Richard; Ramos, Joseph; Kent Fulcher, F.; Patel, Yashomati M.

    2014-03-10

    Insulin stimulated glucose uptake requires the colocalization of myosin IIA (MyoIIA) and the insulin-responsive glucose transporter 4 (GLUT4) at the plasma membrane for proper GLUT4 fusion. MyoIIA facilitates filamentous actin (F-actin) reorganization in various cell types. In adipocytes F-actin reorganization is required for insulin-stimulated glucose uptake. What is not known is whether MyoIIA interacts with F-actin to regulate insulin-induced GLUT4 fusion at the plasma membrane. To elucidate the relationship between MyoIIA and F-actin, we examined the colocalization of MyoIIA and F-actin at the plasma membrane upon insulin stimulation as well as the regulation of this interaction. Our findings demonstrated that MyoIIA and F-actin colocalized at the site of GLUT4 fusion with the plasma membrane upon insulin stimulation. Furthermore, inhibition of MyoII with blebbistatin impaired F-actin localization at the plasma membrane. Next we examined the regulatory role of calcium in MyoIIA-F-actin colocalization. Reduced calcium or calmodulin levels decreased colocalization of MyoIIA and F-actin at the plasma membrane. While calcium alone can translocate MyoIIA it did not stimulate F-actin accumulation at the plasma membrane. Taken together, we established that while MyoIIA activity is required for F-actin localization at the plasma membrane, it alone is insufficient to localize F-actin to the plasma membrane. - Highlights: • Insulin induces colocalization of MyoIIA and F-actin at the cortex in adipocytes. • MyoIIA is necessary but not sufficient to localize F-actin at the cell cortex. • MyoIIA-F-actin colocalization is regulated by calcium and calmodulin.

  2. The v-SNARE Vti1a regulates insulin-stimulated glucose transport and Acrp30 secretion in 3T3-L1 adipocytes.

    PubMed

    Bose, Avirup; Guilherme, Adilson; Huang, Shaohui; Hubbard, Andrea C; Lane, Charles R; Soriano, Neil A; Czech, Michael P

    2005-11-04

    Regulated exocytosis in adipocytes mediates key functions, exemplified by insulin-stimulated secretion of peptides such as adiponectin and recycling of intracellular membranes containing GLUT4 glucose transporters to the cell surface. Using a proteomics approach, the v-SNARE Vti1a (vps10p tail interacting 1a) was identified by mass spectrometry in purified GLUT4-containing membranes. Insulin treatment of 3T3-L1 adipocytes decreased the amounts of both Vti1a and GLUT4 in these membranes, confirming that Vti1a is a component of insulin-sensitive GLUT4-containing vesicles. In the basal state, endogenous Vti1a colocalizes exclusively with perinuclear GLUT4. Although Vti1a has previously been reported to be a v-SNARE localized in the trans-Golgi network, treatment with brefeldin A failed to significantly modify Vti1a or GLUT4 localization while completely dispersing Golgi and trans-Golgi network marker proteins. Furthermore, depletion of Vti1a protein in cultured adipocytes through small interfering RNA-based gene silencing significantly inhibited both adiponectin secretion and insulin-stimulated deoxyglucose uptake. Taken together, these results suggest that the v-SNARE Vti1a may regulate a step common to both GLUT4 and Acrp30 trafficking in 3T3-L1 adipocytes.

  3. The adhesion molecule KAL-1/anosmin-1 regulates neurite branching through a SAX-7/L1CAM–EGL-15/FGFR receptor complex

    PubMed Central

    Díaz-Balzac, Carlos A.; Lázaro-Peña, María I.; Ramos-Ortiz, Gibram A.; Bülow, Hannes E.

    2015-01-01

    Summary Neurite branching is essential for correct assembly of neural circuits, yet remains a poorly understood process. For example, the neural cell adhesion molecule KAL-1/anosmin-1, which is mutated in Kallmann Syndrome regulates neurite branching through mechanisms largely unknown. Here we show that KAL-1/anosmin-1 mediates neurite branching as an autocrine co-factor with EGL-17/FGF through a receptor complex consisting of the conserved cell adhesion molecule SAX-7/L1CAM and the fibroblast growth factor receptor EGL-15/FGFR. This protein complex, which appears conserved in humans, requires the immunoglobulin (Ig) domains of SAX-7/L1CAM and the FN(III) domains of KAL-1/anosmin-1 for formation in vitro as well as function in vivo. The kinase domain of the EGL-15/FGFR is required for branching, and genetic evidence suggests that ras-mediated signaling downstream of EGL-15/FGFR is necessary to effect branching. Our studies establish a molecular pathway that regulates neurite branching during development of the nervous system. PMID:26004184

  4. Nectin-3 (CD113) interacts with Nectin-2 (CD112) to promote lymphocyte transendothelial migration.

    PubMed

    Devilard, Elisabeth; Xerri, Luc; Dubreuil, Patrice; Lopez, Marc; Reymond, Nicolas

    2013-01-01

    Lymphocyte trafficking and migration through vascular endothelial cells (ECs) in secondary lymphoid tissues is critical for immune protection. In the present study, we investigate the role of nectin cell adhesion molecules for the migration of lymphocytes through ECs. Nectins are key players for the establishment of homotypic and heterotypic cell to cell contacts; they are required for cell to cell adherens junction formation and take part in the transendothelial migration of monocytes during the step of diapedesis, when monocytes migrate through EC junctions. We first show that Nectin-3 (CD113) is the only nectin expressed by T lymphocytes and since nectins are expressed on ECs we explored Nectin-3 potential functions in lymphocyte: EC interactions. We demonstrate that Nectin-2, expressed on ECs, is the major counter-receptor of Nectin-3. A soluble form of Nectin-3 binds to Nectin-2 localized at EC junctions and blocking Nectin-2 trans-interactions with monoclonal antibodies abolishes the binding of soluble Nectin-3 to ECs. Nectin-2 is expressed on High Endothelial venules (HEVs), where lymphocyte homing occurs in vivo. Finally, we show that Nectin-3 trans-interaction with Nectin-2 is essential for the process of lymphocyte transendothelial migration in vitro as targeting with blocking monoclonal antibodies either Nectin-3, expressed on lymphocytes, or Nectin-2, expressed on ECs, inhibits lymphocyte extravasation. The nectin family of CAMs is important for the regulation of endothelial barrier functions and transendothelial migration of immune cells. Our results demonstrate for the first time that Nectin-3 trans-interacts with Nectin-2 to promote lymphocyte and monocyte extravasation.

  5. 27-Hydroxycholesterol suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.

    PubMed

    Shirouchi, Bungo; Kashima, Kentaro; Horiuchi, Yasutaka; Nakamura, Yuki; Fujimoto, Yumiko; Tong, Li-Tao; Sato, Masao

    2016-03-17

    Cholesterol oxidation products (oxycholesterols) are produced from cholesterol by automatic and/or enzymatic oxidation of the steroidal backbone and side-chain. Oxycholesterols are present in plasma and serum, suggesting that oxycholesterols are related to the development and progression of various diseases. However, limited information is available about the absolute amounts of oxycholesterols in organs and tissues, and the physiological significance of oxycholesterols in the body. In the present study, we quantified the levels of 13 oxycholesterols in white adipose tissue (WAT) of mice and then evaluated correlations between each oxycholesterol level and WAT weight. The sum of the levels of 13 oxycholesterols in WAT (white adipose tissue) was 15.9 ± 3.4 μg/g of WAT weight and approximately 1 % of cholesterol level. Among oxycholesterols, the levels of 27-hydroxycholesterol (27-OH), an endogenous oxycholesterol produced by enzymatic oxidation, and the relative WAT weights were significantly negatively correlated. Next, we evaluated the effects of 27-OH on lipogenesis and adipogenesis in 3T3-L1 cells. TO901317 (TO), a potent and selective agonist for LXRα, significantly increased intracellular TAG contents, while 27-OH significantly reduced the contents to half when compared with control (DMSO) and completely abolished the effect of TO. In addition, 27-OH significantly reduced the mRNA levels of lipogenic (LXRα and FAS) and adipogenic genes (PPARγ and aP2) during adipocyte maturation of 3T3-L1 cells. In conclusion, our results indicate that 27-OH suppresses lipid accumulation by down-regulating lipogenic and adipogenic gene expression in 3T3-L1 cells.

  6. Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate

    SciTech Connect

    Stadel, J.M.; Poksay, K.S.; Nakada, M.T.; Crooke, S.T.

    1986-05-01

    Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.

  7. Platyphylloside Isolated From Betula platyphylla Inhibit Adipocyte Differentiation and Induce Lipolysis Via Regulating Adipokines Including PPARγ in 3T3-L1 Cells

    PubMed Central

    Lee, Mina; Sung, Sang Hyun

    2016-01-01

    Background: Obesity causes or aggravates many health problems, both independently and in association with several pathological disorders, including Type II diabetes, hypertension, atherosclerosis, and cancer. Therefore, we screened small compounds isolated from natural products for the development of anti-obesity drugs. Objective: The purpose of this study was to investigate the anti-adipogenic activities of platyphylloside, diarylheptanoid isolated from Betula platyphylla, which was selected based on the screening using 3T3-L1 cells. Materials and Methods: To evaluate the inhibition of adipocyte differentiation and lipolysis, lipid contents of BPP on were measured using Oil Red O staining in 3T3-L1 cells. The mRNA and protein expression levels of various adipokines were measured by Quantitative real-time PCR and Western blotting analysis, respectively. Results: Platyphylloside showed significant inhibitory activity on adipocyte differentiation in 3T3-L1 cells and suppressed adipocyte differentiation even in the presence of troglitazone, a PPARγ agonist. Platyphylloside might suppress adipocyte differentiation through PPARγ, C/EBPα, and SREBP1-induced adipogenesis, which is synergistically associated with downstream adipocyte-specific gene promoters such as aP2, FAS, SCD-1, LPL, and Adiponectin. In addition, platyphylloside affected lipolysis by down-regulating perilipin and HSL and up-regulating TNFα. Conclusion: Taken together, the results reveal that platyphylloside has anti-adipogenic activity and highlight its potential in the prevention and treatment of obesity. SUMMARY The extract of B. platyphylla bark and its isolate, BPP, had anti-adipogenic activity in 3T3-L1 cells via suppression of adipocyte differentiation from preadipocytes.Treatment with BPP significantly down-regulated the expression of PPARγ, C/EBP, C/EBPβ, C/EBPδ, SREBP1c, SCD-1, FAS, aP2 and LPL.BPP induced a lipolytic response in mature adipocytes via up-regulation krof TNFá and down-regulation

  8. Germinated brown rice extract inhibits adipogenesis through the down-regulation of adipogenic genes in 3T3-L1 adipocytes.

    PubMed

    Ho, Jin-Nyoung; Son, Mi-Eun; Lim, Won-Chul; Lim, Seung-Taik; Cho, Hong-Yon

    2013-09-01

    The aim of this study was to examine the anti-adipogenic effect of germinated brown rice methanol extract (GBR) in 3T3-L1 adipocytes. The GBR inhibited adipocyte differentiation was measured by Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner without initiating any cytotoxicity. The mRNA levels of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBPα), proliferator-activated receptorγ (PPARγ), and sterol regulatory element-binding protein-1c (SREBP-1c), and adipogenic genes, such as fatty acid synthase (FAS), adipocyte fatty acid-binding protein (aP2), and lipoprotein lipase (LPL), were significantly down-regulated by treatment with GBR when compared to that of untreated control cells. Moreover, tumor necrosis factor-α (TNF-α) and interlukin-6 (IL-6) mRNA expressions were attenuated by GBR in mature adipocytes. These data suggest that GBR exhibits an anti-adipogenic effect through the suppression of adipogenesis in 3T3-L1 adipocytes.

  9. Programmed Death-1 Ligand 2-Mediated Regulation of the PD-L1 to PD-1 Axis Is Essential for Establishing CD4(+) T Cell Immunity.

    PubMed

    Karunarathne, Deshapriya S; Horne-Debets, Joshua M; Huang, Johnny X; Faleiro, Rebecca; Leow, Chiuan Yee; Amante, Fiona; Watkins, Thomas S; Miles, John J; Dwyer, Patrick J; Stacey, Katryn J; Yarski, Michael; Poh, Chek Meng; Lee, Jason S; Cooper, Matthew A; Rénia, Laurent; Richard, Derek; McCarthy, James S; Sharpe, Arlene H; Wykes, Michelle N

    2016-08-16

    Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.

  10. Red yeast rice extracts suppress adipogenesis by down-regulating adipogenic transcription factors and gene expression in 3T3-L1 cells.

    PubMed

    Jeon, Taeil; Hwang, Seong Gu; Hirai, Shizuka; Matsui, Tohru; Yano, Hideo; Kawada, Teruo; Lim, Beoung Ou; Park, Dong Ki

    2004-11-12

    The effects of red yeast rice extracts (RE) on adipocyte differentiation of 3T3-L1 cells were studied. RE were extracted from embryonic rice fermented with red yeast (Monascus ruber). These extracts significantly decreased glycerol-3-phosphate dehydrogenase (GPDH) activity and lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. Moreover, mRNA expression levels of both CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor (PPAR) gamma, the key adipogenic transcription factors, were markedly decreased by RE. RE also inhibited the expression of PPARgamma at protein levels. RE decreased significantly gene expression of adipocyte fatty acid binding protein (aP2) and leptin, which are adipogenic marker proteins and C/EBPalpha and PPARgamma target genes. These results suggest that the inhibitory effect of RE on adipocyte differentiation might be mediated through the down-regulated expression of adipogenic transcription factors and other specific genes.

  11. Exchange protein activated by cyclic AMP is involved in the regulation of adipogenic genes during 3T3-L1 fibroblasts differentiation.

    PubMed

    Gabrielli, Matías; Martini, Claudia N; Brandani, Javier N; Iustman, Laura J R; Romero, Damián G; del C Vila, María

    2014-02-01

    Adipogenesis is stimulated in 3T3-L1 fibroblasts by a combination of insulin, dexamethasone and isobutylmethylxanthine, IBMX, (I+D+M). Two transcription factors are important for the acquisition of the adipocyte phenotype, C/EBP beta (CCAT enhancer-binding protein beta) and PPAR gamma (peroxisome proliferator-activated receptor gamma). IBMX increases cAMP content, which can activate protein kinase A (PKA) and/or EPAC (exchange protein activated by cAMP). To investigate the importance of IBMX in the differentiation mixture, we first evaluated the effect of the addition of IBMX on the increase of C/EBP beta and PPAR gamma and found an enhancement of the amount of both proteins. IBMX addition (I+D+M) or its replacement with a cAMP analogue, dibutyryl-cAMP or 8-(4-chlorophenylthio)-2-O'-methyl-cAMP (8CPT-2-Me-cAMP), the latter activates EPAC and not PKA, remarkably increased PPAR gamma mRNA. However, neither I+D nor any of the inducers alone, increased PPAR gamma mRNA to a similar extent, suggesting the importance of the presence of both IBMX and I+D. It was also found that the addition of IBMX or 8CPT-2-Me-cAMP was able to increase the content of C/EBP beta with respect to I+D. In agreement with these findings, a microarray analysis showed that the presence of either 8CPT-2-Me-cAMP or IBMX in the differentiation mixture was able to upregulate PPAR gamma and PPAR gamma-activated genes as well as other genes involved in lipid metabolism. Our results prove the involvement of IBMX-cAMP-EPAC in the regulation of adipogenic genes during differentiation of 3T3-L1 fibroblasts and therfore contributes to elucidate the role of cyclic AMP in this process.

  12. MicroRNA-410 regulates autophagy-related gene ATG16L1 expression and enhances chemosensitivity via autophagy inhibition in osteosarcoma

    PubMed Central

    Chen, Ren; Li, Xiaohai; He, Bin; Hu, Wei

    2017-01-01

    Osteosarcoma, which is the most common type of primary bone tumor in adolescents, is characterized by complex genetic alterations and frequent resistance to conventional treatments. MicroRNAs (miRs) have emerged as fundamental regulators in gene expression through their ability to silence gene expression at post-transcriptional and translational levels. The present study investigated the role of miR-410 in the progression of osteosarcoma. The results demonstrated that the expression of miR-410 was markedly downregulated in human osteosarcoma tissues, and U2OS and MG-63 osteosarcoma cell lines. Clinicopathological significance suggested that miR-410 may be a potential biomarker for chemotherapy-resistant osteosarcoma. Furthermore, overexpression of miR-410 exhibited a limited effect on cell viability in U2OS and MG-63 cells. Target prediction algorithms (TargetScan and miRanda) indicated that autophagy related 16-like 1 (ATG16L1) was a potential target gene of miR-410. A luciferase reporter assay demonstrated that miR-410 directly decreased ATG16L1 expression by targeting its 3′-untranslated region. In addition, the results revealed that miR-410 was able to markedly inhibit autophagy. Accordingly, autophagy was activated as a protective mechanism when osteosarcoma cells were exposed to three common anticancer drugs, including rapamycin, doxorubicin and cisplatin. Furthermore, the autophagy inhibitor 3-methyladenine and miR-410 expression were able to improve the therapeutic response of the cells to chemotherapy drugs (rapamycin, doxorubicin and cisplatin), thus indicating that miR-410 enhanced chemosensitivity through autophagy inhibition in osteosarcoma cells. In conclusion, studies regarding the function of miR-410 on autophagy provided insight into the biological function of miR-410 in osteosarcoma and may offer a promising approach for the treatment of osteosarcoma. PMID:28138700

  13. The EGFR pathway is involved in the regulation of PD-L1 expression via the IL-6/JAK/STAT3 signaling pathway in EGFR-mutated non-small cell lung cancer.

    PubMed

    Zhang, Nan; Zeng, Yuanyuan; Du, Wenwen; Zhu, Jianjie; Shen, Dan; Liu, Zeyi; Huang, Jian-An

    2016-10-01

    Negative regulation of the signal mediated by the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway can effectively inhibit the function of T and B cells, which play a key role in the regulation of immune response. Recently, emerging evidence has suggested that the expression of PD-L1 is related to the mutation status of the epidermal growth factor receptor (EGFR). Moreover, the activation of the EGFR signaling pathway can induce expression of PD-L1. In the present study, we demonstrated that activated EGFR can upregulate the expression of PD-L1 through the interleukin 6/Janus kinase/signal transducer and activator of transcription 3 (IL-6/JAK/STAT3) signaling pathway in non-small cell lung cancer (NSCLC) cells. Cells treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) can downregulate the activation of the IL-6/JAK/STAT3 pathway, which subsequently reduces the expression of PD-L1. Furthermore, silencing of PD-L1 expression in NSCLC cells correlated with inhibition of cell proliferation and enhanced tumor cell apoptosis. In summary, our research indicates that EGFR is involved in the regulation of PD-L1 expression and cell proliferation via the IL-6/JAK/STAT3 signaling pathway in NSCLC. The present study suggests the potential of combined targeted therapy with immunotherapy in the treatment of NSCLC.

  14. Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

    PubMed

    Schnoor, Michael

    2015-04-15

    The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

  15. Participation of the urokinase-type plasminogen activator receptor (uPAR) in neutrophil transendothelial migration.

    PubMed

    Pliyev, Boris K; Antonova, Olga A; Menshikov, Mikhail

    2011-05-01

    The mechanisms underlying migration of neutrophils across endothelium are not completely understood. The urokinase-type plasminogen activator receptor (uPAR) plays a key role in neutrophil adhesion and migration. In the present study, we addressed whether uPAR regulates neutrophil transendothelial migration. We first showed that siRNA-mediated knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) did not affect neutrophil migration across HUVEC monolayers indicating that endothelial uPAR does not regulate neutrophil transmigration. In contrast, the transmigration was significantly inhibited by Fab' fragment of anti-uPAR monoclonal antibody and proteolytically inactive urokinase (uPA), whereas inhibition of proteolytical activity of endogenous uPA (with amiloride or plasminogen activator inhibitor-1) did not affect the transmigration. Both the anti-uPAR Fab' fragment and proteolytically inactive uPA did not exert significant effects upon the transmigration conducted in the presence of F(ab')(2) fragment of blocking antibody to integrin Mac-1 indicating that uPAR regulates Mac-1-dependent transmigration. Mac-1-dependent, but not Mac-1-independent, transmigration was significantly reduced in the presence of N-acetyl-d-glucosamine and d-mannose, the saccharides that disrupt uPAR/Mac-1 association, but was unaffected in the presence of control saccharides (d-sorbitol and sucrose). We conclude that physical association of uPAR with Mac-1 mediates the regulatory effect of uPAR over the transmigration. Finally, we provide evidence that the functional cooperation between uPAR and Mac-1 is essential at both adhesion and diapedesis steps of neutrophil migration across endothelium. Thus, uPAR expressed on neutrophil plasma membrane regulates transendothelial migration independently of uPA proteolytical activity and acting as a cofactor for integrin Mac-1.

  16. C(2)-ceramide influences the expression and insulin-mediated regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 adipocytes.

    PubMed

    Mei, Jie; Holst, Lena Stenson; Landström, Tova Rahn; Holm, Cecilia; Brindley, David; Manganiello, Vincent; Degerman, Eva

    2002-03-01

    Cyclic nucleotide phosphodiesterase (PDE) 3B plays an important role in the antilipolytic action of insulin and, thereby, the release of fatty acids from adipocytes. Increased concentrations of circulating fatty acids as a result of elevated or unrestrained lipolysis cause insulin resistance. The lipolytic action of tumor necrosis factor (TNF)-alpha is thought to be one of the mechanisms by which TNF-alpha induces insulin resistance. Ceramide is the suggested second messenger of TNF-alpha action, and in this study, we used 3T3-L1 adipocytes to investigate the effects of C(2)-ceramide (a short-chain ceramide analog) on the expression and regulation of PDE3B and lipolysis. Incubation of adipocytes with 100 micromol/l C(2)-ceramide (N-acetyl-sphingosine) resulted in a time-dependent decrease of PDE3B activity, accompanied by decreased PDE3B protein expression. C(2)-ceramide, in a time- and dose-dependent manner, stimulated lipolysis, an effect that was blocked by H-89, an inhibitor of protein kinase A. These ceramide effects were prevented by 20 micromol/l troglitazone, an antidiabetic drug. In addition to downregulation of PDE3B, the antilipolytic action of insulin was decreased by ceramide treatment. These results, together with data from other studies on PDE3B and lipolysis in diabetic humans and animals, suggest a novel pathway by which ceramide induces insulin resistance. Furthermore, PDE3B is demonstrated to be a target for troglitazone action in adipocytes.

  17. Glutamine, insulin and glucocorticoids regulate glutamine synthetase expression in C2C12 myotubes, Hep G2 hepatoma cells and 3T3 L1 adipocytes.

    PubMed

    Wang, Yanxin; Watford, Malcolm

    2007-04-01

    The cell-specific regulation of glutamine synthetase expression was studied in three cell lines. In C2C12 myotubes, glucocorticoids increased the abundance of both glutamine synthetase protein and mRNA. Culture in the absence of glutamine also resulted in very high glutamine synthetase protein abundance but mRNA levels were unchanged. Glucocorticoids also increased the abundance of glutamine synthetase mRNA in Hep G2 hepatoma cells but this was not reflected in changes in protein abundance. Culture of Hep G2 cells without glutamine resulted in very high levels of protein, again with no change in mRNA abundance. Insulin was without effect in both C2C12 and Hep G2 cells. In 3T3 L1 adipocytes glucocorticoids increased the abundance of both glutamine synthetase mRNA and protein, insulin added alone had no effect but in the presence of glucocorticoids resulted in lower mRNA levels than seen with glucocorticoids alone, although protein levels remained high under such conditions. In contrast to the other cell lines glutamine synthetase protein levels were relatively unchanged by culture in the absence of glutamine. The results support the hypothesis that in myocytes, and hepatomas, but not in adipocytes, glutamine acts to moderate glutamine synthetase induction by glucocorticoids.

  18. Fisetin up-regulates the expression of adiponectin in 3T3-L1 adipocytes via the activation of silent mating type information regulation 2 homologue 1 (SIRT1)-deacetylase and peroxisome proliferator-activated receptors (PPARs).

    PubMed

    Jin, Taewon; Kim, Oh Yoen; Shin, Min-Jeong; Choi, Eun Young; Lee, Sung Sook; Han, Ye Sun; Chung, Ji Hyung

    2014-10-29

    Adiponectin, an adipokine, has been described as showing physiological benefits against obesity-related malfunctions and vascular dysfunction. Several natural compounds that promote the expression and secretion of adipokines in adipocytes could be useful for treating metabolic disorders. This study investigated the effect of fisetin, a dietary flavonoid, on the regulation of adiponectin in adipocytes using 3T3-L1 preadipocytes. The expression and secretion of adiponectin increased in 3T3-L1 cells upon treatment with fisetin in a dose-dependent manner. Fisetin-induced adiponectin secretion was inhibited by peroxisome proliferator-activated receptor (PPAR) antagonists. It was also revealed that fisetin increased the activities of PPARs and silent mating type information regulation 2 homologue 1 (SIRT1) in a dose-dependent manner. Furthermore, the up-regulation of adiponectin and the activation of PPARs induced by fisetin were prevented by a SIRT1 inhibitor. Fisetin also promoted deacetylation of PPAR γ coactivator 1 (PGC-1) and its interaction with PPARs. SIRT knockdown by siRNA significantly decreased both adiponectin production and PPARs-PGC-1 interaction. These results provide evidence that fisetin promotes the gene expression of adiponectin through the activation of SIRT1 and PPARs in adipocytes.

  19. The PD-1/PD-L1 inhibitory pathway is altered in pre-eclampsia and regulates T cell responses in pre-eclamptic rats

    PubMed Central

    Tian, Mei; Zhang, Yonghong; Liu, Zhaozhao; Sun, Guoqiang; Mor, Gil; Liao, Aihua

    2016-01-01

    The programmed cell death-1(PD-1)/PD-ligand 1 (PD-L1) pathway is critical to immune homeostasis by promoting regulatory T (Treg) development and inhibiting effector T (such as Th17) cell responses. However, the association between the PD-1/PD-L1 pathway and the Treg/Th17 imbalance has not been fully investigated in pre-eclampsia (PE). In this study, we observed an inverse correlation between the percentages of Treg and Th17 cells, and the expression of PD-1 and PD-L1 on the two subsets also changed in PE compared with normal pregnancy. We further explored their relationship in vivo using the L-NG-Nitroarginine Methyl Ester (L-NAME) induced PE-like rat models, also characterized by Treg/Th17 imbalance. Administration of PD-L1-Fc protein provides a protective effects on the pre-eclamptic models, both to the mother and the fetuses, by reversing Treg/Th17 imbalance through inhibiting PI3K/AKT/m-TOR signaling and enhancing PTEN expression. In addition, we also observed a protective effect of PD-L1-Fc on the placenta by reversing placental damages. These results suggested that altered PD-1/PD-L1 pathway contributed to Treg/Th17 imbalance in PE. Treatment with PD-L1-Fc posed protective effects on pre-eclamptic models, indicating that the use of PD-L1-Fc might be a potential therapeutic target in PE treatment. PMID:27277012

  20. Impairment of transendothelial leukocyte migration by iron complexes.

    PubMed

    Sengoelge, Gürkan; Kletzmayr, Josef; Ferrara, Ilse; Perschl, Agnes; Hörl, Walter H; Sunder-Plassmann, Gere

    2003-10-01

    Although iron sucrose and iron gluconate are generally well tolerated in patients who are treated for renal anemia, recent clinical studies and cell culture experiments suggested significant toxicity and long-term side effects arising from the use of these iron complexes. Because of the possible role of iron in infection or cardiovascular disease, it was theorized that parenteral iron compounds influence endothelial and PMN interaction in vitro. A well-established double-chamber method was used to assess the effect of different concentrations of iron sucrose and iron gluconate (1, 25, 50, and 100 micro g/ml) on the transendothelial migration of PMN. Preincubation of PMN and endothelial cells as well as preincubation of PMN alone with 25, 50, or 100 micro g/ml iron resulted in a significant decrease in PMN migration. In contrast, after incubation of the endothelial cells alone with iron, no reduction in the transendothelial migration of PMN was observed. Preincubation of PMN and/or endothelial cells with 1 micro g/ml iron did not lead to any decrease in the rate of migrated PMN. The only significant change in experiments with 1 micro g/ml was an increase in PMN migration after preincubation of endothelial cells and PMN with iron gluconate. A four-way ANOVA showed a significant effect of the iron concentration (P < 0.000001), of type of iron complex (P < 0.005), of the preincubation of endothelial cell (P < 0.001), and of the preincubation of PMN with iron (P < 0.000001) on PMN diapedesis. It is concluded that iron sucrose and iron gluconate cause a significant inhibition of transendothelial migration of PMN.

  1. Methylene blue modulates transendothelial migration of peripheral blood cells.

    PubMed

    Werner, Isabella; Guo, Fengwei; Bogert, Nicolai V; Stock, Ulrich A; Meybohm, Patrick; Moritz, Anton; Beiras-Fernandez, Andres

    2013-01-01

    Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose

  2. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    SciTech Connect

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui; Cheng, Tian-Lu; Lin, Shinne-Ren; Chang, Long-Sen

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  3. Stathmin mediates neuroblastoma metastasis in a tubulin-independent manner via RhoA/ROCK signaling and enhanced transendothelial migration.

    PubMed

    Fife, C M; Sagnella, S M; Teo, W S; Po'uha, S T; Byrne, F L; Yeap, Y Y C; Ng, D C H; Davis, T P; McCarroll, J A; Kavallaris, M

    2017-01-26

    Neuroblastoma, the most common solid tumor of young children, frequently presents with aggressive metastatic disease and for these children the 5-year survival rates are dismal. Metastasis, the movement of cancer cells from one site to another, involves remodeling of the cytoskeleton including altered microtubule dynamics. The microtubule-destabilizing protein, stathmin, has recently been shown to mediate neuroblastoma metastasis although precise functions remain poorly defined. In this study we investigated stathmin's contribution to the metastatic process and potential mechanism(s) by which it exerts these effects. Stathmin suppression significantly reduced neuroblastoma cell invasion of 3D tumor spheroids into an extracellular matrix. Moreover, inhibiting stathmin expression significantly reduced transendothelial migration in two different neuroblastoma cell lines in vitro. Inhibition of ROCK, a key regulator of cell migration, in neuroblastoma cells highlighted that stathmin regulates transendothelial migration through ROCK signaling. Reduced stathmin expression in neuroblastoma cells significantly increased the activation of the RhoA small GTPase. Notably, re-expression of either wild type or a phospho-mimetic stathmin mutant (4E) made defective in tubulin binding returned cell migration and transendothelial migration back to control levels, indicating that stathmin may influence these processes in neuroblastoma cells independent of tubulin binding. Finally, stathmin suppression in neuroblastoma cells significantly reduced whole body, lung, kidney and liver metastases in an experimental metastases mouse model. In conclusion, stathmin suppression interferes with the metastatic process via RhoA/ROCK signaling in neuroblastoma cells. These findings highlight the importance of stathmin to the metastatic process and its potential as a therapeutic target for the treatment of neuroblastoma.

  4. Monocyte ADAM17 promotes diapedesis during transendothelial migration: identification of steps and substrates targeted by metalloproteinases.

    PubMed

    Tsubota, Yoshiaki; Frey, Jeremy M; Tai, Phillip W L; Welikson, Robert E; Raines, Elaine W

    2013-04-15

    Despite expanded definition of the leukocyte adhesion cascade and mechanisms underlying individual steps, very little is known about regulatory mechanisms controlling sequential shifts between steps. We tested the hypothesis that metalloproteinases provide a mechanism to rapidly transition monocytes between different steps. Our study identifies diapedesis as a step targeted by metalloproteinase activity. Time-lapse video microscopy shows that the presence of a metalloproteinase inhibitor results in a doubling of the time required for human monocytes to complete diapedesis on unactivated or inflamed human endothelium, under both static and physiological-flow conditions. Thus, diapedesis is promoted by metalloproteinase activity. In contrast, neither adhesion of monocytes nor their locomotion over the endothelium is altered by metalloproteinase inhibition. We further demonstrate that metalloproteinase inhibition significantly elevates monocyte cell surface levels of integrins CD11b/CD18 (Mac-1), specifically during transendothelial migration. Interestingly, such alterations are not detected for other endothelial- and monocyte-adhesion molecules that are presumed metalloproteinase substrates. Two major transmembrane metalloproteinases, a disintegrin and metalloproteinase (ADAM)17 and ADAM10, are identified as enzymes that control constitutive cleavage of Mac-1. We further establish that knockdown of monocyte ADAM17, but not endothelial ADAM10 or ADAM17 or monocyte ADAM10, reproduces the diapedesis delay observed with metalloproteinase inhibition. Therefore, we conclude that monocyte ADAM17 facilitates the completion of transendothelial migration by accelerating the rate of diapedesis. We propose that the progression of diapedesis may be regulated by spatial and temporal cleavage of Mac-1, which is triggered upon interaction with endothelium.

  5. MdSOS2L1 forms a complex with MdMYB1 to control vacuolar pH by transcriptionally regulating MdVHA-B1 in apples.

    PubMed

    Sun, Cui-Hui; Zhang, Quan-Yan; Sun, Mei-Hong; Hu, Da-Gang

    2016-01-01

    Vacuolar pH is important and involves in many different physiological processes in plants. A recent paper published in Plant Physiology reveals that MdMYB1 regulates vacuolar pH by directly transcriptionally regulating proton pump genes and malate transporters genes, such as V-ATPase subunit gene MdVHA-B1. Here, we found that MdSOS2L1 in vitro did not directly interact with MdMYB1, however, in vivo formed a complex with MdMYB1 in the nucleus to regulate MdVHA-B1-mediated vacuolar acidification. This finding shed light on the role of MdSOS2L1 in transcriptionally regulating MdVHA-B1 in addition to its post-modified function in apples.

  6. MdSOS2L1 forms a complex with MdMYB1 to control vacuolar pH by transcriptionally regulating MdVHA-B1 in apples

    PubMed Central

    Sun, Cui-Hui; Zhang, Quan-Yan; Sun, Mei-Hong; Hu, Da-Gang

    2016-01-01

    ABSTRACT Vacuolar pH is important and involves in many different physiological processes in plants. A recent paper published in Plant Physiology reveals that MdMYB1 regulates vacuolar pH by directly transcriptionally regulating proton pump genes and malate transporters genes, such as V-ATPase subunit gene MdVHA-B1. Here, we found that MdSOS2L1 in vitro did not directly interact with MdMYB1, however, in vivo formed a complex with MdMYB1 in the nucleus to regulate MdVHA-B1-mediated vacuolar acidification. This finding shed light on the role of MdSOS2L1 in transcriptionally regulating MdVHA-B1 in addition to its post-modified function in apples. PMID:26910596

  7. Metastasis is regulated via microRNA-200/ZEB1 axis control of tumor cell PD-L1 expression and intratumoral immunosuppression

    PubMed Central

    Goswami, Sangeeta; Cortez, Maria Angelica; Ahn, Young-Ho; Byers, Lauren A.; Zhang, Xuejun; Yi, Xiaohui; Dwyer, David; Lin, Wei; Diao, Lixia; Wang, Jing; Roybal, Jonathon; Patel, Mayuri; Ungewiss, Christin; Peng, David; Antonia, Scott; Mediavilla-Varela, Melanie; Robertson, Gordon; Suraokar, Milind; Welsh, James W.; Erez, Baruch; Wistuba, Ignacio I.; Chen, Lieping; Peng, Di; Wang, Shanshan; Ullrich, Stephen E.; Heymach, John V.; Kurie, Jonathan M.; Qin, F. Xiao-Feng

    2014-01-01

    Immunosuppression of tumor-infiltrating lymphocytes (TIL) is a common feature of advanced cancer, but its biological basis has remained obscure. We demonstrate here a molecular link between epithelial-to-mesenchymal transition (EMT) and CD8+ TIL immunosuppression, two key drivers of cancer progression. We show that microRNA-200 (miR-200), a cell-autonomous suppressor of EMT and metastasis, targets PD-L1. Moreover, ZEB1, an EMT activator and transcriptional repressor of miR-200, relieves miR-200 repression of PD-L1 on tumor cells, leading to CD8+ T cell immunosuppression and metastasis. These findings are supported by robust correlations between the EMT score, miR-200 levels and PD-L1 expression in multiple human lung cancer datasets. In addition to revealing a link between EMT and T cell dysfunction, these findings also show that ZEB1 promotes metastasis through a heretofore unappreciated cell non-autonomous mechanism, and suggest that subgroups of patients in whom malignant progression is driven by EMT activators may respond to treatment with PD-L1 antagonists. PMID:25348003

  8. Quantification of transendothelial migration using three-dimensional confocal microscopy.

    PubMed

    Cain, Robert J; d'Água, Bárbara Borda; Ridley, Anne J

    2011-01-01

    Migration of cells across endothelial barriers, termed transendothelial migration (TEM), is an important cellular process that underpins the pathology of many disease states including chronic inflammation and cancer metastasis. While this process can be modeled in vitro using cultured cells, many model systems are unable to provide detailed visual information of cell morphologies and distribution of proteins such as junctional markers, as well as quantitative data on the rate of TEM. Improvements in imaging techniques have made microscopy-based assays an invaluable tool for studying this type of detailed cell movement in physiological processes. In this chapter, we describe a confocal microscopy-based method that can be used to assess TEM of both leukocytes and cancer cells across endothelial barriers in response to a chemotactic gradient, as well as providing information on their migration into a subendothelial extracellular matrix, designed to mimic that found in vivo.

  9. 4-Hydroxyisoleucine ameliorates an insulin resistant-like state in 3T3-L1 adipocytes by regulating TACE/TIMP3 expression

    PubMed Central

    Gao, Feng; Du, Wen; Zafar, Mohammad Ishraq; Shafqat, Raja Adeel; Jian, Liumeng; Cai, Qin; Lu, Furong

    2015-01-01

    Background Obesity-associated insulin resistance (IR) is highly correlated with soluble tumor necrosis factor-α (sTNF-α), which is released from transmembranous TNF-α by TNF-α converting enzyme (TACE). In vivo, TACE activity is suppressed by tissue inhibitor of metalloproteinase 3 (TIMP3). Agents that can interact with TACE/TIMP3 to improve obesity-related IR would be highly valuable. In the current study, we assessed whether (2S,3R,4S)-4-hydroxyisoleucine (4-HIL) could modulate TACE/TIMP3 and ameliorate an obesity-induced IR-like state in 3T3-L1 adipocytes. Materials and methods 3T3-L1 adipocytes were incubated in the presence of 25 mM glucose and 0.6 nM insulin to induce an IR-like state, and were then treated with different concentrations of 4-HIL or 10 µM pioglitazone (positive control). The glucose uptake rate was determined using the 2-deoxy-[3H]-d-glucose method, and the levels of sTNF-α in the cell supernatant were determined using ELISA. The protein expression of TACE, TIMP3, and insulin signaling-related molecules was measured using western blotting. Results Exposure to high glucose and insulin for 18 hours increased the levels of sTNF-α in the cell supernatant. The phosphorylation of insulin receptor substrate-1 (IRS-1) Ser307 and Akt Ser473 was increased, whereas the protein expression of IRS-1, Akt, and glucose transporter-4 was decreased. The insulin-induced glucose uptake was reduced by 67% in 3T3-L1 adipocytes, which indicated the presence of an IR-like state. The above indexes, which demonstrated the successful induction of an IR-like state, were reversed by 4-HIL in a dose-dependent manner by downregulating and upregulating the protein expression of TACE and TIMP3 proteins, respectively. Conclusion 4-HIL improved an obesity-associated IR-like state in 3T3-L1 adipocytes by targeting TACE/TIMP3 and the insulin signaling pathway. PMID:26527864

  10. Traditional Korean Herbal Formula Samsoeum Attenuates Adipogenesis by Regulating the Phosphorylation of ERK1/2 in 3T3-L1 Cells

    PubMed Central

    Jeong, Soo-Jin; Yoo, Sae-Rom; Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Adipogenesis is the cell differentiation process from preadipocytes into adipocytes and the critical action in the development of obesity. In the present study, we conducted in vitro analyses to investigate the inhibitory effects of Samsoeum (SSE), a traditional herbal decoction. SSE had no significant cytotoxic effect against either the undifferentiated or differentiated 3T3-L1 cells. Oil Red O staining results showed that SSE significantly inhibited fat accumulation in adipocytes. SSE treatment consistently reduced the intracellular triglyceride content in the cells. SSE significantly inactivated glycerol-3-phosphate dehydrogenase (GPDH), a major link between carbohydrate and lipid metabolisms in 3T3-L1 adipocytes, and markedly inhibited the production of leptin, an important adipokine, in differentiated cells. SSE markedly suppressed the mRNA expression of the adipogenesis-related genes peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer binding protein-alpha (C/EBP-α), fatty acid synthase (FAS), lipoprotein lipase (LPL), and fatty acid binding protein 4 (FABP4). Importantly, SSE increased the phosphorylation of ERK1/2, but not p38 MAPK and JNK, in adipose cells. Overall, our results indicate that SSE exerts antiadipogenic activity and modulates expressions of adipogenesis-related genes and ERK1/2 activation in adipocytes. PMID:26483846

  11. The autophagy elongation complex (ATG5-12/16L1) positively regulates HCV replication and is required for wild-type membranous web formation

    PubMed Central

    Fahmy, Ahmed M.; Labonté, Patrick

    2017-01-01

    Hepatitis C virus (HCV) infection induces intracellular membrane rearrangements, thus forming a membranous web (MW) in which HCV replication and assembly occur. The HCV-induced MW is primarily composed of double membrane vesicles (DMVs) transfused by multi-membrane vesicles. The autophagy machinery has been proposed to participate in the formation of such vesicles. However, no clear evidence has been found linking autophagy to the formation of these DMVs. In this study, we evaluated the role of the autophagy elongation complex (ATG5-12/16L1) in HCV replication and MW formation. Using a dominant negative form of ATG12 and an siRNA approach, we demonstrated that the ATG5-12 conjugate, but not LC3-II formation, is crucial for efficient viral replication. Furthermore, purification of HCV MW revealed the presence of ATG5-12 and ATG16L1 along with HCV nonstructural proteins. Interestingly, LC3 was not recruited along with the elongation complex to the site of viral replication. Finally, inhibition of the elongation complex, but not LC3, greatly impaired the formation of the wild-type MW phenotype. To our knowledge, this study provides the first evidence of the involvement of autophagy proteins in the formation of wild-type MWs. PMID:28067309

  12. Tubby-like protein superfamily member PLSCR3 functions as a negative regulator of adipogenesis in mouse 3T3-L1 preadipocytes by suppressing induction of late differentiation stage transcription factors

    PubMed Central

    Inokawa, Akira; Inuzuka, Tatsutoshi; Takahara, Terunao; Shibata, Hideki; Maki, Masatoshi

    2015-01-01

    PLSCR3 (phospholipid scramblase 3, Scr3) belongs to the superfamily of membrane-associated transcription regulators named Tubby-like proteins (TULPs). Physiological phospholipid scrambling activities of PLSCRs in vivo have been skeptically argued, and knowledge of the biological functions of Scr3 is limited. We investigated the expression of Scr3 during differentiation of mouse 3T3-L1 preadipocytes by Western blotting (WB) and by reverse-transcription and real-time quantitative PCR (RT-qPCR). The Scr3 protein decreased during 3T3-L1 differentiation accompanied by a reduction in the mRNA level, and there was a significant increase in the amount of Scr3 protein secreted into the culture medium in the form of extracellular microvesicles (exosomes). On the other hand, Scr3 expression did not significantly decrease, and the secretion of Scr3 in 3T3 Swiss-albino fibroblasts (a parental cell-line of 3T3-L1) was not increased by differentiation treatment. Overexpression of human Scr3 during 3T3-L1 differentiation suppressed triacylglycerol accumulation and inhibited induction of the mRNAs of late stage pro-adipogenic transcription factors [CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)] and X-box-binding protein 1 (XBP1). Expression of early stage pro-adipogenic transcription factors (C/EBPβ and C/EBPδ) was not significantly affected. These results suggest that Scr3 functions as a negative regulator of adipogenesis in 3T3-L1 cells at a specific differentiation stage and that decrease in the intracellular amount of Scr3 protein caused by reduction in Scr3 mRNA expression and enhanced secretion of Scr3 protein appears to be important for appropriate adipocyte differentiation. PMID:26677203

  13. Sida rhomboidea. Roxb leaf extract down-regulates expression of PPARγ2 and leptin genes in high fat diet fed C57BL/6J Mice and retards in vitro 3T3L1 pre-adipocyte differentiation.

    PubMed

    Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Ramani, Umed V; Devkar, Ranjitsinh V; Ramachandran, A V

    2011-01-01

    Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity.

  14. Cdc42 promotes transendothelial migration of cancer cells through β1 integrin.

    PubMed

    Reymond, Nicolas; Im, Jae Hong; Garg, Ritu; Vega, Francisco M; Borda d'Agua, Barbara; Riou, Philippe; Cox, Susan; Valderrama, Ferran; Muschel, Ruth J; Ridley, Anne J

    2012-11-12

    Cancer cells interact with endothelial cells during the process of metastatic spreading. Here, we use a small interfering RNA screen targeting Rho GTPases in cancer cells to identify Cdc42 as a critical regulator of cancer cell-endothelial cell interactions and transendothelial migration. We find that Cdc42 regulates β1 integrin expression at the transcriptional level via the transcription factor serum response factor (SRF). β1 integrin is the main target for Cdc42-mediating interaction of cancer cells with endothelial cells and the underlying extracellular matrix, as exogenous β1 integrin expression was sufficient to rescue the Cdc42-silencing phenotype. We show that Cdc42 was required in vivo for cancer cell spreading and protrusion extension along blood vessels and retention in the lungs. Interestingly, transient Cdc42 depletion was sufficient to decrease experimental lung metastases, which suggests that its role in endothelial attachment is important for metastasis. By identifying β1 integrin as a transcriptional target of Cdc42, our results provide new insight into Cdc42 function.

  15. Bisphenol-A impairs insulin action and up-regulates inflammatory pathways in human subcutaneous adipocytes and 3T3-L1 cells.

    PubMed

    Valentino, Rossella; D'Esposito, Vittoria; Passaretti, Federica; Liotti, Antonietta; Cabaro, Serena; Longo, Michele; Perruolo, Giuseppe; Oriente, Francesco; Beguinot, Francesco; Formisano, Pietro

    2013-01-01

    Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1 nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPARγ and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-γ, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function.

  16. cAMP-dependent posttranscriptional regulation of steroidogenic acute regulatory (STAR) protein by the zinc finger protein ZFP36L1/TIS11b.

    PubMed

    Duan, Haichuan; Cherradi, Nadia; Feige, Jean-Jacques; Jefcoate, Colin

    2009-04-01

    Star is expressed in steroidogenic cells as 3.5- and 1.6-kb transcripts that differ only in their 3'-untranslated regions (3'-UTR). In mouse MA10 testis and Y-1 adrenal lines, Br-cAMP preferentially stimulates 3.5-kb mRNA. ACTH is similarly selective in primary bovine adrenocortical cells. The 3.5-kb form harbors AU-rich elements (AURE) in the extended 3'-UTR, which enhance turnover. After peak stimulation of 3.5-kb mRNA, degradation is seen. Star mRNA turnover is enhanced by the zinc finger protein ZFP36L1/TIS11b, which binds to UAUUUAUU repeats in the extended 3'-UTR. TIS11b is rapidly stimulated in each cell type in parallel with Star mRNA. Cotransfection of TIS11b selectively decreases cytomegalovirus-promoted Star mRNA and luciferase-Star 3'-UTR reporters harboring the extended 3'-UTR. Direct complex formation was demonstrated between TIS11b and the extended 3'-UTR of the 3.5-kb Star. AURE mutations revealed that TIS11b-mediated destabilization required the first two UAUUUAUU motifs. HuR, which also binds AURE, did not affect Star expression. Targeted small interfering RNA knockdown of TIS11b specifically enhanced stimulation of 3.5-kb Star mRNA in bovine adrenocortical cells, MA-10, and Y-1 cells but did not affect the reversals seen after peak stimulation. Direct transfection of Star mRNA demonstrated that Br-cAMP stimulated a selective turnover of 3.5-kb mRNA independent of AURE, which may correspond to these reversal processes. Steroidogenic acute regulatory (STAR) protein induction was halved by TIS11b knockdown, concomitant with decreased cholesterol metabolism. TIS11b suppression of 3.5-kb mRNA is therefore surprisingly coupled to enhanced Star translation leading to increased cholesterol metabolism.

  17. Uveal melanoma cells utilize a novel route for transendothelial migration.

    PubMed

    Onken, Michael D; Li, Jinmei; Cooper, John A

    2014-01-01

    Uveal melanoma arises in the eye, and it spreads to distant organs in almost half of patients, leading to a fatal outcome. To metastasize, uveal melanoma cells must transmigrate into and out of the microvasculature, crossing the monolayer of endothelial cells that separates the vessel lumen from surrounding tissues. We investigated how human uveal melanoma cells cross the endothelial cell monolayer, using a cultured cell system with primary human endothelial cell monolayers on hydrogel substrates. We found that uveal melanoma cells transmigrate by a novel and unexpected mechanism. Uveal melanoma cells intercalate into the endothelial cell monolayer and flatten out, assuming a shape and geometry similar to those of endothelial cells in the monolayer. After an extended period of time in the intercalated state, the uveal melanoma cells round up and migrate underneath the monolayer. VCAM is present on endothelial cells, and anti-VCAM antibodies slowed the process of intercalation. Depletion of BAP1, a known suppressor of metastasis in patients, increased the amount of transmigration of uveal melanoma cells in transwell assays; but BAP1 depletion did not affect the rate of intercalation, based on movies of living cells. Our results reveal a novel route of transendothelial migration for uveal melanoma cells, and they provide insight into the mechanism by which loss of BAP1 promotes metastasis.

  18. Transendothelial migration enables subsequent transmigration of neutrophils through underlying pericytes.

    PubMed

    Ayres-Sander, Chantal E; Lauridsen, Holly; Maier, Cheryl L; Sava, Parid; Pober, Jordan S; Gonzalez, Anjelica L

    2013-01-01

    During acute inflammation, neutrophil recruitment into extravascular tissue requires neutrophil tethering and rolling on cytokine-activated endothelial cells (ECs), tight adhesion, crawling towards EC junctions and transendothelial migration (TEM). Following TEM, neutrophils must still traverse the subendothelial basement membrane and network of pericytes (PCs). Until recently, the contribution of the PC layer to neutrophil recruitment was largely ignored. Here we analyze human neutrophil interactions with interleukin (IL)-1β-activated human EC monolayers, PC monolayers and EC/PC bilayers in vitro. Compared to EC, PC support much lower levels of neutrophil binding (54.6% vs. 7.1%, respectively) and transmigration (63.7 vs. 8.8%, respectively) despite comparable levels of IL-8 (CXCL8) synthesis and display. Remarkably, EC/PC bilayers support intermediate levels of transmigration (37.7%). Neutrophil adhesion to both cell types is Mac-1-dependent and while ICAM-1 transduction of PCs increases neutrophil adhesion to (41.4%), it does not increase transmigration through PC monolayers. TEM, which increases neutrophil Mac-1 surface expression, concomitantly increases the ability of neutrophils to traverse PCs (19.2%). These data indicate that contributions from both PCs and ECs must be considered in evaluation of microvasculature function in acute inflammation.

  19. High-resolution fluorescence microscopy to study transendothelial migration.

    PubMed

    Carman, Christopher V

    2012-01-01

    Immune system functions rely heavily on the ability of immune cells (i.e., blood leukocyte) to traffic throughout the body as they conduct immune surveillance and respond to pathogens. A monolayer of vascular endothelial cells (i.e., the "endothelium") provides a critical, selectively permeable barrier between two principal compartments of the body: the blood circulation and the tissue. Thus, knowledge of the basic mechanisms by which leukocytes migrate across the endothelium (i.e., undergo "transendothelial migration"; TEM) is critical for understanding immune system function. Cultured endothelial cell monolayers, used in combination with isolated blood leukocytes, provide a basis for highly useful in vitro models for study of TEM. When used in conjunction with high spatial and temporal resolution imaging approaches, such models have begun to reveal complex and dynamic cell behaviors in leukocytes and endothelial cells that ultimately determine TEM efficiency. In this chapter, we provide protocols for setting up a basic in vitro TEM system and for conducting high-resolution dynamic live-cell and three-dimensional fixed-cell imaging of TEM.

  20. Endothelial monolayers and transendothelial migration depend on mechanical properties of the substrate.

    PubMed

    Onken, Michael D; Mooren, Olivia L; Mukherjee, Suranjana; Shahan, Stefanie T; Li, Jinmei; Cooper, John A

    2014-12-01

    Endothelial cells (ECs) line the microvasculature and constitute a barrier between the vessel lumen and surrounding tissues. ECs inform circulating immune cells of the health and integrity of surrounding tissues, recruiting them in response to pathogens and tissue damage. ECs play an active role in the transmigration of immune cells across the vessel wall. We have discovered important differences in the properties of ECs on soft hydrogel substrates of varying stiffness, in comparison to glass. Primary ECs from several human sources were tested; all formed monolayers normally on soft substrates. EC monolayers formed more mature cell-cell junctions on soft substrates, relative to glass, based on increased recruitment of vinculin and F-actin. EC monolayers supported transendothelial migration (TEM) on soft substrates. Immune cells, including peripheral blood lymphocytes (PBLs) and natural killer cells, showed decreasing numbers of paracellular (between-cell) transmigration events with decreasing substrate stiffness, while the number of transcellular (through-cell) events increased for PBLs. Melanoma cancer cells showed increased transmigration with decreased stiffness. Our findings demonstrate that endothelial monolayers respond to the mechanical properties of their surroundings, which can regulate the integrity and function of the monolayer independently from inflammatory signals. Soft hydrogel substrates are a more appropriate and physiological model for tissue environments than hard substrates, with important implications for the experimental analysis of TEM.

  1. Cell-stiffness-induced mechanosignaling - a key driver of leukocyte transendothelial migration.

    PubMed

    Schaefer, Antje; Hordijk, Peter L

    2015-07-01

    The breaching of cellular and structural barriers by migrating cells is a driving factor in development, inflammation and tumor cell metastasis. One of the most extensively studied examples is the extravasation of activated leukocytes across the vascular endothelium, the inner lining of blood vessels. Each step of this leukocyte transendothelial migration (TEM) process is regulated by distinct endothelial adhesion receptors such as the intercellular adhesion molecule 1 (ICAM1). Adherent leukocytes exert force on these receptors, which sense mechanical cues and transform them into localized mechanosignaling in endothelial cells. In turn, the function of the mechanoreceptors is controlled by the stiffness of the endothelial cells and of the underlying substrate representing a positive-feedback loop. In this Commentary, we focus on the mechanotransduction in leukocytes and endothelial cells, which is induced in response to variations in substrate stiffness. Recent studies have described the first key proteins involved in these mechanosensitive events, allowing us to identify common regulatory mechanisms in both cell types. Finally, we discuss how endothelial cell stiffness controls the individual steps in the leukocyte TEM process. We identify endothelial cell stiffness as an important component, in addition to locally presented chemokines and adhesion receptors, which guides leukocytes to sites that permit TEM.

  2. Recapitulation of in vivo-like neutrophil transendothelial migration using a microfluidic platform.

    PubMed

    Wu, Xiaojie; Newbold, Molly A; Haynes, Christy L

    2015-08-07

    Neutrophil transendothelial migration (TEM) is an essential physiological process that regulates the recruitment of neutrophils in response to inflammatory signals. Herein, a versatile hydrogel scaffold is embedded in a microfluidic platform that supports an endothelial cell layer cultured in the vertical direction and highly stable chemical gradients; this construct is employed to mimic the in vivo neutrophil TEM process. We found that the number of neutrophils migrating across the endothelial cell layer is dependent on the presented chemoattractant concentration and the spatial profile of the chemical gradient. Endothelial cells play a critical role in neutrophil TEM by promoting neutrophil morphological changes as well as expressing surface receptor molecules that are indispensable for inducing neutrophil attachment and migration. Furthermore, the microfluidic device also supports competing chemoattractant gradients to facilitate neutrophil TEM studies in complex microenvironments that more accurately model the in vivo system than simplified microenvironments without the complexity of chemical gradients. This work demonstrates that combinations of any two different chemoattractants induce more significant neutrophil migration than a single chemoattractant in the same total amount, indicating synergistic effects between distinct chemoattractants. The in vitro reconstitution of neutrophil TEM successfully translates planar neutrophil movement into in vivo-like neutrophil recruitment and accelerates understanding of cellular interactions between neutrophils and endothelial cells within the complicated physiological milieu.

  3. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells.

    PubMed

    Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

    2016-01-01

    The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb St

  4. Regulation of StAR by the N-terminal Domain and Coinduction of SIK1 and TIS11b/Znf36l1 in Single Cells

    PubMed Central

    Lee, Jinwoo; Tong, Tiegang; Duan, Haichuan; Foong, Yee Hoon; Musaitif, Ibrahim; Yamazaki, Takeshi; Jefcoate, Colin

    2016-01-01

    The cholesterol transfer function of steroidogenic acute regulatory protein (StAR) is uniquely integrated into adrenal cells, with mRNA translation and protein kinase A (PKA) phosphorylation occurring at the mitochondrial outer membrane (OMM). The StAR C-terminal cholesterol-binding domain (CBD) initiates mitochondrial intermembrane contacts to rapidly direct cholesterol to Cyp11a1 in the inner membrane (IMM). The conserved StAR N-terminal regulatory domain (NTD) includes a leader sequence targeting the CBD to OMM complexes that initiate cholesterol transfer. Here, we show how the NTD functions to enhance CBD activity delivers more efficiently from StAR mRNA in adrenal cells, and then how two factors hormonally restrain this process. NTD processing at two conserved sequence sites is selectively affected by StAR PKA phosphorylation. The CBD functions as a receptor to stimulate the OMM/IMM contacts that mediate transfer. The NTD controls the transit time that integrates extramitochondrial StAR effects on cholesterol homeostasis with other mitochondrial functions, including ATP generation, inter-organelle fusion, and the major permeability transition pore in partnership with other OMM proteins. PKA also rapidly induces two additional StAR modulators: salt-inducible kinase 1 (SIK1) and Znf36l1/Tis11b. Induced SIK1 attenuates the activity of CRTC2, a key mediator of StAR transcription and splicing, but only as cAMP levels decline. TIS11b inhibits translation and directs the endonuclease-mediated removal of the 3.5-kb StAR mRNA. Removal of either of these functions individually enhances cAMP-mediated induction of StAR. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA reveals asymmetric transcription at the gene locus and slow RNA splicing that delays mRNA formation, potentially to synchronize with cholesterol import. Adrenal cells may retain slow transcription to integrate with intermembrane NTD activation. HR-FISH resolves individual 3.5-kb St

  5. Inhibitory Effects of Hwangryunhaedok-Tang in 3T3-L1 Adipogenesis by Regulation of Raf/MEK1/ERK1/2 Pathway and PDK1/Akt Phosphorylation

    PubMed Central

    Lee, Ji-Hye; Kim, Dong-Gun; Kim, Taesoo; Lee, Kwang Jin; Ma, Jin Yeul

    2013-01-01

    Hwangryunhaedok-tang (HRT) has been long used as traditional medicine in Asia. However, inhibitory role of HRT is unclear in early stage of 3T3-L1 adipocyte differentiation related to signaling. In the present study, we investigated the inhibitory effects of HRT on upstream signaling of peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-β (C/EBP-β) expression in differentiation of 3T3-L1 preadipocytes. We found that HRT significantly inhibited the adipocyte differentiation by downregulating several adipocyte-specific transcription factors including PPAR-γ, C/EBP-α, and C/EBP-β in 3T3-L1 preadipocytes. Furthermore, we observed that HRT markedly inhibited the differentiation media-mediated phosphorylation of Raf/extracellular mitogen-activated protein kinase 1 (MEK1)/signal-regulated protein kinase 1/2 (ERK1/2) and phosphorylation of phosphoinositide-dependent kinase 1 (PDK1)/Akt. These results indicate that anti-adipogenesis mechanism involves the downregulation of the major transcription factors of adipogenesis including PPAR-γ and C/EBP-α through inhibition of Raf/MEK1/ERK1/2 phosphorylation and PDK1/Akt phosphorylation by HRT. Furthermore, high performance liquid chromatography (HPLC) analysis showed HRT contains active antiobesity constituents such as palmatine, berberine, geniposide, baicalin, baicalein, and wogonin. Taken together, this study suggested that anti-adipogenesis effects of HRT were accounted by downregulation of Raf/MEK1/ERK1/2 pathway and PDK1/Akt pathway during 3T3-L1 adipocyte differentiation. PMID:23762131

  6. RacGAP1-driven focal adhesion formation promotes melanoma transendothelial migration through mediating adherens junction disassembly.

    PubMed

    Zhang, Pu; Bai, Huiyuan; Fu, Changliang; Chen, Feng; Zeng, Panying; Wu, Chengxiang; Ye, Qichao; Dong, Cheng; Song, Yang; Song, Erqun

    2015-03-27

    Melanoma cell migration across vascular endothelial cells is an essential step of tumor metastasis. Here, we provide evidence that RacGAP1, a cytokinesis-related Rho GTPase-activating protein, contributed to this process. Depletion of RacGAP1 with RacGAP1-targeting siRNA or overexpression of RacGAP1 mutant (T249A) attenuated melanoma cell transendothelial migration and concomitant changes of adherens junctions. In addition, RacGAP1 promoted the activations of RhoA, FAK, paxillin and triggered focal adhesion formation and cytoskeletal rearrangement. By overexpressing FAK-related non-kinase (FRNK) in endothelium, we showed that RacGAP1 mediated endothelial barrier function loss and melanoma transmigration in a focal adhesion-dependent manner. These results suggest that endothelial RacGAP1 may play critical roles in pathogenic processes of cancer by regulating endothelial permeability.

  7. PTH regulates myleoid ELF-1-like factor (MEF)-induced MAB-21-like-1 (MAB21L1) expression through the JNK1 pathway.

    PubMed

    Kim, Byung-Gyu; Park, Youn-Je; Libermann, Towia A; Cho, Je-Yoel

    2011-08-01

    Continuous treatment with parathyroid hormone (PTH) or excess endogenous PTH due to primary hyperparathyroidism causes increased bone resorption and, subsequently, decreased bone volume. Our previous studies showed that myeloid Elf-1-like factor (MEF) not only suppresses osteoblast differentiation through inhibition of Runx2 activity and other osteogenesis-related genes but also specifically increases the expression of Mab21, a potential transcriptional repressor of osteoblast differentiation. Here we show that the JNK1 pathway is involved in the MEF-mediated up-regulation of Mab21 expression due to PTH stimulation. PTH increased the transcription level of Mab21 in MG63 human osteoblastic cells, in contrast to the suppressive effect of TGFβ1. PTH phosphorylates serine residues of MEF as well as c-Jun, a known substrate of JNK1. By in vitro kinase assay, we confirmed that MEF is phosphorylated by JNK1, but not by ERK. Co-transfection of MEF with both MKK4 and JNK1 increased the promoter activity of Mab21 in CV1 cells significantly more than MEF alone. We also identified the phosphorylation of MEF serine 641 by in vitro and in vivo JNK1 kinase assays combined with a proteomics approach. In conclusion, our findings indicate that MEF is involved in PTH suppression of osteoblasts through activating the MKK4/JNK1 pathway and subsequently up-regulating Mab21 expression.

  8. Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots.

    PubMed

    Shulman, Ziv; Cohen, Shmuel J; Roediger, Ben; Kalchenko, Vyacheslav; Jain, Rohit; Grabovsky, Valentin; Klein, Eugenia; Shinder, Vera; Stoler-Barak, Liat; Feigelson, Sara W; Meshel, Tsipi; Nurmi, Susanna M; Goldstein, Itamar; Hartley, Olivier; Gahmberg, Carl G; Etzioni, Amos; Weninger, Wolfgang; Ben-Baruch, Adit; Alon, Ronen

    2011-12-04

    Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (T(H)1 cell) and type 1 cytotoxic T cell (T(C)1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide-binding proteins of the G(i) type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration-promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

  9. T-cell responses against CD19+ pediatric acute lymphoblastic leukemia mediated by bispecific T-cell engager (BiTE) are regulated contrarily by PD-L1 and CD80/CD86 on leukemic blasts

    PubMed Central

    Feucht, Judith; Kayser, Simone; Gorodezki, David; Hamieh, Mohamad; Döring, Michaela; Blaeschke, Franziska; Schlegel, Patrick; Bösmüller, Hans; Quintanilla-Fend, Leticia; Ebinger, Martin; Lang, Peter; Handgretinger, Rupert; Feuchtinger, Tobias

    2016-01-01

    T-cell immunotherapies are promising options in relapsed/refractory B-precursor acute lymphoblastic leukemia (ALL). We investigated the effect of co-signaling molecules on T-cell attack against leukemia mediated by CD19/CD3-bispecific T-cell engager. Primary CD19+ ALL blasts (n≥10) and physiologic CD19+CD10+ bone marrow precursors were screened for 20 co-signaling molecules. PD-L1, PD-1, LAG-3, CD40, CD86, CD27, CD70 and HVEM revealed different stimulatory and inhibitory profiles of pediatric ALL compared to physiologic cells, with PD-L1 and CD86 as most prominent inhibitory and stimulatory markers. PD-L1 was increased in relapsed ALL patients (n=11) and in ALLs refractory to Blinatumomab (n=5). Exhaustion markers (PD-1, TIM-3) were significantly higher on patients' T cells compared to physiologic controls. T-cell proliferation and effector function was target-cell dependent and correlated to expression of co-signaling molecules. Blockade of inhibitory PD-1-PD-L and CTLA-4-CD80/86 pathways enhanced T-cell function whereas blockade of co-stimulatory CD28-CD80/86 interaction significantly reduced T-cell function. Combination of Blinatumomab and anti-PD-1 antibody was feasible and induced an anti-leukemic in vivo response in a 12-year-old patient with refractory ALL. In conclusion, ALL cells actively regulate T-cell function by expression of co-signaling molecules and modify efficacy of therapeutic T-cell attack against ALL. Inhibitory interactions of leukemia-induced checkpoint molecules can guide future T-cell therapies. PMID:27708227

  10. The Herbal Medicine KBH-1 Inhibits Fat Accumulation in 3T3-L1 Adipocytes and Reduces High Fat Diet-Induced Obesity through Regulation of the AMPK Pathway

    PubMed Central

    Lee, Ji-Hye; Kim, Taesoo; Lee, Jung-Jin; Lee, Kwang Jin; Kim, Hyun-Kyu; Yun, Bora; Jeon, Jongwook; Kim, Sang Kyum; Ma, Jin Yeul

    2015-01-01

    The aim of this study was to investigate whether a novel formulation of an herbal extract, KBH-1, has an inhibitory effect on obesity. To determine its anti-obesity effects and its underlying mechanism, we performed anti-obesity-related experiments in vitro and in vivo. 3T3-L1 preadipocytes were analyzed for lipid accumulation as well as the protein and gene expression of molecular targets involved in fatty acid synthesis. To determine whether KBH-1 oral administration results in a reduction in high-fat diet (HFD)-induced obesity, we examined five groups (n = 9) of C57BL/6 mice as follows: 10% kcal fat diet-fed mice (ND), 60% kcal fat diet-fed mice (HFD), HFD-fed mice treated with orlistat (tetrahydrolipstatin, marketed under the trade name Xenical), HFD-fed mice treated with 150 mg/kg KBH-1 (KBH-1 150) and HFD-fed mice treated with 300 mg/kg KBH-1 (KBH-1 300). During adipogenesis of 3T3-L1 cells in vitro, KBH-1 significantly reduced lipid accumulation and down-regulated the expression of master adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferation-activity receptor (PPAR) γ, which led to the suppression of the expression of several adipocyte-specific genes and proteins. KBH-1 also markedly phosphorylated the adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, KBH-1-induced the inhibition effect on lipid accumulation and AMPK-mediated signal activation were decreased by blocking AMPK phosphorylation using AMPK siRNA. Furthermore, daily oral administration of KBH-1 resulted in dose-dependent decreases in body weight, fat pad mass and fat tissue size without systemic toxicity. These results suggest that KBH-1 inhibits lipid accumulation by down-regulating the major transcription factors of the adipogenesis pathway by regulating the AMPK pathway in 3T3-L1 adipocytes and in mice with HFD-induced obesity. These results implicate KBH-1, a safe herbal

  11. Prolonged insulin stimulation down-regulates GLUT4 through oxidative stress-mediated retromer inhibition by a protein kinase CK2-dependent mechanism in 3T3-L1 adipocytes.

    PubMed

    Ma, Jinhui; Nakagawa, Yuko; Kojima, Itaru; Shibata, Hiroshi

    2014-01-03

    Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes GLUT4 depletion that is particularly prominent in the insulin-responsive GLUT4 storage compartment. This effect is caused mainly by accelerated lysosomal degradation of GLUT4, although the mechanism is not fully defined. Here we show that insulin acutely induced dissociation of retromer components from the low density microsomal membranes of 3T3-L1 adipocytes that was accompanied by disruption of the interaction of Vps35 with sortilin. This insulin effect was dependent on the activity of protein kinase CK2 but not phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2. Knockdown of Vps26 decreased GLUT4 to a level comparable with that with insulin stimulation for 4 h. Vps35 with a mutation in the CK2 phosphorylation motif (Vps35-S7A) was resistant to insulin-induced dissociation from the low density microsomal membrane, and its overexpression attenuated GLUT4 down-regulation with insulin. Furthermore, insulin-generated hydrogen peroxide was an upstream mediator of the insulin action on retromer and GLUT4. These results suggested that insulin-generated oxidative stress switches the GLUT4 sorting direction to lysosomes through inhibition of the retromer function in a CK2-dependent manner.

  12. Endothelial cell activation promotes foam cell formation by monocytes following transendothelial migration in an in vitro model.

    PubMed

    Westhorpe, Clare L V; Dufour, Eric M; Maisa, Anna; Jaworowski, Anthony; Crowe, Suzanne M; Muller, William A

    2012-10-01

    Foam cells are a pathological feature present at all stages of atherosclerosis. Foam cells develop from monocytes that enter the nascent atheroma and subsequently ingest modified low density lipoproteins (LDL). The regulation of this process has previously been studied in vitro using cultured macrophage fed modified LDL. We used our existing in vitro model of transendothelial migration (TEM) to study this process in a more physiologically relevant setting. In our model, monocytes undergo TEM across a primary endothelial monolayer into an underlying three-dimensional collagen matrix in the presence of 20% human serum. Foam cells were detected by Oil Red O staining for intracellular lipid droplets. We demonstrate that sub-endothelial monocytes can develop into foam cells within 48 h of TEM across TNF-α activated endothelium, in the absence of additional lipids. Our data indicate a role for both monocyte-endothelial interactions and soluble factors in the regulation of foam cell development, including oxidation of LDL in situ from lipid present in culture medium following TNF-α stimulation of the endothelial cells. Our study provides a simple model for investigating foam cell development in vitro that mimics cell migration in vivo, and demonstrates the critical role of inflammation in regulating early atherogenic events.

  13. Endotoxin activation of endothelium for polymorphonuclear leucocyte transendothelial migration and modulation by interferon-gamma.

    PubMed Central

    Issekutz, A C; Lopes, N

    1993-01-01

    Endotoxin [lipopolysaccharide (LPS)] is a potent inflammatory stimulus and can activate human umbilical vein endothelium (HUVE) for leucocyte adhesiveness and transendothelial migration. Here we investigated the role of HUVE-secreted cytokines in this process. When HUVE monolayers were grown on filters and preincubated for 3 hr with LPS, 51Cr-labelled polymorphonuclear leucocytes (PMNL) migrated across the HUVE in a dose- and time-dependent manner. Maximal PMNL transmigration with LPS (1 ng/ml) was 26 +/- 3% of added PMNL in 75 min. Neutralizing antibodies to interleukin-1 alpha (IL-1 alpha) and IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-8 or recombinant IL-1 receptor antagonist had no effect on the activation by LPS of the HUVE for supporting migration of PMNL. The HUVE 'activated state' declined with prolonged (22 hr) exposure to LPS, as reflected by a decrease in PMNL transendothelial migration to 5.5 +/- 1% and in the expression of the endothelial cell adhesion molecule, E-selectin, as compared to stimulation with LPS for 3 hr. However, simultaneous exposure to interferon-gamma (IFN-gamma) (200 IU/ml) and LPS maintained maximal PMNL transendothelial migration (28 +/- 4%) for at least 24 hr, prolonged E-selectin expression by HUVE and superinduced intracellular adhesion molecule-1 (ICAM-1) expression. The PMNL transendothelial migration was blocked by > 90% by monoclonal antibody (mAb) to CD18 with either 3 hr of LPS or 22 hr LPS + IFN-gamma stimulation. Migration was partially inhibited by mAb to E-selectin (30-40%) or to ICAM-1 (35-45%) and by a combination of both reagents (50-60%) under both stimulation conditions. Thus, LPS activation of HUVE for PMNL transendothelial migration: (a) does not require secretion of IL-1, TNF-alpha or IL-8 by the endothelium, (b) IFN-gamma enhances and prolongs endothelial activation by LPS and may increase leucocyte infiltration in LPS or bacterial inflammatory reactions, and (c) CD18-dependent mechanisms are

  14. Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells In vitro and in the SCID mouse-human rheumatoid arthritis model In vivo.

    PubMed Central

    Oppenheimer-Marks, N; Brezinschek, R I; Mohamadzadeh, M; Vita, R; Lipsky, P E

    1998-01-01

    The capacity of endothelial cells (EC) to produce IL-15 and the capacity of IL-15 to influence transendothelial migration of T cells was examined. Human umbilical vein endothelial cells expressed both IL-15 mRNA and protein. Moreover, endothelial-derived IL-15 enhanced transendothelial migration of T cells as evidenced by the inhibition of this process by blocking monoclonal antibodies to IL-15. IL-15 enhanced transendothelial migration of T cells by activating the binding capacity of the integrin adhesion molecule LFA-1 (CD11a/CD18) and also increased T cell motility. In addition, IL-15 induced expression of the early activation molecule CD69. The importance of IL-15 in regulating migration of T cells in vivo was documented by its capacity to enhance accumulation of adoptively transferred human T cells in rheumatoid arthritis synovial tissue engrafted into immune deficient SCID mice. These results demonstrate that EC produce IL-15 and imply that endothelial IL-15 plays a critical role in stimulation of T cells to extravasate into inflammatory tissue. PMID:9502767

  15. ZFP36L1 — EDRN Public Portal

    Cancer.gov

    ZFP36L1 is a member of the TIS11 family of early response genes, which are induced by various agonists such as the phorbol ester TPA and the polypeptide mitogen EGF. The ZFP36L1 gene is well conserved across species and has a promoter that contains motifs seen in other early-response genes. ZFP36L1 contains a putative zinc finger domain with a repeating cys-his motif. ZFP36L1 is believed to be a nuclear transcription factor most likely functioning in regulating the response to growth factors. Different isoforms encoded by alternatively spliced transcript variants have been isolated.

  16. ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

    PubMed Central

    Song, Huiwen; Pu, Jun; Wang, Lin; Wu, Lihua; Xiao, Jianmin; Liu, Qigong; Chen, Jun; Zhang, Min; Liu, Yang; Ni, Mingke; Mo, Jinggang; Zheng, Yunliang; Wan, Deli; Cai, XiongJiu; Cao, Yaping; Xiao, Weiyi; Ye, Lei; Tu, Enyuan; Lin, Zhihai; Wen, Jianxin; Lu, Xiaoling; He, Jian; Peng, Yi; Su, Jing; Zhang, Heng; Zhao, Yongxiang; Lin, Meihua; Zhang, Zhiyong

    2015-01-01

    Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autophagy induction. PMID:26083323

  17. Ubiquitin carboxyl hydrolase L1 significance for human diseases.

    PubMed

    Suong, Dang Ngoc Anh; Thao, Dang Thi Phuong; Masamitsu, Yamaguchi; Thuoc, Tran Linh

    2014-07-01

    Ubiquitin carboxyl hydrolase L1 (UCH-L1) is an abundant multifunctional neuron protein. It plays an important role in maintaining the ubiquitin proteasome system (UPS), vital for recognizing and degrading dysfunctional proteins in organisms. In recent decades, UCH-L1 has been implicated in the pathogenesis of many diseases, including neurodegenerative disorders, cancer and diabetes. However, the mechanisms of UCH-L1 involvement have yet to be revealed in detail. Since UCH-L1 contributes many different functions to cell metabolism, its role and regulation might be more complex than previously thought and it has become a research target in many laboratories. In this review, we summarize recent findings related to the actions of UCH-L1 in several human diseases.

  18. Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

    PubMed Central

    Hurst-Kennedy, Jennifer; Chin, Lih-Shen; Li, Lian

    2012-01-01

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5) is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis. PMID:22811913

  19. Prolonged transendothelial migration of human haematopoietic stem and progenitor cells (HSPCs) towards hydrogel-released SDF1.

    PubMed

    Sobkow, Lidia; Seib, F Philipp; Prodanov, Ljupco; Kurth, Ina; Drichel, Juliane; Bornhäuser, Martin; Werner, Carsten

    2011-08-01

    The therapeutic success of haematopoetic stem and progenitor cell (HSPC) transplantation is critically dependent on HSPC engraftment in the bone marrow. Gradients of stromal cell-derived factor 1 (SDF1) direct HSPC homing, both in vitro and in vivo. Potentially, regulating the delivery levels of exogenous SDF1 applied to the bone marrow could augment HSPC engraftment. Thus, the aim of the present study was to revise the ability of biocompatible hydrogels to direct HSPC migration in vitro. The delivery system of choice is based on heparin cross-linked with collagen1. We confirm that hydrogel is capable of trapping and releasing SDF1 and using it to generate a protein gradient in transendothelial migration experiments. The use of SDF1-functionalised hydrogel to produce a chemokine gradient revealed, sustained and increased HSPC migration when compared to diffusible SDF1 controls. In conclusion, regulating SDF1 gradients with heparin-containing hydrogels may offer valuable options to direct site-specific migration of HSPC.

  20. A versatile assay for monitoring in vivo-like transendothelial migration of neutrophils.

    PubMed

    Han, Sewoon; Yan, Ji-Jing; Shin, Yoojin; Jeon, Jessie J; Won, Jihee; Jeong, Hyo Eun; Kamm, Roger D; Kim, Young-Joon; Chung, Seok

    2012-10-21

    Spatiotemporal analysis of the inflammatory response has been limited by the difficulties of in vivo imaging and reconstitution of inflammation in vitro. Here, we present a novel method for establishing in vivo-like inflammatory models in a microfluidic device and quantitatively measuring the three-dimensional transmigration of neutrophils during the inflammatory process. This enabled us to concurrently characterize transendothelial migration behaviors of neutrophils under the influence of various inflammatory stimuli.

  1. CD99 expressed on human mobilized peripheral blood CD34+ cells is involved in transendothelial migration.

    PubMed

    Imbert, Anne-Marie; Belaaloui, Ghania; Bardin, Florence; Tonnelle, Cecile; Lopez, Marc; Chabannon, Christian

    2006-10-15

    Hematopoietic progenitor cell trafficking is an important phenomenon throughout life. It is thought to occur in sequential steps, similar to what has been described for mature leukocytes. Molecular actors have been identified for each step of leukocyte migration; recently, CD99 was shown to play a part during transendothelial migration. We explored the expression and role of CD99 on human hematopoietic progenitors. We demonstrate that (1) CD34+ cells express CD99, albeit with various intensities; (2) subsets of CD34+ cells with high or low levels of CD99 expression produce different numbers of erythroid, natural killer (NK), or dendritic cells in the in vitro differentiation assays; (3) the level of CD99 expression is related to the ability to differentiate toward B cells; (4) CD34+ cells that migrate through an endothelial monolayer in response to SDF-1alpha and SCF display the highest level of CD99 expression; (5) binding of a neutralizing antibody to CD99 partially inhibits transendothelial migration of CD34+ progenitors in an in vitro assay; and (6) binding of a neutralizing antibody to CD99 reduces homing of CD34+ progenitors xenotransplanted in NOD-SCID mice. We conclude that expression of CD99 on human CD34+ progenitors has functional significance and that CD99 may be involved in transendothelial migration of progenitors.

  2. Increased vesicular and vacuolar transendothelial transport in traumatic human brain oedema. A review.

    PubMed

    Castejón, Orlando J

    2013-01-01

    The endothelial vacuolar and vesicular transports in traumatic human brain oedema have been reviewed and compared with experimental brain oedema in order to establish their role in both oedema formation and oedema resolution. Normal or "non-activated" and "activated" capillaries are found. The activated capillaries showed predominantly an enhanced abluminally orientated vesicular transport by means of small, medium and large uncoated and clathrin coated vesicles, as well as the presence of endothelial tubular structures. Activation of the endothelial nuclear zone is featured by the increased amount of micropinocytotic vesicles. Vesicles internalizing to the hypertrophic Golgi complex, lysosomes and multivesicular bodies are observed. The protein vacuolar transport is predominant in most cortical capillaries. A wide spectrum of endothelial cell mechanisms is observed increasing the vesicular and vacuolar transport, such as deep invaginations of the luminal surface, large coated vesicles, tubular structures, and transient and incomplete transendothelial channels formed either by chained plasmalemmal vesicles or elongated protein-containing vacuoles. Uncoated vesicles are seen surrounding lysosomes. Vesicular transport might be discriminated between abluminally orientated or transendothelial transport (oedema formation) and intraendothelial transport (oedema resolution) directed towards cell lysosomes to be degraded by lysosomal enzymes. The transendothelial passage via large vacuoles is mainly caused by macromolecular protein transport.

  3. Connexin 43 expression on peripheral blood eosinophils: role of gap junctions in transendothelial migration.

    PubMed

    Vliagoftis, Harissios; Ebeling, Cory; Ilarraza, Ramses; Mahmudi-Azer, Salahaddin; Abel, Melanie; Adamko, Darryl; Befus, A Dean; Moqbel, Redwan

    2014-01-01

    Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

  4. Role of cortactin homolog HS1 in transendothelial migration of natural killer cells.

    PubMed

    Mukherjee, Suranjana; Kim, Joanna; Mooren, Olivia L; Shahan, Stefanie T; Cohan, Megan; Cooper, John A

    2015-01-01

    Natural Killer (NK) cells perform many functions that depend on actin assembly, including adhesion, chemotaxis, lytic synapse assembly and cytolysis. HS1, the hematopoietic homolog of cortactin, binds to Arp2/3 complex and promotes actin assembly by helping to form and stabilize actin filament branches. We investigated the role of HS1 in transendothelial migration (TEM) by NK cells. Depletion of HS1 led to a decrease in the efficiency of TEM by NK cells, as measured by transwell assays with endothelial cell monolayers on porous filters. Transwell assays involve chemotaxis of NK cells across the filter, so to examine TEM more specifically, we imaged live-cell preparations and antibody-stained fixed preparations, with and without the chemoattractant SDF-1α. We found small to moderate effects of HS1 depletion on TEM, including whether the NK cells migrated via the transcellular or paracellular route. Expression of HS1 mutants indicated that phosphorylation of HS1 tyrosines at positions 222, 378 and 397 was required for rescue in the transwell assay, but HS1 mutations affecting interaction with Arp2/3 complex or SH3-domain ligands had no effect. The GEF Vav1, a ligand of HS1 phosphotyrosine, influenced NK cell transendothelial migration. HS1 and Vav1 also affected the speed of NK cells migrating across the surface of the endothelium. We conclude that HS1 has a role in transendothelial migration of NK cells and that HS1 tyrosine phosphorylation may signal through Vav1.

  5. Novel three-dimensional Boyden chamber system for studying transendothelial transport.

    PubMed

    Hebeiss, I; Truckenmüller, R; Giselbrecht, S; Schepers, U

    2012-02-21

    The rapid development in combinatorial chemistry of millions of novel potential drug candidates requires in vitro devices for reliable testing of their transendothelial transport and the uptake in specific cells. To date, this is often achieved in vitro by the use of regular planar Boyden chambers, which are not reflecting the three dimensionality of the blood vessel. This technical note describes the fabrication and biological validation of a novel three-dimensional Boyden chamber system for studying transendothelial transport. The key element of this new system is a porous thin-walled microchannel produced by a SMART (substrate modification and replication by thermoforming) process comprising a combination of microthermoforming and ion track technology. The membrane-like microstructure offers the opportunity to grow endothelial cells on the inner side of the channel resembling a more natural curved organization of vessels. After establishment of a confluent HUVECs layer in the porous microchannel this novel Boyden chamber was successfully applied to study the transendothelial transport of a polycationic cell penetrating peptoid through the 3D- or curved endothelial cell layer. Thus, this system will enable the investigation of such synthetic compounds as drug delivery systems with regard to their bioavailability and functionality under organotypic conditions.

  6. Pr77 and L1TcRz

    PubMed Central

    Sánchez-Luque, Francisco; López, Manuel C.; Macias, Francisco; Alonso, Carlos; Thomas, M. Carmen

    2012-01-01

    The sequence corresponding to the first 77 nucleotides of the L1Tc and NARTc non-LTR retrotransposons from Trypanosoma cruzi is an internal promoter (Pr77) that generates abundant, although poorly translatable, un-spliced transcripts. It has been recently described that L1TcRz, an HDV-like ribozyme, resides within the 5′-end of the RNA from the L1Tc and NARTc retrotransposons. Remarkably, the same first 77 nucleotides of L1Tc/NARTc elements comprise both the Pr77 internal promoter and the HDV-like L1TcRz. The L1TcRz cleaves on the 5′-side of the +1 nucleotide of the L1Tc element insuring that the promoter and the ribozyme functions travel with the transposon during retrotransposition. The ribozyme activity would prevent the mobilization of upstream sequences and insure the individuality of the L1Tc/NARTc copies transcribed from associated tandems. The Pr77/L1TcRz sequence is also found in other trypanosomatid’s non-LTR retrotransposons and degenerated retroposons. The possible conservation of the ribozyme activity in a widely degenerated retrotransposon, as the Leishmania SIDERs, could indicate that the presence of this element and the catalytic activity could play some favorable genetic regulation. The functional implications of the Pr77/L1TcRz dual system in the regulation of the L1Tc/NARTc retrotransposons and in the gene expression of trypanosomatids are also discussed in this paper. PMID:22754746

  7. The RhoA GEF, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration

    PubMed Central

    Lessey-Morillon, Elizabeth C.; Osborne, Lukas D.; Monaghan-Benson, Elizabeth; Guilluy, Christophe; O’Brien, E. Timothy; Superfine, Richard; Burridge, Keith

    2014-01-01

    RhoA-mediated cytoskeletal rearrangements in endothelial cells (ECs) play an active role in leukocyte transendothelial cell migration (TEM), a normal physiological process in which leukocytes cross the endothelium to enter the underlying tissue. While much has been learned about RhoA signaling pathways downstream from ICAM-1 in ECs, little is known about the consequences of the tractional forces that leukocytes generate on ECs as they migrate over the surface before TEM. We have found that after applying mechanical forces to ICAM-1 clusters, there is an increase in cellular stiffening and enhanced RhoA signaling compared to ICAM-1 clustering alone. We have identified that the RhoA GEF LARG/ARHGEF12 acts downstream of clustered ICAM-1 to increase RhoA activity and that this pathway is further enhanced by mechanical force on ICAM-1. Depletion of LARG decreases leukocyte crawling and inhibits TEM. This is the first report of endothelial LARG regulating leukocyte behavior and EC stiffening in response to tractional forces generated by leukocytes. PMID:24585879

  8. Urokinase type plasminogen activator mediates Interleukin-17-induced peripheral blood mesenchymal stem cell motility and transendothelial migration.

    PubMed

    Krstić, Jelena; Obradović, Hristina; Jauković, Aleksandra; Okić-Đorđević, Ivana; Trivanović, Drenka; Kukolj, Tamara; Mojsilović, Slavko; Ilić, Vesna; Santibañez, Juan F; Bugarski, Diana

    2015-02-01

    Mesenchymal stem cells (MSCs) have the potential to migrate toward damaged tissues increasing tissue regeneration. Interleukin-17 (IL-17) is a proinflammatory cytokine with pleiotropic effects associated with many inflammatory diseases. Although IL-17 can modulate MSC functions, its capacity to regulate MSC migration is not well elucidated so far. Here, we studied the role of IL-17 on peripheral blood (PB) derived MSC migration and transmigration across endothelial cells. IL-17 increased PB-MSC migration in a wound healing assay as well as cell mobilization from collagen gel. Concomitantly IL-17 induced the expression of urokinase type plasminogen activator (uPA) without affecting matrix metalloproteinase expression. The incremented uPA expression mediated the capacity of IL-17 to enhance PB-MSC migration in a ERK1,2 MAPK dependent way. Also, IL-17 induced PB-MSC migration alongside with changes in cell polarization and uPA localization in cell protrusions. Moreover, IL-17 increased PB-MSC adhesion to endothelial cells and transendothelial migration, as well as increased the capacity of PB-MSC adhesion to fibronectin, in an uPA-dependent fashion. Therefore, our data suggested that IL-17 may act as chemotropic factor for PB-MSCs by incrementing cell motility and uPA expression during inflammation development.

  9. Mena invasive (MenaINV) promotes multicellular streaming motility and transendothelial migration in a mouse model of breast cancer.

    PubMed

    Roussos, Evanthia T; Balsamo, Michele; Alford, Shannon K; Wyckoff, Jeffrey B; Gligorijevic, Bojana; Wang, Yarong; Pozzuto, Maria; Stobezki, Robert; Goswami, Sumanta; Segall, Jeffrey E; Lauffenburger, Douglas A; Bresnick, Anne R; Gertler, Frank B; Condeelis, John S

    2011-07-01

    We have shown previously that distinct Mena isoforms are expressed in invasive and migratory tumor cells in vivo and that the invasion isoform (Mena(INV)) potentiates carcinoma cell metastasis in murine models of breast cancer. However, the specific step of metastatic progression affected by this isoform and the effects on metastasis of the Mena11a isoform, expressed in primary tumor cells, are largely unknown. Here, we provide evidence that elevated Mena(INV) increases coordinated streaming motility, and enhances transendothelial migration and intravasation of tumor cells. We demonstrate that promotion of these early stages of metastasis by Mena(INV) is dependent on a macrophage-tumor cell paracrine loop. Our studies also show that increased Mena11a expression correlates with decreased expression of colony-stimulating factor 1 and a dramatically decreased ability to participate in paracrine-mediated invasion and intravasation. Our results illustrate the importance of paracrine-mediated cell streaming and intravasation on tumor cell dissemination, and demonstrate that the relative abundance of Mena(INV) and Mena11a helps to regulate these key stages of metastatic progression in breast cancer cells.

  10. Invasive breast carcinoma cells from patients exhibit MenaINV- and macrophage-dependent transendothelial migration.

    PubMed

    Pignatelli, Jeanine; Goswami, Sumanta; Jones, Joan G; Rohan, Thomas E; Pieri, Evan; Chen, Xiaoming; Adler, Esther; Cox, Dianne; Maleki, Sara; Bresnick, Anne; Gertler, Frank B; Condeelis, John S; Oktay, Maja H

    2014-11-25

    Metastasis is a complex, multistep process of cancer progression that has few treatment options. A critical event is the invasion of cancer cells into blood vessels (intravasation), through which cancer cells disseminate to distant organs. Breast cancer cells with increased abundance of Mena [an epidermal growth factor (EGF)-responsive cell migration protein] are present with macrophages at sites of intravasation, called TMEM sites (for tumor microenvironment of metastasis), in patient tumor samples. Furthermore, the density of these intravasation sites correlates with metastatic risk in patients. We found that intravasation of breast cancer cells may be prevented by blocking the signaling between cancer cells and macrophages. We obtained invasive breast ductal carcinoma cells of various subtypes by fine-needle aspiration (FNA) biopsies from patients and found that, in an in vitro transendothelial migration assay, cells that migrated through a layer of human endothelial cells were enriched for the transcript encoding Mena(INV), an invasive isoform of Mena. This enhanced transendothelial migration required macrophages and occurred with all of the breast cancer subtypes. Using mouse macrophages and the human cancer cells from the FNAs, we identified paracrine and autocrine activation of colony-stimulating factor-1 receptor (CSF-1R). The paracrine or autocrine nature of the signal depended on the breast cancer cell subtype. Knocking down Mena(INV) or adding an antibody that blocks CSF-1R function prevented transendothelial migration. Our findings indicate that Mena(INV) and TMEM frequency are correlated prognostic markers and CSF-1 and Mena(INV) may be therapeutic targets to prevent metastasis of multiple breast cancer subtypes.

  11. Tonsil-derived mesenchymal stem cells (T-MSCs) prevent Th17-mediated autoimmune response via regulation of the programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway.

    PubMed

    Kim, Ji-Yon; Park, Minhwa; Kim, Yu-Hee; Ryu, Kyung-Ha; Lee, Kyung Ho; Cho, Kyung-Ah; Woo, So-Youn

    2017-01-20

    Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17 dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil-derived MSCs (T-MSCs) constitutively express both the membrane-bound and soluble forms of programmed death-ligand 1 (PD-L1), which enables T-MSCs to be distinguished from MSCs originating from other organs (i.e., bone marrow or adipose tissue). We also found that T-MSC-derived PD-L1 effectively represses Th17 differentiation via both cell-to-cell contact and a paracrine effect. Further, T-MSCs increase PD-1 expression on T cells by secreting IFN-β, which may enhance engagement with PD-L1. Finally, transplantation of T-MSCs into imiquimod induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD-L1 dependent manner. This study suggests that T-MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features.

  12. Characterization of L1-Ribonucleoprotein Particles

    PubMed Central

    Taylor, Martin S.; LaCava, John; Dai, Lixin; Mita, Paolo; Burns, Kathleen H.; Rout, Michael P.; Boeke, Jef D.

    2016-01-01

    The LINE-1 retrotransposon (L1) encodes two proteins, ORF1p and ORF2p, which bind to the L1 RNA in cis, forming a ribonucleoprotein (RNP) complex that is critical for retrotransposition. Interactions with both permissive and repressive host factors pervade every step of the L1 life cycle. Until recently, limitations in detection and production precluded in-depth characterization of L1 RNPs. Inducible expression and recombinant engineering of epitope tags have made detection of both L1 ORFs routine. Here, we describe large-scale production of L1-expressing HEK-293T cells in suspension cell culture, cryomilling and affinity capture of L1 RNP complexes, sample preparation for analysis by mass spectrometry, and assay using the L1 element amplification protocol (LEAP) and qRT-PCR. PMID:26895062

  13. Characterization of L1-Ribonucleoprotein Particles.

    PubMed

    Taylor, Martin S; LaCava, John; Dai, Lixin; Mita, Paolo; Burns, Kathleen H; Rout, Michael P; Boeke, Jef D

    2016-01-01

    The LINE-1 retrotransposon (L1) encodes two proteins, ORF1p and ORF2p, which bind to the L1 RNA in cis, forming a ribonucleoprotein (RNP) complex that is critical for retrotransposition. Interactions with both permissive and repressive host factors pervade every step of the L1 life cycle. Until recently, limitations in detection and production precluded in-depth characterization of L1 RNPs. Inducible expression and recombinant engineering of epitope tags have made detection of both L1 ORFs routine. Here, we describe large-scale production of L1-expressing HEK-293T cells in suspension cell culture, cryomilling and affinity capture of L1 RNP complexes, sample preparation for analysis by mass spectrometry, and assay using the L1 element amplification protocol (LEAP) and qRT-PCR.

  14. Anion channels, including ClC-3, are required for normal neutrophil oxidative function, phagocytosis, and transendothelial migration.

    PubMed

    Moreland, Jessica G; Davis, A Paige; Bailey, Gail; Nauseef, William M; Lamb, Fred S

    2006-05-05

    NADPH oxidase activity, phagocytosis, and cell migration are essential functions of polymorphonuclear leukocytes (PMNs) in host defense. The cytoskeletal reorganization necessary to perform these functions has been extensively studied, but the role of cell volume regulation, which is likely dependent upon anion channels, has not been defined. Mice lacking the anion channel ClC-3 (Clcn3(-/-)) died from presumed sepsis following intravascular catheter placement, whereas Clcn3(+/+) littermates survived. We hypothesized that ClC-3 has a critical role in host defense and reasoned that PMN function would be compromised in these mice. Clcn3(-/-) PMNs displayed markedly reduced NADPH oxidase activity in response to opsonized zymosan and modestly reduced activity after phorbol 12-myristate 13-acetate. Human PMNs treated with the anion channel inhibitors niflumic acid or 5-nitro-2-(3-phenylpropylamino)benzoic acid had a very similar defect. ClC-3 protein was detected in the secretory vesicles and secondary granules of resting PMNs and was up-regulated to the phagosomal membrane. Clcn3(-/-) PMNs and human PMNs lacking normal anion channel function both exhibited reduced uptake of opsonized zymosan at 1, 5, and 10 min in a synchronized phagocytosis assay. Niflumic acid-treated PMNs also had impaired transendothelial migration in vitro, whereas migration in vivo was not altered in Clcn3(-/-) PMNs. Selective inhibition of the swelling-activated chloride channel with tamoxifen profoundly reduced PMN migration but had no effect on NADPH oxidase activity. In summary, PMNs lacking normal anion channel function exhibited reduced NADPH oxidase activity, diminished phagocytosis, and impaired migration. ClC-3 was specifically involved in the respiratory burst and phagocytosis.

  15. DSCOVR_EPIC_L1A

    Atmospheric Science Data Center

    2017-01-04

    DSCOVR_EPIC_L1A Full sun-light Earth images calibrated with ... 551NM 680NM 688NM 764NM 780NM DSCOVR EPIC IMAGERY L1B LAGRANGE Order Data:  Earthdata ... Guide Documents:  DSCOVR Overview EPIC Data Format Control Book DSCOVR EPIC L0L1a Processor V01 ...

  16. Characterization of Engineered L1 Retrotransposition Events: The Recovery Method.

    PubMed

    Cano, David; Morell, Santiago; Pulgarin, Andres J; Amador, Suyapa; Garcia-Pérez, Jose L

    2016-01-01

    Long Interspersed Element class 1 retrotransposons (LINE-1 or L1) are abundant Transposable Elements in mammalian genomes and their mobility continues to impact the human genome. The development of engineered retrotransposition assays has been instrumental to understand how these elements are regulated and to identify domains involved in the process of retrotransposition. Additionally, the modification of a retrotransposition indicator cassette has allowed developing straightforward approaches to characterize the site of new L1 insertions in cultured cells. In this chapter, we describe a method termed "L1-recovery" that has been used to characterize the site of insertion on engineered L1 retrotransposition events in cultured mammalian cells. Notably, the recovery assay is based on a genetic strategy and avoids the use of PCR and thus reduces to a minimum the appearance of false positives/artifacts.

  17. 17 CFR 275.203(l)-1 - Venture capital fund defined.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... 275.203(l)-1 Section 275.203(l)-1 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) RULES AND REGULATIONS, INVESTMENT ADVISERS ACT OF 1940 § 275.203(l)-1 Venture capital fund defined. (a) Venture capital fund defined. For purposes of section 203(l) of the Act (15...

  18. L1 Retrotransposition in Neural Progenitor Cells.

    PubMed

    Muotri, Alysson R

    2016-01-01

    Long interspersed nucleotide element 1 (LINE-1 or L1) is a family of non-LTR retrotransposons that can replicate and reintegrate into the host genome. L1s have considerably influenced mammalian genome evolution by retrotransposing during germ cell development or early embryogenesis, leading to massive genome expansion. For many years, L1 retrotransposons were viewed as a selfish DNA parasite that had no contribution in somatic cells. Historically, L1s were thought to only retrotranspose during gametogenesis and in neoplastic processes, but recent studies have shown that L1s are extremely active in the mouse, rat, and human neuronal progenitor cells (NPCs). These de novo L1 insertions can impact neuronal transcriptional expression, creating unique transcriptomes of individual neurons, possibly contributing to the uniqueness of the individual cognition and mental disorders in humans.

  19. The Pleiotropic Role of L1CAM in Tumor Vasculature

    PubMed Central

    Angiolini, Francesca; Cavallaro, Ugo

    2017-01-01

    Angiogenesis, the formation of new vessels, is a key step in the development, invasion, and dissemination of solid tumors and, therefore, represents a viable target in the context of antitumor therapy. Indeed, antiangiogenic approaches have given promising results in preclinical models and entered the clinical practice. However, in patients, the results obtained so far with antiangiogenic drugs have not completely fulfilled expectations, especially because their effect has been transient with tumors developing resistance and evasion mechanisms. A better understanding of the mechanisms that underlie tumor vascularization and the functional regulation of cancer vessels is a prerequisite for the development of novel and alternative antiangiogenic treatments. The L1 cell adhesion molecule (L1CAM), a cell surface glycoprotein previously implicated in the development and plasticity of the nervous system, is aberrantly expressed in the vasculature of various cancer types. L1CAM plays multiple pro-angiogenic roles in the endothelial cells of tumor-associated vessels, thus emerging as a potential therapeutic target. In addition, L1CAM prevents the maturation of cancer vasculature and its inhibition promotes vessel normalization, a process that is thought to improve the therapeutic response of tumors to cytotoxic drugs. We here provide an overview on tumor angiogenesis and antiangiogenic therapies and summarize the current knowledge on the biological role of L1CAM in cancer vasculature. Finally, we highlight the clinical implications of targeting L1CAM as a novel antiangiogenic and vessel-normalizing approach. PMID:28134764

  20. L1 Splines with Locally Computed Coefficients

    DTIC Science & Technology

    2013-01-01

    Fang. Univariate Cubic L1 Interpolating Splines : Analytical Results for Linearity, Convexity and Oscillation on 5-PointWindows, Algorithms, (07 2010...0. doi: 10.3390/a3030276 07/21/2011 2.00 Lu Yu, Qingwei Jin, John E. Lavery, Shu-Cherng Fang. Univariate Cubic L1 Interpolating Splines : Spline ...Qingwei Jin, Lu Yu, John E. Lavery, Shu-Cherng Fang. Univariate cubic L1 interpolating splines basedon the first derivative and on 5-point windows

  1. Rocking adhesion assay system to study adhesion and transendothelial migration of cancer cells.

    PubMed

    Bapu, Deepashree; Khadim, Munira; Brooks, Susan A

    2014-01-01

    Adhesion of metastatic cancer cells to the vascular endothelium of the target organs and their subsequent transendothelial migration is one of the critical, yet poorly understood, steps of the metastatic cascade. Conventionally, the mechanisms of this complex process have been studied using static adhesion systems or flow assay systems. Static assay systems are easy to set up and perform but do not mimic the physiological conditions of blood flow. Flow assays closely mimic physiological conditions of flow but are time consuming and require specialist equipment. In this chapter we describe the rocking adhesion system which incorporates the key advantages of both the static and flow assay systems and not only is easy to set up and perform but also mimics conditions of blood flow.

  2. Pivotal role for skin transendothelial radio-resistant anti-inflammatory macrophages in tissue repair

    PubMed Central

    Barreiro, Olga; Cibrian, Danay; Clemente, Cristina; Alvarez, David; Moreno, Vanessa; Valiente, Íñigo; Bernad, Antonio; Vestweber, Dietmar; Arroyo, Alicia G; Martín, Pilar; von Andrian, Ulrich H; Sánchez Madrid, Francisco

    2016-01-01

    Heterogeneity and functional specialization among skin-resident macrophages are incompletely understood. In this study, we describe a novel subset of murine dermal perivascular macrophages that extend protrusions across the endothelial junctions in steady-state and capture blood-borne macromolecules. Unlike other skin-resident macrophages that are reconstituted by bone marrow-derived progenitors after a genotoxic insult, these cells are replenished by an extramedullary radio-resistant and UV-sensitive Bmi1+ progenitor. Furthermore, they possess a distinctive anti-inflammatory transcriptional profile, which cannot be polarized under inflammatory conditions, and are involved in repair and remodeling functions for which other skin-resident macrophages appear dispensable. Based on all their properties, we define these macrophages as Skin Transendothelial Radio-resistant Anti-inflammatory Macrophages (STREAM) and postulate that their preservation is important for skin homeostasis. DOI: http://dx.doi.org/10.7554/eLife.15251.001 PMID:27304075

  3. Live Brugia malayi microfilariae inhibit transendothelial migration of neutrophils and monocytes.

    PubMed

    Schroeder, Jan-Hendrik; Simbi, Bigboy H; Ford, Louise; Cole, Sara R; Taylor, Mark J; Lawson, Charlotte; Lawrence, Rachel A

    2012-01-01

    Lymphatic filariasis is a major tropical disease caused by the parasite Brugia malayi. Microfilariae (Mf) circulate in the peripheral blood for 2-3 hours in synchronisation with maximal feeding of the mosquito vector. When absent from the peripheral blood, Mf sequester in the capillaries of the lungs. Mf are therefore in close contact with vascular endothelial cells (EC) and may induce EC immune function and/or wound repair mechanisms such as angiogenesis. In this study, Mf were co-cultured with human umbilical vein EC (HUVEC) or human lung microvascular EC (HLMVEC) and the transendothelial migration of leukocyte subsets was analysed. In addition, the protein and/or mRNA expression of chemokine, cytokine and angiogenic mediators in endothelial cells in the presence of live microfilariae were measured by a combination of cDNA arrays, protein arrays, ELISA and fluorescence antibody tests.Surprisingly, our findings indicate that Mf presence partially blocked transendothelial migration of monocytes and neutrophils, but not lymphocytes. However, Mf exposure did not result in altered vascular EC expression of key mediators of the tethering stage of extravasation, such as ICAM-1, VCAM-1 and various chemokines. To further analyse the immunological function of vascular EC in the presence of Mf, we measured the mRNA and/or protein expression of a number of pro-inflammatory mediators. We found that expression levels of the mediators tested were predominantly unaltered upon B. malayi Mf exposure. In addition, a comparison of angiogenic mediators induced by intact Mf and Wolbachia-depleted Mf revealed that even intact Mf induce the expression of remarkably few angiogenic mediators in vascular EC. Our study suggests that live microfilariae are remarkably inert in their induction and/or activation of vascular cells in their immediate local environment. Overall, this work presents important insights into the immunological function of the vascular endothelium during an infection

  4. DSCOVR_EPIC_L1B

    Atmospheric Science Data Center

    2017-01-04

    DSCOVR_EPIC_L1B Full sun-light Earth images, georectified to the same ... 551NM 680NM 688NM 764NM 780NM DSCOVR EPIC IMAGERY L1B LAGRANGE Order Data:  Earthdata ...   Order Data Readme Files:  EPIC Data Format Control Book SCAR-B Block:  ...

  5. The RGD integrin binding site in human L1-CAM is important for nuclear signaling

    SciTech Connect

    Gast, Daniela; Riedle, Svenja; Kiefel, Helena; Mueerkoester, Susanne Sebens; Schaefer, Heiner; Schaefer, Michael K.E.; Altevogt, Peter

    2008-08-01

    L1 cell adhesion molecule (L1-CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system. L1 is also overexpressed in a variety of human carcinomas and is associated with bad prognosis. In carcinoma cell lines L1 augments cell motility and metastasis, tumor growth in nude mice and induces expression of L1-dependent genes. It is not known whether L1-signaling requires ligand binding. The RGD motif in the sixth Ig domain of L1 is a binding site for integrins. In the present study we analyzed the role of RGDs in L1-signaling using site-directed mutagenesis combined with antibody blocking studies. We observed that L1-RGE expressing HEK293 cells showed reduced cell-cell binding, cell motility, invasiveness and tumor growth in NOD/SCID mice. The RGE-mutation impaired L1-dependent gene regulation and antibodies to {alpha}v{beta}5 integrin had similar effects. Mutant L1 was unable to translocate to the nucleus. Our findings highlight the importance of the RGD site in L1 for human tumors and suggest that nuclear signaling of L1 is dependent on integrins.

  6. Environmental Influence on L1 Retrotransposons in the Adult Hippocampus

    PubMed Central

    Muotri, Alysson R.; Zhao, Chunmei; Marchetto, Maria C.N.; Gage, Fred H.

    2009-01-01

    It is well established that neuronal circuits can be shaped by experience. Neuronal plasticity can be achieved by synaptic competitive interactions and the addition of new neuronal units in neurogenic regions of the adult brain. Recent data have suggested that neuronal progenitor cells can accommodate somatic LINE-1 (Long Interspersed Nuclear Elements-1 or L1) retrotransposition. Genomic L1 insertions may up- or down-regulate transcriptional control of gene expression. Here, we show that exercise has a positive effect on a L1-EGFP reporter in vivo. We found that neurons from mice that experience voluntary exercise are more likely to activate an EGFP reporter marker, representing L1 insertions in the brain, when compared with sedentary animals. In the hippocampus, a neurogenic region of the adult brain, EGFP expression is mainly found in cells localized in the subgranular layer of the dentate gyrus. This observation implies that neuronal progenitor cells may support de novo retrotransposition upon exposure to a new environment. Such evidence suggests that experience-dependent L1 retrotransposition may contribute to the physiological consequences of neuronal plasticity. PMID:19771587

  7. Curcuma longa polyphenols improve insulin-mediated lipid accumulation and attenuate proinflammatory response of 3T3-L1 adipose cells during oxidative stress through regulation of key adipokines and antioxidant enzymes.

    PubMed

    Septembre-Malaterre, Axelle; Le Sage, Fanny; Hatia, Sarah; Catan, Aurélie; Janci, Laurent; Gonthier, Marie-Paule

    2016-07-08

    Plant polyphenols may exert beneficial action against obesity-related oxidative stress and inflammation which promote insulin resistance. This study evaluated the effect of polyphenols extracted from French Curcuma longa on 3T3-L1 adipose cells exposed to H2 O2 -mediated oxidative stress. We found that Curcuma longa extract exhibited high amounts of curcuminoids identified as curcumin, demethoxycurcumin, and bisdemethoxycurcumin, which exerted free radical-scavenging activities. Curcuma longa polyphenols improved insulin-mediated lipid accumulation and upregulated peroxisome proliferator-activated receptor-gamma gene expression and adiponectin secretion which decreased in H2 O2 -treated cells. Curcuminoids attenuated H2 O2 -enhanced production of pro-inflammatory molecules such as interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, and nuclear factor κappa B. Moreover, they reduced intracellular levels of reactive oxygen species elevated by H2 O2 and modulated the expression of genes encoding superoxide dismutase and catalase antioxidant enzymes. Collectively, these findings highlight that Curcuma longa polyphenols protect adipose cells against oxidative stress and may improve obesity-related metabolic disorders. © 2016 BioFactors, 42(4):418-430, 2016.

  8. DSCOVR_NISTAR_L1A

    Atmospheric Science Data Center

    2017-01-04

    ... Raw and calibrated radiometer science and engineering data. Project Title:  DSCOVR Discipline:  ... Level:  L1 Platform:  DEEP SPACE CLIMATE OBSERVATORY Instrument:  PHOTODIODE RADIOMETER ...

  9. L1 libration point manned space habitat

    NASA Technical Reports Server (NTRS)

    Luttges, Marvin; Johnson, Steve; Banks, Gary; Johnson, Richard; Meyer, Christian; Pepin, Scott; Macelroy, Robert

    1989-01-01

    Second generation stations or Manned Space Habitats (MSHs) are discussed for an Earth-Moon libration point and in lunar orbit. The conceptual design of such a station is outlined. Systems and subsystems described reflect anticipation of moderate technology growth. The evolution of the L1 environments is discussed, several selected subsystems are outlined, and how the L1 MSH will complete some of its activities is described.

  10. Description of the L1C signal

    USGS Publications Warehouse

    Betz, J.W.; Blanco, M.A.; Cahn, C.R.; Dafesh, P.A.; Hegarty, C.J.; Hudnut, K.W.; Kasemsri, V.; Keegan, R.; Kovach, K.; Lenahan, L.S.; Ma, H.H.; Rushanan, J.J.; Sklar, D.; Stansell, T.A.; Wang, C.C.; Yi, S.K.

    2006-01-01

    Detailed design of the modernized LI civil signal (L1C) signal has been completed, and the resulting draft Interface Specification IS-GPS-800 was released in Spring 2006. The novel characteristics of the optimized L1C signal design provide advanced capabilities while offering to receiver designers considerable flexibility in how to use these capabilities. L1C provides a number of advanced features, including: 75% of power in a pilot component for enhanced signal tracking, advanced Weilbased spreading codes, an overlay code on the pilot that provides data message synchronization, support for improved reading of clock and ephemeris by combining message symbols across messages, advanced forward error control coding, and data symbol interleaving to combat fading. The resulting design offers receiver designers the opportunity to obtain unmatched performance in many ways. This paper describes the design of L1C. A summary of LIC's background and history is provided. The signal description then proceeds with the overall signal structure consisting of a pilot component and a carrier component. The new L1C spreading code family is described, along with the logic used for generating these spreading codes. Overlay codes on the pilot channel are also described, as is the logic used for generating the overlay codes. Spreading modulation characteristics are summarized. The data message structure is also presented, showing the format for providing time, ephemeris, and system data to users, along with features that enable receivers to perform code combining. Encoding of rapidly changing time bits is described, as are the Low Density Parity Check codes used for forward error control of slowly changing time bits, clock, ephemeris, and system data. The structure of the interleaver is also presented. A summary of L 1C's unique features and their benefits is provided, along with a discussion of the plan for L1C implementation.

  11. L1C signal design options

    USGS Publications Warehouse

    Betz, J.W.; Cahn, C.R.; Dafesh, P.A.; Hegarty, C.J.; Hudnut, K.W.; Jones, A.J.; Keegan, R.; Kovach, K.; Lenahan, L.S.; Ma, H.H.; Rushanan, J.J.; Stansell, T.A.; Wang, C.C.; Yi, S.K.

    2006-01-01

    Design activities for a new civil signal centered at 1575.42 MHz, called L1C, began in 2003, and the Phase 1 effort was completed in 2004. The L1C signal design has evolved and matured during a Phase 2 design activity that began in 2005. Phase 2 has built on the initial design activity, guided by responses to international user surveys conducted during Phase 1. A common core of signal characteristics has been developed to provide advances in robustness and performance. The Phase 2 activity produced five design options, all drawing upon the core signal characteristics, while representing different blends of characteristics and capabilities. A second round of international user surveys was completed to solicit advice concerning these design options. This paper provides an update of the L1C design process, and describes the current L1C design options. Initial performance estimates are presented for each design option, displaying trades between signal tracking robustness, the speed and robustness of clock and ephemeris data, and the rate and robustness of other data message contents. Planned remaining activities are summarized, leading to optimization of the L1C design.

  12. Face recognition with L1-norm subspaces

    NASA Astrophysics Data System (ADS)

    Maritato, Federica; Liu, Ying; Colonnese, Stefania; Pados, Dimitris A.

    2016-05-01

    We consider the problem of representing individual faces by maximum L1-norm projection subspaces calculated from available face-image ensembles. In contrast to conventional L2-norm subspaces, L1-norm subspaces are seen to offer significant robustness to image variations, disturbances, and rank selection. Face recognition becomes then the problem of associating a new unknown face image to the "closest," in some sense, L1 subspace in the database. In this work, we also introduce the concept of adaptively allocating the available number of principal components to different face image classes, subject to a given total number/budget of principal components. Experimental studies included in this paper illustrate and support the theoretical developments.

  13. miR-128 represses L1 retrotransposition by binding directly to L1 RNA.

    PubMed

    Hamdorf, Matthias; Idica, Adam; Zisoulis, Dimitrios G; Gamelin, Lindsay; Martin, Charles; Sanders, Katie J; Pedersen, Irene M

    2015-10-01

    Long interspersed element 1 (LINE-1 or L1) retrotransposons compose 17% of the human genome. Active L1 elements are capable of replicative transposition (mobilization) and can act as drivers of genetic diversity. However, this mobilization is mutagenic and may be detrimental to the host, and therefore it is under strict control. Somatic cells usually silence L1 activity by DNA methylation of the L1 promoter. In hypomethylated cells, such as cancer cells and induced pluripotent stem cells (iPSCs), a window of opportunity for L1 reactivation emerges, and with it comes an increased risk of genomic instability and tumorigenesis. Here we show that miR-128 represses new retrotransposition events in human cancer cells and iPSCs by binding directly to L1 RNA. Thus, we have identified and characterized a new function of microRNAs: mediating genomic stability by suppressing the mobility of endogenous retrotransposons.

  14. Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo

    PubMed Central

    Colom, Bartomeu; Bodkin, Jennifer V.; Beyrau, Martina; Woodfin, Abigail; Ody, Christiane; Rourke, Claire; Chavakis, Triantafyllos; Brohi, Karim; Imhof, Beat A.; Nourshargh, Sussan

    2015-01-01

    Summary Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic. PMID:26047922

  15. Electrical Monitoring Cytotoxic Effect of Cigarette Smoke Condensate on Transendothelial Invasion of Ovarian Cancer Cells

    NASA Astrophysics Data System (ADS)

    Opp, Daniel; Lo, Chun-Min

    2007-03-01

    We investigated the effects of cigarette smoke condensate (CSC) on barrier function and cellular migration of human umbilical vein endothelial cells (HUVEC), and on the invasive activities of ovarian carcinoma cells through HUVEC monolayers as well. Central to this work was the use of electric cell-substrate impedance sensing (ECIS), a cell-based biosensor that monitors motility and other morphology changes of cells adherent on small gold electrodes. Upon addition of different concentrations of CSC, the junctional resistance and the wound healing rate of the HUVEC layers decrease as CSC concentration increases from 0.01 to 0.25 mg/ml, whereas the average cell-substrate separation increases with CSC concentration. Following the addition of OVCA429 ovarian cancer cells to HUVEC layers with the presence of different CSC concentrations, dose-dependent changes of the transcellular resistance drop were observed. Our results suggest that CSC is detrimental to normal endothelial cell function in maintaining vascular integrity. In addition, the chemicals present in CSC may increase transendothelial invasion of ovarian cancer cells.

  16. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin.

    PubMed

    Shulman, Ziv; Shinder, Vera; Klein, Eugenia; Grabovsky, Valentin; Yeger, Orna; Geron, Erez; Montresor, Alessio; Bolomini-Vittori, Matteo; Feigelson, Sara W; Kirchhausen, Tomas; Laudanna, Carlo; Shakhar, Guy; Alon, Ronen

    2009-03-20

    Endothelial chemokines are instrumental for integrin-mediated lymphocyte adhesion and transendothelial migration (TEM). By dissecting how chemokines trigger lymphocyte integrins to support shear-resistant motility on and across cytokine-stimulated endothelial barriers, we found a critical role for high-affinity (HA) LFA-1 integrin in lymphocyte crawling on activated endothelium. Endothelial-presented chemokines triggered HA-LFA-1 and adhesive filopodia at numerous submicron dots scattered underneath crawling lymphocytes. Shear forces applied to endothelial-bound lymphocytes dramatically enhanced filopodia density underneath crawling lymphocytes. A fraction of the adhesive filopodia invaded the endothelial cells prior to and during TEM and extended large subluminal leading edge containing dots of HA-LFA-1 occupied by subluminal ICAM-1. Memory T cells generated more frequent invasive filopodia and transmigrated more rapidly than their naive counterparts. We propose that shear forces exerted on HA-LFA-1 trigger adhesive and invasive filopodia at apical endothelial surfaces and thereby promote lymphocyte crawling and probing for TEM sites.

  17. Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils.

    PubMed

    Kuckleburg, Christopher J; Tilkens, Sarah B; Santoso, Sentot; Newman, Peter J

    2012-03-01

    Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.

  18. Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

    PubMed

    Colom, Bartomeu; Bodkin, Jennifer V; Beyrau, Martina; Woodfin, Abigail; Ody, Christiane; Rourke, Claire; Chavakis, Triantafyllos; Brohi, Karim; Imhof, Beat A; Nourshargh, Sussan

    2015-06-16

    Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic.

  19. The RhoA guanine nucleotide exchange factor, LARG, mediates ICAM-1-dependent mechanotransduction in endothelial cells to stimulate transendothelial migration.

    PubMed

    Lessey-Morillon, Elizabeth C; Osborne, Lukas D; Monaghan-Benson, Elizabeth; Guilluy, Christophe; O'Brien, E Timothy; Superfine, Richard; Burridge, Keith

    2014-04-01

    RhoA-mediated cytoskeletal rearrangements in endothelial cells (ECs) play an active role in leukocyte transendothelial cell migration (TEM), a normal physiological process in which leukocytes cross the endothelium to enter the underlying tissue. Although much has been learned about RhoA signaling pathways downstream from ICAM-1 in ECs, little is known about the consequences of the tractional forces that leukocytes generate on ECs as they migrate over the surface before TEM. We have found that after applying mechanical forces to ICAM-1 clusters, there is an increase in cellular stiffening and enhanced RhoA signaling compared with ICAM-1 clustering alone. We have identified that leukemia-associated Rho guanine nucleotide exchange factor (LARG), also known as Rho GEF 12 (ARHGEF12) acts downstream of clustered ICAM-1 to increase RhoA activity, and that this pathway is further enhanced by mechanical force on ICAM-1. Depletion of LARG decreases leukocyte crawling and inhibits TEM. To our knowledge, this is the first report of endothelial LARG regulating leukocyte behavior and EC stiffening in response to tractional forces generated by leukocytes.

  20. The effect of DMSA-functionalized magnetic nanoparticles on transendothelial migration of monocytes in the murine lung via a beta2 integrin-dependent pathway.

    PubMed

    Valois, Caroline R A; Braz, Juliana M; Nunes, Eloiza S; Vinolo, Marco A R; Lima, Emilia C D; Curi, Rui; Kuebler, Wolfgang M; Azevedo, Ricardo B

    2010-01-01

    Magnetic nanoparticles surface-functionalized with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute an innovative and promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. Transendothelial migration of leukocytes in the lung is a side effect of endovenous administration of MNPs-DMSA. Using cytologic and phenotypic analysis of murine bronchoalveolar lavage cells, we identified monocytes/macrophages as the main subpopulation of leukocytes involved in this process. Moreover, ultrastructural analysis revealed the presence of nanoparticles inside of numerous macrophages from bronchoalveolar lavage. MNPs-DMSA at concentrations as high as 1 x 10(15) nanoparticles/mL had no toxic effects on macrophages, as evidenced by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Notably, MNPs-DMSA up-regulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen in the murine lung. Upregulation of these cell adhesion molecules was associated with an increased concentration of tumor necrosis factor-alpha in lung. Finally, the critical relevance of the beta(2) integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our results characterize mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung, and identify beta(2) integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications.

  1. Ezetimibe-sensitive cholesterol uptake by NPC1L1 protein does not require endocytosis

    PubMed Central

    Johnson, Tory A.; Pfeffer, Suzanne R.

    2016-01-01

    Human NPC1L1 protein mediates cholesterol absorption in the intestine and liver and is the target of the drug ezetimibe, which is used to treat hypercholesterolemia. Previous studies concluded that NPC1L1-GFP protein trafficking is regulated by cholesterol binding and that ezetimibe blocks NPC1L1-GFP function by inhibiting its endocytosis. We used cell surface biotinylation to monitor NPC1L1-GFP endocytosis and show that ezetimibe does not alter the rate of NPC1L1-GFP endocytosis in cultured rat hepatocytes grown under normal growth conditions. As expected, NPC1L1-GFP endocytosis depends in part on C-terminal, cytoplasmically oriented sequences, but endocytosis does not require cholesterol binding to NPC1L1’s N-terminal domain. In addition, two small- molecule inhibitors of general (and NPC1L1-GFP) endocytosis failed to inhibit the ezetimibe-sensitive uptake of [3H]cholesterol from taurocholate micelles. These experiments demonstrate that cholesterol uptake by NPC1L1 does not require endocytosis; moreover, ezetimibe interferes with NPC1L1’s cholesterol adsorption activity without blocking NPC1L1 internalization in RH7777 cells. PMID:27075173

  2. N-Terminal Truncated UCH-L1 Prevents Parkinson's Disease Associated Damage

    PubMed Central

    Kim, Hee-Jung; Kim, Hyun Jung; Jeong, Jae-Eun; Baek, Jeong Yeob; Jeong, Jaeho; Kim, Sun; Kim, Young-Mee; Kim, Youhwa; Nam, Jin Han; Huh, Sue Hee; Seo, Jawon; Jin, Byung Kwan; Lee, Kong-Joo

    2014-01-01

    Ubiquitin C-terminal hydrolase-L1 (UCH-L1) has been proposed as one of the Parkinson's disease (PD) related genes, but the possible molecular connection between UCH-L1 and PD is not well understood. In this study, we discovered an N-terminal 11 amino acid truncated variant UCH-L1 that we called NT-UCH-L1, in mouse brain tissue as well as in NCI-H157 lung cancer and SH-SY5Y neuroblastoma cell lines. In vivo experiments and hydrogen-deuterium exchange (HDX) with tandem mass spectrometry (MS) studies showed that NT-UCH-L1 is readily aggregated and degraded, and has more flexible structure than UCH-L1. Post-translational modifications including monoubiquitination and disulfide crosslinking regulate the stability and cellular localization of NT-UCH-L1, as confirmed by mutational and proteomic studies. Stable expression of NT-UCH-L1 decreases cellular ROS levels and protects cells from H2O2, rotenone and CCCP-induced cell death. NT-UCH-L1-expressing transgenic mice are less susceptible to degeneration of nigrostriatal dopaminergic neurons seen in the MPTP mouse model of PD, in comparison to control animals. These results suggest that NT-UCH-L1 may have the potential to prevent neural damage in diseases like PD. PMID:24959670

  3. Ubiquitin C-terminal hydrolase L1 deficiency decreases bone mineralization.

    PubMed

    Shim, Sehwan; Kwon, Young-Bae; Yoshikawa, Yasuhiro; Kwon, Jungkee

    2008-06-01

    Ubiquitin C-terminal hydrolase L1 is a component of the ubiquitin proteasome system, which evidences unique biological activities. In this study, we report the pattern of UCH-L1 expression, and show that it regulates bone mineralization in osteogenesis. UCH-L1 was expressed in osteoblasts, osteoclasts, and hematopoietic precursor cells of bone marrow in the metaphysis and diaphysis of the femora. To further assess the involvement of UCH-L1 in the regulation of bone mineralization, we evaluated the bone mineral density (BMD) rate of gad mice, using the Latheta computed tomography system. Male gad mice evidenced a significantly decreased BMD rate in the metaphysis and diaphysis of the femora. These findings of decreased BMD rate in the bones of gad mice may suggest that UCH-L1 function regulates bone mineralization during osteogenesis.

  4. Evaluating L1CAM expression in human endometrial cancer using qRT-PCR

    PubMed Central

    Notaro, Sara; Reimer, Daniel; Duggan-Peer, Michaela; Fiegl, Heidi; Wiedermair, Annamarie; Rössler, Julia; Altevogt, Peter; Marth, Christian; Zeimet, Alain Gustave

    2016-01-01

    Background Management of endometrial carcinoma (EC) still needs improvement of risk assessment. Recently, L1CAM immunohistochemical (IHC) evaluation showed a unique value to predict the outcome of early EC. However IHC results are often conflicting for lack of inter-laboratory standardisation. Methods Here, as a proof of concept and to increase reproducibility we assayed eighty-two EC and 26 normal endometrium samples for L1CAM expression (L1CAMEXP) via qRT-PCR. The IHC evaluation was performed in 50 cancer samples. Moreover, we aimed to substantiate the in-vitro findings of L1CAM regulation through its promoter methylation (L1CAMMET), miR-34a expression and miR-34a promoter methylation. DNA methylation was assessed with MethyLight PCR technique. Results High overall concordant results between IHC and RT-PCR evaluations were found. L1CAMEXP was detected in 11% of cancer specimens. These positive cancers exhibited a worse DFS (p=0.032) and OS (p=0.016) in a multivariate COX-regression model. L1CAMEXP predicted distant failure (p=0.007) and L1CAMMET predicted risk-reduction of lymph-node involvement (p=0.005). Inverse correlations between L1CAMEXP and L1CAMMET (p=0.004) and between L1CAMEXP and miR-34a expression (p=0.002) were found. Conclusions In conclusion qRT-PCR analysis is a reliable approach to evaluate L1CAM status in EC and L1CAMEXP was highly predictive for distant failure and poor outcome, confirming the large IHC-based studies. Interestingly, L1CAMMET was able to assess the risk of pelvic lymph-node involvement. Especially the latter finding has to be confirmed in larger prospective series. PMID:27233077

  5. 26 CFR 1.414(l)-1 - Mergers and consolidations of plans or transfers of plan assets.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... of plan assets. 1.414(l)-1 Section 1.414(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(l)-1 Mergers and consolidations of plans or transfers of plan assets. (a) In general—(1) Scope of the regulations. Sections 401(a)(12) and 414(l) apply only to plans to...

  6. 26 CFR 1.414(l)-1 - Mergers and consolidations of plans or transfers of plan assets.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... of plan assets. 1.414(l)-1 Section 1.414(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(l)-1 Mergers and consolidations of plans or transfers of plan assets. (a) In general—(1) Scope of the regulations. Sections 401(a)(12) and 414(l) apply only to plans to...

  7. 26 CFR 1.414(l)-1 - Mergers and consolidations of plans or transfers of plan assets.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... of plan assets. 1.414(l)-1 Section 1.414(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(l)-1 Mergers and consolidations of plans or transfers of plan assets. (a) In general—(1) Scope of the regulations. Sections 401(a)(12) and 414(l) apply only to plans to...

  8. 26 CFR 1.414(l)-1 - Mergers and consolidations of plans or transfers of plan assets.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... of plan assets. 1.414(l)-1 Section 1.414(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.414(l)-1 Mergers and consolidations of plans or transfers of plan assets. (a) In general—(1) Scope of the regulations. Sections 401(a)(12) and 414(l) apply only to plans to...

  9. The L1 adhesion molecule is a cellular ligand for VLA-5

    PubMed Central

    1995-01-01

    The L1 adhesion molecule is a member of the immunoglobulin superfamily shared by neural and immune cells. In the nervous system L1 can mediate cell binding by a homophilic mechanism. To analyze its function on leukocytes we studied whether L1 could interact with integrins. Here we demonstrate that VLA-5, an RGD-specific fibronectin receptor on a wide variety of cell types, can bind to murine L1. Mouse ESb-MP cells expressing VLA-5 and L1 could be induced to aggregate in the presence of specific mAbs to CD24 (heat-stable antigen), a highly and heterogeneously glycosylated glycophosphatidylinositol-linked differentiation antigen of hematopoietic and neural cells. The aggregation was blocked by both mAbs to L1 and VLA-5, respectively. Aggregation was blocked also by a synthetic RGD-containing peptide derived from the Ig-domain VI of the L1 protein. ESb-MP subclones with low L1 expression could not aggregate. In heterotypic binding assays mouse bone marrow cells could adhere in an L1-dependent fashion to platelets that expressed VLA-5. Also purified L1 coated to polystyrene beads could bind to platelets. The binding of L1-beads was again inhibited by mAbs to L1 and VLA-5, by soluble L1 and the L1-RGD peptide in a dose-dependent manner. Thymocytes or human Nalm-6 tumor cells expressing VLA-5 could adhere to affinity-purified L1 and to the L1- derived RGD-containing peptide coated to glass slides. The adhesion was strongly enhanced in the presence of Mn(2+)-ions and blocked by mAbs to VLA-5. We also demonstrate a direct L1-VLA-5 protein interaction. Our results suggest a novel binding pathway, in which the VLA-5 integrin binds to L1 on adjacent cells. Given its rapid downregulation on lymphocytes after induction of cell proliferation, L1 may be important in integrin-mediated and activation-regulated cell-cell interactions. PMID:8557754

  10. Mechanisms for the proliferation of eosinophilic leukemia cells by FIP1L1-PDGFR{alpha}

    SciTech Connect

    Ishihara, Kenji; Kitamura, Hajime; Hiraizumi, Kenji; Kaneko, Motoko; Takahashi, Aki; Zee, OkPyo; Seyama, Toshio; Hong, JangJa; Ohuchi, Kazuo; Hirasawa, Noriyasu

    2008-02-22

    The constitutively activated tyrosine kinase Fip1-like 1 (FIP1L1)-platelet-derived growth factor receptor {alpha} (PDGFR{alpha}) causes eosinophilic leukemia EoL-1 cells to proliferate. Recently, we demonstrated that histone deacetylase inhibitors suppressed this proliferation and induced the differentiation of EoL-1 cells into eosinophils in parallel with a decrease in the level of FIP1L1-PDGFR{alpha}. In this study, we analyzed the mechanism by which FIP1L1-PDGFR{alpha} induces the proliferation and whether the suppression of cell proliferation triggers the differentiation into eosinophils. The FIP1L1-PDGFR{alpha} inhibitor imatinib inhibited the proliferation of EoL-1 cells and decreased the level of the oncoprotein c-Myc as well as the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK). The proliferation of EoL-1 cells and expression of c-Myc were also inhibited by the MEK inhibitor U0126 and JNK inhibitor SP600125. The expression of the eosinophilic differentiation marker CCR3 was not induced by imatinib. These findings suggest that FIP1L1-PDGFR{alpha} induces the proliferation of EoL-1 cells through the induction of c-Myc expression via ERK and JNK signaling pathways, but is not involved in the inhibition of differentiation toward mature eosinophils.

  11. Tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis in tissue by intravital video microscopy.

    PubMed

    Xu, Najia; Lei, Xi; Liu, Lixin

    2011-09-24

    The recruitment of circulating leukocytes from blood stream to the inflamed tissue is a crucial and complex process of inflammation(1,2). In the postcapillary venules of inflamed tissue, leukocytes initially tether and roll on the luminal surface of venular wall. Rolling leukocytes arrest on endothelium and undergo firm adhesion in response to chemokine or other chemoattractants on the venular surface. Many adherent leukocytes relocate from the initial site of adhesion to the junctional extravasation site in endothelium, a process termed intraluminal crawling(3). Following crawling, leukocytes move across endothelium (transmigration) and migrate in extravascular tissue toward the source of chemoattractant (chemotaxis)(4). Intravital microscopy is a powerful tool for visualizing leukocyte-endothelial cell interactions in vivo and revealing cellular and molecular mechanisms of leukocyte recruitment(2,5). In this report, we provide a comprehensive description of using brightfield intravital microscopy to visualize and determine the detailed processes of neutrophil recruitment in mouse cremaster muscle in response to the gradient of a neutrophil chemoattractant. To induce neutrophil recruitment, a small piece of agarose gel (~1-mm(3) size) containing neutrophil chemoattractant MIP-2 (CXCL2, a CXC chemokine) or WKYMVm (Trp-Lys-Tyr-Val-D-Met, a synthetic analog of bacterial peptide) is placed on the muscle tissue adjacent to the observed postcapillary venule. With time-lapsed video photography and computer software ImageJ, neutrophil intraluminal crawling on endothelium, neutrophil transendothelial migration and the migration and chemotaxis in tissue are visualized and tracked. This protocol allows reliable and quantitative analysis of many neutrophil recruitment parameters such as intraluminal crawling velocity, transmigration time, detachment time, migration velocity, chemotaxis velocity and chemotaxis index in tissue. We demonstrate that using this protocol, these

  12. Transendothelial migration of CD16+ monocytes in response to fractalkine under constitutive and inflammatory conditions.

    PubMed

    Ancuta, Petronela; Moses, Ashlee; Gabuzda, Dana

    2004-01-01

    CD16+ monocytes represent 5-10% of circulating monocytes in healthy individuals and are dramatically expanded in several pathological conditions including AIDS and HIV-1-associated dementia (HAD). CD16+ monocytes constitutively produce high levels of pro-inflammatory cytokines and neurotoxic factors that may contribute to the pathogenesis of these disorders. Monocyte recruitment into the central nervous system (CNS) and other peripheral tissues in response to locally produced chemokines is a critical event in immune surveillance and inflammation and involves monocyte arrest onto vascular beds and subsequent diapedesis. Here we investigate the ability of CD16+ monocytes to undergo transendothelial migration (TEM) under constitutive and inflammatory conditions. CD16+ monocytes underwent TEM across unstimulated human umbilical vascular (HUVEC) and brain microvascular endothelial (BMVEC) cell monolayers in response to soluble fractalkine (FKN/CX3CL1). Stimulation with tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma) induced high and low expression of membrane-bound FKN on HUVEC and BMVEC, respectively, together with expression of VCAM-1 and intercellular adhesion molecule-1 (ICAM)-1. By contrast, only HUVEC expressed CD62E while BMVEC remained negative. Both CD16- and CD16+ monocyte subsets adhered to TNF/IFN-gamma-stimulated HUVEC with higher frequency than to unstimulated HUVEC. Monocyte chemoattractant protein-1 (MCP-1) triggered efficient TEM of CD16- monocytes across TNF/IFN-gamma-stimulated HUVEC, whereas soluble FKN failed to induce TEM of CD16+ monocytes across stimulated HUVEC. These results demonstrate that stimulation with TNF and IFN-gamma triggers expression of membrane-bound FKN on both HUVEC and BMVEC, but prevents TEM of CD16+ monocytes in response to soluble FKN. Thus, pro-inflammatory CD16+ monocytes may contribute to the pathogenesis of HAD and other inflammatory CNS diseases by affecting the integrity of the blood-brain barrier as a

  13. Lab-on-a-brane: A novel physiologically relevant planar arterial model to study transendothelial transport

    NASA Astrophysics Data System (ADS)

    Budhwani, Karim Ismail

    The tremendous quality of life impact notwithstanding, cardiovascular diseases and Cancer add up to over US$ 700bn each year in financial costs alone. Aging and population growth are expected to further expand the problem space while drug research and development remain expensive. However, preclinical costs can be substantially mitigated by substituting animal models with in vitro devices that accurately model human cardiovascular transport. Here we present a novel physiologically relevant lab-on-a-brane that simulates in vivo pressure, flow, strain, and shear waveforms associated with normal and pathological conditions in large and small blood vessels for studying molecular transport across the endothelial monolayer. The device builds upon previously demonstrated integrated microfluidic loop design by: (a) introducing nanoscale pores in the substrate membrane to enable transmembrane molecular transport, (b) transforming the substrate membrane into a nanofibrous matrix for 3D smooth muscle cell (SMC) tissue culture, (c) integrating electrospinning fabrication methods, (d) engineering an invertible sandwich cell culture device architecture, and (e) devising a healthy co-culture mechanism for human arterial endothelial cell (HAEC) monolayer and multiple layers of human smooth muscle cells (HSMC) to accurately mimic arterial anatomy. Structural and mechanical characterization was conducted using confocal microscopy, SEM, stress/strain analysis, and infrared spectroscopy. Transport was characterized using FITC-Dextran hydraulic permeability protocol. Structure and transport characterization successfully demonstrate device viability as a physiologically relevant arterial mimic for testing transendothelial transport. Thus, our lab-on-a-brane provides a highly effective and efficient, yet considerably inexpensive, physiologically relevant alternative for pharmacokinetic evaluation; possibly reducing animals used in pre-clinical testing, clinical trials cost from false

  14. Transendothelial migration of effector T cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans.

    PubMed

    Stoler-Barak, Liat; Barzilai, Sagi; Zauberman, Ayelet; Alon, Ronen

    2014-06-01

    Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1β stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.

  15. Absence of PD-L1 on tumor cells is associated with reduced MHC I expression and PD-L1 expression increases in recurrent serous ovarian cancer

    PubMed Central

    Aust, Stefanie; Felix, Sophie; Auer, Katharina; Bachmayr-Heyda, Anna; Kenner, Lukas; Dekan, Sabine; Meier, Samuel M.; Gerner, Christopher; Grimm, Christoph; Pils, Dietmar

    2017-01-01

    Immune-evasion and immune checkpoints are promising new therapeutic targets for several cancer entities. In ovarian cancer, the clinical role of programmed cell death receptor ligand 1 (PD-L1) expression as mechanism to escape immune recognition has not been clarified yet. We analyzed PD-L1 expression of primary ovarian and peritoneal tumor tissues together with several other parameters (whole transcriptomes of isolated tumor cells, local and systemic immune cells, systemic cytokines and metabolites) and compared PD-L1 expression between primary tumor and tumor recurrences. All expressed major histocompatibility complex (MHC) I genes were negatively correlated to PD-L1 abundances on tumor tissues, indicating two mutually exclusive immune-evasion mechanisms in ovarian cancer: either down-regulation of T-cell mediated immunity by PD-L1 expression or silencing of self-antigen presentation by down-regulation of the MHC I complex. In our cohort and in most of published evidences in ovarian cancer, low PD-L1 expression is associated with unfavorable outcome. Differences in immune cell populations, cytokines, and metabolites strengthen this picture and suggest the existence of concurrent pathways for progression of this disease. Furthermore, recurrences showed significantly increased PD-L1 expression compared to the primary tumors, supporting trials of checkpoint inhibition in the recurrent setting. PMID:28266500

  16. UCH-L1 induces podocyte hypertrophy in membranous nephropathy by protein accumulation.

    PubMed

    Lohmann, Frithjof; Sachs, Marlies; Meyer, Tobias N; Sievert, Henning; Lindenmeyer, Maja T; Wiech, Thorsten; Cohen, Clemens D; Balabanov, Stefan; Stahl, R A K; Meyer-Schwesinger, Catherine

    2014-07-01

    Podocytes are terminally differentiated cells of the glomerular filtration barrier that react with hypertrophy in the course of injury such as in membranous nephropathy (MGN). The neuronal deubiquitinase ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed and activated in podocytes of human and rodent MGN. UCH-L1 regulates the mono-ubiquitin pool and induces accumulation of poly-ubiquitinated proteins in affected podocytes. Here, we investigated the role of UCH-L1 in podocyte hypertrophy and in the homeostasis of the hypertrophy associated "model protein" p27(Kip1). A better understanding of the basic mechanisms leading to podocyte hypertrophy is crucial for the development of specific therapies in MGN. In human and rat MGN, hypertrophic podocytes exhibited a simultaneous up-regulation of UCH-L1 and of cytoplasmic p27(Kip1) content. Functionally, inhibition of UCH-L1 activity and knockdown or inhibition of UCH-L1 attenuated podocyte hypertrophy by decreasing the total protein content in isolated glomeruli and in cultured podocytes. In contrast, UCH-L1 levels and activity increased podocyte hypertrophy and total protein content in culture, specifically of cytoplasmic p27(Kip1). UCH-L1 enhanced cytoplasmic p27(Kip1) levels by nuclear export and decreased poly-ubiquitination and proteasomal degradation of p27(Kip1). In parallel, UCH-L1 increased podocyte turnover, migration and cytoskeletal rearrangement, which are associated with known oncogenic functions of cytoplasmic p27(Kip1) in cancer. We propose that UCH-L1 induces podocyte hypertrophy in MGN by increasing the total protein content through altered degradation and accumulation of proteins such as p27(Kip1) in the cytoplasm of podocytes. Modification of both UCH-L1 activity and levels could be a new therapeutic avenue to podocyte hypertrophy in MGN.

  17. Widespread expression of the Supv3L1 mitochondrial RNA helicase in the mouse

    PubMed Central

    Paul, Erin; Kielbasinski, Marissa; Sedivy, John M.; Murga-Zamalloa, Carlos; Khanna, Hemant; Klysik, Jan E.

    2009-01-01

    Supv3L1 is an evolutionarily conserved helicase that plays a critical role in the mitochondrial RNA surveillance and degradation machinery. Conditional ablation of Supv3L1 in adult mice leads to premature aging phenotypes including loss of muscle mass and adipose tissue and severe skin abnormalities. To get insights into the spatial and temporal expression of Supv3L1 in the mouse, we generated knock-in and transgenic strains in which an EGFP reporter was placed under control of the Supv3L1 native promoter. During development, expression of Supv3L1 begins at the blastocyst stage, becomes widespread and strong in all fetal tissues and cell types, and continues during postnatal growth. In mature animals reporter expression is only slightly diminished in most tissues and continues to be highly expressed in the brain, peripheral sensory organs, and testis. Together, these data confirm that Supv3L1 is an important developmentally regulated gene, which continues to be expressed in all mature tissues, particularly the rapidly proliferating cells of testes, but also in the brain and sensory organs. The transgenic mice and cell lines derived from them constitute a valuable tool for the examination of the spatial and temporal aspects of Supv3L1 promoter activity, and should facilitate future screens for small molecules that regulate Supv3L1 expression. PMID:19937380

  18. Novel ex vivo culture method for human monocytes uses shear flow to prevent total loss of transendothelial diapedesis function.

    PubMed

    Tsubota, Yoshiaki; Frey, Jeremy M; Raines, Elaine W

    2014-01-01

    Monocyte recruitment to inflammatory sites and their transendothelial migration into tissues are critical to homeostasis and pathogenesis of chronic inflammatory diseases. However, even short-term suspension culture of primary human monocytes leads to phenotypic changes. In this study, we characterize the functional effects of ex vivo monocyte culture on the steps involved in monocyte transendothelial migration. Our data demonstrate that monocyte diapedesis is impaired by as little as 4 h culture, and the locomotion step is subsequently compromised. After 16 h in culture, monocyte diapedesis is irreversibly reduced by ∼90%. However, maintenance of monocytes under conditions mimicking physiological flow (5-7.5 dyn/cm²) is sufficient to reduce diapedesis impairment significantly. Thus, through the application of shear during ex vivo culture of monocytes, our study establishes a novel protocol, allowing functional analyses of monocytes not currently possible under static culture conditions. These data further suggest that monocyte-based therapeutic applications may be measurably improved by alteration of ex vivo conditions before their use in patients.

  19. The Rho-guanine nucleotide exchange factor Trio controls leukocyte transendothelial migration by promoting docking structure formation.

    PubMed

    van Rijssel, Jos; Kroon, Jeffrey; Hoogenboezem, Mark; van Alphen, Floris P J; de Jong, Renske J; Kostadinova, Elena; Geerts, Dirk; Hordijk, Peter L; van Buul, Jaap D

    2012-08-01

    Leukocyte transendothelial migration involves the active participation of the endothelium through the formation of apical membrane protrusions that embrace adherent leukocytes, termed docking structures. Using live-cell imaging, we find that prior to transmigration, endothelial docking structures form around 80% of all neutrophils. Previously we showed that endothelial RhoG and SGEF control leukocyte transmigration. In this study, our data reveal that both full-length Trio and the first DH-PH (TrioD1) domain of Trio, which can activate Rac1 and RhoG, interact with ICAM-1 and are recruited to leukocyte adhesion sites. Moreover, upon clustering of ICAM-1, the Rho-guanine nucleotide exchange factor Trio activates Rac1, prior to activating RhoG, in a filamin-dependent manner. We further show that docking structure formation is initiated by ICAM-1 clustering into ring-like structures, which is followed by apical membrane protrusion. Interestingly, we find that Rac1 is required for ICAM-1 clustering, whereas RhoG controls membrane protrusion formation. Finally, silencing endothelial Trio expression or reducing TrioD1 activity without affecting SGEF impairs both docking structure formation and leukocyte transmigration. We conclude that Trio promotes leukocyte transendothelial migration by inducing endothelial docking structure formation in a filamin-dependent manner through the activation of Rac1 and RhoG.

  20. Intracerebral GM-CSF contributes to transendothelial monocyte migration in APP/PS1 Alzheimer's disease mice.

    PubMed

    Shang, De S; Yang, Yi M; Zhang, Hu; Tian, Li; Jiang, Jiu S; Dong, Yan B; Zhang, Ke; Li, Bo; Zhao, Wei D; Fang, Wen G; Chen, Yu H

    2016-11-01

    Although tight junctions between human brain microvascular endothelial cells in the blood-brain barrier prevent molecules or cells in the bloodstream from entering the brain, in Alzheimer's disease, peripheral blood monocytes can "open" these tight junctions and trigger subsequent transendothelial migration. However, the mechanism underlying this migration is unclear. Here, we found that the CSF2RB, but not CSF2RA, subunit of the granulocyte-macrophage colony-stimulating factor receptor was overexpressed on monocytes from Alzheimer's disease patients. CSF2RB contributes to granulocyte-macrophage colony-stimulating factor-induced transendothelial monocyte migration. Granulocyte-macrophage colony-stimulating factor triggers human brain microvascular endothelial cells monolayer tight junction disassembly by downregulating ZO-1 expression via transcription modulation and claudin-5 expression via the ubiquitination pathway. Interestingly, intracerebral granulocyte-macrophage colony-stimulating factor blockade abolished the increased monocyte infiltration in the brains of APP/PS1 Alzheimer's disease model mice. Our results suggest that in Alzheimer's disease patients, high granulocyte-macrophage colony-stimulating factor levels in the brain parenchyma and cerebrospinal fluid induced blood-brain barrier opening, facilitating the infiltration of CSF2RB-expressing peripheral monocytes across blood-brain barrier and into the brain. CSF2RB might be useful as an Alzheimer's disease biomarker. Thus, our findings will help to understand the mechanism of monocyte infiltration in Alzheimer's disease pathogenesis.

  1. Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

    PubMed

    Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle.

  2. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Tseng, Min-Jen

    2013-01-18

    Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  3. Vaspin promotes 3T3-L1 preadipocyte differentiation

    PubMed Central

    Liu, Ping; Wu, Jine; Zhou, Xin; Wang, Liping; Han, Wenqi; Lv, Ying; Sun, Chaofeng

    2015-01-01

    Vaspin, a novel adipocyte factor secreted from visceral adipose tissues, is associated with obesity and insulin resistance and can regulate glucose and lipid metabolism, increase insulin sensitivity, and suppress inflammation; however, the underlying mechanisms remain unknown. Proliferation and maladaptive differentiation are important pathological mechanisms underlying obesity. This study aimed to evaluate the effects of vaspin on the proliferation and differentiation of preadipocyte 3T3-L1 cells and to explore the likely mechanisms responsible for 3T3-L1 differentiation. Vaspin was added to cultured 3T3-L1 cells, and the differentiation of adipocytes was evaluated using Oil Red O staining. The AKT signaling pathway and specific differentiation factors related to the differentiation of preadipocyte 3T3-L1 cells, peroxisome proliferator-activated γ and the CCAAT/enhancer-binding protein (C/EBP) family, were evaluated using reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses during the early phase of differentiation. Additionally, adiponectin mRNA, interleukin-6 mRNA (IL-6 mRNA), and glucose transporter-4 (GLUT4) protein levels were measured in the differentiated adipocytes. The results indicated that vaspin promotes the intracellular accumulation of lipids and increases differentiation-related factors, including peroxisome proliferator-activated receptor γ, C/EBPα, and free fatty acid-binding protein 4 (FABP4), in a dose-dependent manner. Additionally, vaspin (200 ng/mL) increased the mRNA and protein levels of C/EBPβ, peroxisome proliferator-activated γ, C/EBPα, and FABP4. Moreover, compared with the control, significantly smaller eight-day differentiated adipocytes were observed, and these cells exhibited decreased IL-6 mRNA and increased GLUT4 mRNA levels; these results also indicated the potential of vaspin to promote the insulin-mediated AKT signaling pathway during the early phase of differentiation. In conclusion

  4. Cyclization of the Urokinase Receptor-Derived Ser-Arg-Ser-Arg-Tyr Peptide Generates a Potent Inhibitor of Trans-Endothelial Migration of Monocytes

    PubMed Central

    Bifulco, Katia; Ingangi, Vincenzo; Di Carluccio, Gioconda; Merlino, Francesco; Motti, Maria Letizia; Grieco, Paolo; Carriero, Maria Vincenza

    2015-01-01

    The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC50 value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases. PMID:25938482

  5. Extended Lagrange interpolation in L1 spaces

    NASA Astrophysics Data System (ADS)

    Occorsio, Donatella; Russo, Maria Grazia

    2016-10-01

    Let w (x )=e-xβxα , w ¯(x )=x w (x ) and denote by {pm(w)}m,{pn(w¯)}n the corresponding sequences of orthonormal polynomials. The zeros of the polynomial Q2 m +1=pm +1(w )pm(w ¯) are simple and are sufficiently far among them. Therefore it is possible to construct an interpolation process essentially based on the zeros of Q2m+1, which is called "Extended Lagrange Interpolation". Here we study the convergence of this interpolation process in suitable weighted L1 spaces. This study completes the results given by the authors in previous papers in weighted Lup((0 ,+∞ )) , for 1≤p≤∞. Moreover an application of the proposed interpolation process in order to construct an e cient product quadrature scheme for weakly singular integrals is given.

  6. Characterizing the Solar Wind at L1

    NASA Astrophysics Data System (ADS)

    Jahn, J.; Elliott, H. A.

    2008-12-01

    The nature of solar wind-magnetosphere energy transfer plays a big role in understanding the time history and types of global-scale magnetospheric phenomena. However, systematic approaches to quantifying how the specific magnetospheric "modes" (if they can be called that) of substorms, SMCs, sawtooth events, and geomagnetic storms could be controlled by the solar wind are still difficult. We present a fresh approach to characterizing the solar wind and its time history using self-organizing maps. The thrust of this effort is geared towards detecting and classifying solar wind structure on time scales relevant for the magnetospheric responses of interest. Performing this characterization at the L1 point is ideal for uncovering solar wind- magnetosphere relationships. It also provides a very long, contiguous time series that helps us explore these relationships over a complete solar cycle. We present the technique and initial results of solar wind comparisons during and leading up to SMCs and sawtooth events.

  7. Excess α-synuclein worsens disease in mice lacking ubiquitin carboxy-terminal hydrolase L1.

    PubMed

    Shimshek, Derya R; Schweizer, Tatjana; Schmid, Peter; van der Putten, P Herman

    2012-01-01

    Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species.

  8. To Grow or Not to Grow: Nutritional Control of Development During Caenorhabditis elegans L1 Arrest

    PubMed Central

    2013-01-01

    It is widely appreciated that larvae of the nematode Caenorhabditis elegans arrest development by forming dauer larvae in response to multiple unfavorable environmental conditions. C. elegans larvae can also reversibly arrest development earlier, during the first larval stage (L1), in response to starvation. “L1 arrest” (also known as “L1 diapause”) occurs without morphological modification but is accompanied by increased stress resistance. Caloric restriction and periodic fasting can extend adult lifespan, and developmental models are critical to understanding how the animal is buffered from fluctuations in nutrient availability, impacting lifespan. L1 arrest provides an opportunity to study nutritional control of development. Given its relevance to aging, diabetes, obesity and cancer, interest in L1 arrest is increasing, and signaling pathways and gene regulatory mechanisms controlling arrest and recovery have been characterized. Insulin-like signaling is a critical regulator, and it is modified by and acts through microRNAs. DAF-18/PTEN, AMP-activated kinase and fatty acid biosynthesis are also involved. The nervous system, epidermis, and intestine contribute systemically to regulation of arrest, but cell-autonomous signaling likely contributes to regulation in the germline. A relatively small number of genes affecting starvation survival during L1 arrest are known, and many of them also affect adult lifespan, reflecting a common genetic basis ripe for exploration. mRNA expression is well characterized during arrest, recovery, and normal L1 development, providing a metazoan model for nutritional control of gene expression. In particular, post-recruitment regulation of RNA polymerase II is under nutritional control, potentially contributing to a rapid and coordinated response to feeding. The phenomenology of L1 arrest will be reviewed, as well as regulation of developmental arrest and starvation survival by various signaling pathways and gene regulatory

  9. Effects of MAPK and PI3K Pathways on PD-L1 Expression in Melanoma

    PubMed Central

    Atefi, Mohammad; Avramis, Earl; Lassen, Amanda; Wong, Deborah; Robert, Lidia; Foulad, David; Cerniglia, Michael; Titz, Bjoern; Chodon, Thinle; Graeber, Thomas G.; Comin-Anduix, Begonya; Ribas, Antoni

    2014-01-01

    Purpose PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study we investigated whether PD-L1 expression is controlled by melanoma driver mutations and modified by oncogenic signaling inhibition. Experimental Design Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors. The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated. Results No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, while the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines the effects of BRAF, MEK and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1-exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines. Conclusions In melanoma cell lines, including BRAF inhibitor-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1-exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies. PMID:24812408

  10. Ubiquitin C-terminal hydrolase L1 (UCH-L1) acts as a novel potentiator of cyclin-dependent kinases to enhance cell proliferation independently of its hydrolase activity.

    PubMed

    Kabuta, Tomohiro; Mitsui, Takeshi; Takahashi, Masaki; Fujiwara, Yuuki; Kabuta, Chihana; Konya, Chiho; Tsuchiya, Yukihiro; Hatanaka, Yusuke; Uchida, Kenko; Hohjoh, Hirohiko; Wada, Keiji

    2013-05-03

    Dysregulation of cell proliferation and the cell cycle are associated with various diseases, such as cancer. Cyclin-dependent kinases (CDKs) play central roles in cell proliferation and the cell cycle. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is expressed in a restricted range of tissues, including the brain and numerous types of cancer. However, the molecular functions of UCH-L1 remain elusive. In this study, we found that UCH-L1 physically interacts with CDK1, CDK4, and CDK5, enhancing their kinase activity. Using several mutants of UCH-L1, we showed that this enhancement is dependent upon interaction levels between UCH-L1 and CDKs but is independent of the known ubiquitin-related functions of UCH-L1. Gain- and loss-of-function studies revealed that UCH-L1 enhances proliferation of multiple cell types, including human cancer cells. Inhibition of the interaction between UCH-L1 and cell cycle-associated CDK resulted in the abolishment of UCH-L1-induced enhancement of cell proliferation. RNA interference of UCH-L1 reduced the growth of human xenograft tumors in mice. We concluded that UCH-L1 is a novel regulator of the kinase activities of CDKs. We believe that our findings from this study will significantly contribute to our understanding of cell cycle-associated diseases.

  11. The 3T3-L1 adipocyte glycogen proteome

    PubMed Central

    2013-01-01

    Background Glycogen is a branched polysaccharide of glucose residues, consisting of α-1-4 glycosidic linkages with α-1-6 branches that together form multi-layered particles ranging in size from 30 nm to 300 nm. Glycogen spatial conformation and intracellular organization are highly regulated processes. Glycogen particles interact with their metabolizing enzymes and are associated with a variety of proteins that intervene in its biology, controlling its structure, particle size and sub-cellular distribution. The function of glycogen in adipose tissue is not well understood but appears to have a pivotal role as a regulatory mechanism informing the cells on substrate availability for triacylglycerol synthesis. To provide new molecular insights into the role of adipocyte glycogen we analyzed the glycogen-associated proteome from differentiated 3T3-L1-adipocytes. Results Glycogen particles from 3T3-L1-adipocytes were purified using a series of centrifugation steps followed by specific elution of glycogen bound proteins using α-1,4 glucose oligosaccharides, or maltodextrins, and tandem mass spectrometry. We identified regulatory proteins, 14-3-3 proteins, RACK1 and protein phosphatase 1 glycogen targeting subunit 3D. Evidence was also obtained for a regulated subcellular distribution of the glycogen particle: metabolic and mitochondrial proteins were abundant. Unlike the recently analyzed hepatic glycogen proteome, no endoplasmic proteins were detected, along with the recently described starch-binding domain protein 1. Other regulatory proteins which have previously been described as glycogen-associated proteins were not detected, including laforin, the AMPK beta-subunit and protein targeting to glycogen (PTG). Conclusions These data provide new molecular insights into the regulation of glycogen-bound proteins that are associated with the maintenance, organization and localization of the adipocyte glycogen particle. PMID:23521774

  12. Cannabidiol promotes browning in 3T3-L1 adipocytes.

    PubMed

    Parray, Hilal Ahmad; Yun, Jong Won

    2016-05-01

    Recruitment of the brown-like phenotype in white adipocytes (browning) and activation of existing brown adipocytes are currently being investigated as a means to combat obesity. Thus, a wide variety of dietary agents that contribute to browning of white adipocytes have been identified. The present study was designed to investigate the effects of cannabidiol (CBD), a major nonpsychotropic phytocannabinoid of Cannabis sativa, on induction of browning in 3T3-L1 adipocytes. CBD enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cited1, Tmem26, Prdm16, Cidea, Tbx1, Fgf21, and Pgc-1α) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of PPARγ and PI3K. In addition, CBD increased protein expression levels of CPT1, ACSL, SIRT1, and PLIN while down-regulating JNK2, SREBP1, and LPL. These data suggest possible roles for CBD in browning of white adipocytes, augmentation of lipolysis, thermogenesis, and reduction of lipogenesis. In conclusion, the current data suggest that CBD plays dual modulatory roles in the form of inducing the brown-like phenotype as well as promoting lipid metabolism. Thus, CBD may be explored as a potentially promising therapeutic agent for the prevention of obesity.

  13. Rendezvous missions with minimoons from L1

    NASA Astrophysics Data System (ADS)

    Chyba, M.; Haberkorn, T.; Patterson, G.

    2014-07-01

    We propose to present asteroid capture missions with the so-called minimoons. Minimoons are small asteroids that are temporarily captured objects on orbits in the Earth-Moon system. It has been suggested that, despite their small capture probability, at any time there are one or two meter diameter minimoons, and progressively greater numbers at smaller diameters. The minimoons orbits differ significantly from elliptical orbits which renders a rendezvous mission more challenging, however they offer many advantages for such missions that overcome this fact. First, they are already on geocentric orbits which results in short duration missions with low Delta-v, this translates in cost efficiency and low-risk targets. Second, beside their close proximity to Earth, an advantage is their small size since it provides us with the luxury to retrieve the entire asteroid and not only a sample of material. Accessing the interior structure of a near-Earth satellite in its morphological context is crucial to an in-depth analysis of the structure of the asteroid. Historically, 2006 RH120 is the only minimoon that has been detected but work is ongoing to determine which modifications to current observation facilities is necessary to provide detection algorithm capabilities. In the event that detection is successful, an efficient algorithm to produce a space mission to rendezvous with the detected minimoon is highly desirable to take advantage of this opportunity. This is the main focus of our work. For the design of the mission we propose the following. The spacecraft is first placed in hibernation on a Lissajoux orbit around the liberation point L1 of the Earth-Moon system. We focus on eight-shaped Lissajoux orbits to take advantage of the stability properties of their invariant manifolds for our transfers since the cost to minimize is the spacecraft fuel consumption. Once a minimoon has been detected we must choose a point on its orbit to rendezvous (in position and velocities

  14. Role of miR-34a as a suppressor of L1CAM in endometrial carcinoma

    PubMed Central

    Schirmer, Uwe; Doberstein, Kai; Rupp, Anne-Kathleen; Bretz, Niko P.; Wuttig, Daniela; Kiefel, Helena; Breunig, Christian; Fiegl, Heidi; Müller-Holzner, Elisabeth; Zeillinger, Robert; Eva, Heidi; Zeimet, Alain G.; SÜltmann, Holger; Altevogt, Peter

    2014-01-01

    L1CAM promotes cell motility, invasion and metastasis formation in various human cancers and can be considered as a driver of tumor progression. Knowledge about genetic processes leading to the presence of L1CAM in cancers is of considerable importance. Experimentally, L1CAM expression can be achieved by various means. Overexpression of the transcription factor SLUG or treatment of cells with TGF-ß1 can induce or augment L1CAM levels in cancer cells. Likewise, hypomethylation of the L1CAM promoter on the X chromosome correlates with L1CAM expression. However, presently no mechanisms that might control transcriptional activity are known. Here we have identified miR-34a as a suppressor of L1CAM. We observed that L1CAM positive endometrial carcinoma (EC) cell lines HEC1B and SPAC1L lost L1CAM protein and mRNA by treatment with demethylating agents or knock-down of the DNA-methyltransferase-1 (DNMT1). Concomitantly, several miRNAs were up-regulated. Using miRNA profiling, luciferase reporter assays and mutagenesis, we identified miR-34a as a putative binder to the L1CAM-3'UTR. Overexpression of miR-34a in HEC1B cells blocked L1CAM expression and inhibited cell migration. In ECC1 cells (wildtype p53) the activation of p53 caused miR-34a up-regulation and loss of L1CAM expression that was miR-34a dependent. We observed an inverse correlation between L1CAM and miR-34a levels in EC cell lines. In primary tumor sections areas expressing high amounts of L1CAM had less miR-34a expression than those with low L1CAM levels. Our data suggest that miR-34a can regulate L1CAM expression by targeting L1CAM mRNA for degradation. These findings shed new light on the complex regulation of L1CAM in human tumors. PMID:24497324

  15. Role of miR-34a as a suppressor of L1CAM in endometrial carcinoma.

    PubMed

    Schirmer, Uwe; Doberstein, Kai; Rupp, Anne-Kathleen; Bretz, Niko P; Wuttig, Daniela; Kiefel, Helena; Breunig, Christian; Fiegl, Heidi; Müller-Holzner, Elisabeth; Zeillinger, Robert; Schuster, Eva; Zeimet, Alain G; Sültmann, Holger; Altevogt, Peter

    2014-01-30

    L1CAM promotes cell motility, invasion and metastasis formation in various human cancers and can be considered as a driver of tumor progression. Knowledge about genetic processes leading to the presence of L1CAM in cancers is of considerable importance. Experimentally, L1CAM expression can be achieved by various means. Over-expression of the transcription factor SLUG or treatment of cells with TGF-β1 can induce or augment L1CAM levels in cancer cells. Likewise, hypomethylation of the L1CAM promoter on the X chromosome correlates with L1CAM expression. However, presently no mechanisms that might control transcriptional activity are known. Here we have identified miR-34a as a suppressor of L1CAM. We observed that L1CAM positive endometrial carcinoma (EC) cell lines HEC1B and SPAC1L lost L1CAM protein and mRNA by treatment with demethylating agents or knock-down of the DNA-methyltransferase-1 (DNMT1). Concomitantly, several miRNAs were up-regulated. Using miRNA profiling, luciferase reporter assays and mutagenesis, we identified miR-34a as a putative binder to the L1CAM-3'UTR. Over-expression of miR-34a in HEC1B cells blocked L1CAM expression and inhibited cell migration. In ECC1 cells (wildtype p53) the activation of p53 caused miR-34a up-regulation and loss of L1CAM expression that was miR-34a dependent. We observed an inverse correlation between L1CAM and miR-34a levels in EC cell lines. In primary tumor sections areas expressing high amounts of L1CAM had less miR-34a expression than those with low L1CAM levels. Our data suggest that miR-34a can regulate L1CAM expression by targeting L1CAM mRNA for degradation. These findings shed new light on the complex regulation of L1CAM in human tumors.

  16. Robust L1 PCA and application in image denoising

    NASA Astrophysics Data System (ADS)

    Gao, Junbin; Kwan, Paul W. H.; Guo, Yi

    2007-11-01

    The so-called robust L1 PCA was introduced in our recent work [1] based on the L1 noise assumption. Due to the heavy tail characteristics of the L1 distribution, the proposed model has been proved much more robust against data outliers. In this paper, we further demonstrate how the learned robust L1 PCA model can be used to denoise image data.

  17. 26 CFR 1.7701(l)-1 - Conduit financing arrangements.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 13 2010-04-01 2010-04-01 false Conduit financing arrangements. 1.7701(l)-1 Section 1.7701(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES General Actuarial Valuations § 1.7701(l)-1 Conduit...

  18. 26 CFR 1.7701(l)-1 - Conduit financing arrangements.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 13 2012-04-01 2012-04-01 false Conduit financing arrangements. 1.7701(l)-1 Section 1.7701(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) General Actuarial Valuations § 1.7701(l)-1...

  19. 26 CFR 1.7701(l)-1 - Conduit financing arrangements.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 13 2013-04-01 2013-04-01 false Conduit financing arrangements. 1.7701(l)-1 Section 1.7701(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) General Actuarial Valuations § 1.7701(l)-1...

  20. 26 CFR 1.7701(l)-1 - Conduit financing arrangements.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 13 2014-04-01 2014-04-01 false Conduit financing arrangements. 1.7701(l)-1 Section 1.7701(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) General Actuarial Valuations § 1.7701(l)-1...

  1. 26 CFR 1.7701(l)-1 - Conduit financing arrangements.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 13 2011-04-01 2011-04-01 false Conduit financing arrangements. 1.7701(l)-1 Section 1.7701(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX (CONTINUED) INCOME TAXES (CONTINUED) General Actuarial Valuations § 1.7701(l)-1...

  2. Purple L1 Milestone Review Panel - MPI

    SciTech Connect

    Jones, T

    2006-12-07

    to bring up the metric up to the target of 45% efficiency for worse case pairings. By using environment tuning, we were able to achieve 47% efficiency for worse case (see Figure 1). Most pairings actually perform much higher. The Robustness category of MPI was demonstrated by the Synthetic Workload application load Stability Test, or SWL-ST. The test results that were documented and archived will be included in the L1 Milestone completion documentation. All MPI related Statement of Work (SOW) target performance objectives have been met. Both MPI-only and Hybrid-MPI codes have successfully met scaling expectations on Purple (including ale3d, yf3d, and other classified applications).

  3. Endoplasmic Reticulum-Localized Transmembrane Protein Dpy19L1 Is Required for Neurite Outgrowth

    PubMed Central

    Watanabe, Keisuke; Bizen, Norihisa; Sato, Noboru; Takebayashi, Hirohide

    2016-01-01

    The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development. PMID:27959946

  4. Characterization of an upstream regulatory element of adenovirus L1 poly (A) site.

    PubMed

    Liu, Li

    2005-06-20

    The transition from early to late stage infection by adenovirus involves a change in mRNA expression from the adenovirus major late transcription unit (AdMLTU). This early to late switch centers around alternative selection of one of five poly (A) sites (L1-L5) that code for the major structural proteins of Adenovirus. During the early stage of infection, steady state mRNA is primarily derived from the L1 poly (A) site. During the late stage of infection, each of the MLTU poly (A) sites is represented in the steady state mRNA pool (Falck-Pedersen, E., Logan, J., 1989. Regulation of poly(A) site selection in adenovirus. J. Virol. 63 (2), 532-541.). Using transient transfection of a plasmid expressing Chloramphenicol Acetyl Transferase with a tandem poly (A) minigene system (L13) (DeZazzo, J.D., Falck-Pedersen, E., Imperiale, M.J., 1991. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol. Cell. Biol. 11 (12), 5977-5984; Prescott, J., Falck-Pedersen, E., 1994. Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites. Mol. Cell. Biol. 14 (7), 4682-4693.), it has been demonstrated that the promoter-proximal L1 poly (A) site which is poorly recognized by the 3' end processing machinery, contains an upstream repressor element (URE) that influences steady state levels of mRNA (Prescott, J.C., Liu, L., Falck-Pedersen, E., 1997. Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation. Mol. Cell. Biol. 17 (4), 2207-2216.). In this study, we have further characterized the elements that mediate L1URE function. These studies indicate that the L1 upstream regulatory element (L1 URE) contains a complex RNA architecture that serves to repress gene expression through multiple sub-effectors. The L1URE functions when located upstream of a heterologous poly (A) site, and is able to strongly suppress steady state m

  5. Relationships among L1 Print Exposure and Early L1 Literacy Skills, L2 Aptitude, and L2 Proficiency

    ERIC Educational Resources Information Center

    Sparks, Richard L.; Patton, Jon; Ganschow, Leonore; Humbach, Nancy

    2012-01-01

    Authors examined the relationship between individual differences in L1 print exposure and differences in early L1 skills and later L2 aptitude, L2 proficiency, and L2 classroom achievement. Participants were administered measures of L1 word decoding, spelling, phonemic awareness, reading comprehension, receptive vocabulary, and listening…

  6. Poly(A) binding protein C1 is essential for efficient L1 retrotransposition and affects L1 RNP formation.

    PubMed

    Dai, Lixin; Taylor, Martin S; O'Donnell, Kathryn A; Boeke, Jef D

    2012-11-01

    Poly(A) binding proteins (PABPs) specifically bind the polyadenosine tail of mRNA and have been shown to be important for RNA polyadenylation, translation initiation, and mRNA stability. Using a modified L1 retrotransposition vector, we examined the effects of two PABPs (encoded by PABPN1 and PABPC1) on the retrotransposition activity of the L1 non-long-terminal-repeat (non-LTR) retrotransposon in both HeLa and HEK293T cells. We demonstrated that knockdown of these two genes by RNA interference (RNAi) effectively reduced L1 retrotransposition by 70 to 80% without significantly changing L1 transcription or translation or the status of the poly(A) tail. We identified that both poly(A) binding proteins were associated with the L1 ribonucleoprotein complex, presumably through L1 mRNA. Depletion of PABPC1 caused a defect in L1 RNP formation. Knockdown of the PABPC1 inhibitor PAIP2 increased L1 retrotransposition up to 2-fold. Low levels of exogenous overexpression of PABPN1 and PABPC1 increased L1 retrotransposition, whereas unregulated overexpression of these two proteins caused pleiotropic effects, such as hypersensitivity to puromycin and decreased L1 activity. Our data suggest that PABPC1 is essential for the formation of L1 RNA-protein complexes and may play a role in L1 RNP translocation in the host cell.

  7. PD-L1 Expression in Lung Cancer

    PubMed Central

    Yu, Hui; Boyle, Theresa A.; Zhou, Caicun; Rimm, David L.; Hirsch, Fred R.

    2017-01-01

    Immunotherapies targeted against programmed death ligand 1 (PD-L1) and its receptor (PD-1) have improved survival in a subset of patients with advanced lung cancer. PD-L1 protein expression has emerged as a biomarker that predicts which patients are more likely to respond to immunotherapy. The understanding of PD-L1 as a biomarker is complicated by the history of use of different immunohistochemistry platforms with different PD-L1 antibodies, scoring systems, and positivity cut-offs for immunotherapy clinical trials with different anti-PD-L1 and anti-PD-1 drugs. Herein, we summarize the brief history of PD-L1 as a biomarker and describe the challenges remaining to harmonize PD-L1 detection and interpretation for best patient care. PMID:27117833

  8. Stop codon mutagenesis for homogenous expression of human papillomavirus L1 protein in Escherichia coli.

    PubMed

    Wang, Daning; Fan, Fei; Li, Zhihai; Liu, Xinlin; Song, Shuo; Wei, Shuangping; He, Maozhou; Lin, Yahua; Li, Zhongyi; Wei, Minxi; Yu, Hai; Gu, Ying; Li, Shaowei; Xia, Ningshao

    2017-03-04

    Human papillomavirus (HPV) is widely accepted to be the major causative pathogen of cervical cancer, warts, and other epithelial tumors. Virus infection and subsequent disease development can be prevented by vaccination with HPV vaccines derived from eukaryotic expression systems. Here, we report the soluble expression of the major capsid protein L1 of HPV31, a dominant carcinogenic HPV genotype, in Escherichia coli. HPV31 L1 protein and its elongated form (L1+) were observed in SDS-PAGE and CE-SDS analysis, generated by the native HPV31 L1 gene with a TAA stop codon. Replacing the TAA with TAG but not TGA could completely terminate protein translation. Mass spectrometry sequencing showed that L1+ comprised L1 with a C-terminal extension of 38 amino acids (aa). RNA folding analysis revealed that the unfaithful L1+ expression may result from translational read-through, as TAG is more stable and accessible than the other stop codons. The 38-aa elongated fragment perturbs self-assembly of HPV31 L1+, as shown in size and morphology analyses. By 3D cryo-electron microscopy structure determination, we show self-assembly of purified HPV31 L1 (TAG) VLPs into T = 7 icosahedral symmetry particles, resembling the native HPV virion. Finally, through additional characterization and antigenicity/immunogenicity assays, we verified that the E.coli-derived HPV31 VLPs are an ideal immunogen for HPV vaccine development. Our findings outline a codon optimization stratagem for protein expression and provide a method for the in-depth investigation of prokaryotic translation regulation.

  9. Interactions of TOM1L1 with the multivesicular body sorting machinery.

    PubMed

    Puertollano, Rosa

    2005-03-11

    Tom1L1 (Tom1-like1) and related proteins Tom1 (Target of Myb1) and Tom1L2 (Tom1-like2) constitute a new protein family characterized by the presence of a VHS (Vps27p/Hrs/Stam) domain in the N-terminal portion followed by a GAT (GGA and Tom) domain. Recently it was demonstrated that the GAT domain of both Tom1 and Tom1L1 binds ubiquitin, suggesting that these proteins might participate in the sorting of ubiquitinated proteins into multivesicular bodies (MVBs). Here we report a novel interaction between Tom1L1 and members of the MVB sorting machinery. Specifically, we found that the VHS domain of Tom1L1 interacts with Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate), whereas a PTAP motif, located between the VHS and GAT domain of Tom1L1, is responsible for binding to TSG101 (tumor susceptibility gene 101). Myc epitope-tagged Tom1L1 showed a cytosolic distribution but was recruited to endosomes following Hrs expression. In addition, Tom1L1 possesses several tyrosine motifs at the C-terminal region that mediate interactions with members of the Src family kinases and other signaling proteins such as Grb2 and p85. We showed that a fraction of Fyn kinase localizes at endosomes and that this distribution becomes more evident after epidermal growth factor internalization. Moreover, expression of a constitutive active form of Fyn also promoted the recruitment of Tom1L1 to enlarged endosomes. Taken together, we propose that Tom1L1 could act as an intermediary between signaling and degradative pathways.

  10. PD-L1 expression and prognostic impact in glioblastoma

    PubMed Central

    Nduom, Edjah K.; Wei, Jun; Yaghi, Nasser K.; Huang, Neal; Kong, Ling-Yuan; Gabrusiewicz, Konrad; Ling, Xiaoyang; Zhou, Shouhao; Ivan, Cristina; Chen, Jie Qing; Burks, Jared K.; Fuller, Greg N.; Calin, George A.; Conrad, Charles A.; Creasy, Caitlin; Ritthipichai, Krit; Radvanyi, Laszlo; Heimberger, Amy B.

    2016-01-01

    Background Therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and PD-1/PD-L1 has demonstrated tumor regression in clinical trials, and phase 2 trials are ongoing in glioblastoma (GBM). Previous reports have suggested that responses are more frequent in patients with tumors that express PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact. Methods Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with GBM. We categorized our results according to the total number of PD-L1-expressing cells within the GBMs and then validated this finding in ex vivo GBM flow cytometry with further analysis of the T cell populations. We then evaluated the association between PD-L1 expression and median survival time using the protein expression datasets and mRNA from The Cancer Genome Atlas. Results The median percentage of PD-L1-expressing cells in GBM by cell surface staining is 2.77% (range: 0%–86.6%; n = 92), which is similar to the percentage found by ex vivo flow cytometry. The majority of GBM patients (61%) had tumors with at least 1% or more PD-L1-positive cells, and 38% had at least 5% or greater PD-L1 expression. PD-L1 is commonly expressed on the GBM-infiltrating T cells. Expression of both PD-L1 and PD-1 are negative prognosticators for GBM outcome. Conclusions The incidence of PD-L1 expression in GBM patients is frequent but is confined to a minority subpopulation, similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome. PMID:26323609

  11. Parkin-mediated K63-polyubiquitination targets ubiquitin C-terminal hydrolase L1 for degradation by the autophagy-lysosome system.

    PubMed

    McKeon, Jeanne E; Sha, Di; Li, Lian; Chin, Lih-Shen

    2015-05-01

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a key neuronal deubiquitinating enzyme which is mutated in Parkinson disease (PD) and in childhood-onset neurodegenerative disorder with optic atrophy. Furthermore, reduced UCH-L1 protein levels are associated with a number of neurodegenerative diseases, whereas up-regulation of UCH-L1 protein expression is found in multiple types of cancer. However, very little is known about how UCH-L1 protein level is regulated in cells. Here, we report that UCH-L1 is a novel interactor and substrate of PD-linked E3 ubiquitin-protein ligase parkin. We find that parkin mediates K63-linked polyubiquitination of UCH-L1 in cooperation with the Ubc13/Uev1a E2 ubiquitin-conjugating enzyme complex and promotes UCH-L1 degradation by the autophagy-lysosome pathway. Targeted disruption of parkin gene expression in mice causes a significant decrease in UCH-L1 ubiquitination with a concomitant increase in UCH-L1 protein level in brain, supporting an in vivo role of parkin in regulating UCH-L1 ubiquitination and degradation. Our findings reveal a direct link between parkin-mediated ubiquitin signaling and UCH-L1 regulation, and they have important implications for understanding the roles of these two proteins in health and disease.

  12. Decreased anxiety, altered place learning, and increased CA1 basal excitatory synaptic transmission in mice with conditional ablation of the neural cell adhesion molecule L1.

    PubMed

    Law, Janice W S; Lee, Alan Y W; Sun, Mu; Nikonenko, Alexander G; Chung, Sookja K; Dityatev, Alexander; Schachner, Melitta; Morellini, Fabio

    2003-11-12

    L1, a neural cell adhesion molecule of the immunoglobulin superfamily, is involved in neuronal migration and differentiation and axon outgrowth and guidance. Mutations in the human and mouse L1 gene result in similarly severe neurological abnormalities. To dissociate the functional roles of L1 in the adult brain from developmental abnormalities, we have generated a mutant in which the L1 gene is inactivated by cre-recombinase under the control of the calcium/calmodulin-dependent kinase II promoter. This mutant (L1fy+) did not show the overt morphological and behavioral abnormalities observed previously in constitutive L1-deficient (L1-/-) mice; however, there was an increase in basal excitatory synaptic transmission that was not apparent in L1-/- mice. Similar to L1-/- mice, no defects in short- and long-term potentiation in the CA1 region of the hippocampus were observed. Interestingly, L1fy+ mice showed decreased anxiety in the open field and elevated plus-maze, contrary to L1-/- mice, and altered place learning in the water maze, similar to L1-/- mice. Thus, mice conditionally deficient in L1 expression in the adult brain share some abnormalities, but also display different ones, as compared with L1-/- mice, highlighting the role of L1 in the regulation of synaptic transmission and behavior in adulthood.

  13. Microglial TNF-α-dependent elevation of MHC class I expression on brain endothelium induced by amyloid-beta promotes T cell transendothelial migration.

    PubMed

    Yang, Yi-Ming; Shang, De-Shu; Zhao, Wei-Dong; Fang, Wen-Gang; Chen, Yu-Hua

    2013-11-01

    The blood-brain barrier (BBB) normally bars peripheral T lymphocytes from entering the cerebrum. Interestingly, activated T cells exist as infiltrates in the brains of Alzheimer's disease (AD) patients, but little is known about the mechanisms involved. In this study, we observed significantly higher MHC class I expression in rat brain endothelial cells compared with controls following the induction of experimental AD models. An in vitro BBB model, which was constructed with human brain microvascular endothelial cells, was established to study the mechanisms underlying the transendothelial migration of T cells. Using in vitro studies, we demonstrated that secretion of TNF-α from Aβ1-42-treated BV2 microglia contributes to the elevated expression of MHC class I on the brain microvessel endothelium. Transmigration assays and adhesion assays confirmed that the upregulation of MHC class I molecules was associated with T cell transendothelial migration. MHC class I knock-down in HBMECs significantly attenuated the migratory and adhesive capability of the T cells. Interestingly, a TNF-α neutralizing antibody effectively blocked the transendothelial migration of T cells triggered by treatment with the supernatant from Aβ1-42-treated BV2 microglia. We propose that microglia-derived TNF-α upregulates MHC class I molecule expression on brain endothelial cells, which represents a mechanism of T cell migration into the brain. This study may provide a new insight into the potential pathomechanism of Alzheimer's disease.

  14. Frequent expression of PD-L1 on circulating breast cancer cells.

    PubMed

    Mazel, Martine; Jacot, William; Pantel, Klaus; Bartkowiak, Kai; Topart, Delphine; Cayrefourcq, Laure; Rossille, Delphine; Maudelonde, Thierry; Fest, Thierry; Alix-Panabières, Catherine

    2015-11-01

    Immune checkpoint regulators such as PD-L1 have become exciting new therapeutic targets leading to long lasting remissions in patients with advanced malignancies. However, in view of the remarkable costs and the toxicity profiles of these therapies, predictive biomarkers able to discriminate responders from non-responders are urgently needed. In the present paper, we provide evidence that PD-L1 is frequently expressed on metastatic cells circulating in the blood of hormone receptor-positive, HER2-negative breast cancer patients. We performed western blot, flow cytometry and immunocytochemical analyses to demonstrate the specificity of the PDL1 antibody used in our study and established immunoscores for PDL1 expression on single tumor cells. We then selected sixteen patients with circulating tumor cells (CTCs) using the CellSearch(®) system and found PD-L1((+)) CTCs in 11 patients (68.8%). The fraction of PD-L1((+)) CTCs varied from 0.2 to 100% in individual patients. This is the first report demonstrating the expression of PD-L1 on CTCs. The established CTC/PD-L1 assay can be used for liquid biopsy in future clinical trials for stratification and monitoring of cancer patients undergoing immune checkpoint blockade.

  15. S100P is a metastasis-associated gene that facilitates transendothelial migration of pancreatic cancer cells.

    PubMed

    Barry, Sayka; Chelala, Claude; Lines, Kate; Sunamura, Makoto; Wang, Amu; Marelli-Berg, Federica M; Brennan, Caroline; Lemoine, Nicholas R; Crnogorac-Jurcevic, Tatjana

    2013-03-01

    Pancreatic ductal adenocarcinoma (PDAC) is the 5th most common cause of cancer death in the UK and the 4th in the US. The vast majority of deaths following pancreatic cancer are due to metastatic spread, hence understanding the metastatic process is vital for identification of critically needed novel therapeutic targets. An enriched set of 33 genes differentially expressed in common between primary PDAC and liver metastases, when compared to normal tissues, was obtained through global gene expression profiling. This metastasis-associated gene set comprises transcripts from both cancer (S100P, S100A6, AGR2, etc.) and adjacent stroma (collagens type I, III, and V, etc.), thus reinforcing the concept of a continuous crosstalk between the two compartments in both primary tumours and their metastases. The expression of S100P, SFN, VCAN and collagens was further validated in additional primary PDACs and matched liver metastatic lesions, while the functional significance of one of the most highly expressed genes, S100P, was studied in more detail. We show that this protein increases the transendothelial migration of PDAC cancer cells in vitro, which was also confirmed in vivo experiments using a zebrafish embryo model. Thus S100P facilitates cancer cell intravasation/extravasation, critical steps in the hematogenous dissemination of pancreatic cancer cells.

  16. Real-time analysis of integrin-dependent transendothelial migration and integrin-independent interstitial motility of leukocytes.

    PubMed

    Shulman, Ziv; Alon, Ronen

    2012-01-01

    The role of integrins in leukocyte migration across endothelial barriers is widely accepted. In contrast, the contribution of integrins to interstitial motility of leukocytes is still elusive. Chemokine binding to G-protein-coupled receptors expressed on the surface of leukocytes plays key roles in both of these processes by directly activating integrin conformations favorable for ligand binding and integrin microclustering. Chemokines can also serve as weak adhesive ligands and potent inducers of actin cytoskeleton remodeling. Real-time assays utilizing live imaging microscopy have been implemented to dissect these versatile roles of chemokines in different leukocyte migration processes. Here, we review several in vitro assays useful for exploring the contribution of chemokine signals and shear forces to integrin activation and function during various stages of leukocyte transendothelial migration. In addition, we describe a new assay that assesses the contribution of chemokines to integrin-independent interstitial leukocyte motility. These assays can also follow the outcome of specific genetic or biochemical manipulations of either the leukocyte or the endothelial barrier on distinct migratory steps. Following fixation, subcellular changes in the distribution of integrin subsets and of specific integrin-associated adaptors can be further dissected by immunofluorescence tools and by ultrastructural electron microscopic analysis.

  17. TCR-driven transendothelial migration of human effector memory CD4 T cells involves Vav, Rac, and myosin IIA.

    PubMed

    Manes, Thomas D; Pober, Jordan S

    2013-04-01

    Human effector memory (EM) CD4 T cells may be recruited from the blood into a site of inflammation in response either to inflammatory chemokines displayed on or specific Ag presented by venular endothelial cells (ECs), designated as chemokine-driven or TCR-driven transendothelial migration (TEM), respectively. We have previously described differences in the morphological appearance of transmigrating T cells as well as in the molecules that mediate T cell-EC interactions distinguishing these two pathways. In this study, we report that TCR-driven TEM requires ZAP-70-dependent activation of a pathway involving Vav, Rac, and myosin IIA. Chemokine-driven TEM also uses ZAP-70, albeit in a quantitatively and spatially different manner of activation, and is independent of Vav, Rac, and mysosin IIA, depending instead on an as-yet unidentified GTP exchange factor that activates Cdc42. The differential use of small Rho family GTPases to activate the cytoskeleton is consistent with the morphological differences observed in T cells that undergo TEM in response to these distinct recruitment signals.

  18. Polarized granzyme release is required for antigen-driven transendothelial migration of human effector memory CD4 T cells.

    PubMed

    Manes, Thomas D; Pober, Jordan S

    2014-12-15

    Human effector memory CD4 T cells may transmigrate across endothelial cell (EC) monolayers either in response to inflammatory chemokines or in response to TCR recognition of Ag presented on the surface of the EC. The kinetics, morphologic manifestations, and molecular requirements of chemokine- and TCR-driven transendothelial migration (TEM) differ significantly. In this study, we report that, whereas the microtubule organizing center (MTOC) and cytosolic granules follow the nucleus across the endothelium in a uropod during chemokine-driven TEM, MTOC reorientation to the contact region between the T cell and the EC, accompanied by dynein-driven transport of granzyme-containing granules to and exocytosis at the contact region, are early events in TCR-driven, but not chemokine-driven TEM. Inhibitors of either granule function or granzyme proteolytic activity can arrest TCR-driven TEM, implying a requirement for granule discharge in the process. In the final stages of TCR-driven TEM, the MTOC precedes, rather than follows, the nucleus across the endothelium. Thus, TCR-driven TEM of effector memory CD4 T cells appears to be a novel process that more closely resembles immune synapse formation than it does conventional chemotaxis.

  19. Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes.

    PubMed

    Yakovlev, Sergiy; Mikhailenko, Irina; Cao, Chunzhang; Zhang, Li; Strickland, Dudley K; Medved, Leonid

    2012-01-12

    While testing the effect of the (β15-66)(2) fragment, which mimics a pair of fibrin βN-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (β15-66)(2), fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance. The experiments showed that very LDL (VLDL) receptor (VLDLR) interacts with high affinity with fibrin through its βN-domains, and this interaction is inhibited by RAP and (β15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing βN-domains, suggesting the involvement of VLDLR in fibrin-dependent leukocyte transmigration. Our experiments with VLDLR-deficient mice confirmed this suggestion by showing that, in contrast to wild-type mice, fibrin-dependent leukocyte transmigration does not occur in such mice. Altogether, the present study identified VLDLR as a novel endothelial cell receptor for fibrin that promotes fibrin-dependent leukocyte transmigration and thereby inflammation. Establishing the molecular mechanism underlying this interaction may result in the development of novel inhibitors of fibrin-dependent inflammation.

  20. Iodothyronine Interactions with the System L1 Amino Acid Exchanger in 3T3-L1 Adipocytes.

    PubMed

    Mitchell, Fiona E; Roy, Lisa A; Taylor, Peter M

    2010-06-24

    Thyroid hormones enter isolated white adipocytes largely by a System L1-type amino acid transporter en route to exerting genomic actions. Differentiated 3T3-L1 mouse adipocytes in culture express mRNA for LAT1 (the catalytic subunit of high-affinity System L1). L-[(125)I]-T(3) uptake into 3T3-L1 adipocytes included a substantial saturable component inhibited by leucine. L-[(3)H]phenylalanine uptake into 3T3-L1 cells was saturable (K(m) of 31 μM), competitively inhibited by T(3) (K(i) of 1.2 μM) and blocked by leucine, BCH, and rT(3) as expected for substrate interactions of System L1. Efflux of preloaded L-[(3)H]phenylalanine from 3T3-L1 adipocytes was trans stimulated by external leucine, demonstrating the obligatory exchange mechanism of System L1 transport. T(3) (10 μM) did not significantly trans stimulate L-[(3)H]phenylalanine efflux, but did competitively inhibit the trans stimulatory effect of 10 μM leucine. The results highlight strong competitive interactions between iodothyronines (T(3), rT(3)) and amino acids for transport by System L1 in adipocytes, which may impact cellular iodothyronine exchanges during altered states of protein nutrition.

  1. L1 integration in a transgenic mouse model

    PubMed Central

    Babushok, Daria V.; Ostertag, Eric M.; Courtney, Christine E.; Choi, Janice M.; Kazazian, Haig H.

    2006-01-01

    To study integration of the human LINE-1 retrotransposon (L1) in vivo, we developed a transgenic mouse model of L1 retrotransposition that displays de novo somatic L1 insertions at a high frequency, occasionally several insertions per mouse. We mapped 3′ integration sites of 51 insertions by Thermal Asymmetric Interlaced PCR (TAIL–PCR). Analysis of integration locations revealed a broad genomic distribution with a modest preference for intergenic regions. We characterized the complete structures of 33 de novo retrotransposition events. Our results highlight the large number of highly truncated L1s, as over 52% (27/51) of total integrants were <1/3 the length of a full-length element. New integrants carry all structural characteristics typical of genomic L1s, including a number with inversions, deletions, and 5′-end microhomologies to the target DNA sequence. Notably, at least 13% (7/51) of all insertions contain a short stretch of extra nucleotides at their 5′ end, which we postulate result from template-jumping by the L1-encoded reverse transcriptase. We propose a unified model of L1 integration that explains all of the characteristic features of L1 retrotransposition, such as 5′ truncations, inversions, extra nucleotide additions, and 5′ boundary and inversion point microhomologies. PMID:16365384

  2. The L1 retrotransposition assay: a retrospective and toolkit.

    PubMed

    Rangwala, Sanjida H; Kazazian, Haig H

    2009-11-01

    LINE1s (L1s) are a class of mammalian non-LTR (long terminal repeat) retroelements that make up nearly 20% of the human genome. Because of the difficulty of studying the mobilization of endogenous L1s, an exogenous cell culture retrotransposition assay has become integral to research in L1 biology. This assay has allowed for investigation of the mechanism and consequences of mobilization of this retroelement, both in cell lines and in whole animal models. In this paper, we outline the genesis of in vitro retrotransposition systems which led to the development of the L1 retrotransposition assay in the mid-1990s. We then provide a retrospective, describing the many uses and variations of this assay, ending with caveats and predictions for future developments. Finally, we provide detailed protocols on the application of the retrotransposition assay, including lists of constructs available in the L1 research community and cell lines in which this assay has been applied.

  3. [L1-2 lumbar disc herniation: a case report].

    PubMed

    Monobe, T; Fujita, T; Nakaue, Y; Nishi, N

    1996-03-01

    A 49-year-old female presented a two-year history of pain in the right thigh and lower back. Neurological examination on admission demonstrated weakness of the right iliopsoas and quadriceps, hypesthesia on the right L1-2 dermatome. Radiological examination including myelography, CT myelography and discography disclosed an L1-2 herniated disc. Sagittal MRI also revealed an L1-2, an L4-5 and L5-S1 protruded disc. A posterior microdiscectomy (Love's method) was performed for the L1-2 disc. A controlateral protruded disc which compressed the L-2 nerve root was identified and partially removed. The postoperative myelography showed residual disc. The patient was free from pain and regained normal sensorimotor function. Love's posterior microdiscectomy has a disadvantage in that the operative field is limited. Careful surgical procedure was needed to avoid injury to nerve roots and the cauda equina in a tight L1-2 lumbar canal.

  4. PD-L1+MDSCs are increased in HCC patients and induced by soluble factor in the tumor microenvironment

    PubMed Central

    Iwata, Tomoaki; Kondo, Yasuteru; Kimura, Osamu; Morosawa, Tatsuki; Fujisaka, Yasuyuki; Umetsu, Teruyuki; Kogure, Takayuki; Inoue, Jun; Nakagome, Yu; Shimosegawa, Tooru

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) could have important roles in immune regulation, and MDSCs can be induced in patients with various malignant tumors. The immune-suppressive functions of MDSCs in hepatocellular carcinoma (HCC) patients have not been clarified. Therefore, we tried to analyze the biological significance of MDSCs in HCC patients. We quantified PD-L1+MDSCs of HCC patients in various conditions by using multi-color flow cytometry analysis. PBMCs from HCC patients contained significantly higher percentages of PD-L1+MDSCs in comparison to those from healthy subjects (p < 0.001). The percentages of PD-L1+MDSCs were reduced by curative treatment for HCC (p < 0.05), and the percentages of PD-L1+MDSCs before treatment were inversely correlated with disease-free survival time. After we cocultivated PBMCs and several liver cancer cell lines in a transwell coculture system, the percentages of PD-L1+MDSCs were significantly increased compared with control (p < 0.05). The expression of M-CSF and VEGFA was higher in the cell lines that strongly induced PD-L1+MDSCs. Peripheral blood from HCC patients had significantly higher percentages of PD-L1+MDSCs in comparison to those of healthy subjects, and the percentages of PD-L1+MDSCs were reduced by HCC treatment, suggesting that we might use PD-L1+MDSCs as a new biomarker of HCC. PMID:27966626

  5. Do L1 Reading Achievement and L1 Print Exposure Contribute to the Prediction of L2 Proficiency?

    ERIC Educational Resources Information Center

    Sparks, Richard L.; Patton, Jon; Ganschow, Leonore; Humbach, Nancy

    2012-01-01

    The study examined whether individual differences in high school first language (L1) reading achievement and print exposure would account for unique variance in second language (L2) written (word decoding, spelling, writing, reading comprehension) and oral (listening/speaking) proficiency after adjusting for the effects of early L1 literacy and…

  6. Ubiquitin C-terminal hydrolase L1 (UCH-L1): structure, distribution and roles in brain function and dysfunction.

    PubMed

    Bishop, Paul; Rocca, Dan; Henley, Jeremy M

    2016-08-15

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an extremely abundant protein in the brain where, remarkably, it is estimated to make up 1-5% of total neuronal protein. Although it comprises only 223 amino acids it has one of the most complicated 3D knotted structures yet discovered. Beyond its expression in neurons UCH-L1 has only very limited expression in other healthy tissues but it is highly expressed in several forms of cancer. Although UCH-L1 is classed as a deubiquitinating enzyme (DUB) the direct functions of UCH-L1 remain enigmatic and a wide array of alternative functions has been proposed. UCH-L1 is not essential for neuronal development but it is absolutely required for the maintenance of axonal integrity and UCH-L1 dysfunction is implicated in neurodegenerative disease. Here we review the properties of UCH-L1, and how understanding its complex structure can provide new insights into its roles in neuronal function and pathology.

  7. Cranberries (Oxycoccus quadripetalus) inhibit adipogenesis and lipogenesis in 3T3-L1 cells.

    PubMed

    Kowalska, Katarzyna; Olejnik, Anna; Rychlik, Joanna; Grajek, Włodzimierz

    2014-04-01

    Cranberries (Oxycoccus quadripetalus) are a valuable source of bioactive substances with high antioxidant potential and well documented beneficial health properties. In the present study, the activity of cranberries, in terms of the inhibiting effects of adipogenesis, was investigated using the 3T3-L1 cell line. The obtained results showed that cranberries reduced proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner. Treatment with cranberries decreased the number of adipocytes and reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an inhibitory effect on lipogenesis. Moreover, it was found that cranberries directly induced lipolysis in adipocytes and down-regulated the expression of major transcription factors of the adipogenesis pathway, such as PPARγ, C/EBPα and SREBP1. These findings indicate that cranberries are capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation.

  8. Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

    PubMed Central

    Bazan, Silvia Boschi; de Alencar Muniz Chaves, Agtha; Aires, Karina Araújo; Cianciarullo, Aurora Marques; Garcea, Robert L.; Ho, Paulo Lee

    2013-01-01

    Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin–sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines. PMID:19756360

  9. Ubiquitin C-terminal hydrolase-L1 potentiates cancer chemosensitivity by stabilizing NOXA.

    PubMed

    Brinkmann, Kerstin; Zigrino, Paola; Witt, Axel; Schell, Michael; Ackermann, Leena; Broxtermann, Pia; Schüll, Stephan; Andree, Maria; Coutelle, Oliver; Yazdanpanah, Benjamin; Seeger, Jens Michael; Klubertz, Daniela; Drebber, Uta; Hacker, Ulrich T; Krönke, Martin; Mauch, Cornelia; Hoppe, Thorsten; Kashkar, Hamid

    2013-03-28

    The BH3-only protein NOXA represents one of the critical mediators of DNA-damage-induced cell death. In particular, its involvement in cellular responses to cancer chemotherapy is increasingly evident. Here, we identify a strategy of cancer cells to escape genotoxic chemotherapy by increasing proteasomal degradation of NOXA. We show that the deubiquitylating enzyme UCH-L1 is a key regulator of NOXA turnover, which protects NOXA from proteasomal degradation by removing Lys(48)-linked polyubiquitin chains. In the majority of tumors from patients with melanoma or colorectal cancer suffering from high rates of chemoresistance, NOXA fails to accumulate because UCH-L1 expression is epigenetically silenced. Whereas UCH-L1/NOXA-positive tumor samples exhibit increased sensitivity to genotoxic chemotherapy, downregulation of UCH-L1 or inhibition of its deubiquitylase activity resulted in reduced NOXA stability and resistance to genotoxic chemotherapy in both human and C. elegans cells. Our data identify the UCH-L1/NOXA interaction as a therapeutic target for overcoming cancer chemoresistance.

  10. Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

    NASA Astrophysics Data System (ADS)

    Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

    2014-03-01

    Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

  11. L1-mediated colon cancer cell metastasis does not require changes in EMT and cancer stem cell markers.

    PubMed

    Gavert, Nancy; Vivanti, Alessia; Hazin, John; Brabletz, Thomas; Ben-Ze'ev, Avri

    2011-01-01

    Aberrant activation of Wnt/β-catenin signaling is common in most sporadic and inherited colorectal cancer (CRC) cells leading to elevated β-catenin/TCF transactivation. We previously identified the neural cell adhesion molecule L1 as a target gene of β-catenin/TCF in CRC cells. Forced expression of L1 confers increased cell motility, invasion, and tumorigenesis, and the induction of human CRC cell metastasis to the liver. In human CRC tissue, L1 is exclusively localized at the invasive front of such tumors in a subpopulation of cells displaying nuclear β-catenin. We determined whether L1 expression confers metastatic capacities by inducing an epithelial to mesenchymal transition (EMT) and whether L1 cosegregates with cancer stem cell (CSC) markers. We found that changes in L1 levels do not affect the organization or expression of E-cadherin in cell lines, or in invading CRC tissue cells, and no changes in other epithelial or mesenchymal markers were detected after L1 transfection. The introduction of major EMT regulators (Slug and Twist) into CRC cell lines reduced the levels of E-cadherin and induced fibronectin and vimentin, but unlike L1, Slug and Twist expression was insufficient for conferring metastasis. In CRC cells L1 did not specifically cosegregate with CSC markers including CD133, CD44, and EpCAM. L1-mediated metastasis required NF-κB signaling in cells harboring either high or low levels of endogenous E-cadherin. The results suggest that L1-mediated metastasis of CRC cells does not require changes in EMT and CSC markers and operates by activating NF-κβ signaling.

  12. Gold nanoparticles functionalized with a fragment of the neural cell adhesion molecule L1 stimulate L1-mediated functions

    NASA Astrophysics Data System (ADS)

    Schulz, Florian; Lutz, David; Rusche, Norman; Bastús, Neus G.; Stieben, Martin; Höltig, Michael; Grüner, Florian; Weller, Horst; Schachner, Melitta; Vossmeyer, Tobias; Loers, Gabriele

    2013-10-01

    The neural cell adhesion molecule L1 is involved in nervous system development and promotes regeneration in animal models of acute and chronic injury of the adult nervous system. To translate these conducive functions into therapeutic approaches, a 22-mer peptide that encompasses a minimal and functional L1 sequence of the third fibronectin type III domain of murine L1 was identified and conjugated to gold nanoparticles (AuNPs) to obtain constructs that interact homophilically with the extracellular domain of L1 and trigger the cognate beneficial L1-mediated functions. Covalent conjugation was achieved by reacting mixtures of two cysteine-terminated forms of this L1 peptide and thiolated poly(ethylene) glycol (PEG) ligands (~2.1 kDa) with citrate stabilized AuNPs of two different sizes (~14 and 40 nm in diameter). By varying the ratio of the L1 peptide-PEG mixtures, an optimized layer composition was achieved that resulted in the expected homophilic interaction of the AuNPs. These AuNPs were stable as tested over a time period of 30 days in artificial cerebrospinal fluid and interacted with the extracellular domain of L1 on neurons and Schwann cells, as could be shown by using cells from wild-type and L1-deficient mice. In vitro, the L1-derivatized particles promoted neurite outgrowth and survival of neurons from the central and peripheral nervous system and stimulated Schwann cell process formation and proliferation. These observations raise the hope that, in combination with other therapeutic approaches, L1 peptide-functionalized AuNPs may become a useful tool to ameliorate the deficits resulting from acute and chronic injuries of the mammalian nervous system.The neural cell adhesion molecule L1 is involved in nervous system development and promotes regeneration in animal models of acute and chronic injury of the adult nervous system. To translate these conducive functions into therapeutic approaches, a 22-mer peptide that encompasses a minimal and functional L1

  13. The Nucleotide Excision Repair Pathway Limits L1 Retrotransposition

    PubMed Central

    Servant, Geraldine; Streva, Vincent A.; Derbes, Rebecca S.; Wijetunge, Madushani I.; Neeland, Marc; White, Travis B.; Belancio, Victoria P.; Roy-Engel, Astrid M.; Deininger, Prescott L.

    2017-01-01

    Long interspersed elements 1 (L1) are active mobile elements that constitute almost 17% of the human genome. They amplify through a “copy-and-paste” mechanism termed retrotransposition, and de novo insertions related to these elements have been reported to cause 0.2% of genetic diseases. Our previous data demonstrated that the endonuclease complex ERCC1-XPF, which cleaves a 3′ DNA flap structure, limits L1 retrotransposition. Although the ERCC1-XPF endonuclease participates in several different DNA repair pathways, such as single-strand annealing, or in telomere maintenance, its recruitment to DNA lesions is best characterized in the nucleotide excision repair (NER) pathway. To determine if the NER pathway prevents the insertion of retroelements in the genome, we monitored the retrotransposition efficiencies of engineered L1 elements in NER-deficient cells and in their complemented versions. Core proteins of the NER pathway, XPD and XPA, and the lesion binding protein, XPC, are involved in limiting L1 retrotransposition. In addition, sequence analysis of recovered de novo L1 inserts and their genomic locations in NER-deficient cells demonstrated the presence of abnormally large duplications at the site of insertion, suggesting that NER proteins may also play a role in the normal L1 insertion process. Here, we propose new functions for the NER pathway in the maintenance of genome integrity: limitation of insertional mutations caused by retrotransposons and the prevention of potentially mutagenic large genomic duplications at the site of retrotransposon insertion events. PMID:28049704

  14. PD-L1 Status in Refractory Lymphomas

    PubMed Central

    Ghosh, Nilanjan; Kimbrough, Jeffery; Bilalovic, Nurija; Bender, Ryan; Arguello, David; Veloso, Yvonne; Dizdarevic, Aida; Gatalica, Zoran

    2016-01-01

    Targeted immunotherapy based on PD-1/PD-L1 suppression has revolutionized the treatment of various solid tumors. A remarkable improvement has also been observed in the treatment of patients with refractory/relapsing classical Hodgkin lymphoma (cHL). We investigated PD-L1 status in a variety of treatment resistant lymphomas. Tumor samples from 78 patients with therapy resistant lymphomas were immunohistochemically (IHC) investigated for the expression of PD-L1 using two antibody clones (SP142 and SP263, Ventana). Thirteen PD-L1+ cases were further analyzed for gene copy number variations (CNV) by NGS and for PD-L1/JAK2/PD-L2 co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity (≥5% positive cancer cells, IHC) was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently negative (3/3). Co-amplification/CNVs of PD-L1/JAK2/PD-L2 were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL. PMID:27861596

  15. Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier.

    PubMed

    Mooren, Olivia L; Li, Jinmei; Nawas, Julie; Cooper, John A

    2014-12-15

    The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells.

  16. PD-L1 expression in EBV-negative diffuse large B-cell lymphoma: clinicopathologic features and prognostic implications

    PubMed Central

    Xing, Wei; Dresser, Karen; Zhang, Rui; Evens, Andrew M.; Yu, Hongbo; Woda, Bruce A.; Chen, Benjamin J.

    2016-01-01

    Programmed cell death ligand 1 (PD-L1) is a cell surface glycoprotein that regulates the cellular immune response and serves as a targetable immune checkpoint molecule. PD-L1 is expressed on tumor cells and the immune microenvironment of several human malignancies, including a subset of aggressive lymphomas. We sought to investigate further the clinical and pathologic features of EBV-negative diffuse large B-cell lymphoma (DLBCL) cases that express PD-L1. Immunohistochemical staining using an anti-PD-L1 monoclonal antibody was performed on DLBCL cases from 86 patients. These patients received standard chemotherapy treatment and were followed for up to 175 months. Overall, 14 cases (16%) were considered positive for PD-L1 in tumor cells. In comparison with PD-L1 negative cases, PD-L1 positive cases had a higher rate of non-GCB type (71% vs. 30%, P=0.0060), and higher Ann Arbor stage (II-IV) (100% vs. 73%, P=0.0327). No significant differences were seen in the immunohistochemical expression of BCL2, MYC, or Ki67. Patients with tumors expressing PD-L1 demonstrated inferior overall survival (OS) upon long term follow up (P=0.0447). Both age/sex-adjusted and multivariate analyses identified PD-L1 as an independent predictor for OS (P=0.0101 and P=0.0424). There was no significant difference, however, in terms of remission rates after first treatment, relapse rates, and progression free survival between the groups. Identification of DLBCL cases that express PD-L1 may serve to select a subset of patients that could further benefit from targeted immunotherapy. PMID:27527850

  17. Mechanisms for the transendothelial migration of HIV-1-infected monocytes into brain.

    PubMed

    Nottet, H S; Persidsky, Y; Sasseville, V G; Nukuna, A N; Bock, P; Zhai, Q H; Sharer, L R; McComb, R D; Swindells, S; Soderland, C; Gendelman, H E

    1996-02-01

    HIV-1 penetration of the brain is a pivotal event in the neuropathogenesis of AIDS-associated dementia. The establishment of productive viral replication or up-regulation of adhesion molecule expression on brain microvascular endothelial cells (BMVEC) could permit entry of HIV into the central nervous system. To investigate the contribution of both, we inoculated primary human BMVEC with high titer macrophage-tropic HIV-1 or cocultured them with virus-infected monocytes. In both instances, BMVEC failed to demonstrate productive viral replication. Cell to cell contact between monocytes and microvascular endothelium resulted in E-selectin expression on BMVEC. BMVEC. cocultured with LPS-activated HIV-infected monocytes expressed even higher levels of E-selectin and vascular cell adhesion molecule-1 (VCAM-1). Transwell assays supported a role of soluble factors, from virus-infected monocytes, for the induction of adhesion molecules on BMVEC. To verify the in vivo relevance of these findings, levels of adhesion molecules were compared with those of proinflammatory cytokines and HIV-1 gene products in brain tissue of AIDS patients with or without encephalitis and HIV-seronegative controls. E-Selectin, and to a lesser degree VCAM-1, paralleled the levels of HIV-1 gene products and proinflammatory cytokines in brain tissue of subjects with encephalitis. Most importantly, an association between macrophage infiltration and increased endothelial cell adhesion molecules was observed in encephalitic brains. Monocyte binding to encephalitic brain tissue was blocked with Abs to VCAM-1 and E-selectin. These data, taken together, suggest that HIV entry into brain is, in part, a consequence of the ability of virus-infected and immune-activated monocytes to induce adhesion molecules on brain endothelium.

  18. Myeloid-specific deletion of tumor suppressor PTEN augments neutrophil transendothelial migration during inflammation.

    PubMed

    Sarraj, Bara; Massberg, Steffen; Li, Yitang; Kasorn, Anongnard; Subramanian, Kulandayan; Loison, Fabien; Silberstein, Leslie E; von Andrian, Ulrich; Luo, Hongbo R

    2009-06-01

    Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) is a second messenger that is involved in a number of cell activities including cell growth, proliferation, and motility. PIP(3) is produced by PI3K and regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP lipid phosphatases. Evidence from our experiments shows that enhanced PIP(3) production results in elevated neutrophil recruitment under inflammatory conditions. However, the mechanism of this elevation is not well understood. We used intravital video microscopy to investigate neutrophil recruitment in the cremaster venules of wild-type and PTEN knockout (KO) mice. Neutrophil transmigration was augmented in PTEN KO mice 4 h after TNF-alpha intrascrotal injection. PTEN KO neutrophils also showed significantly enhanced transmigration 2 h after MIP-2 intrascrotal injection, an effect that dramatically decreased when PI3K or Src kinase inhibitor treatments preceded MIP-2 stimulation. Similarly, fMLP superfusion of the cremaster muscle lead to enhanced emigration in PTEN KO mice. The observed elevation in neutrophil emigration was likely caused by increased speed of crawling, crossing the venular wall, and migrating through the muscular tissue in PTEN KO mice because the effect of PTEN depletion on neutrophil rolling or adhesion was minimal. Interestingly, chemoattractant-induced release of gelatinase and elastase was also elevated in PTEN null neutrophils, providing a potential mechanism for the enhanced neutrophil migration in the PTEN KO mice. Collectively, these results demonstrate that PTEN deletion in neutrophils enhances their invasivity and recruitment to inflamed sites more likely by raising the cell physical capability to cross the vascular and tissue barriers.

  19. Differential expression of immune-regulatory genes associated with PD-L1 display in melanoma: implications for PD-1 pathway blockade

    PubMed Central

    Taube, Janis M.; Young, Geoffrey D.; McMiller, Tracee L.; Chen, Shuming; Salas, January T.; Pritchard, Theresa S.; Xu, Haiying; Meeker, Alan K.; Fan, Jinshui; Cheadle, Chris; Berger, Alan E.; Pardoll, Drew M.; Topalian, Suzanne L.

    2015-01-01

    Purpose Blocking the immunosuppressive PD-1/PD-L1 pathway has anti-tumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was over-expressed by TILs in PD-L1+ vs. PD-L1(−) melanomas, creating adaptive immune resistance by promoting PD-L1 display. The current study was undertaken to identify additional factors in the PD-L1+ melanoma microenvironment coordinately contributing to immunosuppression. Experimental design Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with immunohistochemistry (IHC). Whole genome expression analysis, quantitative (q)RT-PCR, immunohistochemistry, and functional in vitro validation studies were employed to assess factors differentially expressed in PD-L1+ versus PD-L1(−) melanomas. Results Functional annotation clustering based on whole genome expression profiling revealed pathways up-regulated in PD-L1+ melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated over-expression of functionally related genes in PD-L1+ melanomas, involved in CD8+ T cell activation (CD8A, IFNG, PRF1, CCL5), antigen presentation (CD163, TLR3, CXCL1, LYZ), and immunosuppression [PDCD1 (PD-1), CD274(PD-L1), LAG3, IL10]. Functional studies demonstrated that some factors, including IL-10 and IL-32-gamma, induced PD-L1 expression on monocytes but not tumor cells. Conclusions These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti-PD-1/PD-L1 therapy, and should be considered for co-targeting in combinatorial immunomodulation treatment strategies. PMID:25944800

  20. Ubiquitin C-terminal hydrolase-L1 increases cancer cell invasion by modulating hydrogen peroxide generated via NADPH oxidase 4.

    PubMed

    Kim, Hyun Jung; Magesh, Venkataraman; Lee, Jae-Jin; Kim, Sun; Knaus, Ulla G; Lee, Kong-Joo

    2015-06-30

    This study explored the role of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in the production of ROS and tumor invasion. UCH-L1 was found to increase cellular ROS levels and promote cell invasion. Silencing UCH-L1, as well as inhibition of H2O2 generation by catalase or by DPI, a NOX inhibitor, suppressed the migration potential of B16F10 cells, indicating that UCH-L1 promotes cell migration by up-regulating H2O2 generation. Silencing NOX4, which generates H2O2, with siRNA eliminated the effect of UCH-L1 on cell migration. On the other hand, NOX4 overexpressed in HeLa cells happens to be ubiquitinated, and NOX4 following deubiquitination by UCH-L1, restored H2O2-generating activity. These in vitro findings are consistent with the results obtained in vivo with catalase (-/-) C57BL/6J mice. When H2O2 and UCH-L1 levels were independently varied in these animals, the former by infecting with H2O2-scavenging adenovirus-catalase, and the latter by overexpressing or silencing UCH-L1, pulmonary metastasis of B16F10 cells overexpressing UCH-L1 increased significantly in catalase (-/-) mice. In contrast, invasion did not increase when UCH-L1 was silenced in the B16F10 cells. These findings indicate that H2O2 levels regulated by UCH-L1 are necessary for cell invasion to occur and demonstrate that UCH-L1 promotes cell invasion by up-regulating H2O2 via deubiquitination of NOX4.

  1. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production.

    PubMed

    Saitoh, Tatsuya; Fujita, Naonobu; Jang, Myoung Ho; Uematsu, Satoshi; Yang, Bo-Gie; Satoh, Takashi; Omori, Hiroko; Noda, Takeshi; Yamamoto, Naoki; Komatsu, Masaaki; Tanaka, Keiji; Kawai, Taro; Tsujimura, Tohru; Takeuchi, Osamu; Yoshimori, Tamotsu; Akira, Shizuo

    2008-11-13

    Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.

  2. Compressed-sensed-domain L1-PCA video surveillance

    NASA Astrophysics Data System (ADS)

    Liu, Ying; Pados, Dimitris A.

    2015-05-01

    We consider the problem of foreground and background extraction from compressed-sensed (CS) surveillance video. We propose, for the first time in the literature, a principal component analysis (PCA) approach that computes the low-rank subspace of the background scene directly in the CS domain. Rather than computing the conventional L2-norm-based principal components, which are simply the dominant left singular vectors of the CS measurement matrix, we compute the principal components under an L1-norm maximization criterion. The background scene is then obtained by projecting the CS measurement vector onto the L1 principal components followed by total-variation (TV) minimization image recovery. The proposed L1-norm procedure directly carries out low-rank background representation without reconstructing the video sequence and, at the same time, exhibits significant robustness against outliers in CS measurements compared to L2-norm PCA.

  3. Oxidative stress inhibits adhesion and transendothelial migration, and induces apoptosis and senescence of induced pluripotent stem cells.

    PubMed

    Wu, Yi; Zhang, Xueqing; Kang, Xueling; Li, Ning; Wang, Rong; Hu, Tiantian; Xiang, Meng; Wang, Xinhong; Yuan, Wenjun; Chen, Alex; Meng, Dan; Chen, Sifeng

    2013-09-01

    Oxidative stress caused by cellular accumulation of reactive oxygen species (ROS) is a major contributor to disease and cell death. However, how induced pluripotent stem cells (iPSC) respond to different levels of oxidative stress is largely unknown. Here, we investigated the effect of H2 O2 -induced oxidative stress on iPSC function in vitro. Mouse iPSC were treated with H2 O2 (25-100 μmol/L). IPSC adhesion, migration, viability, apoptosis and senescence were analysed. Expression of adhesion-related genes, stress defence genes, and osteoblast- and adipocyte-associated genes were determined by reverse transcription polymerase chain reaction. The present study found that H2 O2 (25-100 μmol/L) decreased iPSC adhesion to matrix proteins and endothelial cells, and downregulated gene expression levels of adhesion-related molecules, such as integrin alpha 7, cadherin 1 and 5, melanoma cell adhesion molecule, vascular cell adhesion molecule 1, and monocyte chemoattractant protein-1. H2 O2 (100 μmol/L) decreased iPSC viability and inhibited the capacity of iPSC migration and transendothelial migration. iPSC were sensitive to H2 O2 -induced G2/M arrest, senescence and apoptosis when exposed to H2 O2 at concentrations above 25 μmol/L. H2 O2 increased the expression of stress defence genes, including catalase, cytochrome B alpha, lactoperoxidase and thioredoxin domain containing 2. H2 O2 upregulated the expression of osteoblast- and adipocyte-associated genes in iPSC during their differentiation; however, short-term H2 O2 -induced oxidative stress did not affect the protein expression of the pluripotency markers, octamer-binding transcription factor 4 and sex-determining region Y-box 2. The present results suggest that iPSC are sensitive to H2 O2 toxicity, and inhibition of oxidative stress might be a strategy for improving their functions.

  4. Differentiation-specific decrease in heat shock protein synthesis in 3T3-L1 cells

    SciTech Connect

    Sorhage, F.; Kim, J.; Liu, A.Y.C.; Chen, K.Y.

    1986-05-01

    The regulation of synthesis of heat shock proteins (HSPs) in 3T3-L1 preadipocytes (fibroblasts) and adipocytes was examined using the techniques of pulse labeling with (/sup 35/S)methionine followed by analysis of the pattern and amount of radioactivity incorporated by SDS-polyacrylamide gel electrophoresis and autoradiography. Exposure of the 3T3-L1 preadipocyte cultures either to elevated temperature (42..mu..C) or to the amino acid analogue canavanine (400 ..mu..g/ml), markedly induced the synthesis of six major HSPs with apparent molecular weights of 105,000, 89,000, 74,000, 72,000, 50,000, and 42,000. The time course of induction of the HSPs by canavanine was significantly delayed as compared to that of heat shock; maximal increase in synthesis of the HSPs was observed at 3-7 hrs after incubation at 42..mu..c and at 22-24 hrs after incubation with 400 ..mu..g/ml canavanine. The magnitude of induction of HSP in the differentiated adipocytes was significantly reduced as compared to that of the undifferentiated fibroblast cells. The reduced expression of HSPs in 3T3-L1 adipocytes appears to be related to the terminal adipogenic differentiation process. The phenomenon was not observed in the control 3T3-C2 cells nor in a transformed variant of the 3T3-L1 cells.

  5. CHI3L1 — EDRN Public Portal

    Cancer.gov

    CHI3L1, also known as chitinase, a secreted protein, is a carbohydrate-binding lectin with a preference for chitin. It may play a role in defense against pathogens, or in tissue remodeling. It may also play an important role in the capacity of cells to respond to and cope with changes in their environment. CHI3L1 is present in activated macrophages, articular chondrocytes, synovial cells as well as in liver. It is undetectable in muscle tissues, lung, pancreas, mononuclear cells, or fibroblasts.

  6. The Total Variation Regularized L1 Model for Multiscale Decomposition

    DTIC Science & Technology

    2006-01-01

    measure theory approach, and Vixie and Esedoglu’s [43] work on characterizing the solutions of (1.2) below. In this paper we extend the existing...no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control ...2r . THE TV-L1 MODEL FOR MULTISCALE DECOMPOSITION 5 In general the minimizer of the TV-L1 is nonunique . In the above disk example, if λ = 2/r

  7. MicroRNA-184 Modulates Doxorubicin Resistance in Osteosarcoma Cells by Targeting BCL2L1

    PubMed Central

    Lin, Bo-chuan; Huang, Dong; Yu, Chao-qun; Mou, Yong; Liu, Yuan-hang; Zhang, Da-wei; Shi, Feng-jun

    2016-01-01

    Background Early metastasis of osteosarcoma (OS) is highly lethal and responds poorly to drug and radiation therapies. MicroRNAs (miRNAs) are a class of small noncoding RNAs that modulate gene expression at the post-transcriptional level. However, the detailed functions of specific miRNAs are not entirely understood. The aim of the present study was to investigate the role of miR-184 as a mediator of drug resistance in human osteosarcoma. Material/Methods qRT-PCR was used to analyze the expression level of miR-184 in OS cell line U-2 OS and MG-63 treated with doxorubicin. MiR-184 agomir or miR-184 antagomir was transferred into cells to regulated miR-184. The target of miR-184 was predicted by TargetScan and confirmed by luciferase reporter assay. Bcl-2-like protein 1 (BCL2L1) expression was detected by Western blot. Cell apoptosis was determined by Annexin V staining and analysis by flow cytometry. Results Doxorubicin induced time-dependent expression of miR-184 in OS cell line U-2 OS and MG-63. Luciferase reporter assay identified BCL2L1 as the direct target gene of miR-184. Furthermore, doxorubicin reduced BCL2L1 expression, which was reversed by miR-184 overexpression and further decreased by miR-184 inhibition in OS cells. In addition, miR-184 agomir reduced doxorubicin-induced cell apoptosis, whereas miR-184 antagomir enhanced apoptosis in OS cells, suggesting that up-regulation of miR-184 contributes to chemoresistance of the OS cell line. Conclusions Our data show that miR-184 was up-regulated in OS patients treated with doxorubicin therapy and leads to poor response to drug therapy by targeting BCL2L1. PMID:27222034

  8. TOM1L1 drives membrane delivery of MT1-MMP to promote ERBB2-induced breast cancer cell invasion

    PubMed Central

    Chevalier, Clément; Collin, Guillaume; Descamps, Simon; Touaitahuata, Heiani; Simon, Valérie; Reymond, Nicolas; Fernandez, Laurent; Milhiet, Pierre-Emmanuel; Georget, Virginie; Urbach, Serge; Lasorsa, Laurence; Orsetti, Béatrice; Boissière-Michot, Florence; Lopez-Crapez, Evelyne; Theillet, Charles; Roche, Serge; Benistant, Christine

    2016-01-01

    ERBB2 overexpression in human breast cancer leads to invasive carcinoma but the mechanism is not clearly understood. Here we report that TOM1L1 is co-amplified with ERBB2 and defines a subgroup of HER2+/ER+ tumours with early metastatic relapse. TOM1L1 encodes a GAT domain-containing trafficking protein and is a SRC substrate that negatively regulates tyrosine kinase signalling. We demonstrate that TOM1L1 upregulation enhances the invasiveness of ERBB2-transformed cells. This pro-tumoural function does not involve SRC, but implicates membrane-bound membrane-type 1 MMP (MT1-MMP)-dependent activation of invadopodia, membrane protrusions specialized in extracellular matrix degradation. Mechanistically, ERBB2 elicits the indirect phosphorylation of TOM1L1 on Ser321. The phosphorylation event promotes GAT-dependent association of TOM1L1 with the sorting protein TOLLIP and trafficking of the metalloprotease MT1-MMP from endocytic compartments to invadopodia for tumour cell invasion. Collectively, these results show that TOM1L1 is an important element of an ERBB2-driven proteolytic invasive programme and that TOM1L1 amplification potentially enhances the metastatic progression of ERBB2-positive breast cancers. PMID:26899482

  9. Transcriptional activity of novel ALDH1L1 promoters in the rat brain following AAV vector-mediated gene transfer

    PubMed Central

    Mudannayake, Janitha M; Mouravlev, Alexandre; Fong, Dahna M; Young, Deborah

    2016-01-01

    Aldehyde dehydrogenase family 1, member L1 (ALDH1L1) is a recently characterized pan-astrocytic marker that is more homogenously expressed throughout the brain than the classic astrocytic marker, glial fibrillary acidic protein. We generated putative promoter sequence variants of the rat ALDH1L1 gene for use in adeno-associated viral vector-mediated gene transfer, with an aim to achieve selective regulation of transgene expression in astrocytes in the rat brain. Unexpectedly, ALDH1L1 promoter variants mediated transcriptional activity exclusively in neurons in the substantia nigra pars compacta as assessed by luciferase reporter expression at 3 weeks postvector infusion. This selectivity for neurons in the substantia nigra pars compacta also persisted in the context of adeno-associated viral serotype 5, 8 or 9 vector-mediated gene delivery. An in vivo promoter comparison showed the highest performing ALDH1L1 promoter variant mediated higher transgene expression than the neuronal-specific synapsin 1 and tyrosine hydroxylase promoters. The ALDH1L1 promoter was also transcriptionally active in dentate granule neurons following intrahippocampal adeno-associated viral vector infusion, whereas transgene expression was detected in both striatal neurons and astrocytes following vector infusion into the striatum. Our results demonstrate the potential suitability of the ALDH1L1 promoter as a new tool in the development of gene therapy and disease modelling applications. PMID:27990448

  10. L1 adaptive output-feedback control architectures

    NASA Astrophysics Data System (ADS)

    Kharisov, Evgeny

    This research focuses on development of L 1 adaptive output-feedback control. The objective is to extend the L1 adaptive control framework to a wider class of systems, as well as obtain architectures that afford more straightforward tuning. We start by considering an existing L1 adaptive output-feedback controller for non-strictly positive real systems based on piecewise constant adaptation law. It is shown that L 1 adaptive control architectures achieve decoupling of adaptation from control, which leads to bounded away from zero time-delay and gain margins in the presence of arbitrarily fast adaptation. Computed performance bounds provide quantifiable performance guarantees both for system output and control signal in transient and steady state. A noticeable feature of the L1 adaptive controller is that its output behavior can be made close to the behavior of a linear time-invariant system. In particular, proper design of the lowpass filter can achieve output response, which almost scales for different step reference commands. This property is relevant to applications with human operator in the loop (for example: control augmentation systems of piloted aircraft), since predictability of the system response is necessary for adequate performance of the operator. Next we present applications of the L1 adaptive output-feedback controller in two different fields of engineering: feedback control of human anesthesia, and ascent control of a NASA crew launch vehicle (CLV). The purpose of the feedback controller for anesthesia is to ensure that the patient's level of sedation during surgery follows a prespecified profile. The L1 controller is enabled by anesthesiologist after he/she achieves sufficient patient sedation level by introducing sedatives manually. This problem formulation requires safe switching mechanism, which avoids controller initialization transients. For this purpose, we used an L1 adaptive controller with special output predictor initialization routine

  11. Chemotaxis and transendothelial migration of CD34(+) hematopoietic progenitor cells induced by the inflammatory mediator leukotriene D4 are mediated by the 7-transmembrane receptor CysLT1.

    PubMed

    Bautz, F; Denzlinger, C; Kanz, L; Möhle, R

    2001-06-01

    Recent studies suggest that bone marrow (BM)-derived chemotactic mediators such as chemokines play key roles in hematopoietic stem cell trafficking. Lipid mediators, particularly leukotrienes, are involved in leukocyte chemotaxis during inflammation but have also been detected in the normal BM. Therefore, the effects of leukotrienes on hematopoietic progenitor cells were analyzed. Cysteinyl leukotrienes, particularly leukotriene D4 (LTD4), induced strong intracellular calcium fluxes and actin polymerization in mobilized and BM CD34(+) progenitors. Chemotaxis and in vitro transendothelial migration of CD34(+) and more primitive CD34(+)/CD38(-) cells were 2-fold increased by LTD4 at an optimum concentration of 25 to 50 nM. Accordingly, CD34(+) cells expressed the 7-transmembrane LTD4 receptor CysLT1 by reverse transcriptase-polymerase chain reaction and Western blot. Effects of LTD4 were suppressed by the CysLT1 receptor antagonist MK-571 and reduced by pertussis toxin. In contrast, LTB4 induced strong responses only in mature granulocytes. LTD4-induced calcium fluxes were also observed in granulocytes but were not reduced by MK-571, suggesting that these effects were mediated by other receptors (eg, CysLT2) rather than by CysLT1. In addition, expression of 5-lipoxygenase, the key enzyme of leukotriene biosynthesis, was detected in both hematopoietic progenitor cells and mature leukocytes. The study concludes that the functionally active LTD4 receptor CysLT1 is preferentially expressed in immature hematopoietic progenitor cells. LTD4 released in the BM might regulate progenitor cell trafficking and could also act as an autocrine mediator of hematopoiesis. This would be a first physiologic effect of cysteinyl leukotrienes apart from the many known pathophysiologic actions related to allergy and inflammation. (Blood. 2001;97:3433-3440)

  12. Berberine reduces the expression of adipogenic enzymes and inflammatory molecules of 3T3-L1 adipocyte.

    PubMed

    Choi, Bong-Hyuk; Ahn, In-Sook; Kim, Yu-Hee; Park, Ji-Won; Lee, So-Young; Hyun, Chang-Kee; Do, Myoung-Sool

    2006-12-31

    Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 micrometer, 5 micrometer and 10 micrometer concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-alpha, PPAR-gamma, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-alpha, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.

  13. Raising Learners' Awareness through L1-L2 Teacher Collaboration

    ERIC Educational Resources Information Center

    Gunning, Pamela; White, Joanna; Busque, Christine

    2016-01-01

    There is considerable interest in teacher collaboration across mother tongue and second language curricula. However, cross-curricular collaboration in reading strategy instruction has seldom been investigated. We report a two-year study involving collaboration between the French first language (L1) and English second language (L2) teachers in an…

  14. A Characterization of Banach Spaces Containing l1

    PubMed Central

    Rosenthal, Haskell P.

    1974-01-01

    It is proved that a Banach space contains a subspace isomorphic to l1 if (and only if) it has a bounded sequence with no weak-Cauchy subsequence. The proof yields that a sequence of subsets of a given set has a subsequence that is either convergent or Boolean independent. PMID:16592162

  15. Conceptualising the Potential Role of L1 in CLIL

    ERIC Educational Resources Information Center

    Lin, Angel M. Y.

    2015-01-01

    Content and language integrated learning (CLIL) is a rapidly growing area of both research and practice in all parts of the world, especially in Europe and Asia. As a young discipline, CLIL has a good potential of distinguishing itself from monolingual L2 immersion education models by becoming more flexible and balanced about the role of L1 in…

  16. On the L1 Attrition of the Spanish Present Tense

    ERIC Educational Resources Information Center

    Cuza, Alejandro

    2010-01-01

    This study examines the potential native language (L1) attrition of the ongoing value of the Spanish present tense among long-term Spanish immigrants. Based on the assumption of second-language (L2) transfer and proposals on the permeability of interface-conditioned structures, it is hypothesized that long-term Spanish immigrants will show…

  17. L1-L2 Sentence Translation in Classroom Grammar Tests

    ERIC Educational Resources Information Center

    Salem, Ilana

    2012-01-01

    L1-L2 translation of separate sentences is one kind of task format used by mainstream EFL teachers to assess their learners' grammatical accuracy. Aimed at improving teacher-written translation items, this study analyses linguistic features potentially causing such decontextualized cues (and their target responses) to sound odd or untypical of…

  18. Discourse Connectives in L1 and L2 Argumentative Writing

    ERIC Educational Resources Information Center

    Hu, Chunyu; Li, Yuanyuan

    2015-01-01

    Discourse connectives (DCs) are multi-functional devices used to connect discourse segments and fulfill interpersonal levels of discourse. This study investigates the use of selected 80 DCs within 11 categories in the argumentative essays produced by L1 and L2 university students. The analysis is based on the International Corpus Network of Asian…

  19. Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes.

    PubMed

    Kim, So Hui; Shin, Eun-Jung; Kim, Eun-Do; Bayaraa, Tsenguun; Frost, Susan Cooke; Hyun, Chang-Kee

    2007-11-01

    It has recently been known that berberine, an alkaloid of medicinal plants, has anti-hyperglycemic effects. To explore the mechanism underlying this effect, we used 3T3-L1 adipocytes for analyzing the signaling pathways that contribute to glucose transport. Treatment of berberine to 3T3-L1 adipocytes for 6 h enhanced basal glucose uptake both in normal and in insulin-resistant state, but the insulin-stimulated glucose uptake was not augmented significantly. Inhibition of phosphatidylinositol 3-kinase (PI 3-K) by wortmannin did not affect the berberine effect on basal glucose uptake. Berberine did not augment tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate (IRS)-1. Further, berberine had no effect on the activity of the insulin-sensitive downstream kinase, atypical protein kinase C (PKCzeta/lambda). However, interestingly, extracellular signal-regulated kinases (ERKs), which have been known to be responsible for the expression of glucose transporter (GLUT)1, were significantly activated in berberine-treated 3T3-L1 cells. As expected, the level of GLUT1 protein was increased both in normal and insulin-resistant cells in response to berberine. But berberine affected the expression of GLUT4 neither in normal nor in insulin-resistant cells. In addition, berberine treatment increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 cells, which has been reported to be associated with GLUT1-mediated glucose uptake. Together, we concluded that berberine increases glucose transport activity of 3T3-L1 adipocytes by enhancing GLUT1 expression and also stimulates the GLUT1-mediated glucose uptake by activating GLUT1, a result of AMPK stimulation.

  20. Enterobacter asburiae Strain L1: Complete Genome and Whole Genome Optical Mapping Analysis of a Quorum Sensing Bacterium

    PubMed Central

    Lau, Yin Yin; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    Enterobacter asburiae L1 is a quorum sensing bacterium isolated from lettuce leaves. In this study, for the first time, the complete genome of E. asburiae L1 was sequenced using the single molecule real time sequencer (PacBio RSII) and the whole genome sequence was verified by using optical genome mapping (OpGen) technology. In our previous study, E. asburiae L1 has been reported to produce AHLs, suggesting the possibility of virulence factor regulation which is quorum sensing dependent. This evoked our interest to study the genome of this bacterium and here we present the complete genome of E. asburiae L1, which carries the virulence factor gene virK, the N-acyl homoserine lactone-based QS transcriptional regulator gene luxR and the N-acyl homoserine lactone synthase gene which we firstly named easI. The availability of the whole genome sequence of E. asburiae L1 will pave the way for the study of the QS-mediated gene expression in this bacterium. Hence, the importance and functions of these signaling molecules can be further studied in the hope of elucidating the mechanisms of QS-regulation in E. asburiae. To the best of our knowledge, this is the first documentation of both a complete genome sequence and the establishment of the molecular basis of QS properties of E. asburiae. PMID:25196111

  1. Rac1 activation driven by 14-3-3ζ dimerization promotes prostate cancer cell-matrix interactions, motility and transendothelial migration.

    PubMed

    Goc, Anna; Abdalla, Maha; Al-Azayzih, Ahmad; Somanath, Payaningal R

    2012-01-01

    14-3-3 proteins are ubiquitously expressed dimeric adaptor proteins that have emerged as key mediators of many cell signaling pathways in multiple cell types. Its effects are mainly mediated by binding to selective phosphoserine/threonine proteins. The importance of 14-3-3 proteins in cancer have only started to become apparent and its exact role in cancer progression as well as the mechanisms by which 14-3-3 proteins mediate cancer cell function remain unknown. While protein 14-3-3σ is widely accepted as a tumor suppressor, 14-3-3ζ, β and γ isoforms have been shown to have tumor promoting effects. Despite the importance of 14-3-3 family in mediating various cell processes, the exact role and mechanism of 14-3-3ζ remain unexplored. In the current study, we investigated the role of protein 14-3-3ζ in prostate cancer cell motility and transendothelial migration using biochemical, molecular biology and electric cell-substrate impedance sensing approaches as well as cell based functional assays. Our study indicated that expression with wild-type protein 14-3-3ζ significantly enhanced Rac activity in PC3 cells. In contrast, expression of dimer-resistant mutant of protein 14-3-3ζ (DM-14-3-3) inhibited Rac activity and associated phosphorylation of p21 activated kinase-1 and 2. Expression with wild-type 14-3-3ζ or constitutively active Rac1 enhanced extracellular matrix recognition, lamellipodia formation, cell migration and trans-endothelial migration by PC3 cells. In contrast, expression with DM 14-3-3ζ or DN-Rac1 in PC3 cells significantly inhibited these cell functions. Our results demonstrate for the first time that 14-3-3ζ enhances prostate cancer cell-matrix interactions, motility and transendothelial migration in vitro via activation of Rac1-GTPase and is an important target for therapeutic interventions for prostate cancer.

  2. Gene Expression Patterns Induced by HPV-16 L1 VLP in Leukocytes from Vaccine Recipients

    PubMed Central

    García-Piñeres, Alfonso J.; Hildesheim, Allan; Dodd, Lori; Kemp, Troy J.; Yang, Jun; Fullmer, Brandie; Harro, Clayton; Lowy, Douglas R.; Lempicki, Richard A.; Pinto, Ligia A.

    2009-01-01

    Human papilloma (HPV) virus-like particle (VLP) vaccines were recently licensed. Though neutralizing antibody titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMC from 17 vaccine and 4 placebo recipients before vaccination, and 1 month after receiving the second immunization. Vaccination with a monovalent HPV-16 L1 VLP vaccine was associated with modulation of genes involved in the inflammatory/defense response, cytokine, interferon and cell cycle pathways in VLP-stimulated PBMC. Additionally, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing antibody titers were found for cyclin d2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with microarray data was seen for over two-thirds of these probesets. Up-regulation of immune/defense response genes by HPV-16 L1 VLP, in particular interferon-induced genes was observed in PBMC collected prior to vaccination, with many of these genes being further induced following vaccination. In conclusion, we identified important innate and adaptive response related- genes induced by vaccination with HPV-16 L1 VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials. PMID:19155521

  3. PD-1 and PD-L1 antibodies for melanoma

    PubMed Central

    Tsai, Katy K; Zarzoso, Inés; Daud, Adil I

    2015-01-01

    Melanoma is the most serious form of skin cancer. Metastatic melanoma historically carries a poor prognosis and until recently there have been few effective agents available to treat widely disseminated disease. Recognition of the immunogenic nature of melanoma has resulted in the development of various immunotherapeutic approaches, especially with regards to the programmed cell death 1 (PD-1) receptor and its ligand (PD-L1). Antibodies targeting the PD-1 axis have shown enormous potential in the treatment of metastatic melanoma. Here, we will review the immune basis for the disease and discuss approved immunotherapeutic options for advanced melanoma, as well as the current state of development of PD-1 and PD-L1 antibodies and their importance in shaping the future of melanoma treatment. PMID:25625924

  4. L1-Regularized Boltzmann Machine Learning Using Majorizer Minimization

    NASA Astrophysics Data System (ADS)

    Ohzeki, Masayuki

    2015-05-01

    We propose an inference method to estimate sparse interactions and biases according to Boltzmann machine learning. The basis of this method is L1 regularization, which is often used in compressed sensing, a technique for reconstructing sparse input signals from undersampled outputs. L1 regularization impedes the simple application of the gradient method, which optimizes the cost function that leads to accurate estimations, owing to the cost function's lack of smoothness. In this study, we utilize the majorizer minimization method, which is a well-known technique implemented in optimization problems, to avoid the non-smoothness of the cost function. By using the majorizer minimization method, we elucidate essentially relevant biases and interactions from given data with seemingly strongly-correlated components.

  5. Mechanistic studies of ubiquitin C-terminal hydrolase L1.

    PubMed

    Case, April; Stein, Ross L

    2006-02-21

    Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

  6. Least-squares RTM with L1 norm regularisation

    NASA Astrophysics Data System (ADS)

    Wu, Di; Yao, Gang; Cao, Jingjie; Wang, Yanghua

    2016-10-01

    Reverse time migration (RTM), for imaging complex Earth models, is a reversal procedure of the forward modelling of seismic wavefields, and hence can be formulated as an inverse problem. The least-squares RTM method attempts to minimise the difference between the observed field data and the synthetic data generated by the migration image. It can reduce the artefacts in the images of a conventional RTM which uses an adjoint operator, instead of an inverse operator, for the migration. However, as the least-squares inversion provides an average solution with minimal variation, the resolution of the reflectivity image is compromised. This paper presents the least-squares RTM method with a model constraint defined by an L1-norm of the reflectivity image. For solving the least-squares RTM with L1 norm regularisation, the inversion is reformulated as a ‘basis pursuit de-noise (BPDN)’ problem, and is solved directly using an algorithm called ‘spectral projected gradient for L1 minimisation (SPGL1)’. Three numerical examples demonstrate the effectiveness of the method which can mitigate artefacts and produce clean images with significantly higher resolution than the least-squares RTM without such a constraint.

  7. Salmonella induces PD-L1 expression in B cells.

    PubMed

    Lopez-Medina, Marcela; Perez-Lopez, Araceli; Alpuche-Aranda, Celia; Ortiz-Navarrete, Vianney

    2015-10-01

    Salmonella persists for a long time in B cells; however, the mechanism(s) through which infected B cells avoid effector CD8 T cell responses has not been characterized. In this study, we show that Salmonella infects and survives within all B1 and B2 cell subpopulations. B cells are infected with a Salmonella typhimurium strain expressing an ovalbumin (OVA) peptide (SIINFEKL) to evaluate whether B cells process and present Salmonella antigens in the context of MHC-I molecules. Our data showed that OVA peptides are presented by MHC class I K(b)-restricted molecules and the presented antigen is generated through proteasomal degradation and vacuolar processing. In addition, Salmonella-infected B cells express co-stimulatory molecules such as CD40, CD80, and CD86 as well as inhibitory molecules such as PD-L1. Thus, the cross-presentation of Salmonella antigens and the expression of activation molecules suggest that infected B cells are able to prime and activate specific CD8(+) T cells. However, the Salmonella infection-stimulated expression of PD-L1 suggests that the PD-1/PD-L1 pathway may be involved in turning off the cytotoxic effector response during Salmonella persistent infection, thereby allowing B cells to become a reservoir for the bacteria.

  8. Effect of Copper Sulfate on Expression of Endogenous L1 Retrotransposons in HepG2 Cells (Hepatocellular Carcinoma).

    PubMed

    Karimi, Abbas; Majidzadeh-A, Keivan; Madjd, Zahra; Akbari, Abolfazl; Habibi, Laleh; Akrami, Seyed Mohammad

    2015-06-01

    The long interspersed element-1 (LINE-1 or L1) constitutes approximately 17% of human genome. The expression of these elements is deregulated upon exposure to environmental exposures resulting to genomic instability and cancer promotion. The effect of copper as essential elements in regulation of L1 expression remained to be elucidated. Using non-cytotoxic concentrations of the copper, the expression of endogenous L1 was analyzed by qPCR after 6 days of copper pretreatment in human hepatocellular carcinoma cells (HepG2). The results indicated that the expression of active L1 elements are significantly downregulated at concentrations of 12.5, 25, and 50 μM (p < 0.005). Our data imply that low-level copper exposure may have a protective effect to suppress the induction of L1 activity and decrease incidence of cancer-associated L1 mutagenesis. If this achievement is confirmed by further studies, it can be applied in the long-term goals of cancer prevention.

  9. L1 retrotransposon expression in circulating tumor cells

    PubMed Central

    Papasotiriou, Ioannis; Pantopikou, Katerina; Apostolou, Panagiotis

    2017-01-01

    Long interspersed nuclear element 1 (LINE-1 or L1) belongs to the non-long terminal repeat (non-LTR) retrotransposon family, which has been implicated in carcinogenesis and disease progression. Circulating tumor cells (CTCs) are also known to be involved in cancer progression. The present study aimed to compare the L1 expression between circulating tumor cells and non-cancerous samples. Blood samples were collected from 10 healthy individuals and 22 patients with different types of cancer. The whole blood cells were isolated using enrichment protocols and the DNA and RNA were extracted. RT-qPCR was performed for L1-ORF1 (open reading frame 1) and L1-ORF2, using 18S rRNA as the reference gene. The data were analyzed with the Livak method and statistical analyses were carried out with the Mann-Whitney and Kruskal-Wallis tests. In parallel with the above molecular biology experiments, FISH experiments were performed on the interphase nuclei of the cells for the detection of ORF2 RNA. DNA analysis revealed the presence of both ORF1 and ORF2 in all samples. RNA expression experiments demonstrated that ORF1 was not expressed in all samples, while ORF2 was expressed at varying levels in the non-cancer samples and the samples representing the different cancer types. A significant difference in ORF2 expression was observed between the CTCs and non-cancer samples (p = 0,00043), and significant differences were also observed between normal and lung (p = 0,034), pancreatic (p = 0,022), prostate (p = 0,014), and unknown primary of origin (p = 0,0039) cancer samples. Cytogenetic analysis revealed higher levels of ORF2 in the nuclei of CTCs than in normal samples. This study highlights the significant difference in L1-ORF2 expression between CTCs and normal samples. The increased expression levels observed for CTCs may be correlated with the characteristic features of these cells. PMID:28166262

  10. Ubiquitin C-terminal hydrolase l1 is expressed in mouse pituitary gonadotropes in vivo and gonadotrope cell lines in vitro.

    PubMed

    Xu, Yang; Hideshima, Makoto; Ishii, Yoshiyuki; Yoshikawa, Yasuhiro; Kyuwa, Shigeru

    2014-01-01

    The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating various biological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly and restrictedly expressed in neural and reproductive tissues and plays significant roles in these organs. Although the expression of UCH-L1 in the anterior pituitary gland has been reported, the detailed localization and the role of UCH-L1 remain obscure. In the present study, we detected UCH-L1 protein exclusively in hormone-producing cells, but not non-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition, the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes and mammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed a comparative analysis using genetically UCH-L1-deficient gad mice. Significant decreases in the numbers of gonadotropes and mammotropes were observed in gad mice, suggesting a close involvement of UCH-L1 in these cells. Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Taken together, this is the first report to definitely demonstrate the presence of UCH-L1 in mouse anterior pituitary gland, and our results might provide a novel insight for better understanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in the reproduction.

  11. Interferon inhibits the conversion of 3T3-L1 mouse fibroblasts into adipocytes.

    PubMed Central

    Keay, S; Grossberg, S E

    1980-01-01

    Confluent Swiss mouse 3T3-L1 fibroblasts slowly differentiate functionally and morphologically into adipocytes, a conversion hastened by insulin. The cells are sensitive (although less than L929 cells) to the antiviral action of mouse fibroblast interferons but not to interferons from heterologous species (human and chicken). Cultures stimulated with insulin in the presence of partially purified or electrophoretically pure mouse interferons have a much lower percentage of cells accumulating lipid than do insulin-treated control cultures. Interferon-treated cell cultures also contain much less triglyceride, cholesterol, and cholesterol esters than do replicate control cultures stimulated by insulin to differentiate. Increased de novo lipid biosynthesis that occurs during differentiation is inhibited, as determined by incorporation of [14C]acetate into lipids extractable by the Folch method. This incorporation is a sensitive bioassay of the antidifferentiation effect of interferon; less than 1 antiviral unit is inhibitory. Variously inactivated or mock interferon preparations as well as interferons from several heterologous species fail to inhibit 3T3-L1 adipocyte conversion. Interferon is inhibitory even when applied as long as 3 days after insulin stimulation. The effect of interferon does not appear to depend upon its competition with insulin for cell surface receptors. Because interferon can alter the program of events involved in conversion of 3T3-L1 fibroblasts into adipose cells, it may be able to affect the regulation of eukaryotic cell differentiation. Images PMID:6159626

  12. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayoshi; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka

    2013-02-01

    Highlights: ► Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ► Adipose lipin-1 expression is reduced in obesity. ► ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ► Activation of PPAR-γ recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-α and interleukin-1β reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

  13. Detection of L1, infectious virions and anti-L1 antibody in domestic rabbits infected with cottontail rabbit papillomavirus.

    PubMed

    Hu, Jiafen; Budgeon, Lynn R; Cladel, Nancy M; Culp, Timothy D; Balogh, Karla K; Christensen, Neil D

    2007-12-01

    Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections.

  14. Programmed Death Ligand 1 (PD-L1)-targeted TRAIL combines PD-L1-mediated checkpoint inhibition with TRAIL-mediated apoptosis induction

    PubMed Central

    Hendriks, Djoke; He, Yuan; Koopmans, Iris; Wiersma, Valerie R.; van Ginkel, Robert J.; Samplonius, Douwe F.; Helfrich, Wijnand; Bremer, Edwin

    2016-01-01

    ABSTRACT Antibodies that block PD-L1/PD-1 immune checkpoints restore the activity of functionally-impaired antitumor T cells. These antibodies show unprecedented clinical benefit in various advanced cancers, particularly in melanoma. However, only a subset of cancer patients responds to current PD-L1/PD-1-blocking strategies, highlighting the need for further advancements in PD-L1/PD-1-based immunotherapy. Here, we report on a novel approach designed to combine PD-L1 checkpoint inhibition with the tumor-selective induction of apoptosis by TNF-related Apoptosis Inducing Ligand (TRAIL). In brief, a new bi-functional fusion protein, designated anti-PD-L1:TRAIL, was constructed comprising a PD-L1-blocking antibody fragment genetically fused to the extracellular domain of the pro-apoptotic tumoricidal protein TRAIL. Treatment of PD-L1-expressing cancer cells with anti-PD-L1:TRAIL induced PD-L1-directed TRAIL-mediated cancer cell death. Treatment of T cells with anti-PD-L1:TRAIL augmented T cell activation, as evidenced by increased proliferation, secretion of IFNγ and enhanced killing of cancer cell lines and primary patient-derived cancer cells in mixed T cell/cancer cell culture experiments. Of note, elevated levels of IFNγ further upregulated PD-L1 on cancer cells and simultaneously sensitized cancer cells to TRAIL-mediated apoptosis by anti-PD-L1:TRAIL. Additionally, anti-PD-L1:TRAIL converted immunosuppressive PD-L1-expressing myeloid cells into pro-apoptotic effector cells that triggered TRAIL-mediated cancer cell death. In conclusion, combining PD-L1 checkpoint inhibition with TRAIL-mediated induction of apoptosis using anti-PD-L1:TRAIL yields promising multi-fold and mutually reinforcing anticancer activity that may be exploited to enhance the efficacy of therapeutic PD-L1/PD-1 checkpoint inhibition. PMID:27622071

  15. The opiorphin gene (ProL1) and its homologues function in erectile physiology

    PubMed Central

    Tong, Yuehong; Tar, Moses; Melman, Arnold; Davies, Kelvin

    2010-01-01

    OBJECTIVE To determine if ProL1, a member of the opiorphin family of genes, can modulate erectile physiology, as it encodes a peptide which acts as a neutral endopeptidase inhibitor, other examples of which (Vcsa1, hSMR3A) modulate erectile physiology. MATERIALS AND METHODS We cloned members of the opiorphin family of genes into the same mammalian expression backbone (pVAX); 100 μg of these plasmids (pVAX-Vcsa1, -hSMR3A, -hSMR3B and -ProL1) were injected intracorporally into retired breeder rats and the affect on erectile physiology assessed visually, by histology and by measuring the intracavernous pressure (ICP) and blood pressure (BP). As a positive control, rats were treated with pVAX-hSlo (expressing the MaxiK potassium channel) and as a negative control the empty backbone plasmid was injected (pVAX). We also compared the level of expression of ProL1 in corporal tissue of patients not reporting erectile dysfunction (ED), ED associated with diabetes and ED not caused by diabetes. RESULTS Gene transfer of plasmids expressing all members of the opiorphin family had a similar and significant effect on erectile physiology. At the concentration used in these experiments (100 μg) they resulted in higher resting ICP, and histological and visual analysis showed evidence of a priapiclike condition. After electrostimulation of the cavernous nerve, rats had significantly better ICP/BP than the negative control (pVAX). Gene transfer of pVAX-hSlo increased the ICP/BP ratio to a similar extent to the opiorphin homologues, but with no evidence for a priapic-like condition. Corpora cavernosa tissue samples obtained from men with ED, regardless of underlying causes, had significant down-regulation of both hSMR3A and ProL1. CONCLUSION All members of the human opiorphin family of genes can potentially modulate erectile physiology. Both hSMR3 and ProL1 are down-regulated in the corpora of men with ED, and therefore both genes can potentially act as markers of ED. PMID:18410445

  16. The FPGA based L1 track finding Tracklet approach

    NASA Astrophysics Data System (ADS)

    Kyriacou, Savvas; CMS Collaboration

    2017-01-01

    The High Luminosity upgraded LHC is expected to deliver proton-proton collisions per 25ns with an estimated 140-200 pile up interactions per bunch crossing. Ultrafast track finding is vital for handling trigger rates in such conditions. An FPGA based road search algorithm is developed, the Tracklet approach one of a few currently under consideration, for the CMS L1 trigger system. Based on low/high transverse momentum track discrimination and designed for the HL upgraded outer tracker, the algorithm achieves microsecond scale track reconstruction in the expected high track multiplicity environment. The Tracklet method overview, implementation, hardware demonstrator and performance results are presented and discussed.

  17. PD-L1 expression on immune cells, but not on tumor cells, is a favorable prognostic factor for head and neck cancer patients

    PubMed Central

    Kim, Hye Ryun; Ha, Sang-Jun; Hong, Min Hee; Heo, Su Jin; Koh, Yoon Woo; Choi, Eun Chang; Kim, Eun Kyung; Pyo, Kyoung Ho; Jung, Inkyung; Seo, Daekwan; Choi, Jaewoo; Cho, Byoung Chul; Yoon, Sun Och

    2016-01-01

    To investigate the expression of programmed death-ligand 1 (PD-L1) and immune checkpoints and their prognostic value for resected head and neck squamous cell cancer (HNSCC). PD-L1 expression on tumor cells (TC) and tumor-infiltrating immune cells (IC), abundance of tumor-infiltrating lymphocytes (TILs), and expression of the immune checkpoints were investigated in 402 HNSCC patients. PD-L1 expression on TC and IC was categorized into four groups according to the percentage of PD-L1-positive cells. PD-L1 positivity was defined as ≥5% of cells based on immunohistochemistry. High PD-L1 expression on IC, but not TC, was an independent favorable prognostic factor for RFS and OS adjusted for age, gender, smoking, stage, and HPV. High frequencies of CD3+ or CD8+ TILs, Foxp3+ Tregs, and PD-1+ TILs were strongly associated with favorable prognosis. PD-L1 was exclusively expressed on either TC or IC. Transcriptome analysis demonstrated that IC3 expressed higher levels of the effector T cell markers than TC3, suggesting that PD-L1 expression is regulated via an adaptive IFNγ-mediated mechanism. High PD-L1 expression on IC, but not TC, and high abundance of PD-1+ T cells and Foxp3+ Tregs are favorable prognostic factors for resected HNSCC. This study highlights the importance of comprehensive assessment of both TC and IC. PMID:27841362

  18. PD-1/PD-L1 signal pathway participates in HCV F protein-induced T cell dysfunction in chronic HCV infection.

    PubMed

    Xiao, Wen; Jiang, Long Feng; Deng, Xiao Zhao; Zhu, Dan Yan; Pei, Jia Ping; Xu, Mao Lei; Li, Bing Jun; Wang, Chang Jun; Zhang, Jing Hai; Zhang, Qi; Zhou, Zhen Xian; Ding, Wei Liang; Xu, Xiao Dong; Yue, Ming

    2016-04-01

    Programmed cell death-1/programmed cell death-1 ligand 1 (PD-1/PD-L1) inhibitory signal pathway has been verified to be involved in the establishment of persistent viral infections. Blockade of PD-1/PD-L1 engagement to reinvigorate T cell activity is supposed to be a potential therapeutic scheme. Studies have verified the participation of PD-1/PD-L1 in hepatitis C virus (HCV) core protein-regulated immune response. To determine the roles of PD-1/PD-L1 signal pathway in HCV F protein-induced immunoreaction in chronic HCV infection, variations in T cells were examined. The results showed that PD-1 expression on CD8(+) and CD4(+) T cells was increased with HCV F stimulation in both chronic HCV patients and healthy controls, and could be reduced partly by PD-1/PD-L1 blocking. Additionally, by PD-1/PD-L1 blocking, HCV F-induced inhibition of T cell proliferation and promotion of cellular apoptosis were partly or even totally recovered. Furthermore, levels of both Th1 and Th2 cytokines were elevated in the presence of anti-PD-L1 antibody. All these results indicated that PD-1/PD-L1 signal pathway also participates in HCV F protein-induced immunoregulation. PD-1/PD-L1 blocking plays important roles in the restoration of effective functionality of the impaired T cells in chronic HCV patients.

  19. Getting to L1 the Hard Way: Triana's Launch Options

    NASA Technical Reports Server (NTRS)

    Houghton, Martin B.; Bauer, Frank H. (Technical Monitor)

    2002-01-01

    Over the past four years, NASA's Goddard Space Flight Center has built and tested the Triana observatory, which will be the first Earth observing science satellite to take advantage of the unique perspective offered by a Lissajous orbit about the first Earth-Sun Lagrange Point (L1). Triana was originally meant to fly on the U.S. Space Transportation System (a.k.a. the Space Shuttle), but complications with the shuttle manifest have forced Triana into a "wait and see" attitude. The observatory is currently being stored at NASA's Goddard Space Flight Center, where it waits for an appropriate launch opportunity to materialize. To that end, several possible alternatives have been considered, including variations on the nominal shuttle deployment scenario, a high inclination Delta-type launch from Vandenberg Air Force Base, a Tsyklon class vehicle launched from Baikonur, Kazakhstan, and a ride on a French Ariane vehicle out of French Guiana into a somewhat arbitrary geostationary transfer orbit (GTO). This paper chronicles and outlines the pros and cons of how each of these opportunities could be used to send Triana on its way to L1.

  20. Aspartame downregulates 3T3-L1 differentiation.

    PubMed

    Pandurangan, Muthuraman; Park, Jeongeun; Kim, Eunjung

    2014-10-01

    Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. Since aspartame is 200 times sweeter than traditional sugar, it can give the same level of sweetness with less substance, which leads to lower-calorie food intake. There are reports that consumption of aspartame-containing products can help obese people lose weight. However, the potential role of aspartame in obesity is not clear. The present study investigated whether aspartame suppresses 3T3-L1 differentiation, by downregulating phosphorylated peroxisome proliferator-activated receptor γ (p-PPARγ), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (FABP4), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1), which are critical for adipogenesis. The 3T3-L1 adipocytes were cultured and differentiated for 6 d in the absence and presence of 10 μg/ml of aspartame. Aspartame reduced lipid accumulation in differentiated adipocytes as evidenced by Oil Red O staining. qRT-PCR analysis showed that the PPARγ, FABP4, and C/EBPα mRNA expression was significantly reduced in the aspartame-treated adipocytes. Western blot analysis showed that the induction of p-PPARγ, PPARγ, SREBP1, and adipsin was markedly reduced in the aspartame-treated adipocytes. Taken together, these data suggest that aspartame may be a potent substance to alter adipocyte differentiation and control obesity.

  1. L1 track finding for a time multiplexed trigger

    NASA Astrophysics Data System (ADS)

    Cieri, D.; Brooke, J.; Grimes, M.; Newbold, D.; Harder, K.; Shepherd-Themistocleous, C.; Tomalin, I.; Vichoudis, P.; Reid, I.; Iles, G.; Hall, G.; James, T.; Pesaresi, M.; Rose, A.; Tapper, A.; Uchida, K.

    2016-07-01

    At the HL-LHC, proton bunches will cross each other every 25 ns, producing an average of 140 pp-collisions per bunch crossing. To operate in such an environment, the CMS experiment will need a L1 hardware trigger able to identify interesting events within a latency of 12.5 μs. The future L1 trigger will make use also of data coming from the silicon tracker to control the trigger rate. The architecture that will be used in future to process tracker data is still under discussion. One interesting proposal makes use of the Time Multiplexed Trigger concept, already implemented in the CMS calorimeter trigger for the Phase I trigger upgrade. The proposed track finding algorithm is based on the Hough Transform method. The algorithm has been tested using simulated pp-collision data. Results show a very good tracking efficiency. The algorithm will be demonstrated in hardware in the coming months using the MP7, which is a μTCA board with a powerful FPGA capable of handling data rates approaching 1 Tb/s.

  2. Acquisition of Japanese contracted sounds in L1 phonology

    NASA Astrophysics Data System (ADS)

    Tsurutani, Chiharu

    2002-05-01

    Japanese possesses a group of palatalized consonants, known to Japanese scholars as the contracted sounds, [CjV]. English learners of Japanese appear to treat them initially as consonant + glide clusters, where there is an equivalent [Cj] cluster in English, or otherwise tend to insert an epenthetic vowel [CVjV]. The acquisition of the Japanese contracted sounds by first language (L1) learners has not been widely studied compared with the consonant clusters in English with which they bear a close phonetic resemblance but have quite a different phonological status. This is a study to investigate the L1 acquisition process of the Japanese contracted sounds (a) in order to observe how the palatalization gesture is acquired in Japanese and (b) to investigate differences in the sound acquisition processes of first and second language (L2) learners: Japanese children compared with English learners. To do this, the productions of Japanese children ranging in age from 2.5 to 3.5 years were transcribed and the pattern of misproduction was observed.

  3. Getting to L1 the Hard Way: Triana's Launch Options

    NASA Technical Reports Server (NTRS)

    Houghton, Martin B.; Bauer, Frank H. (Technical Monitor)

    2002-01-01

    Over the past four years, NASA's Goddard Space Flight Center has built and tested the Triana observatory, which will be the first Earth observing science satellite to take advantage of the unique perspective offered by a Lissajous orbit about the first Earth-Sun Lagrange Point (L1). Triana was originally meant to fly on the U.S. Space Transportation System (a.k.a. the Space Shuttle but complications with the shuttle manifest have forced Triana into a 'wait and see' attitude. The observatory is currently being stored at NASA's Goddard Space Flight Center, where it waits for an appropriate launch opportunity to surface. To that end, several possible alternatives have been considered, including variations on the nominal shuttle deployment scenario, a high inclination Delta-type launch from Vandenberg Air Force Base, a Tsyklon class vehicle launched from Baikonur, Kazakhstan, and a ride on a French Ariane vehicle out of French Guiana into a somewhat arbitrary geostationary transfer orbit (GTO). This paper chronicles and outlines the pros and cons of how each of these opportunities could be used to send Triana on its way to L1.

  4. Handling L2 Input in Phonological STM: The Effect of Non-L1 Phonetic Segments and Non-L1 Phonotactics on Nonword Repetition

    ERIC Educational Resources Information Center

    Kovacs, Gabor; Racsmany, Mihaly

    2008-01-01

    This article reports on an experiment comparing the effects of three discrete types of deviance from native language (L1) phonetics and phonology on verbal short-term memory performance. A nonword repetition task was used to measure the recall of four stimulus types: (a) high-probability L1-sounding nonwords, (b) low-probability L1-sounding…

  5. Ubiquitin C-terminal hydrolase-L1 interacts with adhesion complexes and promotes cell migration, survival, and anchorage independent growth.

    PubMed

    Frisan, Teresa; Coppotelli, Giuseppe; Dryselius, Rikard; Masucci, Maria G

    2012-12-01

    Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120-catenin, β-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.

  6. Intestinal and hepatic Niemann-Pick C1L1 proteins: future therapeutic targets for cholesterol gallstones disease?

    PubMed

    Castro-Torres, Ibrahim Guillermo; De la O-Arciniega, Minarda; Bravo-Duarte, Gustavo Adolfo; Gallegos-Estudillo, Janeth; Domínguez-Ortíz, Miguel Ángel; Martínez-Vázquez, Mariano

    2014-04-05

    The formation of cholesterol gallstones is a very complex and polygenic disorder that involves an alteration of the secretion of bile lipids, cholesterol crystallization, important immunological reactions in the gallbladder tissue, formation of biliary sludge composed of mucin, and inadequate gallbladder motility. The search for a therapeutic target is oriented towards decreasing bile secretion and intestinal absorption of cholesterol, in which Niemann-Pick C1L1 (NPC1L1) proteins play an important role. In basic and clinical studies, regulating the expression of these proteins can reduce intestinal, liver, plasma and bile cholesterol levels, a therapeutic effect that would be useful not only for treating the disease, but to prevent it, given the large quantity of risk factors. We discuss these effects in this review and propose NPC1L1 proteins as future therapeutic targets of cholesterol gallstones disease.

  7. The promotive effects of thymosin beta4 on neuronal survival and neurite outgrowth by upregulating L1 expression.

    PubMed

    Yang, Hao; Cheng, Xipeng; Yao, Qing; Li, Jingwen; Ju, Gong

    2008-11-01

    Thymosin beta(4) (Tbeta4) is a major actin-sequestering peptide widely distributed in mammalian tissues including the nervous system. The presence of this peptide in the nervous system likely plays a role in synaptogensis, axon growth, cell migration, and plastic changes in dendritic spine. However, the effects of Tbeta4 on the survival of neurons and axonal outgrowth have still not been fully understood. So far it is not clear if the effects of Tbeta4 are associated with L1 functions. In the present study, we hypothesized that Tbeta4-induced up-regulation of L1 synthesis could be involved in the survival and axon outgrowth of cultured spinal cord neurons. To test this hypothesis, primarily cultured neurons were prepared from the mouse spinal cord and treated with various concentrations of Tbeta4 ranging from 0.1 to 10 microg/ml. The analysis of L1 mRNA expression and protein synthesis in neurons was then carried out using RT-PCR and western blot assays, respectively. After the addition of Tbeta4 to cultures, cells were then treated with antibodies against distinct domains of L1-Fc. Subsequently, beta-tubulin III and L1 double-labeled indirect immunofluorescence was carried out. Meanwhile, L1 immunofluorescent reactivity was analyzed and compared in cells treated with Tbeta4. Furthermore, the number of beta-tubulin III-positive cells and neurite lengths were measured. We found that Tbeta4 enhanced L1 expression in a dose-dependent manner, and the highest L1 mRNA and protein synthesis in cells increased by more than 2.1- and 2.3-fold in the presence of Tbeta4 at identical concentrations, respectively. Moreover, it also dose dependently enhanced neurite outgrowth and neuronal survival. Compared to conditions without Tbeta4, the length of neurite and neuronal survival increased markedly in presence of 0.5, 1, and 5 microg/ml Tbeta4, respectively, whereas the effects of Tbeta4 were significantly attenuated or inhibited in the process of L1-Fc antibodies treatment

  8. l1-optimal control of multivariable systems with output norm constraints

    NASA Technical Reports Server (NTRS)

    Mcdonald, J. S.; Pearson, J. B.

    1991-01-01

    This paper considers the l1-optimal control problem for general rational plants. It is shown that, for plants with no poles or zeros on the unit circle, an optimal compensator exists and that the resulting closed loop transfer function is polynomial whenever there are at least as many controls as regulated outputs and at least as many measurements as exogeneous inputs. Exactly or approximately, optimal rational compensators can be obtained by solving a sequence of finite linear programs for the coefficients of a polynomial closed-loop transfer function. No assumptions on plant poles or zeros are required to obtain at least approximately optimal compensators. It is shown that constrained problems in which a set of outputs is regulated subject to l(infinity)-norm constraints on another set of outputs can be solved using a slight modification of the same algorithm.

  9. Induction of PD-L1 expression by epidermal growth factor receptor–mediated signaling in esophageal squamous cell carcinoma

    PubMed Central

    Zhang, Wencheng; Pang, Qingsong; Yan, Cihui; Wang, Qifeng; Yang, Jingsong; Yu, Shufei; Liu, Xiao; Yuan, Zhiyong; Wang, Ping; Xiao, Zefen

    2017-01-01

    Purpose The purpose of this study was to investigate the potential effect of activation of epidermal growth factor receptor (EGFR) signaling pathway on the expression of programmed death-ligand 1 (PD-L1) in esophageal squamous cell carcinoma (ESCC) cells with EGFR overexpression. Methods Flow cytometry and Western blot methods were used to assess PD-L1 expression on ESCC cells when EGFR signaling pathway was activated by epidermal growth factor (EGF) with or without EGFR-specific inhibitor AG-1478, and then EGFR signaling array was applied to analyze the potential signaling pathways involved. Results This study found that PD-L1 expression increased significantly in an EGFR-dependent manner by the activation of EGFR signaling and decreased sharply when EGFR signaling was blocked. The upregulated expression of PD-L1 was not associated with EGFR-STAT3 signaling pathway, but may be affected by EGFR–PI3K–AKT, EGFR–Ras–Raf–Erk, and EGR–PLC-γ signaling pathways. Conclusion The expression of PD-L1 can be regulated by EGFR signaling activation in ESCC, which indicates an important role for EGFR-mediated immune escape and potential molecular pathways for EGFR-targeted therapy and immunotherapy. PMID:28243112

  10. L1-Regularized Reconstruction Error as Alpha Matte

    NASA Astrophysics Data System (ADS)

    Johnson, Jubin; Cholakkal, Hisham; Rajan, Deepu

    2017-04-01

    Sampling-based alpha matting methods have traditionally followed the compositing equation to estimate the alpha value at a pixel from a pair of foreground (F) and background (B) samples. The (F,B) pair that produces the least reconstruction error is selected, followed by alpha estimation. The significance of that residual error has been left unexamined. In this letter, we propose a video matting algorithm that uses L1-regularized reconstruction error of F and B samples as a measure of the alpha matte. A multi-frame non-local means framework using coherency sensitive hashing is utilized to ensure temporal coherency in the video mattes. Qualitative and quantitative evaluations on a dataset exclusively for video matting demonstrate the effectiveness of the proposed matting algorithm.

  11. Solar and Heliospheric Data Requirements: Going Further Than L1

    NASA Technical Reports Server (NTRS)

    Szabo, A.

    2011-01-01

    Current operational space weather forecasting relies on solar wind observations made by the ACE spacecraft located at the L1 point providing 30-40 minutes warning time. Some use is also made of SOHO and STEREO solar imaging that potentially can give multiple days of warning time. However, our understanding of the propagation and evolution of solar wind transients is still limited resulting in a typical timing uncertainty of approximately 10 hours. In order to improve this critical understanding, a number of NASA missions are being planned. Specifically the Solar Probe Plus and Solar Orbiter missions will investigate the inner Heliospheric evolution of coronal mass ejections and the acceleration and propagation of solar energetic particles. In addition, a number of multi-spacecraft concepts have been studied that have the potential to significantly improve the accuracy of long-term space weather forecasts.

  12. Reference payload of the ESA L1 mission candidate ATHENA

    NASA Astrophysics Data System (ADS)

    Martin, Didier; Rando, Nicola; Lumb, David; Verhoeve, Peter; Oosterbroek, Tim; Bavdaz, Marcos

    2012-09-01

    The Advanced Telescope for High ENergy Astrophysics (ATHENA) is one of the three candidates that competed for the first large-class mission (L1) in ESA’s Cosmic Vision 2015-2025 programme, with a launch planned by 2022 and is the result of the IXO reformulation activities. ATHENA is an ESA-led project and is conceived as the next generation X-ray observatory. It is meant to address fundamental questions about accretion around black-holes, reveal the physics underpinning cosmic feedback, trace the large scale structure of baryons in galaxy clusters and the cosmic as well as a large number of astrophysics and fundamental physics phenomena. The observatory consists of two identical mirrors each illuminating a fixed focal plane instrument, providing collectively 1 m2 effective area at 1 keV. The reference payload consists of a medium resolution wide field imager (WFI) and a high resolution X-ray micro-calorimeter spectrometer (XMS). The WFI is based on a monolithic Si DepFET array providing imaging over a 24 × 24 arcmin2 field of view and a good PSF oversampling. The sensor will measure X-rays in the range 0.1-15 keV and provides near Fano limited energy resolution (150eV at 6keV). The XMS is based on a micro-calorimeter array operating at its transition temperature of ~100mK and provides <3eV resolution. The detector array consists of 32 × 32 pixels covering a 2.3 × 2.3 arcmin2 field of view, co-aligned with the WFI. This paper summarizes the results of the reformulation exercise and provides details on the payload complement and its accommodation on the spacecraft. Following the ESA Science Programme Committee decision on the L1 mission in May 2012, ATHENA was not selected to enter Definition Phase.

  13. Tocotrienol suppresses adipocyte differentiation and Akt phosphorylation in 3T3-L1 preadipocytes.

    PubMed

    Uto-Kondo, Harumi; Ohmori, Reiko; Kiyose, Chikako; Kishimoto, Yoshimi; Saito, Hisako; Igarashi, Osamu; Kondo, Kazuo

    2009-01-01

    In vivo studies show that alpha-tocotrienol and gamma-tocotrienol accumulate in adipose tissue. Furthermore, a recent study reports that the oral administration of gamma-tocotrienol from a tocotrienol-rich fraction from palm oil (TRF) decreases body fat levels in rats. The objective of this study was to evaluate the effect of TRF and its components on adipocyte differentiation in 3T3-L1 preadipocytes, which differentiated into adipocytes in the presence of 1.8 micromol/L insulin. TRF suppressed the insulin-induced mRNA expression of adipocyte-specific genes such as PPARgamma, adipocyte fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein-alpha (C/EBPalpha) compared with the differentiation of 3T3-L1 preadipocytes into adipocytes only in the presence of insulin. To confirm the suppressive effect of TRF, the major components of TRF, such as alpha-tocotrienol, gamma-tocotrienol, and alpha-tocopherol, were investigated. Alpha-tocotrienol and gamma-tocotrienol decreased the insulin-induced PPARgamma mRNA expression by 55 and 90%, respectively, compared with insulin, whereas alpha-tocopherol increased the mRNA expression. In addition, gamma-tocotrienol suppressed the insulin-induced aP2 and C/EBPalpha mRNA expression, triglyceride accumulation, and PPARgamma protein levels compared with insulin. The current results also revealed that gamma-tocotrienol inhibited the insulin-stimulated phosphorylation of Akt but not extracellular signal-regulated kinase (ERK)1/2 in the insulin signaling pathway of 3T3-L1 preadipocytes. Thus, the antiadipogenic effect of TRF depends on alpha-tocotrienol and gamma-tocotrienol, and gamma-tocotrienol may be a more potent inhibitor of adipogenesis than alpha-tocotrienol. Therefore, the results of this study suggest that tocotrienol suppresses insulin-induced differentiation and Akt phosphorylation in 3T3-L1 preadipocytes. Furthermore, tocotrienol could act as an antiadipogenic vitamin in the nutrient-mediated regulation of body

  14. Piromelatine decreases triglyceride accumulation in insulin resistant 3T3-L1 adipocytes: role of ATGL and HSL.

    PubMed

    Wang, Ping-Ping; She, Mei-Hua; He, Ping-Ping; Chen, Wu-Jun; Laudon, Moshe; Xu, Xuan-Xuan; Yin, Wei-Dong

    2013-08-01

    Piromelatine, a novel investigational multimodal sleep medicine, is developed for the treatment of patients with primary and co-morbid insomnia. Piromelatine has been shown to inhibit weight gain and improve insulin sensitivity in high-fat/high-sucrose-fed (HFHS) rats. Considering that piromelatine has also been implicated in lowering of triglyceride levels in HFHS rats, this work elucidated whether this effect involves in the regulation of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) in triglyceride (TG) metabolism. In this study, we investigated the effects of piromelatine and MT2 receptors inhibition on TG content, insulin-stimulated glucose uptake, and the expressions of ATGL and HSL in 3T3-L1 adipocytes preincubated in high glucose and high insulin (HGI) conditions. Our results showed that culturing 3T3-L1 adipocytes under HGI conditions increased triglyceride accumulation with concomitant decrease of ATGL and HSL expression, inducing insulin resistance in 3T3-L1 adipocytes. We also found that triglyceride accumulation was significantly inhibited and the levels of ATGL/HSL increased after melatonin or piromelatine treatment. The effects of melatonin/piromelatine (10 nM) were counteracted by pretreatment with the relatively selective MT2 receptor antagonist luzindole (100 nM). In this study, our data demonstrate that piromelatine reverses high glucose and high insulin-induced triglyceride accumulation in 3T3-L1 adipocytes, possibly through up-regulating of ATGL and HSL expression via a melatonin-dependent manner.

  15. Expression of PD-L1 on Canine Tumor Cells and Enhancement of IFN-γ Production from Tumor-Infiltrating Cells by PD-L1 Blockade

    PubMed Central

    Maekawa, Naoya; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Adachi, Mami; Takagi, Satoshi; Kagawa, Yumiko; Nakajima, Chie; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2014-01-01

    Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the “exhausted” status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1–expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-γ production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed. PMID:24915569

  16. Expression of PD-L1 on canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade.

    PubMed

    Maekawa, Naoya; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Adachi, Mami; Takagi, Satoshi; Kagawa, Yumiko; Nakajima, Chie; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2014-01-01

    Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the "exhausted" status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1-expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-γ production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed.

  17. MAD1L1 Arg558His and MAD2L1 Leu84Met interaction with smoking increase the risk of colorectal cancer

    PubMed Central

    Zhong, Rong; Chen, Xiaohua; Chen, Xueqin; Zhu, Beibei; Lou, Jiao; Li, Jiaoyuan; Shen, Na; Yang, Yang; Gong, Yajie; Zhu, Ying; Yuan, Jing; Xia, Xiaoping; Miao, Xiaoping

    2015-01-01

    The spindle assembly checkpoint (SAC) has been established as an important mechanism of driving aneuploidy, which occurs at a high frequency in the colorectal tumorigenesis. Two important components of SAC are MAD1L1 and MAD2L1, which function together in an interactive manner to initiate the checkpoint signal. We hypothesize that genetic variants in the binding domains of MAD1L1 and MAD2L1 may modulate protein structures and eventually contribute to CRC susceptibility. A case-control study including 710 CRC cases and 735 controls was performed to examine MAD1L1 Arg558His and MAD2L1 Leu84Met’s conferring susceptibility to CRC. Cytokinesis-block micronucleus cytome assays were applied to assess the effect of two functional variants on chromosomal instability (CIN). Significant associations with CRC risk were observed for MAD1L1 Arg558His (OR = 1.38,95% CI: 1.09–1.75) and MAD2L1 Leu84Met in a dominant model (OR = 1.48,95% CI: 1.09–2.01). Moreover, significant multiplicative gene-smoking interactions were found in MAD1L1 Arg558His (P = 0.019) and MAD2L184 Leu/Met (P = 0.016) to enhance CRC risk. Additionally, the frequencies of lymphocytic micro-nucleated binucleated cells for MAD1L1 Arg558His polymorphism were significantly different in the exposed group (P = 0.013), but not in the control group. The study emphasized that MAD1L1 Arg558His and MAD2L1 Leu84Met can significantly interact with smoking to enhance CRC risk, and the genetic effects of MAD1L1Arg558His on CIN need to be further clarified in follow-up studies. PMID:26183163

  18. Localization of ubiquitin C-terminal hydrolase L1 in mouse ova and its function in the plasma membrane to block polyspermy.

    PubMed

    Sekiguchi, Satoshi; Kwon, Jungkee; Yoshida, Etsuko; Hamasaki, Hiroko; Ichinose, Shizuko; Hideshima, Makoto; Kuraoka, Mutsuki; Takahashi, Akio; Ishii, Yoshiyuki; Kyuwa, Shigeru; Wada, Keiji; Yoshikawa, Yasuhiro

    2006-11-01

    Protein degradation is essential for oogenesis and embryogenesis. The ubiquitin-proteasome system regulates many cellular processes via the rapid degradation of specific proteins. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is exclusively expressed in neurons, testis, ovary, and placenta, each of which has unique biological activities. However, the functional role of UCH-L1 in mouse oocytes remains unknown. Here, we report the expression pattern of UCH-L1 and its isozyme UCH-L3 in mouse ovaries and embryos. Using immunocytochemistry, UCH-L1 was selectively detected on the plasma membrane, whereas UCH-L3 was mainly detected in the cytoplasm, suggesting that these isozymes have distinct functions in mouse eggs. To further investigate the functional role of UCH-L1 in mouse eggs, we analyzed the fertilization rate of UCH-L1-deficient ova of gad female mice. Female gad mice had a significantly increased rate of polyspermy in in vitro fertilization assays, although the rate of fertilization did not differ significantly from wild-type mice. In addition, the litter size of gad female mice was significantly reduced compared with wild-type mice. These results may identify UCH-L1 as a candidate for a sperm-oocyte interactive binding or fusion protein on the plasma membrane that functions during the block to polyspermy in mouse oocytes.

  19. The role of transcriptional corepressor Nif3l1 in early stage of neural differentiation via cooperation with Trip15/CSN2.

    PubMed

    Akiyama, Hirotada; Fujisawa, Naoko; Tashiro, Yousuke; Takanabe, Natsuko; Sugiyama, Akinori; Tashiro, Fumio

    2003-03-21

    Mouse Nif3l1 gene is highly conserved from bacteria to human. Even though this gene is expressed throughout embryonic development, its biological function is still obscure. Here, we show that Nif3l1 participates in retinoic acid-primed neural differentiation of P19 embryonic carcinoma cells through cooperation with Trip15/CSN2, a transcriptional corepressor/component of COP9 signalosome. We isolated Nif3l1 cDNA from P19 cell cDNA library by a yeast two-hybrid screening using Trip15/CSN2 as a bait. This interaction was confirmed by a pull-down assay and an epitope-tagged coimmunoprecipitation. Although Nif3l1 was mainly detected in the cytoplasm, the translocation of Nif3l1 into the nuclei was observed in retinoic acid-primed neural differentiation of P19 cells and enhanced by the enforced expression of Trip15/CSN2. Furthermore, enforced expression of sense Nif3l1 RNA, but not antisense RNA, enhanced the neural differentiation of P19 cells accompanying the intense down-regulation of Oct-3/4 mRNA expression and the rapid induction of Mash-1 mRNA expression. Luciferase reporter assay showed that Nif3l1 could act as a transcriptional repressor and synergized the transcriptional repression by Trip15/CSN2. These results indicate that Nif3l1 implicates in neural differentiation through the cooperation with Trip15/CSN2.

  20. Tissue-Specific Methylation of Long Interspersed Nucleotide Element-1 of Homo Sapiens (L1Hs) During Human Embryogenesis and Roles in Neural Tube Defects.

    PubMed

    Wang, L; Chang, S; Guan, J; Shangguan, S; Lu, X; Wang, Z; Wu, L; Zou, J; Zhao, H; Bao, Y; Qiu, Z; Niu, B; Zhang, T

    2015-01-01

    Epigenetic regulation of long interspersed nucleotide element-1 (LINE-1) retrotransposition events plays crucial roles during early development. Previously we showed that LINE-1 hypomethylation in neuronal tissues is associated with pathogenesis of neural tube defect (NTD). Herein, we further evaluated LINE-1 Homo sapiens (L1Hs) methylation in tissues derived from three germ layers of stillborn NTD fetuses, to define patterns of tissue specific methylation and site-specific hypomethylation at CpG sites within an L1Hs promoter region. Stable, tissue-specific L1Hs methylation patterns throughout three germ layer lineages of the fetus, placenta, and maternal peripheral blood were observed. Samples from maternal peripheral blood exhibited the highest level of L1Hs methylation (64.95%) and that from placenta showed the lowest (26.82%). Between samples from NTDs and controls, decrease in L1Hs methylation was only significant in NTD-affected brain tissue at 7.35%, especially in females (8.98%). L1Hs hypomethylation in NTDs was also associated with a significant increase in expression level of an L1Hs-encoded transcript in females (r = -0.846, p = 0.004). This could be due to genomic DNA instability and alternation in chromatins accessibility resulted from abnormal L1Hs hypomethylation, as showed in this study with HCT-15 cells treated with methylation inhibitor 5-Aza.

  1. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    SciTech Connect

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai; Yang, Ying; Shen, Weili

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

  2. Gsalpha signalling suppresses PPARgamma2 generation and inhibits 3T3L1 adipogenesis.

    PubMed

    Zhang, Lei; Paddon, Carol; Lewis, Mark D; Grennan-Jones, Fiona; Ludgate, Marian

    2009-08-01

    Since TSH receptor (TSHR) expression increases during adipogenesis and signals via cAMP/phospho-cAMP-response element binding protein (CREB), reported to be necessary and sufficient for adipogenesis, we hypothesised that TSHR activation would induce preadipocyte differentiation. Retroviral vectors introduced constitutively active TSHR (TSHR*) into 3T3L1 preadipocytes; despite increased cAMP (RIA) and phospho-CREB (western blot) there was no spontaneous adipogenesis (assessed morphologically, using oil red O and QPCR measurement of adipogenesis markers). We speculated that Gbetagamma signalling may be inhibitory but failed to induce adipogenesis using activated Gsalpha (gsp*). Inhibition of phosphodiesterases did not promote adipogenesis in TSHR* or gsp* populations. Furthermore, differentiation induced by adipogenic medium with pioglitazone was reduced in TSHR* and abolished in gsp* expressing 3T3L1 cells. TSHR* and gsp* did not inactivate PPARgamma (PPARG as listed in the HUGO database) by phosphorylation but expression of PPARgamma1 was reduced and PPARgamma2 undetectable in gsp*. FOXO1 phosphorylation (required to inactivate this repressor of adipogenesis) was lowest in gsp* despite the activation of AKT by phosphorylation. PROF is a mediator that facilitates FOXO1 phosphorylation by phospho-Akt. Its transcript levels remained constantly low in the gsp* population. In most measurements, the TSHR* cells were between the gsp* and control 3T3L1 preadipocytes. The enhanced down-regulation of PREF1 (adipogenesis inhibitor) permits retention of some adipogenic potential in the TSHR* population. We conclude that Gsalpha signalling impedes FOXO1 phosphorylation and thus inhibits PPARgamma transcription and the alternative promoter usage required to generate PPARgamma2, the fat-specific transcription factor necessary for adipogenesis.

  3. Therapeutic uses of anti-PD-1 and anti-PD-L1 antibodies.

    PubMed

    Philips, George K; Atkins, Michael

    2015-01-01

    Despite extensive investigation over the past three decades, cancer immunotherapy has produced limited success, with few agents achieving approval by the Food and Drug Administration and even the most effective helping only a minority of patients, primarily with melanoma or renal cancer. In recent years, immune checkpoints that maintain physiologic self-tolerance have been implicated in the down-regulation of anti-tumor immunity. Efforts to restore latent anti-tumor immunity have focused on antibody-based interventions targeting CTL antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1) on T lymphocytes and its principal ligand (PD-L1) on tumor cells. Ipilimumab, an antibody targeting CTLA-4, appears to restore tumor immunity at the priming phase, whereas anti-PD-1/PD-L1 antibodies restore immune function in the tumor microenvironment. Although ipilimumab can produce durable long-term responses in patients with advanced melanoma, it is associated with significant immune-related toxicities. By contrast, antibodies targeting either PD-1 or PD-L1 have produced significant anti-tumor activity with considerably less toxicity. Activity was seen in patients with melanoma and renal cancer, as well as those with non-small-cell lung, bladder and head and neck cancers, tumors not previously felt to be sensitive to immunotherapy. The tolerability of PD-1-pathway blockers and their unique mechanism of action have made them ideal backbones for combination regimen development. Combination approaches involving cytotoxic chemotherapy, anti-angiogenic agents, alternative immune-checkpoint inhibitors, immunostimulatory cytokines and cancer vaccines are currently under clinical investigation. Current efforts focus on registration trials of single agents and combinations in various diseases and disease settings and identifying predictive biomarkers of response.

  4. [Efficacy of PD-1/PD-L1 immune checkpoint inhibitors and PD-L1 testing in thoracic cancers].

    PubMed

    Duruisseaux, Michaël; Rouquette, Isabelle; Adam, Julien; Cortot, Alexis; Cazes, Aurélie; Gibault, Laure; Damotte, Diane; Lantuejoul, Sylvie

    2017-02-01

    Tumoral immune environment is a major component of cancer. Its composition and its organization represent a reproducible characteristic of tumors and a validated prognostic factor. In non-small cell lung cancer (NSCLC), cytotoxic T CD8+ lymphocyte density, associated with a Th1 environment and tertiary lymphoid structures impacts survival. Tumor cell-immune cell interaction is targeted by PD1/PD-L1 inhibitors. In advanced NSCLC, PD1/PD-L1 inhibitors are more effective than second-line chemotherapy. Pembrolizumab outperforms first-line chemotherapy in NSCLC strongly positive for PD-L1. PD1/PD-L1 inhibitors are currently tested in mesothelioma and thymic tumors. PD-L1 expression evaluated with immunochemistry is the most studied predictive biomarker of PD1/PD-L1 inhibitor efficacy. Tumor and immune cell expression of PD-L1 is still difficult to evaluate because of intra-tumoral heterogeneity and expression modulation by the microenvironment. Four commercial diagnostic antibodies are in development, with differences concerning recognized epitopes, methodology of evaluation of PD-L1 expression, positivity threshold, kit and platforms used. Clinical trials in NSCLC have shown that patients with tumors strongly positive for PD-L1 derived the best clinical benefit with PD1/PD-L1 inhibitors whereas clinical benefit is less common in tumors negative for PD-L1. PD-L1 expression is not a perfect biomarker since some PD-L1 negative NSCLC respond to PD1/PD-L1 inhibitors and some PD-L1 positive NSCLC do not. PD-L1 testing is likely to be implemented in daily practice for selection of advanced NSCLC that will be treated with pembrolizumab, underscoring the relevance of ongoing harmonization studies of the use of the different antibodies available for PD-L1 testing.

  5. Ultra-thin L1{sub 0}-FePt for perpendicular anisotropy L1{sub 0}-FePt/Ag/[Co/Pd]{sub 30} pseudo spin valves

    SciTech Connect

    Ho, Pin; Chow, Gan Moog; Chen, Jing-Sheng; Han, Guchang; He, Kaihua

    2014-05-07

    Perpendicular anisotropy L1{sub 0}-FePt/Ag/[Co/Pd]{sub 30} pseudo spin valves (PSVs) with ultra-thin L1{sub 0}-FePt alloy free layer possessing high anisotropy and thermal stability have been fabricated and studied. The thickness of the L1{sub 0}-FePt layer was varied between 2 and 4 nm. The PSV became increasingly decoupled with reduced L1{sub 0}-FePt thickness due to the larger difference between the coercivity of the L1{sub 0}-FePt and [Co/Pd]{sub 30} films. The PSV with an ultra-thin L1{sub 0}-FePt free layer of 2 nm displayed a high K{sub u} of 2.21 × 10{sup 7} ergs/cm{sup 3}, high thermal stability of 84 and a largest giant magnetoresistance of 0.54%.

  6. Brain abnormality segmentation based on l1-norm minimization

    NASA Astrophysics Data System (ADS)

    Zeng, Ke; Erus, Guray; Tanwar, Manoj; Davatzikos, Christos

    2014-03-01

    We present a method that uses sparse representations to model the inter-individual variability of healthy anatomy from a limited number of normal medical images. Abnormalities in MR images are then defined as deviations from the normal variation. More precisely, we model an abnormal (pathological) signal y as the superposition of a normal part ~y that can be sparsely represented under an example-based dictionary, and an abnormal part r. Motivated by a dense error correction scheme recently proposed for sparse signal recovery, we use l1- norm minimization to separate ~y and r. We extend the existing framework, which was mainly used on robust face recognition in a discriminative setting, to address challenges of brain image analysis, particularly the high dimensionality and low sample size problem. The dictionary is constructed from local image patches extracted from training images aligned using smooth transformations, together with minor perturbations of those patches. A multi-scale sliding-window scheme is applied to capture anatomical variations ranging from fine and localized to coarser and more global. The statistical significance of the abnormality term r is obtained by comparison to its empirical distribution through cross-validation, and is used to assign an abnormality score to each voxel. In our validation experiments the method is applied for segmenting abnormalities on 2-D slices of FLAIR images, and we obtain segmentation results consistent with the expert-defined masks.

  7. Very low latitude (L = 1.08) whistlers

    NASA Astrophysics Data System (ADS)

    Singh, Rajesh; Cohen, Morris B.; Maurya, Ajeet K.; Veenadhari, B.; Kumar, Sushil; Pant, P.; Said, Ryan K.; Inan, Umran S.

    2012-12-01

    For decades, whistlers observed on the ground at mid and high latitudes have been used for diagnostics of Earth's plasmasphere. Whistlers have also been observed at low latitudes however, the propagation characteristics of low latitude whistlers are poorly understood thus they have not been used effectively as a diagnostic for the low latitude ionosphere. One key limitation with past studies has been lack of knowledge of the whistler source lightning location. Here we present the first cases of low latitude ground whistlers most likely linked with their causative lightning discharges in the conjugate zone. The Global Lightning Dataset 360 (GLD360) detected lightning discharges were found to be located close to the conjugate location of the recording stations, providing direct evidence of inter-hemispheric propagation at the low latitudes. A total of 864 whistlers were observed at Allahabad, India (Geomag. lat. 16.05°N Geomag. long. 155.34°E L = 1.08) during the night of 26 January 2011. Using GLD360 network data, we show the occurrence of thunderstorm activity between 200 and 450 km from the conjugate point of Allahabad. We also report the distribution of peak currents of whistler-producing lightning, which demonstrate a cutoff at 30 kA.

  8. Status of the ESA L1 mission candidate ATHENA

    NASA Astrophysics Data System (ADS)

    Rando, N.; Martin, D.; Lumb, D.; Verhoeve, P.; Oosterbroek, T.; Bavdaz, M.; Fransen, S.; Linder, M.; Peyrou-Lauga, R.; Voirin, T.; Braghin, M.; Mangunsong, S.; van Pelt, M.; Wille, E.

    2012-09-01

    ATHENA (Advanced Telescope for High Energy Astrophysics) was an L class mission candidate within the science programme Cosmic Vision 2015-2025 of the European Space Agency, with a planned launch by 2022. ATHENA was conceived as an ESA-led project, open to the possibility of focused contributions from JAXA and NASA. By allowing astrophysical observations between 100 eV and 10 keV, it would represent the new generation X-ray observatory, following the XMM-Newton, Astro-H and Chandra heritage. The main scientific objectives of ATHENA include the study of large scale structures, the evolution of black holes, strong gravity effects, neutron star structure as well as investigations into dark matter. The ATHENA mission concept would be based on focal length of 12m achieved via a rigid metering tube and a twoaperture, x-ray telescope. Two identical x-ray mirrors would illuminate fixed focal plane instruments: a cryogenic imaging spectrometer (XMS) and a wide field imager (WFI). The S/C is designed to be fully compatible with Ariane 5 ECA. The observatory would operate at SE-L2, with a nominal lifetime of 5 yr. This paper provides a summary of the reformulation activities, completed in December 2011. An overview of the spacecraft design and of the payload is provided, including both telescope and instruments. Following the ESA Science Programme Committee decision on the L1 mission in May 2012, ATHENA was not selected to enter Definition Phase.

  9. The Apolipoprotein L1 Gene and Cardiovascular Disease

    PubMed Central

    Robinson, Todd W.; Freedman, Barry I.

    2016-01-01

    Relative to those with European ancestry, African Americans have an excess incidence of nondiabetic chronic kidney disease predominantly due to two coding renal-risk variants in the apolipoprotein L1 gene (APOL1). This APOL1–kidney disease association is independent of systemic hypertension or blood pressure. Recent reports describe extra-renal effects of the APOL1 G1 and G2 renal-risk variants on cardiovascular disease (CVD), subclinical atherosclerosis, lipoprotein particle concentrations, and survival. However, results have been less consistent than those seen in kidney disease, and the observed APOL1 associations with CVD vary from risk to protective. This manuscript reviews the relationships between APOL1 renal-risk variants and CVD, with an emphasis on study-specific factors that may have contributed to disparate observations. It is possible that APOL1 renal-risk variants impact the systemic vasculature, not only the kidneys. As novel therapies for APOL1-associated nephropathy are developed, APOL1 variant protein effects on large blood vessels and risk of CVD will need to be considered. PMID:28298955

  10. Stabilizing l1-norm prediction models by supervised feature grouping.

    PubMed

    Kamkar, Iman; Gupta, Sunil Kumar; Phung, Dinh; Venkatesh, Svetha

    2016-02-01

    Emerging Electronic Medical Records (EMRs) have reformed the modern healthcare. These records have great potential to be used for building clinical prediction models. However, a problem in using them is their high dimensionality. Since a lot of information may not be relevant for prediction, the underlying complexity of the prediction models may not be high. A popular way to deal with this problem is to employ feature selection. Lasso and l1-norm based feature selection methods have shown promising results. But, in presence of correlated features, these methods select features that change considerably with small changes in data. This prevents clinicians to obtain a stable feature set, which is crucial for clinical decision making. Grouping correlated variables together can improve the stability of feature selection, however, such grouping is usually not known and needs to be estimated for optimal performance. Addressing this problem, we propose a new model that can simultaneously learn the grouping of correlated features and perform stable feature selection. We formulate the model as a constrained optimization problem and provide an efficient solution with guaranteed convergence. Our experiments with both synthetic and real-world datasets show that the proposed model is significantly more stable than Lasso and many existing state-of-the-art shrinkage and classification methods. We further show that in terms of prediction performance, the proposed method consistently outperforms Lasso and other baselines. Our model can be used for selecting stable risk factors for a variety of healthcare problems, so it can assist clinicians toward accurate decision making.

  11. cAMP signaling by anthrax edema toxin induces transendothelial cell tunnels, which are resealed by MIM via Arp2/3-driven actin polymerization.

    PubMed

    Maddugoda, Madhavi P; Stefani, Caroline; Gonzalez-Rodriguez, David; Saarikangas, Juha; Torrino, Stéphanie; Janel, Sebastien; Munro, Patrick; Doye, Anne; Prodon, François; Aurrand-Lions, Michel; Goossens, Pierre L; Lafont, Frank; Bassereau, Patricia; Lappalainen, Pekka; Brochard, Françoise; Lemichez, Emmanuel

    2011-11-17

    RhoA-inhibitory bacterial toxins, such as Staphylococcus aureus EDIN toxin, induce large transendothelial cell macroaperture (TEM) tunnels that rupture the host endothelium barrier and promote bacterial dissemination. Host cells repair these tunnels by extending actin-rich membrane waves from the TEM edges. We reveal that cyclic-AMP signaling produced by Bacillus anthracis edema toxin (ET) also induces TEM formation, which correlates with increased vascular permeability. We show that ET-induced TEM formation resembles liquid dewetting, a physical process of nucleation and growth of holes within a thin liquid film. We also identify the cellular mechanisms of tunnel closure and reveal that the I-BAR domain protein Missing in Metastasis (MIM) senses de novo membrane curvature generated by the TEM, accumulates at the TEM edge, and triggers Arp2/3-dependent actin polymerization, which induces actin-rich membrane waves that close the TEM. Thus, the balance between ET-induced TEM formation and resealing likely determines the integrity of the host endothelium barrier.

  12. A prototypic modified risk tobacco product exhibits reduced effects on chemotaxis and transendothelial migration of monocytes compared with a reference cigarette.

    PubMed

    van der Toorn, Marco; Frentzel, Stefan; Goedertier, Didier; Peitsch, Manuel; Hoeng, Julia; De Leon, Hector

    2015-06-01

    Monocyte adhesion and migration to the subendothelial space represent critical steps in atherogenesis. Here, we investigated whether extracts from the aerosol of a prototypic modified risk tobacco product (pMRTP), based on heating rather than combusting tobacco, exhibited differential effects on the migratory behavior of monocytes compared with that from the reference cigarette, 3R4F. THP-1 cells, a monocytic cell line, and human coronary arterial endothelial cells (HCAECs) were used to investigate chemotaxis and transendothelial migration (TEM) of monocytes in conventional and impedance-based systems. THP-1 cells migrated through a monolayer of HCAECs in response to C-X-C motif ligand 12 (CXCL12), a chemokine involved in diverse cellular functions including chemotaxis and survival of stem cells. Treatment of THP-1 cells with extracts from 3R4F or pMRTP induced concentration-dependent increases in cytotoxicity (7-aminoactinomycin D), and inflammation (IL-8 and TNF-α). CXCL12-mediated chemotaxis and TEM were decreased in extract-treated THP-1 cells. Extracts from 3R4F were ~21 times more potent than those from pMRTP in all examined endpoints. Extracts from 3R4F and pMRTP induced concentration-dependent responses in assays of inflammation, cytotoxicity, chemotaxis, and TEM. Furthermore, our findings indicate that extracts from a pMRTP are significantly less cytotoxic and induce less inflammation than those from the reference cigarette, 3R4F.

  13. Targeted NF-kappaB inhibition of asthmatic serum-mediated human monocyte-derived dendritic cell differentiation in a transendothelial trafficking model.

    PubMed

    Gu, Xiao-Yan; Zhou, Lin-Fu; Zhang, Ming-Shun; Dai, Wen-Jing; Chen, Sai-Ying; He, Shao-Heng; Ji, Xiao-Hui; Yin, Kai-Sheng

    2009-01-01

    Transendothelial trafficking model mimics in vivo differentiation of monocytes into dendritic cells (DC). The serum from patients with systemic lupus erythematosus promotes the differentiation of monocytes into mature DC. We have shown that selective inhibition of NF-kappaB by adenoviral gene transfer of a novel mutated IkappaBalpha (AdIkappaBalphaM) in DC contributes to T cell tolerance. Here we demonstrated for the first time that asthmatic serum facilitated human monocyte-derived DC (MDDC) maturation associated with increased NF-kappaB activation in this model. Furthermore, selective blockade of NF-kappaB by AdIkappaBalphaM in MDDC led to increased apoptosis, and decreased levels of CD80, CD83, CD86, and IL-12 p70 but not IL-10 in asthmatic serum-stimulated MDDC, accompanied by reduced proliferation of T cells. These results suggest that AdIkappaBalphaM-transferred MDDC are at a more immature stage which is beneficial to augment the immune tolerance in asthma.

  14. Intravenous immunoglobulin G (IVIG) inhibits IL-1- and TNF-α-dependent, but not chemotactic-factor-stimulated, neutrophil transendothelial migration.

    PubMed

    Issekutz, Andrew C; Rowter, Derek; Macmillan, Heather F

    2011-11-01

    High-dose intravenous immunoglobulin (IVIG) has anti-inflammatory effects via incompletely understood mechanisms. By investigating whether IVIG might modulate neutrophil (PMN) recruitment, we observed that IVIG dose-dependently inhibited (by 30-50%) PMN transendothelial migration (TEM) across human umbilical vein endothelial cells (EC) stimulated with IL-1α, IL-1β, TNF-α or IL-1β+TNF-α. Inhibition required the presence of IVIG with the responding PMNs, was attributable to the F(ab)(2) portion and was unrelated to putative contaminants in IVIG. IVIG did not inhibit IL-1β- or TNF-α-induced increase of PMN adhesion to EC, nor did it affect C5a- or IL-8-induced PMN TEM across unstimulated EC. Effects of IVIG and F(ab)(2) fragments were not associated with PMN activation, assessed by CD62L shedding, CD11b upregulation or PMN shape. Thus, IVIG selectively inhibits PMN TEM across inflammatory-cytokine-stimulated - but not unstimulated - EC, perhaps contributing to therapeutic benefit in chronic inflammation with minimal impact on chemotactic-factor-induced PMN recruitment during acute infection.

  15. Aerosol from a candidate modified risk tobacco product has reduced effects on chemotaxis and transendothelial migration compared to combustion of conventional cigarettes.

    PubMed

    van der Toorn, Marco; Frentzel, Stefan; De Leon, Hector; Goedertier, Didier; Peitsch, Manuel C; Hoeng, Julia

    2015-12-01

    Reduction of harmful constituents by heating rather than combusting tobacco is a promising new approach to reduce harmful effects associated with cigarette smoking. We investigated the effect from a new candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, on the migratory behavior of monocytes in comparison with combustible 3R4F reference cigarettes. The monocytic cell line (THP-1) and human coronary arterial endothelial cells (HCAECs) were used to analyze chemotaxis and transendothelial migration (TEM). To assess the influence of aerosol extract from THS2.2 and smoke extract from 3R4F on toxicity and inflammation, flow cytometry and ELISA assays were performed. The results show that treatment of THP-1 cells with extract from 3R4F or THS2.2 induced concentration-dependent increases in cytotoxicity and inflammation. The inhibitory effects of THS2.2 extract for chemotaxis and TEM were ∼18 times less effective compared to 3R4F extract. Furthermore, extract from 3R4F or THS2.2 induced concentration-dependent decreases in the integrity of HCAEC monolayer. For all examined endpoints, the extract from 3R4F showed more than one order of magnitude stronger effects than that from THS2.2 extract. These data indicate the potential of a heat not burn tobacco product to reduce the risk for cardiovascular disease compared to combustible cigarettes.

  16. Activated T cell trans-endothelial migration relies on myosin-IIA contractility for squeezing the cell nucleus through endothelial cell barriers.

    PubMed

    Jacobelli, Jordan; Estin Matthews, Miriam; Chen, Stephanie; Krummel, Matthew F

    2013-01-01

    Following activation, T cells are released from lymph nodes to traffic via the blood to effector sites. The re-entry of these activated T cells into tissues represents a critical step for them to carry out local effector functions. Here we have assessed defects in effector T cells that are acutely depleted in Myosin-IIA (MyoIIA) and show a T cell intrinsic requirement for this motor to facilitate the diapedesis step of extravasation. We show that MyoIIA accumulates at the rear of T cells undergoing trans-endothelial migration. T cells can extend protrusions and project a substantial portion of their cytoplasm through the endothelial wall in the absence of MyoIIA. However, this motor protein plays a crucial role in allowing T cells to complete the movement of their relatively rigid nucleus through the endothelial junctions. In vivo, this defect manifests as poor entry into lymph nodes, tumors and into the spinal cord, during tissue-specific autoimmunity, but not the spleen. This suggests that therapeutic targeting of this molecule may allow for differential attenuation of tissue-specific inflammatory responses.

  17. Development of phonological awareness in English-Mandarin bilinguals: a comparison of English-L1 and Mandarin-L1 kindergarten children.

    PubMed

    Yeong, Stephanie H M; Rickard Liow, Susan J

    2012-06-01

    Phoneme awareness is critical for literacy acquisition in English, but relatively little is known about the early development of phonological awareness in ESL (English as a second language) bilinguals when their two languages have different phonological structures. Using parallel tasks in English and Mandarin, we tracked the development of L1 (first language) and L2 (second language) syllable and phoneme awareness longitudinally in English-L1 and Mandarin-L1 prereaders (n=70, 4- and 5-year-olds) across three 6-month intervals. In English, the English-L1 children's performance was better in phoneme awareness at all three time points, but the Mandarin-L1 children's syllable awareness was equivalent to the English-L1 children's syllable awareness by Time 3. In Mandarin, the English-L1 children's phoneme awareness, but not their syllable awareness, was also significantly better than that of the Mandarin-L1 children at all three time points. Cross-lagged correlations revealed that only the English-L1 children applied their L1 syllable and phoneme awareness to their L2 (Mandarin) processing by Time 2 and that the Mandarin-L1 children seemed to require exposure to English (L2) before they developed phoneme awareness in either language. The data provide further evidence that phonological awareness is a language-general ability but that cross-language application depends on the similarity between the phonological structures of a child's L1 and L2. Implications for classroom teaching are briefly discussed.

  18. L1 syndrome diagnosis complemented with functional analysis of L1CAM variants located to the two N-terminal Ig-like domains.

    PubMed

    Christaller, W A A; Vos, Y; Gebre-Medhin, S; Hofstra, R M W; Schäfer, M K E

    2017-01-01

    L1CAM gene mutations cause neurodevelopmental disorders collectively termed L1 syndrome. Insufficient information about L1CAM variants complicates clinical prognosis, genetic diagnosis and genetic counseling. We combined clinical data, in silico effect predictions and functional analysis of four L1CAM variants, p.I37N, p.T38M, p.M172I and p.D202Y, located to the two N-terminal Ig-like domains present in five families with symptoms of L1 syndrome. Software tools predicted destabilizing effects of p.I37N and p.D202Y but results for p.T38M and p.M172I were inconsistent. Cell surface expression of mutant proteins L1-T38M, L1-M172I and L1-D202Y was normal. Conversely, L1-I37N accumulated in the endoplasmic reticulum (ER) and showed temperature-sensitive protein maturation suggesting that p.I37N induces protein misfolding. L1CAM-mediated cell-cell aggregation was severely impaired by L1CAM variants p.I37N, p.M172I and p.D202Y but was preserved by the variant p.T38M. Our experimental data indicate that protein misfolding and accumulation in the ER affect function of the L1CAM variant p.I37N whereas the variants p.M172I and p.D202Y impair homophilic interaction at the cell surface.

  19. A new role for the neuronal ubiquitin C-terminal hydrolase-L1 (UCH-L1) in podocyte process formation and podocyte injury in human glomerulopathies.

    PubMed

    Meyer-Schwesinger, C; Meyer, T N; Münster, S; Klug, P; Saleem, M; Helmchen, U; Stahl, R A K

    2009-02-01

    Glomerular epithelial cell (podocyte) injury is characterized by foot process retraction, slit diaphragm reorganization, and degradation of podocyte-specific proteins. However, the mechanisms underlying podocyte injury are largely unknown. The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a key modulator of ubiquitin modification in neurons. Like neurons, UCH-L1 expression was associated with an undifferentiated status in cultured human podocytes, whereas differentiation and arborization decreased UCH-L1 and monoUb expression. Inhibition of UCH-L1 induced time and concentration-dependent process formation with alpha-actinin-4 distribution to the cell membrane and processes. An immunohistochemical approach was used to evaluate whether UCH-L1 expression was associated with podocyte injury in 15 different human glomerular diseases. Whereas normal kidneys expressed no UCH-L1 and little ubiquitin, a subset of human glomerulopathies associated with podocyte foot process effacement (membranous nephropathy, SLE class V, FSGS) de novo expressed UCH-L1 in podocyte cell bodies, nuclei, and processes. Interestingly, UCH-L1 expression correlated with podocyte ubiquitin content and internalization of the podocyte-specific proteins nephrin and alpha-actinin-4. In contrast, minimal change glomerulonephritis, a reversible disease, demonstrated minimal UCH-L1 and ubiquitin expression with intact alpha-actinin-4 but internalized nephrin. Glomerular kidney diseases typically not associated with foot process effacement (SLE class IV, ANCA+ necrotizing GN, amyloidosis, IgA nephritis) expressed intermediate to no UCH-L1 and ubiquitin. These studies show a role for UCH-L1 and ubiquitin modification in podocyte differentiation and injury.

  20. Combination therapy with catechins and caffeine inhibits fat accumulation in 3T3-L1 cells

    PubMed Central

    Zhu, Xiaojuan; Yang, Licong; Xu, Feng; Lin, Lezhen; Zheng, Guodong

    2017-01-01

    Catechins and caffeine, which are green tea components, have a slimming effect; however, the combinational effect of fat metabolism in 3T3-L1 cells remains unclear. In the present study, 3T3-L1 cells were treated with catechins and caffeine in combination, and it was found that combination therapy with catechins and caffeine markedly reduced intracellular fat accumulation, mRNA expression levels of peroxisome proliferator-activated receptor-γ and CCAAT/enhancer-binding protein α in the early stage of cell differentiation were significantly reduced, and mRNA expression of fatty acid synthetase(FAS) andglycerol-3-phosphate dehydrogenase protein expression levels of FAS were downregulated. Noradrenaline-induced lipolysis was enhanced by caffeine, which markedly increased the protein expression of adipose triglyceride lipase and hormone sensitive lipase. These results indicated that combination therapy with catechins and caffeine synergistically inhibited lipid accumulation by regulating the gene and protein expression levels of lipid metabolism-related enzymes. Therefore, catechins and caffeine combination therapy has potential as a functional food that may be used to prevent obesity and lifestyle-associated diseases. PMID:28352352

  1. Berberine inhibits 3T3-L1 adipocyte differentiation through the PPARgamma pathway.

    PubMed

    Huang, Cheng; Zhang, Yuebo; Gong, Zhenwei; Sheng, Xiaoyan; Li, Zongmeng; Zhang, Wei; Qin, Ying

    2006-09-22

    Berberine (BBR), a compound purified from Cortidis rhizoma, reduces serum cholesterol, triglycerides, and LDL-cholesterol of hypercholesterolemic patients and high fat diet fed animals, and increases hepatic LDLR mRNA and protein levels through a post-transcriptional mechanism. BBR also enhances the hypoglycemic action of insulin in diabetic animal models. Here, we show that BBR inhibits the differentiation of 3T3-L1 preadipocytes induced by DM and suppresses the mitotic clonal expansion of 3T3-L1 preadipocytes in a time- and dose-dependent manner. Gene expression analysis and Western blot analysis reveal that the BBR inhibits the mRNA and protein levels of adipogenesis related transcription factors PPARgamma and C/EBPalpha and their upstream regulator, C/EBPbeta. Reporter gene assays demonstrate that the full-length PPARgamma and alpha transcription activities are inhibited by BBR. Using real-time PCR, we have also found that the PPAR target genes that are involved in adipocyte differentiation, such as aP2, CD36, ACO, LPL, and other adipocyte markers, are suppressed by BBR. These studies suggest that BBR works on multiple molecular targets as an inhibitor of PPARgamma and alpha, and is a potential weight reducing, hypolipidemic, and hypoglycemic drug.

  2. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy: analysis of the organization of the Atg16L1 complex in fibroblasts.

    PubMed

    Fujita, Naonobu; Saitoh, Tatsuya; Kageyama, Shun; Akira, Shizuo; Noda, Takeshi; Yoshimori, Tamotsu

    2009-11-20

    A single nucleotide polymorphism in Atg16L1, an autophagy-related gene (ATG), is a risk factor for Crohn disease, a major form of chronic inflammatory bowel disease. However, it is still unknown how the Atg16L1 variant contributes to disease development. The Atg16L1 protein possesses a C-terminal WD repeat domain whose function is entirely unknown, and the Crohn disease-associated mutation (T300A) is within this domain. To elucidate the function of the WD repeat domain, we established an experimental system in which a WD repeat domain mutant of Atg16L1 is stably expressed in Atg16L1-deficient mouse embryonic fibroblasts. Using the system, we show that the Atg16L1 complex forms a dimeric complex and that the total Atg16L1 protein level is strictly maintained, possibly by the ubiquitin proteasome system. Furthermore, we show that an Atg16L1 WD repeat domain deletion and the T300A mutant have little impact on canonical autophagy and autophagy against Salmonella enterica serovar Typhimurium. Therefore, we propose that Atg16L1 T300A is differentially involved in Crohn disease and canonical autophagy.

  3. Dimethylfumarate suppresses adipogenic differentiation in 3T3-L1 preadipocytes through inhibition of STAT3 activity.

    PubMed

    Kang, Hyeon-Ji; Seo, Hyun-Ae; Go, Younghoon; Oh, Chang Joo; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2013-01-01

    The excessive accumulation of adipocytes contributes to the development of obesity and obesity-related diseases. The interactions of several transcription factors, such as C/EBPβ, PPARγ, C/EBPα, Nrf2, and STAT3, are required for adipogenic differentiation. Dimethylfumarate (DMF), an immune modulator and antioxidant, may function as an inhibitor of STAT3 and an activator of Nrf2. This study examined whether DMF inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting STAT3 or activating Nrf2. DMF suppressed 3T3-L1 preadipocyte differentiation to mature adipocytes in a dose-dependent manner as determined by Oil Red O staining. The mRNA and protein levels of adipogenic genes, including C/EBPβ, C/EBPα, PPARγ, SREBP-1c, FAS, and aP2, were significantly lower in DMF-treated 3T3-L1 preadipocytes. Suppression of adipogenic differentiation by DMF treatment resulted primarily from inhibition of the early stages of differentiation. DMF inhibits clonal expansion during adipogenic differentiation through induction of a G1 cell cycle arrest. Additionally, DMF regulates cell cycle-related proteins, such as p21, pRb, and cyclin D. DMF treatment markedly inhibited differentiation medium-induced STAT3 phosphorylation and inhibited STAT3 transcriptional activation of a reporter construct composed of four synthetic STAT3-response elements. Moreover, inhibition of endogenous Nrf2 activity using a dominant negative Nrf2 did not abolish the DMF-induced inhibition of adipogenic differentiation of 3T3-L1 preadipocytes. In summary, DMF is a negative regulator of adipogenic differentiation based on its regulation of adipogenic transcription factors and cell cycle proteins. This negative regulation by DMF is mediated by STAT3 inhibition, but is unlikely to involve Nrf2 activation.

  4. Effects of C-reactive protein on adipokines genes expression in 3T3-L1 adipocytes

    SciTech Connect

    Yuan, Guoyue; Jia, Jue; Di, Liangliang; Zhou, Libin; Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang; Li, Lianxi; Yang, Ying; Mao, Chaoming; Chen, Mingdao

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer CRP increases TNF-{alpha} and IL-6 genes expression in matured 3T3-L1 adipocytes. Black-Right-Pointing-Pointer CRP suppresses adiponectin, leptin and PPAR-{gamma} mRNA levels in matured 3T3-L1 cells. Black-Right-Pointing-Pointer Wortmannin reverses effects of CRP on adiponectin, TNF-{alpha} and leptin mRNA levels. Black-Right-Pointing-Pointer CRP may regulate IR, obesity and metabolic syndrome by this mechanism. -- Abstract: Adipose tissue is now recognized to be an important endocrine organ, secreting a variety of adipokines that are involved in the regulation of energy metabolism, insulin resistance and metabolic syndrome. C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. A number of studies have shown that elevation of CRP concentrations is an independent predictive parameter of type 2 diabetes mellitus, which is also strongly associated with various components of the metabolic syndrome. The aim of the present study is to investigate the effects of CRP on adipokines genes expression in 3T3-L1 adipocytes. Quantitative real-time PCR analysis revealed that CRP inhibited adiponectin, leptin and peroxisome proliferator-activated receptor-gamma (PPAR-{gamma}) genes expression and raised tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) mRNA levels in matured 3T3-L1 adipocytes in a dose and time-dependent manner. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by wortmannin partially reversed the effects of CRP on adiponectin, TNF-{alpha} and leptin genes expression. These results collectively suggest that CRP regulates adiponectin, TNF-{alpha}, leptin, IL-6 and PPAR-{gamma} genes expression, and that might represent a mechanism by which CRP regulates insulin resistance, obesity and metabolic syndrome.

  5. PD-L1 is a critical mediator of regulatory B cells and T cells in invasive breast cancer

    PubMed Central

    Guan, Honggeng; Wan, Yuqiu; Lan, Jing; Wang, Qin; Wang, Zhangyu; Li, Yecheng; Zheng, Jiqing; Zhang, Xueguang; Wang, Zemin; Shen, Yueping; Xie, Fang

    2016-01-01

    Regulatory T cells (Tregs), a key mediator in regulating anti-tumor immune suppression, tumor immune escape, metastasis and relapse, are considered an important therapeutic target in immunotherapy of human cancers. In the present investigation, elevated CD19+ CD24+ CD38+ regulatory B cells (Bregs) were observed in PBMCs of invasive carcinoma of breast (IBCa) patients compared with that in patients with fibroadenoma (FIBma) or healthy individuals, and the positive correlation existed between Bregs and CD4+ CD25+ CD127− Tregs (r = 0.316, P = 0.001). We found that PD-L1 expression was higher on Bregs in IBCa patients compared with patients with FIBma or healthy individuals (P < 0.05, respectively), and that a tight correlation exists between CD19+ CD24+ CD38+ PD-L1+ Bregs and CD19+ CD24+ CD38+ Bregs (r = 0.267, P = 0.007), poor TNM phases and up-regulated expression of PD-L1 on Bregs. The pattern of PD-1 expression on CD4+ T cells indicated that high level of PD-1hi expressed on CD4+ CD25+ CD127+ effector T cells (P < 0.001). More importantly, the presence of PD-L1 on Bregs was positively correlated with Tregs (r = 0.299, P = 0.003), but negatively correlated with PD-1hi effector T cells (r = −0.22, P = 0.031). Together, results of the present study indicated that PD-L1 is an important molecule on Bregs, mediated the generation of Tregs in IBCa. PMID:27762298

  6. L1 CELL ADHESION MOLECULE IS NEUROPROTECTIVE OF ALCOHOL INDUCED CELL DEATH

    PubMed Central

    Gubitosi-Klug, Rose; Larimer, Corena G.; Bearer, Cynthia F.

    2009-01-01

    L1 cell adhesion molecule (L1), a protein critical for appropriate development of the central nervous system, is a target for ethanol teratogenicity. Ethanol inhibits both L1 mediated cell adhesion as well as L1 mediated neurite outgrowth. L1 has been shown to increase cell survival in cerebellar granule cells while ethanol has been shown to increase cell death. We sought to determine if L1 protected cells from ethanol induced cell death. Cerebellar granule cells from postnatal day 6 rat pups were cultured on either poly L-lysine with or without an L1 substratum. Alcohol was added at 2 hours post plating and cell survival was measured at various times. L1 substratum significantly increased cell survival at 72 and 120 hours. Ethanol significantly reduced cell survival at 48 hours, with no effect at 72 or 120 hours, both in the presence and absence of L1. At 48 hours, L1 significantly increased cell survival in the presence of ethanol. We conclude that ethanol interferes with processes other than L1-L1 interactions in causing cell death, and that ethanol effects would be more severe in the absence of L1. PMID:17267039

  7. PD-L1 expression in human cancers and its association with clinical outcomes

    PubMed Central

    Wang, Xin; Teng, Feifei; Kong, Li; Yu, Jinming

    2016-01-01

    PD-L1 is an immunoinhibitory molecule that suppresses the activation of T cells, leading to the progression of tumors. Overexpression of PD-L1 in cancers such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer is associated with poor clinical outcomes. In contrast, PD-L1 expression correlates with better clinical outcomes in breast cancer and merkel cell carcinoma. The prognostic value of PD-L1 expression in lung cancer, colorectal cancer, and melanoma is controversial. Blocking antibodies that target PD-1 and PD-L1 have achieved remarkable response rates in cancer patients who have PD-L1-overexpressing tumors. However, using PD-L1 as an exclusive predictive biomarker for cancer immunotherapy is questionable due to the low accuracy of PD-L1 immunohistochemistry staining. Factors that affect the accuracy of PD-L1 immunohistochemistry staining are as follows. First, antibodies used in different studies have different sensitivity. Second, in different studies, the cut-off value of PD-L1 staining positivity is different. Third, PD-L1 expression in tumors is not uniform, and sampling time and location may affect the results of PD-L1 staining. Therefore, better understanding of tumor microenvironment and use of other biomarkers such as gene marker and combined index are necessary to better identify patients who will benefit from PD-1/PD-L1 checkpoint blockade therapy. PMID:27574444

  8. AnL1 smoothing spline algorithm with cross validation

    NASA Astrophysics Data System (ADS)

    Bosworth, Ken W.; Lall, Upmanu

    1993-08-01

    We propose an algorithm for the computation ofL1 (LAD) smoothing splines in the spacesWM(D), with . We assume one is given data of the formyiD(f(ti) +ɛi, iD1,...,N with {itti}iD1N ⊂D, theɛi are errors withE(ɛi)D0, andf is assumed to be inWM. The LAD smoothing spline, for fixed smoothing parameterλ?;0, is defined as the solution,sλ, of the optimization problem (1/N)∑iD1N yi-g(ti +λJM(g), whereJM(g) is the seminorm consisting of the sum of the squaredL2 norms of theMth partial derivatives ofg. Such an LAD smoothing spline,sλ, would be expected to give robust smoothed estimates off in situations where theɛi are from a distribution with heavy tails. The solution to such a problem is a "thin plate spline" of known form. An algorithm for computingsλ is given which is based on considering a sequence of quadratic programming problems whose structure is guided by the optimality conditions for the above convex minimization problem, and which are solved readily, if a good initial point is available. The "data driven" selection of the smoothing parameter is achieved by minimizing aCV(λ) score of the form .The combined LAD-CV smoothing spline algorithm is a continuation scheme in λ↘0 taken on the above SQPs parametrized inλ, with the optimal smoothing parameter taken to be that value ofλ at which theCV(λ) score first begins to increase. The feasibility of constructing the LAD-CV smoothing spline is illustrated by an application to a problem in environment data interpretation.

  9. Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes.

    PubMed

    Fleuren, Wilco W M; Linssen, Margot M L; Toonen, Erik J M; van der Zon, Gerard C M; Guigas, Bruno; de Vlieg, Jacob; Dokter, Wim H A; Ouwens, D Margriet; Alkema, Wynand

    2013-05-01

    Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.

  10. Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes

    PubMed Central

    Fleuren, Wilco W. M.; Linssen, Margot M. L.; Toonen, Erik J. M.; van der Zon, Gerard C. M.; Guigas, Bruno; de Vlieg, Jacob; Dokter, Wim H. A.; Ouwens, D. Margriet

    2013-01-01

    Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids. PMID:23506355

  11. Ubiquitin C-terminal hydrolase-L1 (Uch-L1) correlates with gonadal transformation in the rice field eel.

    PubMed

    Sun, Jinhua; Shang, Xuan; Tian, Yihao; Zhao, Wei; He, Yan; Chen, Ke; Cheng, Hanhua; Zhou, Rongjia

    2008-01-01

    The ubiquitin-proteasome pathway is crucial for a variety of biological processes, including spermatogenesis. Ubiquitin C-terminal hydrolase-L1 (Uch-L1) is thought to associate with monoubiquitin to control ubiquitin levels. Here, we report the identification of Uch-L1 cDNA from the testis of the rice field eel, a natural sex reversal vertebrate, by using cDNA microarray analysis. Uch-L1 encodes a protein of 220 amino acids that shows high homology to Uch-L1 of vertebrates, especially fish species. Both mRNA and protein are mainly expressed in testis, ovotestis and ovary, as well as in the brain. Immunohistochemistry analysis revealed differential expression of Uch-L1 in three kinds of gonads. In the ovary, expression of Uch-L1 was observed mainly in the developing ovary and slightly in the mature ovary. In ovotestis during the intersex stage, Uch-L1 was expressed in the male gonad epithelium and degraded ovary. In testis, expression was observed in developing germ cells, including spermatogonia and spermatocytes. Furthermore, Uch-L1 was upregulated during gonadal transformation, especially from the beginning of the intersex stage onwards. Native-PAGE showed that Uch-L1 underwent dimerization and oligomerization in gonads, and that the relative level of dimerization/oligomerization decreased during gonadal transformation. Simultaneously, ubiquitin polypeptide expression was upregulated during this process. These results suggest that Uch-L1, via the ubiquitin-proteasome system, may play an important role not only in gametogenesis, but also in the gonadal transformation process in the rice field eel.

  12. PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation.

    PubMed

    Noman, Muhammad Zaeem; Desantis, Giacomo; Janji, Bassam; Hasmim, Meriem; Karray, Saoussen; Dessen, Philippe; Bronte, Vincenzo; Chouaib, Salem

    2014-05-05

    Tumor-infiltrating myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) form an important component of the hypoxic tumor microenvironment. Here, we investigated the influence of hypoxia on immune checkpoint receptors (programmed death [PD]-1 and CTLA-4) and their respective ligands (PD-1 ligand 1 [PD-L1], PD-L2, CD80, and CD86) on MDSCs. We demonstrate that MDSCs at the tumor site show a differential expression of PD-L1 as compared with MDSCs from peripheral lymphoid organ (spleen). Hypoxia caused a rapid, dramatic, and selective up-regulation of PD-L1 on splenic MDSCs in tumor-bearing mice. This was not limited to MDSCs, as hypoxia also significantly increased the expression of PD-L1 on macrophages, dendritic cells, and tumor cells. Furthermore, PD-L1 up-regulation under hypoxia was dependent on hypoxia-inducible factor-1α (HIF-1α) but not HIF-2α. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of HIF-1α to a transcriptionally active hypoxia-response element (HRE) in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation and was accompanied by the down-regulation of MDSCs IL-6 and IL-10. Finally, neutralizing antibodies against IL-10 under hypoxia significantly abrogated the suppressive activity of MDSCs. Simultaneous blockade of PD-L1 along with inhibition of HIF-1α may thus represent a novel approach for cancer immunotherapy.

  13. The PD1:PD-L1/2 Pathway from Discovery to Clinical Implementation

    PubMed Central

    Bardhan, Kankana; Anagnostou, Theodora; Boussiotis, Vassiliki A.

    2016-01-01

    The immune system maintains a critically organized network to defend against foreign particles, while evading self-reactivity simultaneously. T lymphocytes function as effectors and play an important regulatory role to orchestrate the immune signals. Although central tolerance mechanism results in the removal of the most of the autoreactive T cells during thymic selection, a fraction of self-reactive lymphocytes escapes to the periphery and pose a threat to cause autoimmunity. The immune system evolved various mechanisms to constrain such autoreactive T cells and maintain peripheral tolerance, including T cell anergy, deletion, and suppression by regulatory T cells (TRegs). These effects are regulated by a complex network of stimulatory and inhibitory receptors expressed on T cells and their ligands, which deliver cell-to-cell signals that dictate the outcome of T cell encountering with cognate antigens. Among the inhibitory immune mediators, the pathway consisting of the programed cell death 1 (PD-1) receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) plays an important role in the induction and maintenance of peripheral tolerance and for the maintenance of the stability and the integrity of T cells. However, the PD-1:PD-L1/L2 pathway also mediates potent inhibitory signals to hinder the proliferation and function of T effector cells and have inimical effects on antiviral and antitumor immunity. Therapeutic targeting of this pathway has resulted in successful enhancement of T cell immunity against viral pathogens and tumors. Here, we will provide a brief overview on the properties of the components of the PD-1 pathway, the signaling events regulated by PD-1 engagement, and their consequences on the function of T effector cells. PMID:28018338

  14. The PD1:PD-L1/2 Pathway from Discovery to Clinical Implementation.

    PubMed

    Bardhan, Kankana; Anagnostou, Theodora; Boussiotis, Vassiliki A

    2016-01-01

    The immune system maintains a critically organized network to defend against foreign particles, while evading self-reactivity simultaneously. T lymphocytes function as effectors and play an important regulatory role to orchestrate the immune signals. Although central tolerance mechanism results in the removal of the most of the autoreactive T cells during thymic selection, a fraction of self-reactive lymphocytes escapes to the periphery and pose a threat to cause autoimmunity. The immune system evolved various mechanisms to constrain such autoreactive T cells and maintain peripheral tolerance, including T cell anergy, deletion, and suppression by regulatory T cells (TRegs). These effects are regulated by a complex network of stimulatory and inhibitory receptors expressed on T cells and their ligands, which deliver cell-to-cell signals that dictate the outcome of T cell encountering with cognate antigens. Among the inhibitory immune mediators, the pathway consisting of the programed cell death 1 (PD-1) receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) plays an important role in the induction and maintenance of peripheral tolerance and for the maintenance of the stability and the integrity of T cells. However, the PD-1:PD-L1/L2 pathway also mediates potent inhibitory signals to hinder the proliferation and function of T effector cells and have inimical effects on antiviral and antitumor immunity. Therapeutic targeting of this pathway has resulted in successful enhancement of T cell immunity against viral pathogens and tumors. Here, we will provide a brief overview on the properties of the components of the PD-1 pathway, the signaling events regulated by PD-1 engagement, and their consequences on the function of T effector cells.

  15. TCF7L1 Modulates Colorectal Cancer Growth by Inhibiting Expression of the Tumor-Suppressor Gene EPHB3

    PubMed Central

    Murphy, Matthew; Chatterjee, Sujash S.; Jain, Sidharth; Katari, Manpreet; DasGupta, Ramanuj

    2016-01-01

    Dysregulation of the Wnt pathway leading to accumulation of β-catenin (CTNNB1) is a hallmark of colorectal cancer (CRC). Nuclear CTNNB1 acts as a transcriptional coactivator with TCF/LEF transcription factors, promoting expression of a broad set of target genes, some of which promote tumor growth. However, it remains poorly understood how CTNNB1 interacts with different transcription factors in different contexts to promote different outcomes. While some CTNNB1 target genes are oncogenic, others regulate differentiation. Here, we found that TCF7L1, a Wnt pathway repressor, buffers CTNNB1/TCF target gene expression to promote CRC growth. Loss of TCF7L1 impaired growth and colony formation of HCT116 CRC cells and reduced tumor growth in a mouse xenograft model. We identified a group of CTNNB1/TCF target genes that are activated in the absence of TCF7L1, including EPHB3, a marker of Paneth cell differentiation that has also been implicated as a tumor suppressor in CRC. Knockdown of EPHB3 partially restores growth and normal cell cycle progression of TCF7L1-Null cells. These findings suggest that while CTNNB1 accumulation is critical for CRC progression, activation of specific Wnt target genes in certain contexts may in fact inhibit tumor growth. PMID:27333864

  16. Quantifying the Quality Difference between L1 and L2 Essays: A Rating Procedure with Bilingual Raters and L1 and L2 Benchmark Essays

    ERIC Educational Resources Information Center

    Tillema, Marion; van den Bergh, Huub; Rijlaarsdam, Gert; Sanders, Ted

    2013-01-01

    It is the consensus that, as a result of the extra constraints placed on working memory, texts written in a second language (L2) are usually of lower quality than texts written in the first language (L1) by the same writer. However, no method is currently available for quantifying the quality difference between L1 and L2 texts. In the present…

  17. The L2 Acquisition of Spanish Rhotics by L1 English Speakers: The Effect of L1 Articulatory Routines and Phonetic Context for Allophonic Variation

    ERIC Educational Resources Information Center

    Olsen, Michael K.

    2012-01-01

    This article offers a fine-grained investigation of how first-language (L1) phonetics involving English rhotics affect Spanish rhotic production by second-language (L2) learners. Specifically, this study investigates how different L1 English rhotic articulatory routines (retroflex-like and bunched-like) and the phonetic context that produces…

  18. Do L2 Writing Courses Affect the Improvement of L1 Writing Skills via Skills Transfer from L2 to L1?

    ERIC Educational Resources Information Center

    Gonca, Altmisdort

    2016-01-01

    This study investigates the relationship of second language (L2) writing skills proficiency with the first language (L1) writing skills, in light of the language transfer. The study aims to analyze the positive effects of L2 writing proficiency on L1 writing proficiency. Forty native Turkish-speaking university students participated in the study.…

  19. Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

    PubMed Central

    Si, Yaguang; Shi, Hai; Lee, Kyongbum

    2009-01-01

    Background Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear. Methodology/Principal Findings This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects. Conclusions/Significance The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism. PMID:19746157

  20. Accent, Listening Assessment and the Potential for a Shared-L1 Advantage: A DIF Perspective

    ERIC Educational Resources Information Center

    Harding, Luke

    2012-01-01

    This paper reports on an investigation of the potential for a shared-L1 advantage on an academic English listening test featuring speakers with L2 accents. Two hundred and twelve second-language listeners (including 70 Mandarin Chinese L1 listeners and 60 Japanese L1 listeners) completed three versions of the University Test of English as a Second…

  1. L1 Glosses: Effects on EFL Learners' Reading Comprehension and Vocabulary Retention

    ERIC Educational Resources Information Center

    Cheng, Ying-Hsueh; Good, Robert L.

    2009-01-01

    The present study examines the effects of 3 kinds of glosses--first-language (L1) Chinese glosses plus second-language (L2) English example sentences, L1 in-text glosses, and L1 marginal glosses--in comparison with a no-gloss condition in reading an English passage, to explore whether providing glosses can facilitate reading comprehension and…

  2. 26 CFR 36.3121(l)(1)-3 - Effect of agreement.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Effect of agreement. 36.3121(l)(1)-3 Section 36.3121(l)(1)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-3 Effect of agreement. (a) Liability for amounts equivalent to tax—(1) In...

  3. 26 CFR 31.3402(l)-1 - Determination and disclosure of marital status.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    .... 31.3402(l)-1 Section 31.3402(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... COLLECTION OF INCOME TAX AT SOURCE Collection of Income Tax at Source § 31.3402(l)-1 Determination and... married person. (c) Determination of marital status. For the purposes of section 3402(l)(2) and...

  4. 26 CFR 31.3402(l)-1 - Determination and disclosure of marital status.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    .... 31.3402(l)-1 Section 31.3402(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... COLLECTION OF INCOME TAX AT SOURCE Collection of Income Tax at Source § 31.3402(l)-1 Determination and... married person. (c) Determination of marital status. For the purposes of section 3402(l)(2) and...

  5. 26 CFR 36.3121(l)(1)-3 - Effect of agreement.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 15 2012-04-01 2012-04-01 false Effect of agreement. 36.3121(l)(1)-3 Section 36.3121(l)(1)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-3 Effect of agreement. (a) Liability for amounts equivalent to tax—(1) In...

  6. On the Effects of L2 on Iranian Bilinguals' L1 Writing Ability

    ERIC Educational Resources Information Center

    Agheshteh, Hessam

    2015-01-01

    While the effects of L1 on L2 have been extensively investigated, the effects of L2 on L1 have been largely ignored. The present study attempted to address this issue by investigating the effects of L2 English on Iranian Bilinguals' L1 writing ability. For this end, 61 participants, 30 bilinguals and 31 monolinguals, were assessed on an…

  7. 26 CFR 36.3121(l)(1)-2 - Amendment of agreement.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 15 2014-04-01 2014-04-01 false Amendment of agreement. 36.3121(l)(1)-2 Section 36.3121(l)(1)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-2 Amendment of agreement. (a) An agreement entered into by a domestic...

  8. 26 CFR 36.3121(l)(1)-3 - Effect of agreement.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 15 2013-04-01 2013-04-01 false Effect of agreement. 36.3121(l)(1)-3 Section 36.3121(l)(1)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-3 Effect of agreement. (a) Liability for amounts equivalent to tax—(1) In...

  9. 26 CFR 36.3121(l)(1)-2 - Amendment of agreement.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 15 2011-04-01 2011-04-01 false Amendment of agreement. 36.3121(l)(1)-2 Section 36.3121(l)(1)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-2 Amendment of agreement. (a) An agreement entered into by a domestic...

  10. 26 CFR 31.3402(l)-1 - Determination and disclosure of marital status.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    .... 31.3402(l)-1 Section 31.3402(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... COLLECTION OF INCOME TAX AT SOURCE Collection of Income Tax at Source § 31.3402(l)-1 Determination and... married person. (c) Determination of marital status. For the purposes of section 3402(l)(2) and...

  11. 26 CFR 1.401(l)-1 - Permitted disparity in employer-provided contributions or benefits.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... contributions or benefits. 1.401(l)-1 Section 1.401(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.401(l)-1 Permitted disparity in employer-provided contributions or benefits... permitted under section 401(l). Thus, if a plan satisfies section 401(l), permitted disparities in...

  12. 26 CFR 1.401(l)-1 - Permitted disparity in employer-provided contributions or benefits.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... contributions or benefits. 1.401(l)-1 Section 1.401(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT... Plans, Etc. § 1.401(l)-1 Permitted disparity in employer-provided contributions or benefits. (a... section 401(l). Thus, if a plan satisfies section 401(l), permitted disparities in...

  13. 26 CFR 36.3121(l)(1)-2 - Amendment of agreement.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 26 Internal Revenue 15 2013-04-01 2013-04-01 false Amendment of agreement. 36.3121(l)(1)-2 Section 36.3121(l)(1)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-2 Amendment of agreement. (a) An agreement entered into by a domestic...

  14. 26 CFR 1.401(l)-1 - Permitted disparity in employer-provided contributions or benefits.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... contributions or benefits. 1.401(l)-1 Section 1.401(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.401(l)-1 Permitted disparity in employer-provided contributions or benefits... permitted under section 401(l). Thus, if a plan satisfies section 401(l), permitted disparities in...

  15. 26 CFR 36.3121(l)(1)-2 - Amendment of agreement.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 26 Internal Revenue 15 2012-04-01 2012-04-01 false Amendment of agreement. 36.3121(l)(1)-2 Section 36.3121(l)(1)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-2 Amendment of agreement. (a) An agreement entered into by a domestic...

  16. 26 CFR 1.401(l)-1 - Permitted disparity in employer-provided contributions or benefits.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... contributions or benefits. 1.401(l)-1 Section 1.401(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.401(l)-1 Permitted disparity in employer-provided contributions or benefits... permitted under section 401(l). Thus, if a plan satisfies section 401(l), permitted disparities in...

  17. 26 CFR 36.3121(l)(1)-3 - Effect of agreement.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 15 2011-04-01 2011-04-01 false Effect of agreement. 36.3121(l)(1)-3 Section 36.3121(l)(1)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-3 Effect of agreement. (a) Liability for amounts equivalent to tax—(1) In...

  18. 26 CFR 31.3402(l)-1 - Determination and disclosure of marital status.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    .... 31.3402(l)-1 Section 31.3402(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... COLLECTION OF INCOME TAX AT SOURCE Collection of Income Tax at Source § 31.3402(l)-1 Determination and... married person. (c) Determination of marital status. For the purposes of section 3402(l)(2) and...

  19. 26 CFR 31.3402(l)-1 - Determination and disclosure of marital status.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    .... 31.3402(l)-1 Section 31.3402(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE... COLLECTION OF INCOME TAX AT SOURCE Collection of Income Tax at Source § 31.3402(l)-1 Determination and... married person. (c) Determination of marital status. For the purposes of section 3402(l)(2) and...

  20. 26 CFR 1.401(l)-1 - Permitted disparity in employer-provided contributions or benefits.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... contributions or benefits. 1.401(l)-1 Section 1.401(l)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT..., Stock Bonus Plans, Etc. § 1.401(l)-1 Permitted disparity in employer-provided contributions or benefits... permitted under section 401(l). Thus, if a plan satisfies section 401(l), permitted disparities in...

  1. 26 CFR 36.3121(l)(1)-3 - Effect of agreement.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 26 Internal Revenue 15 2014-04-01 2014-04-01 false Effect of agreement. 36.3121(l)(1)-3 Section 36.3121(l)(1)-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-3 Effect of agreement. (a) Liability for amounts equivalent to tax—(1) In...

  2. 26 CFR 36.3121(l)(1)-2 - Amendment of agreement.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Amendment of agreement. 36.3121(l)(1)-2 Section 36.3121(l)(1)-2 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED... SUBSIDIARIES § 36.3121(l)(1)-2 Amendment of agreement. (a) An agreement entered into by a domestic...

  3. English Teachers' Use of Learners' L1 (Arabic) in College Classrooms in Kuwait

    ERIC Educational Resources Information Center

    Alrabah, Sulaiman; Wu, Shu-hua; Alotaibi, Abdullah M.; Aldaihani, Hussein A.

    2016-01-01

    This study investigated English teachers' use of learners' L1 (Arabic) in college classrooms in Kuwait. The purpose of the study was three-fold: (1) to describe the functions for which L1 was employed by the teachers, (2) to explore the affective, sociolinguistic, and psycholinguistic factors that may have led teachers to use L1 in L2 teaching,…

  4. A Study of Relationships between L1 Pragmatic Transfer and L2 Proficiency

    ERIC Educational Resources Information Center

    Bu, Jiemin

    2012-01-01

    Studies in interlanguage pragmatics have shown that L2 learners' proficiency has an influence on the occurrences of L1 pragmatic transfer. However, questions remain whether the relationship between L1 pragmatic transfer and L2 proficiency is positive or negative. This paper is designed to study L1 pragmatic transfer in requests made by Chinese…

  5. CALL versus Paper: In Which Context Are L1 Glosses More Effective?

    ERIC Educational Resources Information Center

    Taylor, Alan M.

    2013-01-01

    CALL glossing in first language (L1) or second language (L2) texts has been shown by previous studies to be more effective than traditional, paper-and-pen L1 glossing. Using a pool of studies with much more statistical power and more accurate results, this meta-analysis demonstrates more precisely the degree to which CALL L1 glossing can be more…

  6. Metacognitive Online Reading Strategy Use: Readers' Perceptions in L1 and L2

    ERIC Educational Resources Information Center

    Taki, Saeed

    2016-01-01

    This study aimed to explore whether first-language (L1) readers of different language backgrounds would employ similar metacognitive online reading strategies and whether reading online in a second language (L2) could be influenced by L1 reading strategies. To this end, 52 Canadian college students as English L1 readers and 38 Iranian university…

  7. MicroRNA-24 Modulates Staphylococcus aureus-Induced Macrophage Polarization by Suppressing CHI3L1.

    PubMed

    Jingjing, Zhang; Nan, Zhang; Wei, Wu; Qinghe, Guo; Weijuan, Wang; Peng, Wang; Xiangpeng, Wang

    2017-03-16

    Macrophages play a crucial role in host innate anti-Staphylococcus aureus defense, which is tightly regulated by multiple factors, including microRNAs. A recent study showed that miR-24 plays an important role in macrophage polarization. Here, we investigated the biological function of miR-24 in S. aureus-stimulated macrophages. The results revealed that miR-24 expression was significantly decreased in both human and mouse macrophage cell lines with S. aureus stimulation in a time-dependent manner. Moreover, miR-24 overexpression significantly decreased the production of M1 phenotype markers, such as IL-6, iNOS, TNF-α, CD86, and CD80, whereas it increased the production of M2 markers, such as Arg1, CCL17, CCL22, CD163, and CD206, in S. aureus-stimulated macrophages. Conversely, knockdown of miR-24 promoted M1 macrophage polarization but diminished M2 macrophage polarization in S. aureus-stimulated macrophages. Furthermore, CHI3L1 was predicted as a target gene of miR-24 using bioinformatics software and identified by luciferase reporter assay. Additionally, miR-24 overexpression inhibited CHI3L1 expression and downregulated the downstream MAPK pathway in S. aureus-stimulated macrophages. Finally, CHI3L1 overexpression rescued macrophage polarization and MAPK pathway inhibition induced by miR-24 mimic transfection in S. aureus-stimulated macrophages. In conclusion, the data suggest that miR-24 serves as a molecular regulator in S. aureus-induced macrophage polarization through targeting of CHI3L1 and regulation of the MAPK pathway, which may provide a promising therapeutic target for S. aureus-related infections and inflammatory diseases.

  8. Interferon-γ-induced activation of JAK1 and JAK2 suppresses tumor cell susceptibility to NK cells through upregulation of PD-L1 expression.

    PubMed

    Bellucci, Roberto; Martin, Allison; Bommarito, Davide; Wang, Kathy; Hansen, Steen H; Freeman, Gordon J; Ritz, Jerome

    2015-06-01

    Inhibition of JAK1 or JAK2 in human tumor cells was previously shown to increase susceptibility of these cells to NK cell lysis. In the present study, we examined the cellular mechanisms that mediate this effect in hematopoietic tumor cell lines and primary tumor cells. Incubation of tumor cells with supernatant from activated NK cells or interferon-gamma (IFNγ)-induced activation of pSTAT1 and increased expression of PD-L1 without altering expression of other activating or inhibitory NK cell ligands. These functional effects were blocked by chemical JAK inhibition or shRNAs targeting JAK1, JAK2 or STAT1. Inhibition of IFNγ signaling also prevented the upregulation of PD-L1 and blocking PD-L1 resulted in increased tumor lysis by NK cells. These results show that NK cell activation and secretion of IFNγ results in activation of JAK1, JAK2 and STAT1 in tumor cells, resulting in rapid up-regulation of PD-L1 expression. Increased expression of PD-L1 results in increased resistance to NK cell lysis. Blockade of JAK pathway activation prevents increased PD-L1 expression resulting in increased susceptibility of tumor cells to NK cell activity. These observations suggest that JAK pathway inhibitors as well as PD-1 and PD-L1 antibodies may work synergistically with other immune therapies by preventing IFN-induced inhibition of NK cell-mediated tumor cell lysis.

  9. Generation, characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

    PubMed Central

    Cho, Seulki; Park, Insoo; Kim, Haejung; Jeong, Mun Sik; Lim, Mooney; Lee, Eung Suk; Kim, Jin Hong; Kim, Semi; Hong, Hyo Jeong

    2016-01-01

    ABSTRACT L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (KD) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent. PMID:26785809

  10. Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.) 1

    PubMed Central

    Harrison, Marcia A.; Kaufman, Peter B.

    1980-01-01

    Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. `Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10−5 and 10−6 molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10−5 and 10−6 molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon-gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin α (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system. PMID:16661589

  11. L1 retrotransposition alters the hippocampal genomic landscape enabling memory formation.

    PubMed

    Bachiller, Sara; Del-Pozo-Martín, Yaiza; Carrión, Ángel Manuel

    2016-12-21

    Somatic LINE-1 (L1) retrotransposition is a source of genomic mosaicism and potential phenotypic diversity among neurons during brain development. In the adult brain, L1 expression can be triggered by different environmental alterations, but its functional role in this context remains unknown. Here we demonstrate a neural activation-dependent increase in the number of L1 retrotransposon insertions in the hippocampus. Using both pharmacologic and genetic approaches in mice, we demonstrate that L1 expression in the adult hippocampus enables long-term memory formation. These results provide experimental evidence that L1 retrotransposition-induced genomic mosaicism is involved in cognitive processes such as memory formation.

  12. A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors.

    PubMed

    Chatterjee, Samit; Lesniak, Wojciech G; Gabrielson, Matthew; Lisok, Ala; Wharram, Bryan; Sysa-Shah, Polina; Azad, Babak Behnam; Pomper, Martin G; Nimmagadda, Sridhar

    2016-03-01

    Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.

  13. Chapter 14. Real-time in vitro assays for studying the role of chemokines in lymphocyte transendothelial migration under physiologic flow conditions.

    PubMed

    Shulman, Ziv; Alon, Ronen

    2009-01-01

    The mechanisms underlying leukocyte migration across endothelial barriers are still largely elusive. Integrin activation by chemokine signals is a key checkpoint in this process. Most of the current knowledge on transendothelial migration (TEM) of leukocytes has been derived from in vitro modified Boyden-chamber transfilter migration assays. In these assays, leukocyte migration toward chemokine gradients established across an endothelial barrier is measured under shear-free conditions. Consequently, these assays do not address the critical contribution of shear forces to dynamic integrin activation and redistribution at focal lymphocyte-endothelial contacts. Endothelial chemokines are displayed at high levels on blood vessel walls in vivo and play critical roles in both integrin activation and polarization of leukocytes on blood vessels, yet transwell assays do not assess the role of these chemokines in leukocyte TEM. To overcome these two drawbacks, several laboratories, including our group, developed assays based on in vitro live imaging microscopy to follow leukocyte migration across endothelial barriers that display defined compositions of integrin-stimulatory chemokines. These assays not only successfully simulate physiologic TEM processes but also enable the tracking and dissection of leukocyte adhesion, motility, and crossing of endothelial barriers in real time and under physiologic flow conditions. In addition, fluorescent tagging of membranes, adhesion molecules, and cytoskeletal regulatory elements on the endothelial barrier or the leukocyte can provide key spatial and temporal information on the mode of activity of these elements during distinct stages of leukocyte TEM. After fixation, subcellular changes in the redistribution of these key molecules can be further dissected by immunofluorescence tools and by ultrastructural analysis based on scanning and transmission electron microscopy.

  14. Adapting coculture in vitro models of the blood-brain barrier for use in cancer research: maintaining an appropriate endothelial monolayer for the assessment of transendothelial migration.

    PubMed

    Vandenhaute, Elodie; Drolez, Aurore; Sevin, Emmanuel; Gosselet, Fabien; Mysiorek, Caroline; Dehouck, Marie-Pierre

    2016-05-01

    Although brain metastases are the most common brain tumors in adults, there are few treatment options in this setting. To colonize the brain, circulating tumor cells must cross the blood-brain barrier (BBB), which is situated within specialized, restrictive microvascular endothelium. Understanding how cancer cells manage to transmigrate through the BBB might enable this process to be prevented. In vitro models are dedicated tools for characterizing the cellular and molecular mechanisms that underlie transendothelial migration process, as long as they accurately mimic the brain endothelium's in vivo characteristics. The objective of the present study was to adapt an existing in vitro model of the human BBB for use in studying cancer cell transmigration. The model is based on the coculture of endothelial cells (ECs, derived from cord blood hematopoietic stem cells) and brain pericytes. To allow the migration of cancer cells into the lower compartment, our model had to be transposed onto inserts with a larger pore size. However, we encountered a problem when culturing ECs on large (3-μm)-pore inserts: the cells crossed the membrane and formed a non-physiological second layer on the lower face of the insert. Using 3-μm-pore inserts (in a 12-well plate format), we report here on a method that enables the maintenance of a single monolayer of ECs on the insert's upper face only. Under these chosen conditions, the ECs exhibited typical BBB properties found in the original model (including restricted paracellular permeability and the expression of continuous tight junctions). This modified in vitro model of the human BBB enabled us to investigate the migratory potential of the MDA-MB-231 cell line (derived from highly metastatic human breast cancer cells). Last, the results obtained were compared with the rate of transmigration through endothelia with no BBB features.

  15. The Ribosomal Protein Rpl22 Controls Ribosome Composition by Directly Repressing Expression of Its Own Paralog, Rpl22l1

    PubMed Central

    O'Leary, Monique N.; Schreiber, Katherine H.; Zhang, Yong; Duc, Anne-Cécile E.; Rao, Shuyun; Hale, J. Scott; Academia, Emmeline C.; Shah, Shreya R.; Morton, John F.; Holstein, Carly A.; Martin, Dan B.; Kaeberlein, Matt; Ladiges, Warren C.; Fink, Pamela J.; MacKay, Vivian L.; Wiest, David L.; Kennedy, Brian K.

    2013-01-01

    Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22−/− mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22−/− mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog. PMID:23990801

  16. [Optimized expression of the L1 protein of human papillomavirus in Hansenula polymorpha].

    PubMed

    Li, Weiwei; He, Xiuping; Guo, Xuena; Zhang, Zhenying; Zhang, Borun

    2009-10-01

    The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.

  17. Isorhamnetin represses adipogenesis in 3T3-L1 cells.

    PubMed

    Lee, Jongsung; Jung, Eunsun; Lee, Jienny; Kim, Saebom; Huh, Sungran; Kim, Youngsoo; Kim, Yongwoo; Byun, Sang Yo; Kim, Yeong-Shik; Park, Deokhoon

    2009-02-01

    Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders including diabetes, hypertension, and heart disease. It is generally accepted that the regulation of adipogenesis or adipokines expression prevents obesity. In this study, we show that isorhamnetin inhibits adipocyte differentiation, as evidenced by reduced triglyceride (TG) accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity. At the molecular level, the mRNA expression levels of peroxidase proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer-binding protein-alpha (C/EBP-alpha), which are the major adipogenic transcription factors, were markedly reduced by isorhamnetin. However, the mRNA levels of C/EBP-beta and -delta, the upstream regulators of PPAR-gamma and C/EBP-alpha, were not reduced by isorhamnetin. Moreover, the mRNA levels of PPAR-gamma target genes such as lipoprotein lipase (LPL), CD36, aP2, and liver X receptor-alpha (LXR-alpha) were downregulated by isorhamnetin. We also showed that isorhamnetin inhibits the expression and secretion of adiponectin, and the results of adiponectin promoter assays suggest the inhibition of PPAR-gamma expression as a possible mechanism underlying the isorhamnetin-mediated effects. Taken together, these results indicate that isorhamnetin inhibits adipogenesis through downregulation of PPAR-gamma and C/EBP-alpha.

  18. cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis.

    PubMed

    Ahn, J I; Yoo, J-Y; Kim, T H; Kim, Y I; Ferguson, S D; Fazleabas, A T; Young, S L; Lessey, B A; Ahn, J Y; Lim, J M; Jeong, J-W

    2016-01-01

    Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner.

  19. PD-L1 Expression and Combined Status of PD-L1/PD-1–Positive Tumor Infiltrating Mononuclear Cell Density Predict Prognosis in Glioblastoma Patients

    PubMed Central

    Han, Jiheun; Hong, Yongkil; Lee, Youn Soo

    2017-01-01

    Background Programmed death ligand 1 (PD-L1) in tumor cells is known to promote immune escape of cancer by interacting with programmed cell death 1 (PD-1) in tumor infiltrating immune cells. Immunotherapy targeting these molecules is emerging as a new strategy for the treatment of glioblastoma (GBM). Understanding the relationship between the PD-L1/PD-1 axis and prognosis in GBM patients may be helpful to predict the effects of immunotherapy. Methods PD-L1 expression and PD-1–positive tumor infiltrating mononuclear cell (PD-1+tumor infiltrating mononuclear cell [TIMC]) density were evaluated using tissue microarray containing 54 GBM cases by immunohistochemical analysis; the associations with patient clinical outcomes were evaluated. Results PD-L1 expression and high PD-1+TIMC density were observed in 31.5% and 50% of GBM cases, respectively. High expression of PD-L1 in tumor cells was an independent and significant predictive factor for worse overall survival (OS; hazard ratio, 4.958; p = .007) but was not a significant factor in disease-free survival (DFS). PD-1+TIMC density was not correlated with OS or DFS. When patients were classified based on PD-1 expression and PD-1+TIMC density, patients with PD-L1+/PD-1+TIMC low status had the shortest OS (13 months, p = .009) and DFS (7 months, p = .053). Conclusions PD-L1 expression in GBM was an independent prognostic factor for poor OS. In addition, combined status of PD-L1 expression and PD-1+TIMC density also predicted patient outcomes, suggesting that the therapeutic role of the PD-1/PD-L1 axis should be considered in the context of GBM immunity. PMID:27989100

  20. Vaccine potential of recombinant pro- and mature cathepsinL1 against fasciolosis gigantica in mice.

    PubMed

    Kueakhai, Pornanan; Changklungmoa, Narin; Chaichanasak, Pannigan; Jaikua, Wipaphorn; Itagaki, Tadashi; Sobhon, Prasert

    2015-10-01

    In Fasciola gigantica cathepsin L1 (CatL1) is a family of predominant proteases that is expressed in caecal epithelial cells and secreted into the excretory-secretory products (ES). CatL1 isotypes are expressed in both early and late stages of the life cycle and the parasites use them for migration and digestion. Therefore, CatL1 is a plausible target for vaccination against this parasite. Recombinant pro-F.gigantica CatL1 (rproFgCatL1) and recombinant mature F.gigantica CatL1 (rmatFgCatL1) were expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rproFgCatL1 and rmatFgCatL1 combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The level of protection of rproFgCatL1 and rmatFgCatL1 vaccines was estimated to be 39.1, 41.7% and 44.9, 47.2% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immuno-blotting to react with the native FgCatL1 in the extract of newly excysted juveniles (NEJ), 4-week-old juveniles and the ES products of 4 week-old juveniles. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune response, respectively, it was found that both Th1 and Th2 responses were significantly increased in rproFgCatL1- and rmatFgCatL1-immunized groups compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rmatFgCatL1-immunized group showed a significant decrease when compared to rproFgCatL1-immunized group, indicating that rmatFgCatL1-vaccinated mice had reduced liver parenchyma damage. The pathological lesions of liver in vaccinated groups were significantly decreased when compared with control groups. This study indicates that rFgCatL

  1. Backbone assignments of the 26 kDa neuron-specific ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1).

    PubMed

    Andersson, Fredrik I; Jackson, Sophie E; Hsu, Shang-Te Danny

    2010-04-01

    UCH-L1 is a member of the family of ubiquitin C-terminal hydrolases whose primary role is to hydrolyze small C-terminal adducts of ubiquitin to generate free ubiquitin monomers. Expression of UCH-L1 is highly specific to neurons and point mutations in this enzyme are associated with a hereditary form of Parkinson's disease. Herein, we present the NMR backbone assignments of human UCH-L1, thus enabling future solution-state NMR spectroscopic studies on the structure and function of this important protein.

  2. The role and possible mechanism of lncRNA U90926 in modulating 3T3-L1 preadipocyte differentiation

    PubMed Central

    Chen, J; Liu, Y; Lu, S; Yin, L; Zong, C; Cui, S; Qin, D; Yang, Y; Guan, Q; Li, X; Wang, X

    2017-01-01

    Background: Obesity is a risk factor for metabolic diseases, while preadipocyte differentiation or adipogenesis is closely related to obesity occurrence. Long noncoding RNAs (lncRNAs) are a unique class of transcripts in regulation of a variety of biological processes. Using cDNA microarray, we found lncRNA U90926 is negatively correlated with 3T3-L1 preadipocyte differentiation. Objective: The aim of this study was to explore the role of lncRNA U90926 (lnc-U90926) in adipogenesis and the underlying mechanisms. Methods: Quantitative real-time PCR (qPCR) was performed to determine lnc-U90926 expression in 3T3-L1 preadipocytes, differentiated adipocytes, and in adipose tissues form mice. RNA fluorescent in situ hybridization (FISH) was performed to determine the localization of lnc-U90926 in 3T3-L1 preadipocytes. The effects of lnc-U90926 on 3T3-L1 adipogenesis were analyzed with lentivirus-mediated gain- and loss-of-function experiments. Lipid accumulation was evaluated by oil red O staining; several adipogenesis makers were analyzed by qPCR and western blotting. Dual luciferase assay was applied to explore the transactivation of target genes modulated by lnc-U90926. All measurements were performed at least for three times. Results: Lnc-U90926 expression decreased along the differentiation of 3T3-L1 preadipocytes. In mice, lnc-U90926 is predominantly expressed in adipose tissue. Obese mice have lower lnc-U90926 expression in subcutaneous and visceral adipose tissue than non-obese mice. FISH results showed that lnc-U90926 was mainly located in the cytoplasm. Overexpression lnc-U90926 attenuated 3T3-L1 adipocyte differentiation as evidenced by its ability to inhibit lipid accumulation, to decrease the mRNA levels of peroxisome proliferator-activated receptor gamma 2 (PPARγ2), fatty acid binding protein 4 (FABP4) and adiponectin (AdipoQ) as well as to reduce the protein levels of PPARγ and FABP4 (P<0.05). Knockdown of lnc-U90926 showed opposite effects, which

  3. Capsaicin Induces “Brite” Phenotype in Differentiating 3T3-L1 Preadipocytes

    PubMed Central

    Baboota, Ritesh K.; Singh, Dhirendra P.; Sarma, Siddhartha M.; Kaur, Jaspreet; Sandhir, Rajat; Boparai, Ravneet K.; Kondepudi, Kanthi K.; Bishnoi, Mahendra

    2014-01-01

    Objective Targeting the energy storing white adipose tissue (WAT) by pharmacological and dietary means in order to promote its conversion to energy expending “brite” cell type holds promise as an anti-obesity approach. Present study was designed to investigate/revisit the effect of capsaicin on adipogenic differentiation with special reference to induction of “brite” phenotype during differentiation of 3T3-L1 preadipocytes. Methods Multiple techniques such as Ca2+ influx assay, Oil Red-O staining, nutrigenomic analysis in preadipocytes and matured adipocytes have been employed to understand the effect of capsaicin at different doses. In addition to in-vitro experiments, in-vivo studies were carried out in high-fat diet (HFD) fed rats treated with resiniferatoxin (RTX) (a TRPV1 agonist) and in mice administered capsaicin. Results TRPV1 channels are expressed in preadipocytes but not in adipocytes. In preadipocytes, both capsaicin and RTX stimulate Ca2+ influx in dose-dependent manner. This stimulation may be prevented by capsazepine, a TRPV1 antagonist. At lower doses, capsaicin inhibits lipid accumulation and stimulates TRPV1 gene expression, while at higher doses it enhances accumulation of lipids and suppresses expression of its receptor. In doses of 0.1–100 µM, capsaicin promotes expression of major pro-adipogenic factor PPARγ and some of its downstream targets. In concentrations of 1 µM, capsaicin up-regulates anti-adipogenic genes. Low-dose capsaicin treatment of 3T3-L1 preadipocytes differentiating into adipocytes results in increased expression of brown fat cell marker genes. In white adipose of mice, capsaicin administration leads to increase in browning-specific genes. Global TRPV1 ablation (i.p. by RTX administration) leads to increase in locomotor activity with no change in body weight. Conclusion Our findings suggest the dual modulatory role of capsaicin in adipogenesis. Capsaicin inhibits adipogenesis in 3T3-L1 via TRPV1 activation and

  4. L1 regularization facilitates detection of cell type-specific parameters in dynamical systems

    PubMed Central

    Steiert, Bernhard; Timmer, Jens; Kreutz, Clemens

    2016-01-01

    Motivation: A major goal of drug development is to selectively target certain cell types. Cellular decisions influenced by drugs are often dependent on the dynamic processing of information. Selective responses can be achieved by differences between the involved cell types at levels of receptor, signaling, gene regulation or further downstream. Therefore, a systematic approach to detect and quantify cell type-specific parameters in dynamical systems becomes necessary. Results: Here, we demonstrate that a combination of nonlinear modeling with L1 regularization is capable of detecting cell type-specific parameters. To adapt the least-squares numerical optimization routine to L1 regularization, sub-gradient strategies as well as truncation of proposed optimization steps were implemented. Likelihood-ratio tests were used to determine the optimal regularization strength resulting in a sparse solution in terms of a minimal number of cell type-specific parameters that is in agreement with the data. By applying our implementation to a realistic dynamical benchmark model of the DREAM6 challenge we were able to recover parameter differences with an accuracy of 78%. Within the subset of detected differences, 91% were in agreement with their true value. Furthermore, we found that the results could be improved using the profile likelihood. In conclusion, the approach constitutes a general method to infer an overarching model with a minimum number of individual parameters for the particular models. Availability and Implementation: A MATLAB implementation is provided within the freely available, open-source modeling environment Data2Dynamics. Source code for all examples is provided online at http://www.data2dynamics.org/. Contact: bernhard.steiert@fdm.uni-freiburg.de PMID:27587694

  5. Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes.

    PubMed

    Choi, Sun-Sil; Cha, Byung-Yoon; Iida, Kagami; Sato, Masako; Lee, Young-Sil; Teruya, Toshiaki; Yonezawa, Takayuki; Nagai, Kazuo; Woo, Je-Tae

    2011-07-01

    Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10 μM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.

  6. Overexpression of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) delays Alzheimer's progression in vivo.

    PubMed

    Zhang, Mingming; Cai, Fang; Zhang, Shuting; Zhang, Si; Song, Weihong

    2014-12-03

    Deposition of amyloid β protein (Aβ) to form neuritic plaques in the brain is the pathological hallmark of Alzheimer's disease (AD). Aβ is produced by β- and γ-cleavages of amyloid β precursor protein (APP). Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme that cleaves ubiquitin at its carboxyl terminal. Dysfunction of UCHL1 has been reported in neurodegenerative diseases. However, whether UCHL1 affects Aβ production and AD progression remains unknown. Here we report that UCHL1 interacts with APP and regulates Aβ production. UCHL1 increases free ubiquitin level and accelerates the lysosomal degradation of APP by promoting its ubiquitination. Furthermore, we demonstrate that overexpression of UCHL1 by intracranial injection of UCHL1-expressing rAAV reduces Aβ production, inhibits neuritic plaque formation and improves memory deficits in AD transgenic model mice. Our study suggests that UCHL1 may delay Alzheimer's progression by regulating APP degradation in a long-term fashion, and that overexpression of UCHL1 may be a safe and effective disease-modifying strategy to treat AD.

  7. Gene expression patterns induced by HPV-16 L1 virus-like particles in leukocytes from vaccine recipients.

    PubMed

    García-Piñeres, Alfonso J; Hildesheim, Allan; Dodd, Lori; Kemp, Troy J; Yang, Jun; Fullmer, Brandie; Harro, Clayton; Lowy, Douglas R; Lempicki, Richard A; Pinto, Ligia A

    2009-02-01

    Human papillomavirus (HPV) virus-like particle (VLP) vaccines were recently licensed. Although neutralizing Ab titers are thought to be the main effectors of protection against infection, early predictors of long-term efficacy are not yet defined and a comprehensive understanding of innate and adaptive immune responses to vaccination is still lacking. Here, microarrays were used to compare the gene expression signature in HPV-16 L1 VLP-stimulated PBMCs from 17 vaccine and 4 placebo recipients before vaccination and 1 mo after receiving the second immunization. Vaccination with a monovalent HPV-16 L1 VLP vaccine was associated with modulation of genes involved in the inflammatory/defense response, cytokine, IFN, and cell cycle pathways in VLP-stimulated PBMCs. Additionally, there was up-regulation of probesets associated with cytotoxic (GZMB, TNFSF10) and regulatory (INDO, CTLA4) activities. The strongest correlations with neutralizing Ab titers were found for cyclin D2 (CCND2) and galectin (LGALS2). Twenty-two differentially expressed probesets were selected for confirmation by RT-PCR in an independent sample set. Agreement with microarray data was seen for more than two-thirds of these probesets. Up-regulation of immune/defense response genes by HPV-16 L1 VLP, in particular, IFN-induced genes, was observed in PBMCs collected before vaccination, with many of these genes being further induced following vaccination. In conclusion, we identified important innate and adaptive response-related genes induced by vaccination with HPV-16 L1 VLP. Further studies are needed to identify gene expression signatures of immunogenicity and long-term protection with potential utility in prediction of long-term HPV vaccination outcomes in clinical trials.

  8. MicroRNA-125b-5p inhibits proliferation and promotes adipogenic differentiation in 3T3-L1 preadipocytes.

    PubMed

    Ouyang, Dan; Ye, Yaqiong; Guo, Dongguang; Yu, Xiaofang; Chen, Jian; Qi, Junjie; Tan, Xiaotong; Zhang, Yuan; Ma, Yongjiang; Li, Yugu

    2015-05-01

    Previous evidence has indicated that the microRNA-125b (miR-125b) family plays important roles in the regulation of cancer cell growth, development, differentiation, and apoptosis. However, whether they contribute to the process of adipocyte differentiation remains unclear. In the present study, we revealed that the expression level of miR-125b-5p, a member of miR-125b family, was dramatically up-regulated during differentiation of 3T3-L1 preadipocyte into mature adipocyte. Supplement of miR-125b-5p into 3T3-L1 cells promoted adipogenic differentiation as evidenced by increased lipid droplets and mRNA levels of adipocyte-specific molecular markers, including peroxisome proliferators-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, and lipoprotein lipase, and by triglyceride accumulation. CCK-8 assay showed that miR-125b-5p supplementation significantly inhibited cell proliferation. Flow cytometry analysis showed that miR-125b-5p impaired G1/S phase transition as well as the mRNA and protein expression of G1/S-related genes, such as Cyclin D2, Cyclin D3, and CDK4. Nevertheless, it had no effect on apoptosis. Additionally, by target gene prediction, we demonstrated that smad4 may be a potential target of miR-125b-5p in mouse 3T3-L1 preadipocytes, accounting for some of miR-125b-5p's functions. Taken together, these data indicated that miR-125b-5p may serve as an important positive regulator in adipocyte differentiation, at least partially through down-regulating smad4.

  9. Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Promotes Neuritogenesis and Cell Survival*

    PubMed Central

    Lutz, David; Loers, Gabriele; Kleene, Ralf; Oezen, Iris; Kataria, Hardeep; Katagihallimath, Nainesh; Braren, Ingke; Harauz, George; Schachner, Melitta

    2014-01-01

    The cell adhesion molecule L1 is a Lewisx-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewisx-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg687 yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system. PMID:24671420

  10. Inverse changes in L1 retrotransposons between blood and brain in major depressive disorder

    PubMed Central

    Liu, Shu; Du, Tingfu; Liu, Zeyue; Shen, Yan; Xiu, Jianbo; Xu, Qi

    2016-01-01

    Long interspersed nuclear element-1 (LINE-1 or L1) is a type of retrotransposons comprising 17% of the human and mouse genome, and has been found to be associated with several types of neurological disorders. Previous post-mortem brain studies reveal increased L1 copy number in the prefrontal cortex from schizophrenia patients. However, whether L1 retrotransposition occurs similarly in major depressive disorder (MDD) is unknown. Here, L1 copy number was measured by quantitative PCR analysis in peripheral blood of MDD patients (n = 105) and healthy controls (n = 105). The results showed that L1 copy number was increased in MDD patients possibly due to its hypomethylation. Furthermore, L1 copy number in peripheral blood and five brain regions (prefrontal cortex, hippocampus, amygdala, nucleus accumbens and paraventricular hypothalamic nucleus) was measured in the chronic unpredictable mild stress (CUMS) model of depression in mice. Intriguingly, increased L1 copy number in blood and the decreased L1 copy number in the prefrontal cortex were observed in stressed mice, while no change was found in other brain regions. Our results suggest that the changes of L1 may be associated with the pathophysiology of MDD, but the biological mechanism behind dysfunction of L1 retrotransposition in MDD remains to be further investigated. PMID:27874048

  11. PD-L1 expression as predictive biomarker in patients with NSCLC: a pooled analysis

    PubMed Central

    Natoli, Clara; Rizzo, Sergio; Galvano, Antonio; Listì, Angela; Cicero, Giuseppe; Rolfo, Christian; Santini, Daniele; Russo, Antonio

    2016-01-01

    Background Clinical trials of immune checkpoints modulators, including both programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors, have recently shown promising activity and tolerable toxicity in pre-treated NSCLC patients. However the predictive role of PD-L1 expression is still controversial. This pooled analysis aims to clarify the association of clinical objective responses to anti PD-1/PD-L1 monoclonal antibodies (MoAbs) and tumor PD-L1 expression in pre-treated NSCLC patients. Methods Data from published studies, that evaluated efficacy and safety of PD-1/PD-L1 inhibitors in pre-treated NSCLC patients, stratified by tumor PD-L1 expression status (immunohistochemistry, cut-off point 1%), were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, European Society of Medical Oncology and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the Overall Response Rate (ORR) (as evaluated by Response Evaluation Criteria in Solid Tumors, version 1.1), according to PD-L1 expression status. Results A total of seven studies, with 914 patients, were eligible. Pooled analysis showed that patients with PD-L1 positive tumors (PD-L1 tumor cell staining ≥1%), had a significantly higher ORR, compared to patients with PD-L1 negative tumors (OR: 2.44; 95% CIs: 1.61-3.68). Conclusions PD-L1 tumor over-expression seems to be associated with higher clinical activity of anti PD-1/PD-L1 MoAbs, in pre-treated NSCLC patients, suggesting a potential role of PD-L1 expression, IHC cut-off point 1%, as predictive biomarker for the selection of patients to treat with immune-checkpoint inhibitors. PMID:26918451

  12. Robust L1 principal component analysis and its Bayesian variational inference.

    PubMed

    Gao, Junbin

    2008-02-01

    We introduce a robust probabilistic L1-PCA model in which the conventional gaussian distribution for the noise in the observed data was replaced by the Laplacian distribution (or L1 distribution). Due to the heavy tail characteristics of the L1 distribution, the proposed model is supposed to be more robust against data outliers. In this letter, we demonstrate how a variational approximation scheme enables effective inference of key parameters in the probabilistic L1-PCA model. As the L1 density can be expanded as a superposition of infinite number of gaussian densities, we express the L1-PCA model as a marginalized model over the superpositions. By doing so, a tractable Bayesian inference can be achieved based on the variational expectation-maximization-type algorithm.

  13. Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1)

    PubMed Central

    Zak, Krzysztof M.; Grudnik, Przemyslaw; Guzik, Katarzyna; Zieba, Bartosz J.; Musielak, Bogdan; Dömling, Alexander; Dubin, Grzegorz; Holak, Tad A.

    2016-01-01

    Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptidic chemical inhibitors that target the PD-1/PD-L1 interaction have only been recently disclosed by Bristol-Myers Squibb. Here, we show that these small-molecule compounds bind directly to PD-L1 and that they potently block PD-1 binding. Structural studies reveal a dimeric protein complex with a single small molecule which stabilizes the dimer thus occluding the PD-1 interaction surface of PD-L1s. The small-molecule interaction “hot spots” on PD-L1 surfaces suggest approaches for the PD-1/PD-L1 antagonist drug discovery. PMID:27083005

  14. PD-1/PD-L1 expression in extra-medullary lesions of multiple myeloma.

    PubMed

    Crescenzi, Anna; Annibali, Ombretta; Bianchi, Antonella; Pagano, Anastasia; Donati, Michele; Grifoni, Alba; Avvisati, Giuseppe

    2016-10-01

    Multiple myeloma patients may develop extraosseous involvement in the course of the disease making prognosis very poor and new drugs clearly needed. The PD-1/PD-L1 axis has emerged as a master immune checkpoint in antitumor responses and recent studies investigated the role of PD-L1 in multiple myeloma cells; no data however are still available about PD-L1 expression in extramedullary localizations. We demonstrate PD-L1 expression in 4/12 cases of extraosseous myeloma suggesting that these lesions represent a specialized microenvironment. We found presence of PD-1+ infiltrating lymphocytes in all observed cases supporting the relevance of PD-1/PD-L1 checkpoint in extramedullary myeloma. We also investigated the correlation in PD1/PD-L1 staining between marrow staining and EMP lesions.

  15. miR-424(322) reverses chemoresistance via T-cell immune response activation by blocking the PD-L1 immune checkpoint

    PubMed Central

    Xu, Shaohua; Tao, Zhen; Hai, Bo; Liang, Huagen; Shi, Ying; Wang, Tao; Song, Wen; Chen, Yong; OuYang, Jun; Chen, Jinhong; Kong, Fanfei; Dong, Yishan; Jiang, Shi-Wen; Li, Weiyong; Wang, Ping; Yuan, Zhiyong; Wan, Xiaoping; Wang, Chenguang; Li, Wencheng; Zhang, Xiaoping; Chen, Ke

    2016-01-01

    Immune checkpoint blockade of the inhibitory immune receptors PD-L1, PD-1 and CTLA-4 has emerged as a successful treatment strategy for several advanced cancers. Here we demonstrate that miR-424(322) regulates the PD-L1/PD-1 and CD80/CTLA-4 pathways in chemoresistant ovarian cancer. miR-424(322) is inversely correlated with PD-L1, PD-1, CD80 and CTLA-4 expression. High levels of miR-424(322) in the tumours are positively correlated with the progression-free survival of ovarian cancer patients. Mechanistic investigations demonstrated that miR-424(322) inhibited PD-L1 and CD80 expression through direct binding to the 3′-untranslated region. Restoration of miR-424(322) expression reverses chemoresistance, which is accompanied by blockage of the PD-L1 immune checkpoint. The synergistic effect of chemotherapy and immunotherapy is associated with the proliferation of functional cytotoxic CD8+ T cells and the inhibition of myeloid-derived suppressive cells and regulatory T cells. Collectively, our data suggest a biological and functional interaction between PD-L1 and chemoresistance through the microRNA regulatory cascade. PMID:27147225

  16. Crystal clear: visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies.

    PubMed

    Tan, Shuguang; Chen, Danqing; Liu, Kefang; He, Mengnan; Song, Hao; Shi, Yi; Liu, Jun; Zhang, Catherine W-H; Qi, Jianxun; Yan, Jinghua; Gao, Shan; Gao, George F

    2016-12-01

    Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

  17. CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1–infected individuals

    PubMed Central

    Ahuja, Sunil K; Kulkarni, Hemant; Catano, Gabriel; Agan, Brian K; Camargo, Jose F; He, Weijing; O'Connell, Robert J; Marconi, Vincent C; Delmar, Judith; Eron, Joseph; Clark, Robert A; Frost, Simon; Martin, Jeffrey; Ahuja, Seema S; Deeks, Steven G; Little, Susan; Richman, Douglas; Hecht, Frederick M; Dolan, Matthew J

    2008-01-01

    The basis for the extensive variability seen in the reconstitution of CD4+ T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4+ T cells/mm3. The CCL3L1-CCR5 genotypes favoring CD4+ T cell recovery are similar to those that blunted CD4+ T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4+ cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution. PMID:18376407

  18. The Effects of L2 Reading Skills on L1 Reading Skills through Transfer

    ERIC Educational Resources Information Center

    Altmisdort, Gonca

    2016-01-01

    This study investigated whether transfer from L2 to L1 in reading occurs, and if so, which reading sub-skills are transferred into L1 reading. The aim is to identify the role of second language reading skills in L1 reading skills by means of transfer. In addition, the positive effects of the second language transfer to the first language in the…

  19. Heterozygous L1-deficient mice express an autism-like phenotype.

    PubMed

    Sauce, Bruno; Wass, Christopher; Netrakanti, Meera; Saylor, Joshua; Schachner, Melitta; Matzel, Louis D

    2015-10-01

    The L1CAM (L1) gene encodes a cell adhesion molecule that contributes to several important processes in the developing and adult nervous system, including neuronal migration, survival, and plasticity. In humans and mice, mutations in the X chromosome-linked gene L1 cause severe neurological defects in males. L1 heterozygous female mice with one functional copy of the L1 gene show complex morphological features that are different from L1 fully-deficient and wild-type littermate mice. However, almost no information is available on the behavior of L1 heterozygous mice and humans. Here, we investigated the behavior of heterozygous female mice in which the L1 gene is constitutively inactivated. These mice were compared to wild-type littermate females. Animals were assessed in five categories of behavioral tests: five tests for anxiety/stress/exploration, four tests for motor abilities, two tests for spatial learning, three tests for social behavior, and three tests for repetitive behavior. We found that L1 heterozygous mice express an autism-like phenotype, comprised of reduced social behaviors and excessive self-grooming (a repetitive behavior also typical in animal models of autism). L1 heterozygous mice also exhibited an increase in sensitivity to light, assessed by a reluctance to enter the lighted areas of novel environments. However, levels of anxiety, stress, motor abilities, and spatial learning in L1 heterozygous mice were similar to those of wild-type mice. These observations raise the possibility that using molecules known to trigger L1 functions may become valuable in the treatment of autism in humans.

  20. L1Base 2: more retrotransposition-active LINE-1s, more mammalian genomes

    PubMed Central

    Penzkofer, Tobias; Jäger, Marten; Figlerowicz, Marek; Badge, Richard; Mundlos, Stefan; Robinson, Peter N.; Zemojtel, Tomasz

    2017-01-01

    LINE-1 (L1) insertions comprise as much as 17% of the human genome sequence, and similar proportions have been recorded for other mammalian species. Given the established role of L1 retrotransposons in shaping mammalian genomes, it becomes an important task to track and annotate the sources of this activity: full length elements, able to encode the cis and trans acting components of the retrotransposition machinery. The L1Base database (http://l1base.charite.de) contains annotated full-length sequences of LINE-1 transposons including putatively active L1s. For the new version of L1Base, a LINE-1 annotation tool, L1Xplorer, has been used to mine potentially active L1 retrotransposons from the reference genome sequences of 17 mammals. The current release of the human genome, GRCh38, contains 146 putatively active L1 elements or full length intact L1 elements (FLIs). The newest versions of the mouse, GRCm38 and the rat, Rnor_6.0, genomes contain 2811 and 492 FLIs, respectively. Most likely reflecting the current level of completeness of the genome project, the latest reference sequence of the common chimpanzee genome, PT 2.19, only contains 19 FLIs. Of note, the current assemblies of the dog, CF 3.1 and the sheep, OA 3.1, genomes contain 264 and 598 FLIs, respectively. Further developments in the new version of L1Base include an updated website with implementation of modern web server technologies. including a more responsive design for an improved user experience, as well as the addition of data sharing capabilities for L1Xplorer annotation. PMID:27924012

  1. An adaptive support driven reweighted L1-regularization algorithm for fluorescence molecular tomography

    PubMed Central

    Shi, Junwei; Liu, Fei; Pu, Huangsheng; Zuo, Simin; Luo, Jianwen; Bai, Jing

    2014-01-01

    Fluorescence molecular tomography (FMT) is a promising in vivo functional imaging modality in preclinical study. When solving the ill-posed FMT inverse problem, L1 regularization can preserve the details and reduce the noise in the reconstruction results effectively. Moreover, compared with the regular L1 regularization, reweighted L1 regularization is recently reported to improve the performance. In order to realize the reweighted L1 regularization for FMT, an adaptive support driven reweighted L1-regularization (ASDR-L1) algorithm is proposed in this work. This algorithm has two integral parts: an adaptive support estimate and the iteratively updated weights. In the iteratively reweighted L1-minimization sub-problem, different weights are equivalent to different regularization parameters at different locations. Thus, ASDR-L1 can be considered as a kind of spatially variant regularization methods for FMT. Physical phantom and in vivo mouse experiments were performed to validate the proposed algorithm. The results demonstrate that the proposed reweighted L1-reguarization algorithm can significantly improve the performance in terms of relative quantitation and spatial resolution. PMID:25426329

  2. L1-gain analysis and control synthesis of positive switched systems

    NASA Astrophysics Data System (ADS)

    Zhang, Junfeng; Han, Zhengzhi; Zhu, Fubo

    2015-09-01

    This paper investigates the problems of L1-gain analysis and control synthesis of positive switched systems. Linear supply rates and L1-gain notations are introduced to analyse the performance of the underlying systems. Stability with a weighted L1-gain for autonomous systems are solved by using multiple linear copositive Lyapunov functions incorporated with the average dwell time approach. Then, state-feedback and output-feedback controllers are designed to guarantee the stabilisation with a weighted L1-gain for non-autonomous systems. All present conditions are solvable in terms of linear programming. Finally, a practical example is given to illustrate the validity of theoretical findings.

  3. Apolipoprotein L1 Variant Associated with Increased Susceptibility to Trypanosome Infection

    PubMed Central

    Cuypers, Bart; Lecordier, Laurence; Meehan, Conor J.; Van den Broeck, Frederik; Imamura, Hideo; Büscher, Philippe; Dujardin, Jean-Claude; Laukens, Kris; Schnaufer, Achim; Dewar, Caroline; Lewis, Michael; Balmer, Oliver; Azurago, Thomas; Kyei-Faried, Sardick; Ohene, Sally-Ann; Duah, Boateng; Homiah, Prince; Mensah, Ebenezer Kofi; Anleah, Francis; Franco, Jose Ramon; Pays, Etienne

    2016-01-01

    ABSTRACT African trypanosomes, except Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause human African trypanosomiasis, are lysed by the human serum protein apolipoprotein L1 (ApoL1). These two subspecies can resist human ApoL1 because they express the serum resistance proteins T. b. gambiense glycoprotein (TgsGP) and serum resistance-associated protein (SRA), respectively. Whereas in T. b. rhodesiense, SRA is necessary and sufficient to inhibit ApoL1, in T. b. gambiense, TgsGP cannot protect against high ApoL1 uptake, so different additional mechanisms contribute to limit this uptake. Here we report a complex interplay between trypanosomes and an ApoL1 variant, revealing important insights into innate human immunity against these parasites. Using whole-genome sequencing, we characterized an atypical T. b. gambiense infection in a patient in Ghana. We show that the infecting trypanosome has diverged from the classical T. b. gambiense strains and lacks the TgsGP defense mechanism against human serum. By sequencing the ApoL1 gene of the patient and subsequent in vitro mutagenesis experiments, we demonstrate that a homozygous missense substitution (N264K) in the membrane-addressing domain of this ApoL1 variant knocks down the trypanolytic activity, allowing the trypanosome to avoid ApoL1-mediated immunity. PMID:27073096

  4. The PD-L1/PD-1 pathway promotes dysfunction, but not "exhaustion", in tumor-responding T cells from pleural effusions in lung cancer patients.

    PubMed

    Prado-Garcia, Heriberto; Romero-Garcia, Susana; Puerto-Aquino, Alejandra; Rumbo-Nava, Uriel

    2017-03-13

    Malignant pleural effusions are frequent in patients with advanced stages of lung cancer and are commonly infiltrated by lymphocytes and tumor cells. CD8+ T cells from these effusions have reduced effector functions. The programmed death receptor 1(PD-1)/programmed death ligand 1 (PD-L1) pathway is involved in T-cell exhaustion, and it might be responsible for T-cell dysfunction in lung cancer patients. Here, we show that PD-L1 is expressed on tumor cell samples from malignant effusions, on lung cancer cell lines, and, interestingly, on MRC-5 lung fibroblasts. PD-L1 was up-regulated in lung cancer cell lines upon treatment with IFN-gamma, but not under hypoxic conditions, as detected by RT-qPCR and flow cytometry. Blockade of PD-L1 on tumor cells restored granzyme-B expression in allogenic CD8+ T cells in vitro. Remarkably, pleural effusion CD8+ T cells that responded to the tumor antigens MAGE-3A and WT-1 (identified as CD137+ cells) were lower in frequency than CMV pp65-responding CD8+ T cells and did not have an exhausted phenotype (PD-1+ TIM-3+). Nonetheless, tumor-responding CD8+ T cells had a memory phenotype and expressed higher levels of PD-1. A PD-L1 blocking antibody increased the expression of granzyme-B and perforin on polyclonal- and tumor-stimulated CD8+ T cells. Taken together, our data show that rather than being exhausted, tumor-responding CD8+ T cells are not completely differentiated into effector cells and are prone to negative regulation by PD-L1. Hence, our study provides evidence that lung cancer patients respond to immunotherapy due to blockade of the PD-L1/PD-1 pathway.

  5. Interference with PD-L1/PD-1 co-stimulation during antigen presentation enhances the multifunctionality of antigen-specific T cells.

    PubMed

    Pen, J J; Keersmaecker, B D; Heirman, C; Corthals, J; Liechtenstein, T; Escors, D; Thielemans, K; Breckpot, K

    2014-03-01

    The release of cytokines by T cells strongly defines their functional activity in vivo. The ability to produce multiple cytokines has been associated with beneficial immune responses in cancer and infectious diseases, while their progressive loss is associated with T-cell exhaustion, senescence and anergy. Consequently, strategies that enhance the multifunctional status of T cells are a key for immunotherapy. Dendritic cells (DCs) are professional antigen presenting cells that regulate T-cell functions by providing positive and negative co-stimulatory signals. A key negative regulator of T-cell activity is provided by binding of programmed death-1 (PD-1) receptor on activated T cells, to its ligand PD-L1, expressed on DCs. We investigated the impact of interfering with PD-L1/PD-1 co-stimulation on the multifunctionality of T cells, by expression of the soluble extracellular part of PD-1 (sPD-1) or PD-L1 (sPD-L1) in human monocyte-derived DCs during antigen presentation. Expression, secretion and binding of these soluble molecules after mRNA electroporation were demonstrated. Modification of DCs with sPD-1 or sPD-L1 mRNA resulted in increased levels of the co-stimulatory molecule CD80 and a distinct cytokine profile, characterized by the secretion of IL-10 and TNF-α, respectively. Co-expression in DCs of sPD-1 and sPD-L1 with influenza virus nuclear protein 1 (Flu NP1) stimulated Flu NP1 memory T cells, with a significantly higher number of multifunctional T cells and increased cytokine secretion, while it did not induce regulatory T cells. These data provide a rationale for the inclusion of interfering sPD-1 or sPD-L1 in DC-based immunotherapeutic strategies.

  6. Suppressive Effects of Barley β-Glucans with Different Molecular Weight on 3T3-L1 Adipocyte Differentiation.

    PubMed

    Zhu, Yingying; Yao, Yang; Gao, Yue; Hu, Yibo; Shi, Zhenxing; Ren, Guixing

    2016-03-01

    In this study, 2 β-glucans with different molecular weight were prepared and purified from hull-less barley bran. The aim was to evaluate their effects on the differentiation of 3T3-L1 pre-adipocytes. Results showed that barley β-glucans inhibited the differentiation of 3T3-L1 pre-adipocytes induced by differentiation medium in a dose-dependent manner, the suppressive effect of high-molecular-weight barley β-glucans (552 kDa, BGH) was stronger (P < 0.05) than that of low-molecular-weight barley β-glucan (32 kDa, BGL), evidenced by the significantly decrease (P < 0.05) of Oil-red O staining and intracellular triglyceride content in the mature adipocytes. Besides, gene expression analysis and Western Blot analysis revealed that both BGH and BGL inhibited the mRNA and protein levels of adipogenesis related transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα) which are principal regulators of adipogenesis. Furthermore, the mRNA and protein expression levels of PPARγ target genes in adipose tissue including adipocyte fatty acid binding protein (ap2), lipoprotein lipase (LPL), uncoupling protein-2 (UCP-2), and glucose-transporter 4 (Glut4) in 3T3-L1 cells was also markedly downregulated (P < 0.05). These findings were anticipated to help develop barley β-glucans based functional food for the management of obesity.

  7. MiR-124 represses vasculogenic mimicry and cell motility by targeting amotL1 in cervical cancer cells.

    PubMed

    Wan, Hai-Ying; Li, Qin-Qin; Zhang, Yan; Tian, Wei; Li, Ya-Nan; Liu, Min; Li, Xin; Tang, Hua

    2014-12-01

    miRNAs have extensive functions in differentiation, metabolism, programmed cell death, and tumor metastasis by post-transcriptional regulation. Vasculogenic mimicry is an important pathway in tumor metastasis. Many factors can regulate vasculogenic mimicry, including miRNAs. In previous studies, miR-124 was found to repress proliferation and metastasis in different types of cancers, but whether it functions in cervical cancer remained unknown. Here, we demonstrate that miR-124 can repress vasculogenic mimicry, migration and invasion in HeLa and C33A cells in vitro. Furthermore, we reveal that the effect of miR-124 on vasculogenic mimicry, migration and invasion results from its interaction with AmotL1. MiR-124 regulates AmotL1 negatively by targeting its 3'untranslated region (3'UTR). We found that miR-124 can repress the EMT process. Together, these results improve our understanding of the function of miR-124 in tumor metastasis and will help to provide new potential target sites for cervical cancer treatment.

  8. L1/2 regularization: a thresholding representation theory and a fast solver.

    PubMed

    Xu, Zongben; Chang, Xiangyu; Xu, Fengmin; Zhang, Hai

    2012-07-01

    The special importance of L1/2 regularization has been recognized in recent studies on sparse modeling (particularly on compressed sensing). The L1/2 regularization, however, leads to a nonconvex, nonsmooth, and non-Lipschitz optimization problem that is difficult to solve fast and efficiently. In this paper, through developing a threshoding representation theory for L1/2 regularization, we propose an iterative half thresholding algorithm for fast solution of L1/2 regularization, corresponding to the well-known iterative soft thresholding algorithm for L1 regularization, and the iterative hard thresholding algorithm for L0 regularization. We prove the existence of the resolvent of gradient of ||x||1/2(1/2), calculate its analytic expression, and establish an alternative feature theorem on solutions of L1/2 regularization, based on which a thresholding representation of solutions of L1/2 regularization is derived and an optimal regularization parameter setting rule is formulated. The developed theory provides a successful practice of extension of the well- known Moreau's proximity forward-backward splitting theory to the L1/2 regularization case. We verify the convergence of the iterative half thresholding algorithm and provide a series of experiments to assess performance of the algorithm. The experiments show that the half algorithm is effective, efficient, and can be accepted as a fast solver for L1/2 regularization. With the new algorithm, we conduct a phase diagram study to further demonstrate the superiority of L1/2 regularization over L1 regularization.

  9. A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

    PubMed

    Lai, Wei-Yun; Huang, Bo-Tsang; Wang, Jen-Wei; Lin, Pei-Ying; Yang, Pan-Chyr

    2016-12-13

    The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4(+) and CD8(+) T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8(+) T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31(+) intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

  10. A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

    PubMed

    Lai, Wei-Yun; Huang, Bo-Tsang; Wang, Jen-Wei; Lin, Pei-Ying; Yang, Pan-Chyr

    2016-01-01

    The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4(+) and CD8(+) T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8(+) T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31(+) intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

  11. HPV L1 and P16 Expression in CIN1 to Predict Future CIN2.

    PubMed

    Liu, Chong; Du, Hui; Wang, Chun; Belinson, Jerome L; Yang, Bin; Zhang, Wei; Tang, Jinlong; Wu, Ruifang

    2017-03-08

    To use the biomarkers human papillomavirus (HPV) L1 and p16 to develop an algorithm that could triage the individual patient with CIN1 for the risk of progression. A total of 82 patients initially diagnosed with CIN1 at Peking University Shenzhen Hospital in China had their initial and follow-up paraffin-embedded tissue blocks immune-stained for HPV L1 capsid protein and p16. For CIN1, any staining of abnormal epithelium was considered positive. All patients were followed until they developed CIN2+ or for ≥3 years. About 38 patients regressed (HPV-, Cytology-), 17 persisted (CIN1), and 27 progressed (≥CIN2+). At initial diagnosis, HPV L1 capsid protein was expressed in 42.7% of the CIN1 cases. There was no difference in L1 expression among the 3 groups. However, p16-positive staining in the progression group was significantly higher than in the regression group (P<0.05). In the regression group, the proportion of HPV L1-/p16- category was significantly higher than that in the progression group. In the progression group, when CIN1 lesions progressed to CIN2+, the L1-positive rate was significantly decreased from 51.9% to 18.5%, the p16+/HPV L1+ rate decreased from the initial (44.4%) to the final diagnosis (14.8%), and the p16+/HPV L1- rate increased from the initial (25.9%) to the final diagnosis (66.7%). P16 expression is a clear risk factor for the progression of CIN1. The p16-/HPV L1- pattern was significantly associated with the regression of CIN1. Moving from CIN1 to CIN2+ over time, p16+/HPV L1+ decreased, and p16+/HPV L1- increased. Unfortunately, our objective of finding a sensitive and specific triage algorithm for the individual patient with CIN1 was still not achieved.

  12. L1CAM and HER2 Expression in Early Endometrioid Uterine Cancer.

    PubMed

    Abdel Azim, Samira; Sprung, Susanne; Mutz-Dehbalaie, Irene; Fessler, Siegfried; Zeimet, Alain G; Marth, Christian

    2017-02-17

    Recently L1CAM was shown to be a promising biomarker for early-stage endometrial carcinoma (EC). As L1CAM expression was found to be related to serous ECs and areas of serous differentiation in endometrioid carcinomas, there is evidence that L1CAM-positive cancers more likely resemble type II carcinomas. Furthermore, expression of growth factor receptor HER2 has been found to be closely associated with serous ECs. We conducted a retrospective study on 142 patients in FIGO stages I and II with endometrioid EC and analyzed L1CAM and HER2 expression by double-staining immunohistochemistry. The association between these 2 transmembrane molecules and their impact on patient outcome was analyzed. Both L1CAM and HER2 showed a significant association with recurrent disease (P<0.001 and P=0.007, respectively). We found 39 (27%) L1CAM-positive cases and 17 (12%) HER2-positive cases. About 6.3% of cases were positive for both biomarkers. Survival of L1CAM-positive patients showed a significant difference between HER2-positive and HER2-negative patients (P=0.019) regarding disease-free survival. The most unfavorable disease-free survival and overall survival was found for patients with L1CAM and HER2 double-positive tumors (P<0.001). Double immunostaining revealed a mutually exclusive staining pattern for L1CAM and HER2 expression on the level of tumor cells. In early endometrioid uterine carcinoma, an additional expression of HER2 to L1CAM seems to further worsen disease-free survival and overall survival. In terms of "personalized medicine," detection of these molecules in endometrioid ECs may open new avenues for targeted therapies with the newly available anti-HER2 drugs and/or with the upcoming humanized anti-L1CAM antibodies.

  13. Clinical impact of L1CAM expression measured on the transcriptome level in ovarian cancer

    PubMed Central

    Azim, Samira Abdel; Duggan-Peer, Michaela; Sprung, Susanne; Reimer, Daniel; Fiegl, Heidi; Soleiman, Afschin; Marth, Christian; Zeimet, Alain G.

    2016-01-01

    Background High expression of L1 cell adhesion molecules (L1CAM) has been repeatedly shown to be associated with aggressive disease behavior, which translates in poor clinical outcome in various cancer entities. However, in ovarian cancer results based either on immunohistochemistry or cytosolic protein quantifications remained conflicting regarding clinical behavior. In the present work we assessed L1CAM expression on the transcriptome level with the highly sensitive quantitative real-time PCR (qRT-PCR) to define its relevance in ovarian cancer biology. Results There was a significant difference in L1CAM high and low mRNA expressing cancers with regard to disease-free (p=0.002) and overall survival (p=0.008). L1CAM proofed to be an independent predictor for disease progression (HR 1.8, p=0.01) and overall survival (HR 1.6, p=0.04). Furthermore, a significant positive correlation between the level of L1CAM and the grade of tumor differentiation (p=0.04), the FIGO stage (p=0.025) as well as the histological subtype (p= 0.002) was found. Methods This study included fresh frozen tissue samples of 138 patients with FIGO I-IV stage ovarian cancer. L1CAM mRNA expression was determined using qRT-PCR. In the calculations special attention was put on the various histological subtypes. In survival analysis median L1CAM mRNA expression obtained in the entire cohort of ovarian cancer samples was used as a cut-off to distinguish between high and low L1CAM mRNA expression. Conclusion L1CAM mRNA expression appears to play a substantial role in the pathophysiology of ovarian cancer that is translated into poor clinical outcome. Additionally humanized L1CAM antibodies, which can serve as potential future treatment options are under testing. PMID:27174921

  14. Prognostic value of PD-L1 and PD-1 expression in pulmonary neuroendocrine tumors

    PubMed Central

    Fan, Yangwei; Ma, Ke; Wang, Chuying; Ning, Jing; Hu, Yuan; Dong, Danfeng; Dong, Xuyuan; Geng, Qianqian; Li, Enxiao; Wu, Yinying

    2016-01-01

    Purpose Programmed death 1 (PD-1) receptor and its ligand, programmed death ligand-1 (PD-L1), play critical roles in the immune invasion of various tumors. This study aimed to explore the clinical significance of PD-L1/PD-1 expression in the progression of pulmonary neuroendocrine tumors (PNETs). Methods The expression of PD-L1 and PD-1 in 80 patients diagnosed with PNETs were investigated. Immunohistochemical analysis was performed on 80 formalin-fixed paraffin-embedded tissue specimens from PNETs and 20 corresponding cancer-adjacent tissue specimens. Results Tissues from PNETs had higher levels of PD-L1 (58.8%) and PD-1 (51.3%) compared to the cancer-adjacent tissues (25% and 20%, respectively). Meanwhile, PD-L1 expression was associated with PD-1 expression (P=0.007). PD-L1 expression was significantly associated with histological type (P=0.014) and tumor stage (P=0.014). Univariate analyses showed that the overall survival time of PNETs patients was significantly associated with PD-L1 expression in cancer cells (P=0.003), PD-1 expression in tumor-infiltrating lymphocytes (P=0.001), tumor node metastasis stage (P<0.05), and distant metastasis (P<0.001). Additionally, multivariate analysis revealed that PD-L1 expression, PD1 expression, and distant metastasis of PNETs were independently associated with survival time. Moreover, Kaplan–Meier survival curves analysis revealed that patients with negative PD-L1 and PD-1 expression had better prognoses. Conclusion Data suggested that PD-L1 and PD-1 can be useful prognostic biomarkers for survival and can pave the way toward new immunotherapy regimens against PNETs through targeting the PD-L1/PD-1 pathway. PMID:27785054

  15. Clinical Neuropathology mini-review 6-2015: PD-L1: emerging biomarker in glioblastoma?

    PubMed

    Preusser, Matthias; Berghoff, Anna S; Wick, Wolfgang; Weller, Michael

    2015-01-01

    Programmed death 1 (PD-1, CD279) and programmed death ligand 1 (PD-L1, CD274) are involved in generating tumor-associated immunosuppression by suppression of T-cell proliferation and interleukin 2 (IL-2) production and immune checkpoint inhibitors targeting these molecules are showing compelling activity against a variety of human cancers. PD-L1 expression has shown a positive association with response to PD-1 inhibition in noncentral nervous system (CNS) tumors, e.g., melanoma or non-small cell lung cancer, and is discussed as a potential predictive biomarker for patient selection in these tumor types. This review summarizes current knowledge and potential clinical implications of PD-L1 expression in glioblastoma. At present, the following conclusions are drawn: (a) functional data support a role for PD-1/PD-L1 in tumor-associated immunosuppression in glioblastoma; (b) the incidence of PD-L1-expressing glioblastomas seems to be relatively high in comparison to other tumor types, however, the reported rates of glioblastomas with PD-L1 protein expression vary and range from 61 to 88%; (c) there is considerable variability in the methodology of PD-L1 assessment in glioblastoma across studies with heterogeneity in utilized antibodies, tissue sampling strategies, immunohistochemical staining protocols, cut-off definitions, and evaluated staining patterns; (d) there are conflicting data on the prognostic role and so far no data on the predictive role of PD-L1 gene and protein expression in glioblastoma. In summary, the ongoing clinical studies evaluating the activity of PD-1/PD-L1 inhibitors in glioblastoma need to be complemented with well designed and stringently executed studies to understand the influence of PD-1/PD-L1 expression on therapy response or failure and to develop robust means of PD-L1 assessment for meaningful biomarker development.

  16. Aberrant PD-L1 expression through 3'-UTR disruption in multiple cancers.

    PubMed

    Kataoka, Keisuke; Shiraishi, Yuichi; Takeda, Yohei; Sakata, Seiji; Matsumoto, Misako; Nagano, Seiji; Maeda, Takuya; Nagata, Yasunobu; Kitanaka, Akira; Mizuno, Seiya; Tanaka, Hiroko; Chiba, Kenichi; Ito, Satoshi; Watatani, Yosaku; Kakiuchi, Nobuyuki; Suzuki, Hiromichi; Yoshizato, Tetsuichi; Yoshida, Kenichi; Sanada, Masashi; Itonaga, Hidehiro; Imaizumi, Yoshitaka; Totoki, Yasushi; Munakata, Wataru; Nakamura, Hiromi; Hama, Natsuko; Shide, Kotaro; Kubuki, Yoko; Hidaka, Tomonori; Kameda, Takuro; Masuda, Kyoko; Minato, Nagahiro; Kashiwase, Koichi; Izutsu, Koji; Takaori-Kondo, Akifumi; Miyazaki, Yasushi; Takahashi, Satoru; Shibata, Tatsuhiro; Kawamoto, Hiroshi; Akatsuka, Yoshiki; Shimoda, Kazuya; Takeuchi, Kengo; Seya, Tsukasa; Miyano, Satoru; Ogawa, Seishi

    2016-06-16

    Successful treatment of many patients with advanced cancer using antibodies against programmed cell death 1 (PD-1; also known as PDCD1) and its ligand (PD-L1; also known as CD274) has highlighted the critical importance of PD-1/PD-L1-mediated immune escape in cancer development. However, the genetic basis for the immune escape has not been fully elucidated, with the exception of elevated PD-L1 expression by gene amplification and utilization of an ectopic promoter by translocation, as reported in Hodgkin and other B-cell lymphomas, as well as stomach adenocarcinoma. Here we show a unique genetic mechanism of immune escape caused by structural variations (SVs) commonly disrupting the 3' region of the PD-L1 gene. Widely affecting multiple common human cancer types, including adult T-cell leukaemia/lymphoma (27%), diffuse large B-cell lymphoma (8%), and stomach adenocarcinoma (2%), these SVs invariably lead to a marked elevation of aberrant PD-L1 transcripts that are stabilized by truncation of the 3'-untranslated region (UTR). Disruption of the Pd-l1 3'-UTR in mice enables immune evasion of EG7-OVA tumour cells with elevated Pd-l1 expression in vivo, which is effectively inhibited by Pd-1/Pd-l1 blockade, supporting the role of relevant SVs in clonal selection through immune evasion. Our findings not only unmask a novel regulatory mechanism of PD-L1 expression, but also suggest that PD-L1 3'-UTR disruption could serve as a genetic marker to identify cancers that actively evade anti-tumour immunity through PD-L1 overexpression.

  17. Bilingual lexical access during L1 sentence reading: The effects of L2 knowledge, semantic constraint, and L1-L2 intermixing.

    PubMed

    Titone, Debra; Libben, Maya; Mercier, Julie; Whitford, Veronica; Pivneva, Irina

    2011-11-01

    Libben and Titone (2009) recently observed that cognate facilitation and interlingual homograph interference were attenuated by increased semantic constraint during bilingual second language (L2) reading, using eye movement measures. We now investigate whether cross-language activation also occurs during first language (L1) reading as a function of age of L2 acquisition and task demands (i.e., inclusion of L2 sentences). In Experiment 1, participants read high and low constraint English (L1) sentences containing interlingual homographs, cognates, or control words. In Experiment 2, we included French (L2) filler sentences to increase salience of the L2 during L1 reading. The results suggest that bilinguals reading in their L1 show nonselective activation to the extent that they acquired their L2 early in life. Similar to our previous work on L2 reading, high contextual constraint attenuated cross-language activation for cognates. The inclusion of French filler items promoted greater cross-language activation, especially for late stage reading measures. Thus, L1 bilingual reading is modulated by L2 knowledge, semantic constraint, and task demands.

  18. Inhibition of adipogenesis and leptin production in 3T3-L1 adipocytes by a derivative of meridianin C

    SciTech Connect

    Park, Yu-Kyoung; Lee, Tae-Yoon; Choi, Jong-Soon; Hong, Victor Sukbong; Lee, Jinho; Park, Jong-Wook; Jang, Byeong-Churl

    2014-10-03

    Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-α, PPAR-γ, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of these meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.

  19. Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells.

    PubMed

    Kim, Sang Chon; Kim, Yoo Hoon; Son, Sung Wook; Moon, Eun-Yi; Pyo, Suhkneung; Um, Sung Hee

    2015-11-27

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPARγ in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association