Kaposi's-sarcoma-associated-herpesvirus-activated dendritic cells promote HIV-1 trans-infection and suppress CD4{sup +} T cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Wan; Qin, Yan; Bai, Lei

2013-06-05

Infection of Kaposi's sarcoma-associated herpesvirus (KSHV) is commonly occurred in AIDS patients. KSHV and HIV-1 act cooperatively in regulating infection with each other and in human carcinogenesis. Dendritic cells (DCs), as the pivotal cells in host immunity, may be modulated by both viruses, for immunoevasion and dissemination, therefore, the interaction between DCs and each virus has been a prior focus for pathogenesis elucidation. Here, we assessed the potential effect of KSHV on DC–HIV-1 interaction. We found that KSHV stimulation could promote maturation of monocyte-derived DCs (MDDCs) and impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} Tmore » cells, demonstrating the immunosuppression induced by KSHV. More importantly, KSHV-stimulated MDDCs could capture more HIV-1 and efficiently transferred these infectious viruses to Hut/CCR5 T cell line. Our results reveal the novel modulation of DC-mediated HIV-1 dissemination by KSHV, and highlight the importance of studying DC–HIV-1 interaction to elucidate HIV/AIDS pathogenesis. - Highlights: ► KSHV impaired the ability of MDDCs to drive proliferation of resting CD4{sup +} T cells. ► KSHV stimulation matured MDDCs and enhanced HIV-1 endocytosis. ► KSHV stimulated MDDCs increased ICAM-1 expression and tighten contact with T cells. ► KSHV-stimulated MDDCs promoted HIV-1 trans-infection of CD4{sup +} T cells.« less

Circulating angiogenic cell function is inhibited by cortisol in vitro and associated with psychological stress and cortisol in vivo.

PubMed

Aschbacher, Kirstin; Derakhshandeh, Ronak; Flores, Abdiel J; Narayan, Shilpa; Mendes, Wendy Berry; Springer, Matthew L

2016-05-01

Psychological stress and glucocorticoids are associated with heightened cardiovascular disease risk. We investigated whether stress or cortisol would be associated with reduced circulating angiogenic cell (CAC) function, an index of impaired vascular repair. We hypothesized that minority-race individuals who experience threat in interracial interactions would exhibit reduced CAC function, and that this link might be explained by cortisol. To test this experimentally, we recruited 106 African American participants for a laboratory interracial interaction task, in which they received socially evaluative feedback from Caucasian confederates. On a separate day, a subset of 32 participants (mean age=26years, 47% female) enrolled in a separate biological substudy and provided blood samples for CAC isolation and salivary samples to quantify the morning peak in cortisol (the cortisol awakening response, CAR). CAC function was quantified using cell culture assays of migration to vascular endothelial growth factor (VEGF) and secretion of VEGF into the culture medium. Heightened threat in response to an interracial interaction and trait anxiety in vivo were both associated with poorer CAC migratory function in vitro. Further, threat and poorer sustained attention during the interracial interaction were associated with a higher CAR, which in turn, was related to lower CAC sensitivity to glucocorticoids. In vitro, higher doses of cortisol impaired CAC migratory function and VEGF protein secretion. The glucocorticoid receptor antagonist RU486 reversed this functional impairment. These data identify a novel, neuroendocrine pathway by which psychological stress may reduce CAC function, with potential implications for cardiovascular health. Copyright © 2016 Elsevier Ltd. All rights reserved.

Circulating Angiogenic Cell Function is Inhibited by Cortisol in Vitro and Associated with Psychological Stress and Cortisol in Vivo

PubMed Central

Aschbacher, Kirstin; Derakhshandeh, Ronak; Flores, Abdiel J.; Narayan, Shilpa; Mendes, Wendy Berry; Springer, Matthew L.

2016-01-01

Psychological stress and glucocorticoids are associated with heightened cardiovascular disease risk. We investigated whether stress or cortisol would be associated with reduced circulating angiogenic cell (CAC) function, an index of impaired vascular repair. We hypothesized that minority-race individuals who experience threat in interracial interactions would exhibit reduced CAC function, and that this link might be explained by cortisol. To test this experimentally, we recruited 106 African American participants for a laboratory interracial interaction task, in which they received socially evaluative feedback from Caucasian confederates. On a separate day, a subset of 32 participants (mean age = 26 years, 47% female) enrolled in a separate biological substudy and provided blood samples for CAC isolation and salivary samples to quantify the morning peak in cortisol (the cortisol awakening response, CAR). CAC function was quantified using cell culture assays of migration to vascular endothelial growth factor (VEGF) and secretion of VEGF into the culture medium. Heightened threat in response to an interracial interaction and trait anxiety in vivo were both associated with poorer CAC migratory function in vitro. Further, threat and poorer sustained attention during the interracial interaction were associated with a higher CAR, which in turn, was related to lower CAC sensitivity to glucocorticoids. In vitro, higher doses of cortisol impaired CAC migratory function and VEGF protein secretion. The glucocorticoid receptor antagonist RU486 reversed this functional impairment. These data identify a novel, neuroendocrine pathway by which psychological stress may reduce CAC function, with potential implications for cardiovascular health. PMID:26925833

Wound Healing in Mac-1 Deficient Mice

DTIC Science & Technology

2017-05-01

36. Rosenkranz AR, Coxon A, Maurer M, Gurish MF, Austen KF, Friend DS, Galli SJ, Mayadas TN. Impaired mast cell development and innate immunity in Mac...genetically deficient mice. 3 INTRODUCTION Wound healing is a complex yet well-regulated process in which multiple resident cells ...recruited inflammatory cells , and stem cells interact to create an environment that supports the healing process. An optimal inflammatory response is a

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

PubMed

Campodónico, Paola B; de Kier Joffé, Elisa D Bal; Urtreger, Alejandro J; Lauria, Lilia S; Lastiri, José M; Puricelli, Lydia I; Todaro, Laura B

2010-04-01

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

Host-parasite interactions and purifying selection in a microsporidian parasite of honey bees

USDA-ARS?s Scientific Manuscript database

Nosema ceranae is a microsporidian parasite that infects honey bee mid-gut epithelial cells. Infection can impair honey bee cognitive function, shorten lifespan, suppress the innate immune response and inhibit the apoptosis of infected gut cells. However, the virulence factors of this parasite are s...

Major Histocompatibility Complex in Human - HLA System: Biological Role and Impact for Practical Medicine.

PubMed

Alexeev, Leonid P.

1999-10-01

Interactions of HLA constitute the key basis for development of the whole number of pathologies, starting from oncological and infectious diseases, and ending with autoimmune disorders and allergies. The most demonstrable example is oncopathology. The fact is that HLA class I (namely, its non-polymorphic determinants) have recently been shown to be the main target for so called natural (or non-specific) killer cells (NK). Naturally, the profound decrease of class I histocompatibility antigens on the surface of pathologically changed cells, impairing cellular interaction between NK and target cells, "takes them out" from the control of NK. As a result, the body looses one of the most important protective functions. Quite another type of impairment of HLA role in cellular interaction may be the basis of autoimmune diseases. The most successful results were obtained in studies of insulin dependent diabetes. One of the main pathogenic factors was shown to be marked elevation (aberrant expression) of HLA on islet cells (insulin producers). This, in its turn, is the consequence of dysfunction and activation of genes, responsible for "assembly and transport" of HLA class II. The problem about role of HLA in cell interactions in allergy is rather novel, but poor studied trend, however some obtained results are encouraging. The point is that the unique feature in expression of class II histocompatibility antigens, specific for allergy, was revealed for recent years. Expression of class II histocompatibility antigens is appeared to be sharply increased on B lymphocytes of allergic patients.

Impairment of Fas-ligand-caveolin-1 interaction inhibits Fas-ligand translocation to rafts and Fas-ligand-induced cell death.

PubMed

Glukhova, Xenia A; Trizna, Julia A; Proussakova, Olga V; Gogvadze, Vladimir; Beletsky, Igor P

2018-01-22

Fas-ligand/CD178 belongs to the TNF family proteins and can induce apoptosis through death receptor Fas/CD95. The important requirement for Fas-ligand-dependent cell death induction is its localization to rafts, cholesterol- and sphingolipid-enriched micro-domains of membrane, involved in regulation of different signaling complexes. Here, we demonstrate that Fas-ligand physically associates with caveolin-1, the main protein component of rafts. Experiments with cells overexpressing Fas-ligand revealed a FasL N-terminal pre-prolin-rich region, which is essential for the association with caveolin-1. We found that the N-terminal domain of Fas-ligand bears two caveolin-binding sites. The first caveolin-binding site binds the N-terminal domain of caveolin-1, whereas the second one appears to interact with the C-terminal domain of caveolin-1. The deletion of both caveolin-binding sites in Fas-ligand impairs its distribution between cellular membranes, and attenuates a Fas-ligand-induced cytotoxicity. These results demonstrate that the interaction of Fas-ligand and caveolin-1 represents a molecular basis for Fas-ligand translocation to rafts, and the subsequent induction of Fas-ligand-dependent cell death. A possibility of a similar association between other TNF family members and caveolin-1 is discussed.

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

PubMed Central

Col, Edwige; Caron, Cécile; Chable-Bessia, Christine; Legube, Gaelle; Gazzeri, Sylvie; Komatsu, Yasuhiko; Yoshida, Minoru; Benkirane, Monsef; Trouche, Didier; Khochbin, Saadi

2005-01-01

HIV-1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP-associated E4-type ubiquitin-ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60-dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV-1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination. PMID:16001085

AML1/ETO accelerates cell migration and impairs cell-to-cell adhesion and homing of hematopoietic stem/progenitor cells

PubMed Central

Saia, Marco; Termanini, Alberto; Rizzi, Nicoletta; Mazza, Massimiliano; Barbieri, Elisa; Valli, Debora; Ciana, Paolo; Gruszka, Alicja M.; Alcalay, Myriam

2016-01-01

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression. PMID:27713544

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duangtum, Natapol; Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700; Junking, Mutita

Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{supmore » -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.« less

Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

PubMed Central

Boursalian, Tamar E.; Fink, Pamela J.

2003-01-01

Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I– and class II–restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection. PMID:12860933

Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation

PubMed Central

Gross, Catharina C.; Schulte-Mecklenbeck, Andreas; Rünzi, Anna; Kuhlmann, Tanja; Posevitz-Fejfár, Anita; Schwab, Nicholas; Schneider-Hohendorf, Tilman; Herich, Sebastian; Held, Kathrin; Konjević, Matea; Hartwig, Marvin; Dornmair, Klaus; Hohlfeld, Reinhard; Ziemssen, Tjalf; Klotz, Luisa; Meuth, Sven G.; Wiendl, Heinz

2016-01-01

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood–brain barrier, CD56bright NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4+ T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4+ T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor’s ligand CD155 on CD4+ T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4+ T cells and the cytolytic activity of NK cells. PMID:27162345

Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

PubMed

Woodham, Andrew W; Sanna, Adriana M; Taylor, Julia R; Skeate, Joseph G; Da Silva, Diane M; Dekker, Lodewijk V; Kast, W Martin

2016-11-18

During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.

Killing of Staphylococci by θ-Defensins Involves Membrane Impairment and Activation of Autolytic Enzymes

PubMed Central

Wilmes, Miriam; Stockem, Marina; Bierbaum, Gabriele; Schlag, Martin; Götz, Friedrich; Tran, Dat Q.; Schaal, Justin B.; Ouellette, André J.; Selsted, Michael E.; Sahl, Hans-Georg

2014-01-01

θ-Defensins are cyclic antimicrobial peptides expressed in leukocytes of Old world monkeys. To get insight into their antibacterial mode of action, we studied the activity of RTDs (rhesus macaque θ-defensins) against staphylococci. We found that in contrast to other defensins, RTDs do not interfere with peptidoglycan biosynthesis, but rather induce bacterial lysis in staphylococci by interaction with the bacterial membrane and/or release of cell wall lytic enzymes. Potassium efflux experiments and membrane potential measurements revealed that the membrane impairment by RTDs strongly depends on the energization of the membrane. In addition, RTD treatment caused the release of Atl-derived cell wall lytic enzymes probably by interaction with membrane-bound lipoteichoic acid. Thus, the premature and uncontrolled activity of these enzymes contributes strongly to the overall killing by θ-defensins. Interestingly, a similar mode of action has been described for Pep5, an antimicrobial peptide of bacterial origin. PMID:25632351

Zinc improves learning and memory abilities of fetal growth restriction rats and promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 axis activity.

PubMed

Zong, Lu; Wei, Xiaohua; Gou, Wenli; Huang, Pu; Lv, Ye

2017-12-29

Fetal growth restriction (FGR) is a well-known risk factor for cognitive dysfunction, especially for learning and memory abilities. However, knowledge about prevention and treatment methods of learning and memory abilities of fetal are limit. Here, Morris water maze and passive avoidance tests showed zinc supplementation could protect the impairment of the learning and memory abilities caused by FGR. As accumulating evidence suggested that insufficiency of placental trophoblast cell invasion was closely related to FGR fetal neurodevelopmental dysplasia, we further explored the relationship between zinc supplementation during pregnancy and placental trophoblast. Microarray identified 346 differently expressed genes in placental tissues with and without zinc supplementation, and GO and KEGG analyses showed these differently expressed genes were highly enriched in cell invasion and migration and STAT3 pathway. Protein-protein interaction(PPI) analysis found that STAT3 interacted with matrix metalloproteinase-2/9 (MMP-2/9). In vivo , western blot results authenticated that the expression levels of phospho-STAT3, STAT3, MMP-2 and MMP-9 were up-regulated in placental tissues after zinc treatment. To validate whether zinc could promotes trophoblast cell invasion and migration via enhancing STAT3-MMP-2/9 activity. In vitro , Transwell assay was performed, and we observed that abilities of invasion and migration were obviously increased in zinc treated trophoblast cells. And phospho-STAT3, STAT3, MMP-2 and MMP-9 expression levels were correspondingly increased in zinc treated trophoblast cells, which were dose-dependent. Moreover, gain-of-function and loss-of-function of STAT3 confirmed that zinc promotes cell invasion and migration via regulating STAT3 mediated up-regulation of MMP-2/9 activity. We propose that activation of MMP-2/9 mediated by STAT3 may contribute to invasion and migration of trophoblast cells, which improved neurodevelopmental impairment of FGR rats probably via contributing to placental development. Our findings are the first to show a possible mechanism of reversing neurodevelopmental impairment of FGR rats by zinc supplementation, holding promise for the development of novel therapeutic modalities for learning and memory abilities impairment caused by FGR.

Evidence that β7 Integrin Regulates Hematopoietic Stem Cell Homing and Engraftment Through Interaction with MAdCAM-1.

PubMed

Murakami, Jodi L; Xu, Baohui; Franco, Christopher B; Hu, Xingbin; Galli, Stephen J; Weissman, Irving L; Chen, Ching-Cheng

2016-01-01

α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis is controversial. We identified a subset of hematopoietic stem cells (HSCs) in the bone marrow (BM) that expressed β7 integrin. These β7(+) HSCs were capable of multilineage, long-term reconstitution and had an inherent competitive advantage over β7(-) HSCs. On the other hand, HSCs that lacked β7 integrin (β7KO) had reduced engraftment potential. Interestingly, quantitative RT-PCR and flow cytometry revealed that β7KO HSCs expressed lower levels of the chemokine receptor CXCR4. Accordingly, β7KO HSCs exhibited impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on BM endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with its endothelial ligand MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment.

Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells.

PubMed

Sasaki, Natsuki; Nakamura, Masayuki; Kodama, Akiko; Urata, Yuka; Shiokawa, Nari; Hayashi, Takehiro; Sano, Akira

2016-11-01

The autophagy pathway has recently been implicated in several neurodegenerative diseases. Recently, it was reported that chorein-depleted cells showed accumulation of autophagic markers and impaired autophagic flux. Here, we demonstrate that chorein overexpression preserves cell viability from starvation-induced cell death in human embryonic kidney 293 (HEK293) cells. Subsequent coimmunoprecipitation and reverse coimmunoprecipitation assays using extracts from chorein that stably overexpressed HEK293 cells revealed that chorein interacts with α-tubulin and histone deacetylase 6, a known α-tubulin deacetylater and central component of basal autophagy. Indeed, acetylated α-tubulin immunoreactivity was significantly decreased in chorein that stably overexpressed HEK293 cells. These results suggest that chorein/histone deacetylase 6/α-tubulin interactions may play an important role in starvation-induced cell stress, and their disruption may be one of the molecular pathogenic mechanisms of chorea-acanthocytosis.-Sasaki, N., Nakamura, M., Kodama, A., Urata, Y., Shiokawa, N., Hayashi, T., Sano, A. Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells. © FASEB.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

PubMed

Engelsdorf, Timo; Will, Cornelia; Hofmann, Jörg; Schmitt, Christine; Merritt, Brian B; Rieger, Leonie; Frenger, Marc S; Marschall, André; Franke, Rochus B; Pattathil, Sivakumar; Voll, Lars M

2017-01-01

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

The mucin MUC4 and its membrane partner ErbB2 regulate biological properties of human CAPAN-2 pancreatic cancer cells via different signalling pathways.

PubMed

Jonckheere, Nicolas; Skrypek, Nicolas; Merlin, Johann; Dessein, Anne Frédérique; Dumont, Patrick; Leteurtre, Emmanuelle; Harris, Ann; Desseyn, Jean-Luc; Susini, Christiane; Frénois, Frédéric; Van Seuningen, Isabelle

2012-01-01

The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.

Involvement of the interaction of afadin with ZO-1 in the formation of tight junctions in Madin-Darby canine kidney cells.

PubMed

Ooshio, Takako; Kobayashi, Reiko; Ikeda, Wataru; Miyata, Muneaki; Fukumoto, Yuri; Matsuzawa, Naomi; Ogita, Hisakazu; Takai, Yoshimi

2010-02-12

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with beta- and alpha-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-DeltaPR1-2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.

Involvement of the Interaction of Afadin with ZO-1 in the Formation of Tight Junctions in Madin-Darby Canine Kidney Cells*

PubMed Central

Ooshio, Takako; Kobayashi, Reiko; Ikeda, Wataru; Miyata, Muneaki; Fukumoto, Yuri; Matsuzawa, Naomi; Ogita, Hisakazu; Takai, Yoshimi

2010-01-01

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with β- and α-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-ΔPR1–2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells. PMID:20008323

Mesenchymal Stem and Progenitor Cells in Normal and Dysplastic Hematopoiesis—Masters of Survival and Clonality?

PubMed Central

Pleyer, Lisa; Valent, Peter; Greil, Richard

2016-01-01

Myelodysplastic syndromes (MDS) are malignant hematopoietic stem cell disorders that have the capacity to progress to acute myeloid leukemia (AML). Accumulating evidence suggests that the altered bone marrow (BM) microenvironment in general, and in particular the components of the stem cell niche, including mesenchymal stem cells (MSCs) and their progeny, play a pivotal role in the evolution and propagation of MDS. We here present an overview of the role of MSCs in the pathogenesis of MDS, with emphasis on cellular interactions in the BM microenvironment and related stem cell niche concepts. MSCs have potent immunomodulatory capacities and communicate with diverse immune cells, but also interact with various other cellular components of the microenvironment as well as with normal and leukemic stem and progenitor cells. Moreover, compared to normal MSCs, MSCs in MDS and AML often exhibit altered gene expression profiles, an aberrant phenotype, and abnormal functional properties. These alterations supposedly contribute to the “reprogramming” of the stem cell niche into a disease-permissive microenvironment where an altered immune system, abnormal stem cell niche interactions, and an impaired growth control lead to disease progression. The current article also reviews molecular targets that play a role in such cellular interactions and possibilities to interfere with abnormal stem cell niche interactions by using specific targeted drugs. PMID:27355944

Mesenchymal Stem and Progenitor Cells in Normal and Dysplastic Hematopoiesis-Masters of Survival and Clonality?

PubMed

Pleyer, Lisa; Valent, Peter; Greil, Richard

2016-06-27

Myelodysplastic syndromes (MDS) are malignant hematopoietic stem cell disorders that have the capacity to progress to acute myeloid leukemia (AML). Accumulating evidence suggests that the altered bone marrow (BM) microenvironment in general, and in particular the components of the stem cell niche, including mesenchymal stem cells (MSCs) and their progeny, play a pivotal role in the evolution and propagation of MDS. We here present an overview of the role of MSCs in the pathogenesis of MDS, with emphasis on cellular interactions in the BM microenvironment and related stem cell niche concepts. MSCs have potent immunomodulatory capacities and communicate with diverse immune cells, but also interact with various other cellular components of the microenvironment as well as with normal and leukemic stem and progenitor cells. Moreover, compared to normal MSCs, MSCs in MDS and AML often exhibit altered gene expression profiles, an aberrant phenotype, and abnormal functional properties. These alterations supposedly contribute to the "reprogramming" of the stem cell niche into a disease-permissive microenvironment where an altered immune system, abnormal stem cell niche interactions, and an impaired growth control lead to disease progression. The current article also reviews molecular targets that play a role in such cellular interactions and possibilities to interfere with abnormal stem cell niche interactions by using specific targeted drugs.

Impairment of regulatory capacity of CD4+CD25+ regulatory T cells mediated by dendritic cell polarization and hyperthyroidism in Graves' disease.

PubMed

Mao, Chaoming; Wang, Shu; Xiao, Yichuan; Xu, Jingwei; Jiang, Qian; Jin, Min; Jiang, Xiaohua; Guo, Hua; Ning, Guang; Zhang, Yanyun

2011-04-15

Graves' disease (GD) is one of the most common autoimmune diseases. The immune dysfunction in GD involves the generation of thyroid-stimulating hormone receptor (TSHR) autoantibodies that presumably arise consequent to interactions among dendritic cells (DCs), T cells, and regulatory T (Treg) cells. However, the immunological mechanisms of interactions between them that lead to the induction and regulation of this autoimmune disease are poorly defined. In this study, we investigated whether DCs are the main cause of the defective activity of Treg cells in GD patients. We found a significant decrease in the percentage of circulating CD4(+)CD25(+)FOXP3(+) Treg cells in untreated GD patients (uGD), which was negatively correlated with the concentration of TSHR autoantibodies. uGD-derived DCs were polarized to increase the number of plasmacytoid DCs (pDCs) and conferred the ability to abrogate the suppressive function of Treg cells through inducing apoptosis of CD4(+)CD25(+) Treg cells in an IFN-α-dependent manner, and elevated thyroid hormones further exacerbated the effect. The nucleotide UDP, which inhibits IFN-α secretion of pDCs through P2Y6 receptor signaling, restored the suppressive function of CD4(+)CD25(+) Treg cells. Collectively, uGD-derived DCs through pDC polarization and elevated thyroid hormones act in concert to impair the regulatory capacity of Treg cells, facilitating the production of TSHR autoantibodies in the pathogenesis of GD.

Numb controls E-cadherin endocytosis through p120 catenin with aPKC

PubMed Central

Sato, Kazuhide; Watanabe, Takashi; Wang, Shujie; Kakeno, Mai; Matsuzawa, Kenji; Matsui, Toshinori; Yokoi, Keiko; Murase, Kiyoko; Sugiyama, Ikuko; Ozawa, Masayuki; Kaibuchi, Kozo

2011-01-01

Cadherin trafficking controls tissue morphogenesis and cell polarity. The endocytic adaptor Numb participates in apicobasal polarity by acting on intercellular adhesions in epithelial cells. However, it remains largely unknown how Numb controls cadherin-based adhesion. Here, we found that Numb directly interacted with p120 catenin (p120), which is known to interact with E-cadherin and prevent its internalization. Numb accumulated at intercellular adhesion sites and the apical membrane in epithelial cells. Depletion of Numb impaired E-cadherin internalization, whereas depletion of p120 accelerated internalization. Expression of the Numb-binding fragment of p120 inhibited E-cadherin internalization in a dominant-negative fashion, indicating that Numb interacts with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induced mislocalization of E-cadherin from the lateral membrane to the apical membrane. Atypical protein kinase C (aPKC), a member of the PAR complex, phosphorylated Numb and inhibited its association with p120 and α-adaptin. Depletion or inhibition of aPKC accelerated E-cadherin internalization. Wild-type Numb restored E-cadherin internalization in the Numb-depleted cells, whereas a phosphomimetic mutant or a mutant with defective α-adaptin-binding ability did not restore the internalization. Thus, we propose that aPKC phosphorylates Numb to prevent its binding to p120 and α-adaptin, thereby attenuating E-cadherin endocytosis to maintain apicobasal polarity. PMID:21775625

Nanoparticle-Cell Interaction: A Cell Mechanics Perspective.

PubMed

Septiadi, Dedy; Crippa, Federica; Moore, Thomas Lee; Rothen-Rutishauser, Barbara; Petri-Fink, Alke

2018-05-01

Progress in the field of nanoparticles has enabled the rapid development of multiple products and technologies; however, some nanoparticles can pose both a threat to the environment and human health. To enable their safe implementation, a comprehensive knowledge of nanoparticles and their biological interactions is needed. In vitro and in vivo toxicity tests have been considered the gold standard to evaluate nanoparticle safety, but it is becoming necessary to understand the impact of nanosystems on cell mechanics. Here, the interaction between particles and cells, from the point of view of cell mechanics (i.e., bionanomechanics), is highlighted and put in perspective. Specifically, the ability of intracellular and extracellular nanoparticles to impair cell adhesion, cytoskeletal organization, stiffness, and migration are discussed. Furthermore, the development of cutting-edge, nanotechnology-driven tools based on the use of particles allowing the determination of cell mechanics is emphasized. These include traction force microscopy, colloidal probe atomic force microscopy, optical tweezers, magnetic manipulation, and particle tracking microrheology. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Programmed disorders of beta-cell development and function as one cause for type 2 diabetes? The GK rat paradigm.

PubMed

Portha, Bernard

2005-01-01

Now that the reduction in beta-mass has been clearly established in humans with type 2 diabetes mellitus (T2DM) 1-4, the debate focuses on the possible mechanisms responsible for decreased beta-cell number and impaired beta-cell function and their multifactorial etiology. Appropriate inbred rodent models are essential tools for identification of genes and environmental factors that increase the risk of abnormal beta-cell function and of T2DM. The information available in the Goto-Kakizaki (GK) rat, one of the best characterized animal models of spontaneous T2DM, are reviewed in such a perspective. We propose that the defective beta-cell mass and function in the GK model reflect the complex interactions of three pathogenic players: (1) several independent loci containing genes causing impaired insulin secretion; (2) gestational metabolic impairment inducing a programming of endocrine pancreas (decreased beta-cell neogenesis) which is transmitted to the next generation; and (3) secondary (acquired) loss of beta-cell differentiation due to chronic exposure to hyperglycemia (glucotoxicity). An important message is that the 'heritable' determinants of T2DM are not simply dependant on genetic factors, but probably involve transgenerational epigenetic responses. Copyright (c) 2005 John Wiley & Sons, Ltd.

T Follicular Helper Cells and B Cell Dysfunction in Aging and HIV-1 Infection

PubMed Central

Pallikkuth, Suresh; de Armas, Lesley; Rinaldi, Stefano; Pahwa, Savita

2017-01-01

T follicular helper (Tfh) cells are a subset of CD4 T cells that provide critical signals to antigen-primed B cells in germinal centers to undergo proliferation, isotype switching, and somatic hypermutation to generate long-lived plasma cells and memory B cells during an immune response. The quantity and quality of Tfh cells therefore must be tightly controlled to prevent immune dysfunction in the form of autoimmunity and, on the other hand, immune deficiency. Both Tfh and B cell perturbations appear during HIV infection resulting in impaired antibody responses to vaccines such as seasonal trivalent influenza vaccine, also seen in biologic aging. Although many of the HIV-associated defects improve with antiretroviral therapy (ART), excess immune activation and antigen-specific B and T cell responses including Tfh function are still impaired in virologically controlled HIV-infected persons on ART. Interestingly, HIV infected individuals experience increased risk of age-associated pathologies. This review will discuss Tfh and B cell dysfunction in HIV infection and highlight the impact of chronic HIV infection and aging on Tfh–B cell interactions. PMID:29109730

T Follicular Helper Cells and B Cell Dysfunction in Aging and HIV-1 Infection.

PubMed

Pallikkuth, Suresh; de Armas, Lesley; Rinaldi, Stefano; Pahwa, Savita

2017-01-01

T follicular helper (Tfh) cells are a subset of CD4 T cells that provide critical signals to antigen-primed B cells in germinal centers to undergo proliferation, isotype switching, and somatic hypermutation to generate long-lived plasma cells and memory B cells during an immune response. The quantity and quality of Tfh cells therefore must be tightly controlled to prevent immune dysfunction in the form of autoimmunity and, on the other hand, immune deficiency. Both Tfh and B cell perturbations appear during HIV infection resulting in impaired antibody responses to vaccines such as seasonal trivalent influenza vaccine, also seen in biologic aging. Although many of the HIV-associated defects improve with antiretroviral therapy (ART), excess immune activation and antigen-specific B and T cell responses including Tfh function are still impaired in virologically controlled HIV-infected persons on ART. Interestingly, HIV infected individuals experience increased risk of age-associated pathologies. This review will discuss Tfh and B cell dysfunction in HIV infection and highlight the impact of chronic HIV infection and aging on Tfh-B cell interactions.

Pyruvate kinase M2 interacts with DNA damage-binding protein 2 and reduces cell survival upon UV irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xie, Xiao; Wang, Mingsong; Mei, Ju, E-mail: jumei_xinhua@163.com

Pyruvate Kinase M2 (PKM2) is highly expressed in many solid tumors and associated with metabolism reprogramming and proliferation of tumors. Here, we report that PKM2 can bind to DNA Damage-Binding Protein 2 (DDB2), which is necessary for global nucleotide excision repair of UV induced DNA damage. The binding is promoted by UV irradiation and K433 acetylation of PKM2. Over expression of PKM2 facilitates phosphorylation of DDB2 and impairs DDB2-DDB1 binding. Furthermore, knocking down of PKM2 increases cell survival upon UV irradiation, while over expression of PKM2 reduces cell survival and over expression of DDB2-DDB1 reverts this effect. These results revealmore » a previously unknown regulation of PKM2 on DDB2 and provide a possible mechanism for UV induced tumorigenesis. - Highlights: • PKM2 interacts with DDB2. • UV irradiation increases PKM2-DDB2 binding via up regulation of PKM2 K433 acetylation. • PKM2 facilitates DDB2 phosphorylation and impairs DDB2-DDB1 binding. • PKM2 reduces cell survival upon UV irradiation.« less

Nip the HPV encoded evil in the cancer bud: HPV reshapes TRAILs and signaling landscapes

PubMed Central

2013-01-01

HPV encoded proteins can elicit ectopic protein–protein interactions that re-wire signaling pathways, in a mode that promotes malignancy. Moreover, accumulating data related to HPV is now providing compelling substantiation of a central role played by HPV in escaping immunosurveillance and impairment of apoptotic response. What emerges is an intricate network of Wnt, TGF, Notch signaling cascades that forms higher-order ligand–receptor complexes routing downstream signaling in HPV infected cells. These HPV infected cells are regulated both extracellularly by ligand receptor axis and intracellularly by HPV encoded proteins and impair TRAIL mediated apoptosis. We divide this review into different sections addressing how linear signaling pathways integrate to facilitate carcinogenesis and compounds that directly or indirectly reverse these aberrant interactions offer new possibilities for therapy in cancer. Although HPV encoded proteins mediated misrepresentation of pathways is difficult to target, improved drug-discovery platforms and new technologies have facilitated the discovery of agents that can target dysregulated pathways in HPV infected cervical cancer cells, thus setting the stage for preclinical models and clinical trials. PMID:23773282

Interrelations Between Mitochondrial DNA Copy Number and Inflammation in Older Adults.

PubMed

Wu, I-Chien; Lin, Cheng-Chieh; Liu, Chin-San; Hsu, Chih-Cheng; Chen, Ching-Yu; Hsiung, Chao A

2017-07-01

Interplays between inflammation and mitochondrial biology are reported. Here, we examined the cross-sectional interrelationships of mitochondrial DNA copy number (mtDNACN) and inflammation and their interaction with physical functioning. A total of 1990 community-dwelling adults aged 65 years and older who were participating in the Healthy Aging Longitudinal Study in Taiwan underwent measurements of peripheral-blood leukocytes MtDNACN, multiple inflammatory markers, grip strength, and gait speed. Principal components analysis revealed two inflammatory factors: factor 1 (high-sensitivity C-reactive protein [hs-CRP], white blood cell count, fibrinogen and interleukin-6 [IL-6]); factor 2 (tumor necrosis factor receptor 1, D-dimer and soluble interleukin-6 receptor). Participants with severe physical functioning impairment (low grip strength and gait speed) had higher (p < .05) levels of factor 1 and 2, but not mtDNACN, than did those with moderately impaired (low grip strength or gait speed) and normal physical functioning. MtDNACN was negatively related to factor 1 (r = -.221, p < .001) but not factor 2 (r = -.002, p = .938). Increased factor 1 was strongly associated with higher odds of physical functioning impairment in those with a low mtDNACN (adjusted odds ratios [OR] of moderate physical function impairment 1.21, 95% CI 1.01-1.44; adjusted OR of severe physical function impairment 1.52, 95% CI 1.25-1.85) but not in those with a high mtDNACN (p for interaction = .016). A low mtDNACN was associated with an inflammation exhibiting elevated hs-CRP, IL-6, fibrinogen, and white blood cell count, and strengthened the association of this inflammation with physical functioning impairment. © The Author 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells

PubMed Central

Ibabao, Christopher N.; Bunaciu, Rodica P.; Schaefer, Deanna M.W.; Yen, Andrew

2015-01-01

In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14+CD11b+ monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47phox. Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr. PMID:25941627

The AhR agonist VAF347 augments retinoic acid-induced differentiation in leukemia cells.

PubMed

Ibabao, Christopher N; Bunaciu, Rodica P; Schaefer, Deanna M W; Yen, Andrew

2015-01-01

In binary cell-fate decisions, driving one lineage and suppressing the other are conjoined. We have previously reported that aryl hydrocarbon receptor (AhR) promotes retinoic acid (RA)-induced granulocytic differentiation of lineage bipotent HL-60 myeloblastic leukemia cells. VAF347, an AhR agonist, impairs the development of CD14(+)CD11b(+) monocytes from granulo-monocytic (GM) stage precursors. We thus hypothesized that VAF347 propels RA-induced granulocytic differentiation and impairs D3-induced monocytic differentiation of HL-60 cells. Our results show that VAF347 enhanced RA-induced cell cycle arrest, CD11b integrin expression and neutrophil respiratory burst. Granulocytic differentiation is known to be driven by MAPK signaling events regulated by Fgr and Lyn Src-family kinases, the CD38 cell membrane receptor, the Vav1 GEF, the c-Cbl adaptor, as well as AhR, all of which are embodied in a putative signalsome. We found that the VAF347 AhR ligand regulates the signalsome. VAF347 augments RA-induced expression of AhR, Lyn, Vav1, and c-Cbl as well as p47(phox). Several interactions of partners in the signalsome appear to be enhanced: Fgr interaction with c-Cbl, CD38, and with pS259c-Raf and AhR interaction with c-Cbl and Lyn. Thus, we report that, while VAF347 impedes monocytic differentiation induced by 1,25-dihydroxyvitamin D3, VAF347 promotes RA-induced differentiation. This effect seems to involve but not to be limited to Lyn, Vav1, c-Cbl, AhR, and Fgr.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

PubMed

Gurevich, David B; Severn, Charlotte E; Twomey, Catherine; Greenhough, Alexander; Cash, Jenna; Toye, Ashley M; Mellor, Harry; Martin, Paul

2018-06-04

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Identification of a subset of human natural killer cells expressing high levels of programmed death 1: A phenotypic and functional characterization.

PubMed

Pesce, Silvia; Greppi, Marco; Tabellini, Giovanna; Rampinelli, Fabio; Parolini, Silvia; Olive, Daniel; Moretta, Lorenzo; Moretta, Alessandro; Marcenaro, Emanuela

2017-01-01

Programmed death 1 (PD-1) is an immunologic checkpoint that limits immune responses by delivering potent inhibitory signals to T cells on interaction with specific ligands expressed on tumor/virus-infected cells, thus contributing to immune escape mechanisms. Therapeutic PD-1 blockade has been shown to mediate tumor eradication with impressive clinical results. Little is known about the expression/function of PD-1 on human natural killer (NK) cells. We sought to clarify whether human NK cells can express PD-1 and analyze their phenotypic/functional features. We performed multiparametric cytofluorimetric analysis of PD-1 + NK cells and their functional characterization using degranulation, cytokine production, and proliferation assays. We provide unequivocal evidence that PD-1 is highly expressed (PD-1 bright ) on an NK cell subset detectable in the peripheral blood of approximately one fourth of healthy subjects. These donors are always serologically positive for human cytomegalovirus. PD-1 is expressed by CD56 dim but not CD56 bright NK cells and is confined to fully mature NK cells characterized by the NKG2A - KIR + CD57 + phenotype. Proportions of PD-1 bright NK cells were higher in the ascites of a cohort of patients with ovarian carcinoma, suggesting their possible induction/expansion in tumor environments. Functional analysis revealed a reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production on interaction with tumor targets. We have identified and characterized a novel subpopulation of human NK cells expressing high levels of PD-1. These cells have the phenotypic characteristics of fully mature NK cells and are increased in patients with ovarian carcinoma. They display low proliferative responses and impaired antitumor activity that can be partially restored by antibody-mediated disruption of PD-1/programmed death ligand interaction. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Androgen-Induced Cell Migration: Role of Androgen Receptor/Filamin A Association

PubMed Central

Castoria, Gabriella; D'Amato, Loredana; Ciociola, Alessandra; Giovannelli, Pia; Giraldi, Tiziana; Sepe, Leandra; Paolella, Giovanni; Barone, Maria Vittoria; Migliaccio, Antimo; Auricchio, Ferdinando

2011-01-01

Background Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. Methodology/Principal Findings Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. Conclusions/Significance The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis. PMID:21359179

Genetic resistance to malaria, oxidative stress and hemoglobin oxidation.

PubMed

Destro Bisol, G

1999-09-01

I describe a model which posits the molecular basis of some malaria-resistance genes in the interaction between oxidized hemoglobin and membrane components. The model is supported by a considerable body of evidence which indicates that erythrocytes of genetically protected individuals (carriers of sickle cell trait, alpha- and beta-thalassemia, and G6PD deficiency) are susceptible to the increase of oxidation of hemoglobin following H2O2 release in the host cell by Plasmodium falciparum. I suggest that the irreversible interaction between oxidized hemoglobin and the red cell membrane could trigger mechanisms that: (i) reduce invasion of erythrocytes by the falciparum parasite; (ii) impair parasite survival and development within the cell; (iii) accelerate infected erythrocyte clearance by phagocytosis.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants

PubMed Central

Engelsdorf, Timo; Will, Cornelia; Hofmann, Jörg; Schmitt, Christine; Merritt, Brian B.; Rieger, Leonie; Frenger, Marc S.; Marschall, André; Franke, Rochus B.; Pattathil, Sivakumar

2017-01-01

Abstract Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance. PMID:28204541

Abnormalities in iNKT cells are associated with impaired ability of monocytes to produce IL-10 and suppress T-cell proliferation in sarcoidosis.

PubMed

Crawshaw, Anjali; Kendrick, Yvonne R; McMichael, Andrew J; Ho, Ling-Pei

2014-07-01

Sarcoidosis is a multisystem granulomatous disorder characterized by marked T-cell expansion of T helper 1 (Th1) cells. The cause of T-cell overactivity is unknown. We hypothesized that interleukin-10 (IL-10) production by a yet undefined cell type might be defective, resulting in loss of regulation of T-cell activity. Focusing on IL-10-producing monocytes, we first showed that monocytes isolated from the peripheral blood of corticosteroid-naïve sarcoidosis patients (n = 51) produced less IL-10 compared to controls, and were less able to suppress T-cell proliferation. In addition, monocytic IL-10 production correlated negatively with disease activity score. As invariant natural killer T (iNKT) cells are known to both interact with monocytes and be reduced in sarcoidosis patients, we then asked whether iNKT-specific defects might be responsible for this reduced IL-10 production. We found that greater numbers of circulating iNKT cells was associated with higher IL-10 production. Moreover, iNKT cells enhanced monocytic IL-10 production in vitro. Defective IL-10 production and T-cell suppression by sarcoidosis monocytes could be restored following their coculture with iNKT cells, in a CD1d- and cell contact-dependent process. We suggest that reduced iNKT-cell numbers in sarcoidosis may lead to impaired monocytic IL-10 production and unchecked T-cell expansion in sarcoidosis. These findings provide fresh insight into the mechanism of sarcoidosis disease, and interaction between iNKT cells and monocytes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impairment of autophagosome-lysosome fusion in the buff mutant mice with the VPS33AD251E mutation

PubMed Central

Zhen, Yuanli; Li, Wei

2015-01-01

The HOPS (homotypic fusion and protein sorting) complex functions in endocytic and autophagic pathways in both lower eukaryotes and mammalian cells through its involvement in fusion events between endosomes and lysosomes or autophagosomes and lysosomes. However, the differential molecular mechanisms underlying these fusion processes are largely unknown. Buff (bf) is a mouse mutant that carries an Asp251-to-Glu point mutation (D251E) in the VPS33A protein, a tethering protein and a core subunit of the HOPS complex. Bf mice showed impaired spontaneous locomotor activity, motor learning, and autophagic activity. Although the gross anatomy of the brain was apparently normal, the number of Purkinje cells was significantly reduced. Furthermore, we found that fusion between autophagosomes and lysosomes was defective in bf cells without compromising the endocytic pathway. The direct association of mutant VPS33AD251E with the autophagic SNARE complex, STX17 (syntaxin 17)-VAMP8-SNAP29, was enhanced. In addition, the VPS33AD251E mutation enhanced interactions with other HOPS subunits, namely VPS41, VPS39, VPS18, and VPS11, except for VPS16. Reduction of the interactions between VPS33AY440D and several other HOPS subunits led to decreased association with STX17. These results suggest that the VPS33AD251E mutation plays dual roles by increasing the HOPS complex assembly and its association with the autophagic SNARE complex, which selectively affects the autophagosome-lysosome fusion that impairs basal autophagic activity and induces Purkinje cell loss. PMID:26259518

The hnRNP-Htt axis regulates necrotic cell death induced by transcriptional repression through impaired RNA splicing.

PubMed

Mao, Y; Tamura, T; Yuki, Y; Abe, D; Tamada, Y; Imoto, S; Tanaka, H; Homma, H; Tagawa, K; Miyano, S; Okazawa, H

2016-04-28

In this study, we identify signaling network of necrotic cell death induced by transcriptional repression (TRIAD) by α-amanitin (AMA), the selective RNA polymerase II inhibitor, as a model of neurodegenerative cell death. We performed genetic screen of a knockdown (KD) fly library by measuring the ratio of transformation from pupa to larva (PL ratio) under TRIAD, and selected the cell death-promoting genes. Systems biology analysis of the positive genes mapped on protein-protein interaction databases predicted the signaling network of TRIAD and the core pathway including heterogeneous nuclear ribonucleoproteins (hnRNPs) and huntingtin (Htt). RNA sequencing revealed that AMA impaired transcription and RNA splicing of Htt, which is known as an endoplasmic reticulum (ER)-stabilizing molecule. The impairment in RNA splicing and PL ratio was rescued by overexpresion of hnRNP that had been also affected by transcriptional repression. Fly genetics with suppressor or expresser of Htt and hnRNP worsened or ameliorated the decreased PL ratio by AMA, respectively. Collectively, these results suggested involvement of RNA splicing and a regulatory role of the hnRNP-Htt axis in the process of the transcriptional repression-induced necrosis.

Elevated HLA-A expression impairs HIV control through inhibition of NKG2A-expressing cells.

PubMed

Ramsuran, Veron; Naranbhai, Vivek; Horowitz, Amir; Qi, Ying; Martin, Maureen P; Yuki, Yuko; Gao, Xiaojiang; Walker-Sperling, Victoria; Del Prete, Gregory Q; Schneider, Douglas K; Lifson, Jeffrey D; Fellay, Jacques; Deeks, Steven G; Martin, Jeffrey N; Goedert, James J; Wolinsky, Steven M; Michael, Nelson L; Kirk, Gregory D; Buchbinder, Susan; Haas, David; Ndung'u, Thumbi; Goulder, Philip; Parham, Peter; Walker, Bruce D; Carlson, Jonathan M; Carrington, Mary

2018-01-05

The highly polymorphic human leukocyte antigen ( HLA ) locus encodes cell surface proteins that are critical for immunity. HLA-A expression levels vary in an allele-dependent manner, diversifying allele-specific effects beyond peptide-binding preference. Analysis of 9763 HIV-infected individuals from 21 cohorts shows that higher HLA-A levels confer poorer control of HIV. Elevated HLA-A expression provides enhanced levels of an HLA-A-derived signal peptide that specifically binds and determines expression levels of HLA-E, the ligand for the inhibitory NKG2A natural killer (NK) cell receptor. HLA-B haplotypes that favor NKG2A-mediated NK cell licensing (i.e., education) exacerbate the deleterious effect of high HLA-A on HIV control, consistent with NKG2A-mediated inhibition impairing NK cell clearance of HIV-infected targets. Therapeutic blockade of HLA-E:NKG2A interaction may yield benefit in HIV disease. Copyright © 2017, American Association for the Advancement of Science.

Interaction of pathogens with host cholesterol metabolism.

PubMed

Sviridov, Dmitri; Bukrinsky, Michael

2014-10-01

Pathogens of different taxa, from prions to protozoa, target cellular cholesterol metabolism to advance their own development and to impair host immune responses, but also causing metabolic complications, for example, atherosclerosis. This review describes recent findings of how pathogens do it. A common theme in interaction between pathogens and host cholesterol metabolism is pathogens targeting lipid rafts of the host plasma membrane. Many intracellular pathogens use rafts as an entry gate, taking advantage of the endocytic machinery and high abundance of outward-looking molecules that can be used as receptors. At the same time, disruption of the rafts' functional capacity, achieved by the pathogens through a number of various means, impairs the ability of the host to generate immune response, thus helping pathogen to thrive. Pathogens cannot synthesize cholesterol, and salvaging host cholesterol helps pathogens build advanced cholesterol-containing membranes and assembly platforms. Impact on cholesterol metabolism is not limited to the infected cells; proteins and microRNAs secreted by infected cells affect lipid metabolism systemically. Given an essential role that host cholesterol metabolism plays in pathogen development, targeting this interaction may be a viable strategy to fight infections, as well as metabolic complications of the infections.

The necroptosis adaptor RIPK3 promotes injury-induced cytokine expression and tissue repair.

PubMed

Moriwaki, Kenta; Balaji, Sakthi; McQuade, Thomas; Malhotra, Nidhi; Kang, Joonsoo; Chan, Francis Ka-Ming

2014-10-16

Programmed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor-interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation by regulating cytokine expression in dendritic cells (DCs). Ripk3(-/-) bone-marrow-derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and by impaired caspase 1-mediated processing of interleukin-1β (IL-1β). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3(-/-) mice exhibited an impaired axis of injury-induced IL-1β, IL-23, and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild-type DCs, but not Ripk3(-/-) DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory-cytokine expression, and injury-induced tissue repair.

HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

PubMed Central

Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

2008-01-01

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

Elevation of SHARPIN Protein Levels in Prostate Adenocarcinomas Promotes Metastasis and Impairs Patient Survivals.

PubMed

Huang, Hai; Du, Tao; Zhang, Yiming; Lai, Yiming; Li, Kaiwen; Fan, Xinxing; Zhu, Dingjun; Lin, Tianxin; Xu, Kewei; Huang, Jian; Liu, Leyuan; Guo, Zhenghui

2017-05-01

SHARPIN, SHANK-associated RH domain interacting protein, associates with a linear ubiquitin chain assembly complex (LUBAC) to regulate inflammation and immunity. It has been reported that SHARPIN is highly expressed in several human tumors including ovarian cancer and liver cancer. We found that SHARPIN is also highly expressed in prostate cancer cell lines of DU145, LNCAP, and PC-3. Suppression of SHARPIN caused an inhibition of NF-κB signal and decreases in tumorigenesis of cultured cells in NOD/SCID mouse model. Overexpression of SHARPIN in prostate cancer cells promoted cell growth and reduced apoptosis through NF-kB/ERK/Akt pathway and apoptosis-associated proteins. We analyzed the expression of SHARPIN in prostate cancer tissues from 95 patients and its relationship with other clinical characteristics associated with PCA malignancies and patient survivals, and examined the impacts of SHARPIN suppression with siRNA on proliferation, angiogenesis, invasion, and expression levels of MMP-9 of prostate cancer cells and metastasis to lung by these cells in nude mice. High levels of SHARPIN were associated with high malignancies of PCA and predicted shorter survivals of PCA patients. Suppression of SHARPIN impaired cell proliferation, angiogenesis, and invasion and reduced levels of MMP-9 in prostate cancer cells and reduced the size of metastatic lung tumors induced by these cells in mice. SHARPIN enhances the metastasis of prostate cancer and impair patient survivals. Prostate 77:718-728, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Cortical Structure Alterations and Social Behavior Impairment in p50-Deficient Mice.

PubMed

Bonini, Sara Anna; Mastinu, Andrea; Maccarinelli, Giuseppina; Mitola, Stefania; Premoli, Marika; La Rosa, Luca Rosario; Ferrari-Toninelli, Giulia; Grilli, Mariagrazia; Memo, Maurizio

2016-06-01

Alterations in genes that regulate neurodevelopment can lead to cortical malformations, resulting in malfunction during postnatal life. The NF-κB pathway has a key role during neurodevelopment by regulating the maintenance of the neural progenitor cell pool and inhibiting neuronal differentiation. In this study, we evaluated whether mice lacking the NF-κB p50 subunit (KO) present alterations in cortical structure and associated behavioral impairment. We found that, compared with wild type (WT), KO mice at postnatal day 2 present an increase in radial glial cells, an increase in Reelin protein expression levels, in addition to an increase of specific layer thickness. Moreover, adult KO mice display abnormal columnar organization in the somatosensory cortex, a specific decrease in somatostatin- and parvalbumin-expressing interneurons, altered neurite orientation, and a decrease in Synapsin I protein levels. Concerning behavior, KO mice, in addition to an increase in locomotor and exploratory activity, display impairment in social behaviors, with a reduction in social interaction. Finally, we found that risperidone treatment decreased hyperactivity of KO mice, but had no effect on defective social interaction. Altogether, these data add complexity to a growing body of data, suggesting a link between dysregulation of the NF-κB pathway and neurodevelopmental disorders pathogenesis. © The Author 2016. Published by Oxford University Press.

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation.

PubMed

Cho, Chris; Smallwood, Philip M; Nathans, Jeremy

2017-08-30

Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck, combined with loss of Norrin, impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck, and a soluble Reck(CC1-5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB. Copyright © 2017 Elsevier Inc. All rights reserved.

Blockade of PD-1/B7-H1 Interaction Restores Effector CD8+ T Cell Responses in a Hepatitis C Virus Core Murine Model1

PubMed Central

Lukens, John R.; Cruise, Michael W.; Lassen, Matthew G.; Hahn, Young S.

2010-01-01

The impaired function of CD8+ T cells is characteristic of hepatitis C virus (HCV) persistent infection. HCV core protein has been reported to inhibit CD8+ T cell responses. To determine the mechanism of the HCV core in suppressing Ag-specific CD8+ T cell responses, we generated a transgenic mouse, core(+) mice, where the expression of core protein is directed to the liver using the albumin promoter. Using a recombinant adenovirus to deliver Ag, we demonstrated that core(+) mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. The effector function of LacZ-specific CD8+ T cells was particularly impaired in the livers of core(+) mice, with suppression of IFN-γ, TNF-α, and granzyme B production by CD8+ T cells. In addition, the impaired CD8+ T cell responses in core(+) mice were accompanied by the enhanced expression of the inhibitory receptor programmed death-1 (PD-1) by LacZ-specific CD8+ T cells and its ligand B7-H1 on liver dendritic cells following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway (using a B7-H1 blocking antibody) in core(+) mice enhanced effector function of CD8+ T cells and cleared Ad-LacZ-infection as compared with that in mice treated with control Ab. This suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence in the liver. This also suggests that manipulation of the PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent HCV infection. PMID:18354211

Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation

PubMed Central

Sainski, Amy M.; Dai, Haiming; Natesampillai, Sekar; Pang, Yuan-Ping; Bren, Gary D.; Cummins, Nathan W.; Correia, Cristina; Meng, X. Wei; Tarara, James E.; Ramirez-Alvarado, Marina; Katzmann, David J.; Ochsenbauer, Christina; Kappes, John C.

2014-01-01

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein. PMID:25246614

MHCII-Mediated Dialog between Group 2 Innate Lymphoid Cells and CD4+ T Cells Potentiates Type 2 Immunity and Promotes Parasitic Helminth Expulsion

PubMed Central

Oliphant, Christopher J.; Hwang, You Yi; Walker, Jennifer A.; Salimi, Maryam; Wong, See Heng; Brewer, James M.; Englezakis, Alexandros; Barlow, Jillian L.; Hams, Emily; Scanlon, Seth T.; Ogg, Graham S.; Fallon, Padraic G.; McKenzie, Andrew N.J.

2014-01-01

Summary Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity. PMID:25088770

The Mucin MUC4 and Its Membrane Partner ErbB2 Regulate Biological Properties of Human CAPAN-2 Pancreatic Cancer Cells via Different Signalling Pathways

PubMed Central

Jonckheere, Nicolas; Skrypek, Nicolas; Merlin, Johann; Dessein, Anne Frédérique; Dumont, Patrick; Leteurtre, Emmanuelle; Harris, Ann; Desseyn, Jean-Luc; Susini, Christiane; Frénois, Frédéric; Van Seuningen, Isabelle

2012-01-01

The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells. PMID:22393391

Interaction of Low Molecular Weight Hyaluronan (LMW-HA) with CD44 and Toll-Like Receptors Promotes the Actin Filament-Associated Protein (AFAP-110)-Actin Binding and MyD88-NFκB Signaling Leading to Pro-inflammatory Cytokine/Chemokine Production and Breast Tumor Invasion

PubMed Central

Bourguignon, Lilly Y.W.; Wong, Gabriel; Earle, Christine A.; Xia, Weiliang

2011-01-01

Both high and low molecular weight hyaluronan (HMW-HA vs. LMW-HA) exist in various tissues and cells. In this study we investigated LMW-HA-mediated CD44 interaction with Toll-like receptors (TLRs), the actin filament-associated protein (AFAP-110) and a myeloid differentiation factor (MyD88) in breast tumor cells (MDA-MB-231 cells). Our data indicate that LMW-HA (but not HMW-HA) preferentially stimulates a physical association between CD44 and TLRs followed by a concomitant recruitment of AFAP-110 and MyD88 into receptor-containing complexes in breast tumor cells. LMW-HA-activated AFAP-110 then binds to F-actin resulting in MyD88/NF-κB nuclear translocation, NF-κB-specific transcription and target gene (IL-1β and IL-8) expression. These signaling events lead to pro-inflammatory cytokine/chemokine production in the breast tumor cells. AFAP-110-F-actin (activated by LMW-HA) also promotes tumor cell invasion. Downregulation of AFAP-110 or MyD88 by transfecting breast tumor cells with AFAP-110 siRNA or MyD88 siRNA, respectively not only blocks the ability of LMW-HA to stimulate AFAP-110-actin function, but also impairs MyD88-NF-κB nuclear translocation and NF-κB transcriptional activation. Consequently, both IL-1β/IL-8 production and tumor cell invasion are impaired. Taken together, these findings suggest that LMW-HA plays an important role in CD44-TLR-associated AFAP-110-actin interaction and MyD88-NF-κB signaling required for tumor cell behaviors which may contribute to the progression of breast cancer. PMID:22031535

Mycobacterium avium subspecies impair dendritic cell maturation.

PubMed

Basler, Tina; Brumshagen, Christina; Beineke, Andreas; Goethe, Ralph; Bäumer, Wolfgang

2013-10-01

Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic, granulomatous enteritis of ruminants. Dendritic cells (DC) of the gut are ideally placed to combat invading mycobacteria; however, little is known about their interaction with MAP. Here, we investigated the interaction of MAP and the closely related M. avium ssp. avium (MAA) with murine DC and the effect of infected macrophages on DC maturation. The infection of DC with MAP or MAA induced DC maturation, which differed to that of LPS as maturation was accompanied by higher production of IL-10 and lower production of IL-12. Treatment of maturing DC with supernatants from mycobacteria-infected macrophages resulted in impaired DC maturation, leading to a semi-mature, tolerogenic DC phenotype expressing low levels of MHCII, CD86 and TNF-α after LPS stimulation. Though the cells were not completely differentiated they responded with an increased IL-10 and a decreased IL-12 production. Using recombinant cytokines we provide evidence that the semi-mature DC phenotype results from a combination of secreted cytokines and released antigenic mycobacterial components of the infected macrophage. Our results indicate that MAP and MAA are able to subvert DC function directly by infecting and indirectly via the milieu created by infected macrophages.

NHERF Links the N-Cadherin/Catenin Complex to the Platelet-derived Growth Factor Receptor to Modulate the Actin Cytoskeleton and Regulate Cell Motility

PubMed Central

Theisen, Christopher S.; Wahl, James K.; Johnson, Keith R.

2007-01-01

Using phage display, we identified Na+/H+ exchanger regulatory factor (NHERF)-2 as a novel binding partner for the cadherin-associated protein, β-catenin. We showed that the second of two PSD-95/Dlg/ZO-1 (PDZ) domains of NHERF interacts with a PDZ-binding motif at the very carboxy terminus of β-catenin. N-cadherin expression has been shown to induce motility in a number of cell types. The first PDZ domain of NHERF is known to bind platelet-derived growth factor-receptor β (PDGF-Rβ), and the interaction of PDGF-Rβ with NHERF leads to enhanced cell spreading and motility. Here we show that β-catenin and N-cadherin are in a complex with NHERF and PDGF-Rβ at membrane ruffles in the highly invasive fibrosarcoma cell line HT1080. Using a stable short hairpin RNA system, we showed that HT1080 cells knocked down for either N-cadherin or NHERF had impaired ability to migrate into the wounded area in a scratch assay, similar to cells treated with a PDGF-R kinase inhibitor. Cells expressing a mutant NHERF that is unable to associate with β-catenin had increased stress fibers, reduced lamellipodia, and impaired cell migration. Using HeLa cells, which express little to no PDGF-R, we introduced PDGF-Rβ and showed that it coimmunoprecipitates with N-cadherin and that PDGF-dependent cell migration was reduced in these cells when we knocked-down expression of N-cadherin or NHERF. These studies implicate N-cadherin and β-catenin in cell migration via PDGF-R–mediated signaling through the scaffolding molecule NHERF. PMID:17229887

SPG20 Protein Spartin Is Recruited to Midbodies by ESCRT-III Protein Ist1 and Participates in Cytokinesis

PubMed Central

Renvoisé, Benoît; Parker, Rell L.; Yang, Dong; Bakowska, Joanna C.; Hurley, James H.

2010-01-01

Hereditary spastic paraplegias (HSPs, SPG1-46) are inherited neurological disorders characterized by lower extremity spastic weakness. Loss-of-function SPG20 gene mutations cause an autosomal recessive HSP known as Troyer syndrome. The SPG20 protein spartin localizes to lipid droplets and endosomes, and it interacts with tail interacting protein 47 (TIP47) as well as the ubiquitin E3 ligases atrophin-1-interacting protein (AIP)4 and AIP5. Spartin harbors a domain contained within microtubule-interacting and trafficking molecules (MIT) at its N-terminus, and most proteins with MIT domains interact with specific ESCRT-III proteins. Using yeast two-hybrid and in vitro surface plasmon resonance assays, we demonstrate that the spartin MIT domain binds with micromolar affinity to the endosomal sorting complex required for transport (ESCRT)-III protein increased sodium tolerance (Ist)1 but not to ESCRT-III proteins charged multivesicular body proteins 1–7. Spartin colocalizes with Ist1 at the midbody, and depletion of Ist1 in cells by small interfering RNA significantly decreases the number of cells where spartin is present at midbodies. Depletion of spartin does not affect Ist1 localization to midbodies but markedly impairs cytokinesis. A structure-based amino acid substitution in the spartin MIT domain (F24D) blocks the spartin–Ist1 interaction. Spartin F24D does not localize to the midbody and acts in a dominant-negative manner to impair cytokinesis. These data suggest that Ist1 interaction is important for spartin recruitment to the midbody and that spartin participates in cytokinesis. PMID:20719964

Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection

PubMed Central

Muir, Roshell; Metcalf, Talibah; Tardif, Virginie; Takata, Hiroshi; Phanuphak, Nittaya; Kroon, Eugene; Colby, Donn J.; Trichavaroj, Rapee; Valcour, Victor; Robb, Merlin L.; Michael, Nelson L.; Ananworanich, Jintanat; Trautmann, Lydie; Haddad, Elias K.

2016-01-01

The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory. PMID:27463374

Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

PubMed Central

Solis, Gonzalo P.; Hülsbusch, Nikola; Radon, Yvonne; Katanaev, Vladimir L.; Plattner, Helmut; Stuermer, Claudia A. O.

2013-01-01

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1–decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA–mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin–transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca2+ switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca2+ repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved. PMID:23825023

Tomato leaf curl Yunnan virus-encoded C4 induces cell division through enhancing stability of Cyclin D 1.1 via impairing NbSKη -mediated phosphorylation in Nicotiana benthamiana

PubMed Central

Mei, Yuzhen; Yang, Xiuling; Huang, Changjun

2018-01-01

The whitefly-transmitted geminiviruses induce severe developmental abnormalities in plants. Geminivirus-encoded C4 protein functions as one of viral symptom determinants that could induce abnormal cell division. However, the molecular mechanism by which C4 contributes to cell division induction remains unclear. Here we report that tomato leaf curl Yunnan virus (TLCYnV) C4 interacts with a glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinase, designed NbSKη, in Nicotiana benthamiana. Pro32, Asn34 and Thr35 of TLCYnV C4 are critical for its interaction with NbSKη and required for C4-induced typical symptoms. Interestingly, TLCYnV C4 directs NbSKη to the membrane and reduces the nuclear-accumulation of NbSKη. The relocalization of NbSKη impairs phosphorylation dependent degradation on its substrate-Cyclin D1.1 (NbCycD1;1), thereby increasing the accumulation level of NbCycD1;1 and inducing the cell division. Moreover, NbSKη-RNAi, 35S::NbCycD1;1 transgenic N. benthamiana plants have the similar phenotype as 35S::C4 transgenic N. benthamiana plants on callus-like tissue formation resulted from abnormal cell division induction. Thus, this study provides new insights into mechanism of how a viral protein hijacks NbSKη to induce abnormal cell division in plants. PMID:29293689

TRPM8 inhibits endothelial cell migration via a non-channel function by trapping the small GTPase Rap1

PubMed Central

Grolez, Guillaume P.; Bernardini, Michela; Richard, Elodie; Scianna, Marco; Lemonnier, Loic; Munaron, Luca; Mattot, Virginie; Prevarskaya, Natalia; Gkika, Dimitra

2017-01-01

Endothelial cell adhesion and migration are critical steps of the angiogenic process, whose dysfunction is associated with tumor growth and metastasis. The TRPM8 channel has recently been proposed to play a protective role in prostate cancer by impairing cell motility. However, the mechanisms by which it could influence vascular behavior are unknown. Here, we reveal a novel non-channel function for TRPM8 that unexpectedly acts as a Rap1 GTPase inhibitor, thereby inhibiting endothelial cell motility, independently of pore function. TRPM8 retains Rap1 intracellularly through direct protein–protein interaction, thus preventing its cytoplasm–plasma membrane trafficking. In turn, this mechanism impairs the activation of a major inside-out signaling pathway that triggers the conformational activation of integrin and, consequently, cell adhesion, migration, in vitro endothelial tube formation, and spheroid sprouting. Our results bring to light a novel, pore-independent molecular mechanism by which endogenous TRPM8 expression inhibits Rap1 GTPase and thus plays a critical role in the behavior of vascular endothelial cells by inhibiting migration. PMID:28550110

Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits

PubMed Central

Messa, Mirko; Fernández-Busnadiego, Rubén; Sun, Elizabeth Wen; Chen, Hong; Czapla, Heather; Wrasman, Kristie; Wu, Yumei; Ko, Genevieve; Ross, Theodora; Wendland, Beverly; De Camilli, Pietro

2014-01-01

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits. DOI: http://dx.doi.org/10.7554/eLife.03311.001 PMID:25122462

Mycobacterium tuberculosis impairs dendritic cell functions through the serine hydrolase Hip1.

PubMed

Madan-Lala, Ranjna; Sia, Jonathan Kevin; King, Rebecca; Adekambi, Toidi; Monin, Leticia; Khader, Shabaana A; Pulendran, Bali; Rengarajan, Jyothi

2014-05-01

Mycobacterium tuberculosis is a highly successful human pathogen that primarily resides in host phagocytes, such as macrophages and dendritic cells (DCs), and interferes with their functions. Although multiple strategies used by M. tuberculosis to modulate macrophage responses have been discovered, interactions between M. tuberculosis and DCs are less well understood. DCs are the primary APCs of the immune system and play a central role in linking innate and adaptive immune responses to microbial pathogens. In this study, we show that M. tuberculosis impairs DC cytokine secretion, maturation, and Ag presentation through the cell envelope-associated serine hydrolase, Hip1. Compared to wild-type, a hip1 mutant strain of M. tuberculosis induced enhanced levels of the key Th1-inducing cytokine IL-12, as well as other proinflammatory cytokines (IL-23, IL-6, TNF-α, IL-1β, and IL-18) in DCs via MyD88- and TLR2/9-dependent pathways, indicating that Hip1 restricts optimal DC inflammatory responses. Infection with the hip1 mutant also induced higher levels of MHC class II and costimulatory molecules CD40 and CD86, indicating that M. tuberculosis impairs DC maturation through Hip1. Further, we show that M. tuberculosis promotes suboptimal Ag presentation, as DCs infected with the hip1 mutant showed increased capacity to present Ag to OT-II- and early secreted antigenic target 6-specific transgenic CD4 T cells and enhanced Th1 and Th17 polarization. Overall, these data show that M. tuberculosis impairs DC functions and modulates the nature of Ag-specific T cell responses, with important implications for vaccination strategies.

Interactions of Human Endothelial and Multipotent Mesenchymal Stem Cells in Cocultures

PubMed Central

Ern, Christina; Krump-Konvalinkova, Vera; Docheva, Denitsa; Schindler, Stefanie; Rossmann, Oliver; Böcker, Wolfgang; Mutschler, Wolf; Schieker, Matthias

2010-01-01

Current strategies for tissue engineering of bone rely on the implantation of scaffolds, colonized with human mesenchymal stem cells (hMSC), into a recipient. A major limitation is the lack of blood vessels. One approach to enhance the scaffold vascularisation is to supply the scaffolds with endothelial cells (EC). The main goal of this study was to establish a coculture system of hMSC and EC for the purposes of bone tissue engineering. Therefore, the cell behaviour, proliferation and differentiation capacity in various cell culture media as well as cell interactions in the cocultures were evaluated. The differentiation capacity of hMSC along osteogenic, chondrogenic, and adipogenic lineage was impaired in EC medium while in a mixed EC and hMSC media, hMSC maintained osteogenic differentiation. In order to identify and trace EC in the cocultures, EC were transduced with eGFP. Using time-lapse imaging, we observed that hMSC and EC actively migrated towards cells of their own type and formed separate clusters in long term cocultures. The scarcity of hMSC and EC contacts in the cocultures suggest the influence of growth factor-mediated cell interactions and points to the necessity of further optimization of the coculture conditions. PMID:21625373

Low-expression of E-cadherin in leukaemia cells causes loss of homophilic adhesion and promotes cell growth.

PubMed

Rao, Qing; Wang, Ji-Ying; Meng, Jihong; Tang, Kejing; Wang, Yanzhong; Wang, Min; Xing, Haiyan; Tian, Zheng; Wang, Jianxiang

2011-09-01

E-cadherin (epithelial cadherin) belongs to the calcium-dependent adhesion molecule superfamily and is implicated in the interactions of haematopoietic progenitors and bone marrow stromal cells. Adhesion capacity to bone marrow stroma was impaired for leukaemia cells, suggesting that a breakdown of adhesive mechanisms governed by an adhesion molecule may exist in leukaemic microenvironment. We previously found that E-cadherin was low expressed in primary acute leukaemia cells compared with normal bone marrow mononuclear cells. In this study, we investigate the functional importance of low E-cadherin expression in leukaemia cell behaviours and investigate its effects in the abnormal interaction of leukaemic cells with stromal cells. After expression of E-cadherin was restored by a demethylating agent in leukaemia cells, E-cadherin-specific adhesion was enhanced. Additionally, siRNA (small interfering RNA)-mediated silencing of E-cadherin in Raji cells resulted in a reduction of cell homophilic adhesion and enhancement of cell proliferation and colony formation. These results suggest that low expression of E-cadherin contributes to the vigorous growth and transforming ability of leukaemic cells.

Cadm1-Expressing Synapses on Purkinje Cell Dendrites Are Involved in Mouse Ultrasonic Vocalization Activity

PubMed Central

Fujita, Eriko; Tanabe, Yuko; Imhof, Beat A.; Momoi, Mariko Y.; Momoi, Takashi

2012-01-01

Foxp2(R552H) knock-in (KI) mouse pups with a mutation related to human speech–language disorders exhibit poor development of cerebellar Purkinje cells and impaired ultrasonic vocalization (USV), a communication tool for mother-offspring interactions. Thus, human speech and mouse USV appear to have a Foxp2-mediated common molecular basis in the cerebellum. Mutations in the gene encoding the synaptic adhesion molecule CADM1 (RA175/Necl2/SynCAM1/Cadm1) have been identified in people with autism spectrum disorder (ASD) who have impaired speech and language. In the present study, we show that both Cadm1-deficient knockout (KO) pups and Foxp2(R552H) KI pups exhibit impaired USV and smaller cerebellums. Cadm1 was preferentially localized to the apical–distal portion of the dendritic arbor of Purkinje cells in the molecular layer of wild-type pups, and VGluT1 level decreased in the cerebellum of Cadm1 KO mice. In addition, we detected reduced immunoreactivity of Cadm1 and VGluT1 on the poorly developed dendritic arbor of Purkinje cells in the Foxp2(R552H) KI pups. However, Cadm1 mRNA expression was not altered in the Foxp2(R552H) KI pups. These results suggest that although the Foxp2 transcription factor does not target Cadm1, Cadm1 at the synapses of Purkinje cells and parallel fibers is necessary for USV function. The loss of Cadm1-expressing synapses on the dendrites of Purkinje cells may be associated with the USV impairment that Cadm1 KO and Foxp2(R552H) KI mice exhibit. PMID:22272290

Connecting Malfunctioning Glial Cells and Brain Degenerative Disorders.

PubMed

Kaminsky, Natalie; Bihari, Ofer; Kanner, Sivan; Barzilai, Ari

2016-06-01

The DNA damage response (DDR) is a complex biological system activated by different types of DNA damage. Mutations in certain components of the DDR machinery can lead to genomic instability disorders that culminate in tissue degeneration, premature aging, and various types of cancers. Intriguingly, malfunctioning DDR plays a role in the etiology of late onset brain degenerative disorders such as Parkinson's, Alzheimer's, and Huntington's diseases. For many years, brain degenerative disorders were thought to result from aberrant neural death. Here we discuss the evidence that supports our novel hypothesis that brain degenerative diseases involve dysfunction of glial cells (astrocytes, microglia, and oligodendrocytes). Impairment in the functionality of glial cells results in pathological neuro-glial interactions that, in turn, generate a "hostile" environment that impairs the functionality of neuronal cells. These events can lead to systematic neural demise on a scale that appears to be proportional to the severity of the neurological deficit. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA.

PubMed

Torseth, Kathrin; Doseth, Berit; Hagen, Lars; Olaisen, Camilla; Liabakk, Nina-Beate; Græsmann, Heidi; Durandy, Anne; Otterlei, Marit; Krokan, Hans E; Kavli, Bodil; Slupphaug, Geir

2012-06-01

In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Mouse CD23 regulates monocyte activation through an interaction with the adhesion molecule CD11b/CD18.

PubMed

Lecoanet-Henchoz, S; Plater-Zyberk, C; Graber, P; Gretener, D; Aubry, J P; Conrad, D H; Bonnefoy, J Y

1997-09-01

CD23 is expressed on a variety of hemopoietic cells. Recently, we have reported that blocking CD23 interactions in a murine model of arthritis resulted in a marked improvement of disease severity. Here, we demonstrate that CD11b, the alpha chain of the beta 2 integrin adhesion molecule complex CD11b/CD18 expressed on monocytes interacts with CD23. Using a recombinant fusion protein (ZZ-CD23), murine CD23 was shown to bind to peritoneal macrophages and peripheral blood cells isolated from mice as well as the murine macrophage cell line, RAW. The interactions between mouse ZZ-CD23 and CD11b/CD18-expressing cells were significantly inhibited by anti-CD11b monoclonal antibodies. A functional consequence was then demonstrated by inducing an up-regulation of interleukin-6 (IL-6) production following ZZ-CD23 incubation with monocytes. The addition of Fab fragments generated from the monoclonal antibody CD11b impaired this cytokine production by 50%. Interestingly, a positive autocrine loop was identified as IL-6 was shown to increase CD23 binding to macrophages. These results demonstrate that similar to findings using human cells, murine CD23 binds to the surface adhesion molecule, CD11b, and these interactions regulate biological activities of murine myeloid cells.

Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors

PubMed Central

Lanfumey, Laurence; Cordomí, Arnau; Pastor, Antoni; de La Torre, Rafael; Gasperini, Paola; Navarro, Gemma; Howell, Lesley A.; Pardo, Leonardo; Lluís, Carmen; Canela, Enric I.; McCormick, Peter J.; Maldonado, Rafael; Robledo, Patricia

2015-01-01

Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties. PMID:26158621

Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors.

PubMed

Viñals, Xavier; Moreno, Estefanía; Lanfumey, Laurence; Cordomí, Arnau; Pastor, Antoni; de La Torre, Rafael; Gasperini, Paola; Navarro, Gemma; Howell, Lesley A; Pardo, Leonardo; Lluís, Carmen; Canela, Enric I; McCormick, Peter J; Maldonado, Rafael; Robledo, Patricia

2015-07-01

Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.

BHC80 is Critical in Suppression of Snail-LSD1 Interaction and Breast Cancer Metastasis

DTIC Science & Technology

2013-01-01

characteristics such as invasion and metastasis. The most common chemotherapeutic drugs function by introducing DNA damage to impair cell division...expression confers drug resistancy on cancer cells indicates that Snail can function as a survival factor. Recently we performed a sequential protein...one of alanine, isoleucine , leucine and valine (Figure 3A). While the sequence surrounding Arg151, Lys152 and Ala153 does not exactly follow the rule

Necroptosis Resumes Apoptosis in Hippocampus but Not in Frontal Cortex.

PubMed

Nikseresht, Sara; Khodagholi, Fariba; Dargahi, Leila; Ahmadiani, Abolhassan

2017-12-01

Cell death subsequent to or concurrent with neuroinflammation results in some damages like neuron loss and spatial memory impairment. In this study, we demonstrated the temporal pattern of neuroinflammation, necroptotic, and apoptotic cell deaths in hippocampus and frontal cortex following intracerebroventricular administration of lipopolysaccharide (LPS). We evaluated receptor interacting protein kinase 1 (RIP1), RIP3, and two related metabolic enzymes including glutamate-ammonia ligase (GLUL) and glutamate dehydrogenase (GLUD) as necroptosis factors. Apoptosis pathway, antioxidant status and inflammatory cytokines were also assessed. Based on the probable role of these brain regions in working memory performance, spontaneous alternation was evaluated through the Y-maze apparatus. RIP1, RIP3, and then GLUL and GLUD, as well as apoptosis markers, inflammatory regulators, and antioxidant defense demonstrated different time-dependent patterns in hippocampus and frontal cortex. Interestingly, in hippocampus but not in frontal cortex, necroptosis resumed apoptosis. Our results in behavioral section revealed that neuroinflammation along with apoptosis and necroptosis pathways could lead to reversible short-term memory impairment after LPS injection. In conclusion, it can be suggested that there is a region-specific response of cell deaths regulators activation in hippocampus and frontal cortex. In addition, elucidating the time profile of events in response to neuroinflammation would be of great help in mechanistic studies and understanding of pathways interaction. J. Cell. Biochem. 118: 4628-4638, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Interaction between dendritic cells and natural killer cells during pregnancy in mice.

PubMed

Blois, Sandra M; Barrientos, Gabriela; Garcia, Mariana G; Orsal, Arif S; Tometten, Mareike; Cordo-Russo, Rosalia I; Klapp, Burghard F; Santoni, Angela; Fernández, Nelson; Terness, Peter; Arck, Petra C

2008-07-01

A complex regulation of innate and adaptive immune responses at the maternal fetal interface promotes tolerance of trophoblast cells carrying paternally derived antigens. Such regulatory functions involve uterine dendritic cells (uDC) and natural killer (uNK) cells. The existence of a NK and DC "cross talk" has been revealed in various experimental settings; its biological significance ranging from cooperative stimulation to cell lysis. Little is known about the presence or role of NK and DC cross talk at the maternal fetal interface. The present study shows that mouse NK and DC interactions are subject to modulation by trophoblast cells in vitro. This interaction promotes a tolerogenic microenvironment characterized by downregulation of the expression of activation markers on uNK cells and uDC and dominance of Th2 cytokines. NK and DC interactions would also influence uterine cell proliferation and this process would be strongly modulated by trophoblast-derived signals. Indeed; while low proliferation rates were observed upon regular coculture allowing direct contact between uterine cells and trophoblasts, incubation in a transwell culture system markedly increased uterine cell proliferation suggesting that soluble factors are key mediators in the molecular "dialog" between the mother and the conceptus during the establishment of mouse pregnancy. Our data further reveal that the regulatory functions of trophoblast cells associated with tolerance induction are impaired in high abortion murine matings. Interestingly, we observed that secretion of interleukin-12p70 by uDC is dramatically abrogated in the presence of uNK cells. Taken together, our results provide the first evidence that a delicate balance of interactions involving NK cells, DC, and trophoblasts at the mouse maternal fetal interface supports a successful pregnancy outcome.

HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca

HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process bymore » recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.« less

Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity.

PubMed

Ueno, Akiko; Omori, Yoshihiro; Sugita, Yuko; Watanabe, Satoshi; Chaya, Taro; Kozuka, Takashi; Kon, Tetsuo; Yoshida, Satoyo; Matsushita, Kenji; Kuwahara, Ryusuke; Kajimura, Naoko; Okada, Yasushi; Furukawa, Takahisa

2018-03-27

In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Cell cycle regulator E2F4 is essential for the development of the ventral telencephalon.

PubMed

Ruzhynsky, Vladimir A; McClellan, Kelly A; Vanderluit, Jacqueline L; Jeong, Yongsu; Furimsky, Marosh; Park, David S; Epstein, Douglas J; Wallace, Valerie A; Slack, Ruth S

2007-05-30

Early forebrain development is characterized by extensive proliferation of neural precursors coupled with complex structural transformations; however, little is known regarding the mechanisms by which these processes are integrated. Here, we show that deficiency of the cell cycle regulatory protein, E2F4, results in the loss of ventral telencephalic structures and impaired self-renewal of neural precursor cells. The mechanism underlying aberrant ventral patterning lies in a dramatic loss of Sonic hedgehog (Shh) expression specifically in this region. The E2F4-deficient phenotype can be recapitulated by interbreeding mice heterozygous for E2F4 with those lacking one allele of Shh, suggesting a genetic interaction between these pathways. Treatment of E2F4-deficient cells with a Hh agonist rescues stem cell self-renewal and cells expressing the homeodomain proteins that specify the ventral telencephalic structures. Finally, we show that E2F4 deficiency results in impaired activity of Shh forebrain-specific enhancers. In conclusion, these studies establish a novel requirement for the cell cycle regulatory protein, E2F4, in the development of the ventral telencephalon.

Herpes simplex virus type 2 glycoprotein H interacts with integrin αvβ3 to facilitate viral entry and calcium signaling in human genital tract epithelial cells.

PubMed

Cheshenko, Natalia; Trepanier, Janie B; González, Pablo A; Eugenin, Eliseo A; Jacobs, William R; Herold, Betsy C

2014-09-01

Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca2+) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion.

PubMed

Oliphant, Christopher J; Hwang, You Yi; Walker, Jennifer A; Salimi, Maryam; Wong, See Heng; Brewer, James M; Englezakis, Alexandros; Barlow, Jillian L; Hams, Emily; Scanlon, Seth T; Ogg, Graham S; Fallon, Padraic G; McKenzie, Andrew N J

2014-08-21

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Dynamin-mediated Nephrin Phosphorylation Regulates Glucose-stimulated Insulin Release in Pancreatic Beta Cells*

PubMed Central

Jeon, Jongmin; Leibiger, Ingo; Moede, Tilo; Walter, Britta; Faul, Christian; Maiguel, Dony; Villarreal, Rodrigo; Guzman, Johanna; Berggren, Per-Olof; Mundel, Peter; Ricordi, Camillo; Merscher-Gomez, Sandra; Fornoni, Alessia

2012-01-01

We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function. PMID:22718751

Germinal Cell Aplasia in Kif18a Mutant Male Mice Due to Impaired Chromosome Congression and Dysregulated BubR1 and CENP-E

PubMed Central

Liu, Xue-song; Zhao, Xu-dong; Wang, Xiaoxing; Yao, Yi-xin; Zhang, Liang-liang; Shu, Run-zhe; Ren, Wei-hua; Huang, Ying; Huang, Lei; Gu, Ming-min; Kuang, Ying; Wang, Long; Lu, Shun-yuan; Chi, Jun; Fen, Jing-sheng; Wang, Yi-fei; Fei, Jian; Dai, Wei; Wang, Zhu-Gang

2010-01-01

Chromosomal instability during cell division frequently causes cell death or malignant transformation. Orderly chromosome congression at the metaphase plate, a paramount process to vertebrate mitosis and meiosis, is controlled by a number of molecular regulators, including kinesins. Kinesin-8 (Kif18A) functions to control mitotic chromosome alignment at the mid-zone by negative regulation of kinetochore oscillation. Here the authors report that disrupting Kif18a function results in complete sterility in male but not in female mice. Histological examination reveals that Kif18a−/− testes exhibit severe developmental impairment of seminiferous tubules. Testis atrophy in Kif18a−/− mice is caused by perturbation of microtubule dynamics and spindle pole integrity, leading to chromosome congression defects during mitosis and meiosis. Depletion of KIF18A via RNAi causes mitotic arrest accompanied by unaligned chromosomes and increased microtubule nucleating centers in both GC-1 and HeLa cells. Prolonged depletion of KIF18A causes apoptosis due to perturbed microtubule dynamics. Further studies reveal that KIF18A silencing results in degradation of CENP-E and BubR1, which is accompanied by premature sister chromatid separation. KIF18A physically interacts with BubR1 and CENP-E, and this interaction is modulated during mitosis. Combined, the studies indicate that KIF18A is essential for normal chromosome congression during cell division and that the absence of KIF18A function causes severe defects in microtubule dynamics, spindle integrity, and checkpoint activation, leading to germinal cell aplasia in mice. PMID:20981276

Mycobacterium tuberculosis impairs dendritic cell functions through the serine hydrolase Hip1

PubMed Central

Madan-Lala, Ranjna; Sia, Jonathan Kevin; King, Rebecca; Adekambi, Toidi; Monin, Leticia; Khader, Shabaana A; Pulendran, Bali; Rengarajan, Jyothi

2014-01-01

Mycobacterium tuberculosis (Mtb) is a highly successful human pathogen that primarily resides in host phagocytes, such as macrophages and dendritic cells (DCs), and interferes with their functions. While multiple strategies used by Mtb to modulate macrophage responses have been discovered, interactions between Mtb and DCs are less well understood. DCs are the primary antigen presenting acells (APCs) of the immune system and play a central role in linking innate and adaptive immune responses to microbial pathogens. In this study we show that Mtb impairs DC cytokine secretion, maturation and antigen presentation through the cell envelope-associated serine hydrolase Hip1. Compared to wild type, a hip1 mutant strain of Mtb induced enhanced levels of the key T helper 1 (Th1)-inducing cytokine IL-12, as well as other proinflammatory cytokines (IL-23, IL-6, TNF-α, IL-1β, IL-18) in DCs via MyD88- and TLR2/9-dependent pathways, indicating that Hip1 restricts optimal DC inflammatory responses. Infection with the hip1 mutant also induced higher levels of MHC class II and co-stimulatory molecules, CD40 and CD86, indicating that Mtb impairs DC maturation through Hip1. Further, we show that Mtb promotes sub-optimal antigen presentation, as DCs infected with the hip1 mutant showed increased capacity to present antigen to OT-II- and early secreted antigenic target 6 (ESAT-6)-specific transgenic CD4 T cells and enhanced Th1 and Th17 polarization. Overall, these data show that Mtb impairs DC functions and modulates the nature of antigen-specific T cell responses, with important implications for vaccination strategies. PMID:24659689

Treatment of hypertension by increasing impaired endothelial TRPV4-KCa2.3 interaction.

PubMed

He, Dongxu; Pan, Qiongxi; Chen, Zhen; Sun, Chunyuan; Zhang, Peng; Mao, Aiqin; Zhu, Yaodan; Li, Hongjuan; Lu, Chunxiao; Xie, Mingxu; Zhou, Yin; Shen, Daoming; Tang, Chunlei; Yang, Zhenyu; Jin, Jian; Yao, Xiaoqiang; Nilius, Bernd; Ma, Xin

2017-11-01

The currently available antihypertensive agents have undesirable adverse effects due to systemically altering target activity including receptors, channels, and enzymes. These effects, such as loss of potassium ions induced by diuretics, bronchospasm by beta-blockers, constipation by Ca 2+ channel blockers, and dry cough by ACEI, lead to non-compliance with therapies (Moser, 1990). Here, based on new hypertension mechanisms, we explored a new antihypertensive approach. We report that transient receptor potential vanilloid 4 (TRPV4) interacts with Ca 2+ -activated potassium channel 3 (KCa2.3) in endothelial cells (ECs) from small resistance arteries of normotensive humans, while ECs from hypertensive patients show a reduced interaction between TRPV4 and KCa2.3. Murine hypertension models, induced by high-salt diet, N(G)-nitro-l-arginine intake, or angiotensin II delivery, showed decreased TRPV4-KCa2.3 interaction in ECs. Perturbation of the TRPV4-KCa2.3 interaction in mouse ECs by overexpressing full-length KCa2.3 or defective KCa2.3 had hypotensive or hypertensive effects, respectively. Next, we developed a small-molecule drug, JNc-440, which showed affinity for both TRPV4 and KCa2.3. JNc-440 significantly strengthened the TRPV4-KCa2.3 interaction in ECs, enhanced vasodilation, and exerted antihypertensive effects in mice. Importantly, JNc-440 specifically targeted the impaired TRPV4-KCa2.3 interaction in ECs but did not systemically activate TRPV4 and KCa2.3. Together, our data highlight the importance of impaired endothelial TRPV4-KCa2.3 coupling in the progression of hypertension and suggest a novel approach for antihypertensive drug development. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

PubMed Central

Wang, Jun; Rouse, Clay; Jasper, Jeff S.; Pendergast, Ann Marie

2016-01-01

Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. Here, we report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast activating factors interleukin 6 (IL6) and matrix metalloproteinase-1 (MMP1). Furthermore, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for targeting both tumor and the bone microenvironment with ABL-specific inhibitors. PMID:26838548

Immune Checkpoint Blockade for Breast Cancer.

PubMed

Swoboda, April; Nanda, Rita

An effective antitumor immune response requires interaction between cells of the adaptive and innate immune system. Three key elements are required: generation of activated tumor-directed T cells, infiltration of activated T cells into the tumor microenvironment, and killing of tumor cells by activated T cells. Tumor immune evasion can occur as a result of the disruption of each of these three key T cell activities, resulting in three distinct cancer-immune phenotypes. The immune inflamed phenotype, characterized by the presence of a robust tumor immune infiltrate, suggests impaired activated T cell killing of tumor cells related to the presence of inhibitory factors. Programmed death receptor-1 (PD-1) is an inhibitory transmembrane protein expressed on T cells, B cells, and NK cells. The interaction between PD-1 and its ligands (PD-L1/L2) functions as an immune checkpoint against unrestrained cytotoxic T effector cell activity-it promotes peripheral T effector cell exhaustion and conversion of T effector cells to immunosuppressive T regulatory (Treg) cells. Immune checkpoint inhibitors, which block the PD-1/PD-L1 axis and reactivate cytotoxic T effector cell function, are actively being investigated for the treatment of breast cancer.

Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells.

PubMed

Dolnik, Olga; Kolesnikova, Larissa; Welsch, Sonja; Strecker, Thomas; Schudt, Gordian; Becker, Stephan

2014-10-01

Endosomal sorting complex required for transport (ESCRT) machinery supports the efficient budding of Marburg virus (MARV) and many other enveloped viruses. Interaction between components of the ESCRT machinery and viral proteins is predominantly mediated by short tetrapeptide motifs, known as late domains. MARV contains late domain motifs in the matrix protein VP40 and in the genome-encapsidating nucleoprotein (NP). The PSAP late domain motif of NP recruits the ESCRT-I protein tumor susceptibility gene 101 (Tsg101). Here, we generated a recombinant MARV encoding NP with a mutated PSAP late domain (rMARV(PSAPmut)). rMARV(PSAPmut) was attenuated by up to one log compared with recombinant wild-type MARV (rMARV(wt)), formed smaller plaques and exhibited delayed virus release. Nucleocapsids in rMARV(PSAPmut)-infected cells were more densely packed inside viral inclusions and more abundant in the cytoplasm than in rMARV(wt)-infected cells. A similar phenotype was detected when MARV-infected cells were depleted of Tsg101. Live-cell imaging analyses revealed that Tsg101 accumulated in inclusions of rMARV(wt)-infected cells and was co-transported together with nucleocapsids. In contrast, rMARV(PSAPmut) nucleocapsids did not display co-localization with Tsg101, had significantly shorter transport trajectories, and migration close to the plasma membrane was severely impaired, resulting in reduced recruitment into filopodia, the major budding sites of MARV. We further show that the Tsg101 interacting protein IQGAP1, an actin cytoskeleton regulator, was recruited into inclusions and to individual nucleocapsids together with Tsg101. Moreover, IQGAP1 was detected in a contrail-like structure at the rear end of migrating nucleocapsids. Down regulation of IQGAP1 impaired release of MARV. These results indicate that the PSAP motif in NP, which enables binding to Tsg101, is important for the efficient actin-dependent transport of nucleocapsids to the sites of budding. Thus, the interaction between NP and Tsg101 supports several steps of MARV assembly before virus fission.

Neurexin dysfunction in adult neurons results in autistic-like behavior in mice.

PubMed

Rabaneda, Luis G; Robles-Lanuza, Estefanía; Nieto-González, José Luis; Scholl, Francisco G

2014-07-24

Autism spectrum disorders (ASDs) comprise a group of clinical phenotypes characterized by repetitive behavior and social and communication deficits. Autism is generally viewed as a neurodevelopmental disorder where insults during embryonic or early postnatal periods result in aberrant wiring and function of neuronal circuits. Neurexins are synaptic proteins associated with autism. Here, we generated transgenic βNrx1ΔC mice in which neurexin function is selectively impaired during late postnatal stages. Whole-cell recordings in cortical neurons show an impairment of glutamatergic synaptic transmission in the βNrx1ΔC mice. Importantly, mutant mice exhibit autism-related symptoms, such as increased self-grooming, deficits in social interactions, and altered interaction for nonsocial olfactory cues. The autistic-like phenotype of βNrx1ΔC mice can be reversed after removing the mutant protein in aged animals. The defects resulting from disruption of neurexin function after the completion of embryonic and early postnatal development suggest that functional impairment of mature circuits can trigger autism-related phenotypes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Communicative interactions between visually impaired mothers and their sighted children: analysis of gaze, facial expressions, voice and physical contacts.

PubMed

Chiesa, S; Galati, D; Schmidt, S

2015-11-01

Social and emotional development of infants and young children is largely based on the communicative interaction with their mother, or principal caretaker (Trevarthen ). The main modalities implied in this early communication are voice, facial expressions and gaze (Stern ). This study aims at analysing early mother-child interactions in the case of visually impaired mothers who do not have access to their children's gaze and facial expressions. Spontaneous play interactions between seven visually impaired mothers and their sighted children aged between 6 months and 3 years were filmed. These dyads were compared with a control group of sighted mothers and children analysing four modalities of communication and interaction regulation: gaze, physical contacts, verbal productions and facial expressions. The visually impaired mothers' facial expressions differed from the ones of sighted mothers mainly with respect to forehead movements, leading to an impoverishment of conveyed meaning. Regarding the other communicative modalities, results suggest that visually impaired mothers and their children use compensatory strategies to guaranty harmonic interaction despite the mother's impairment: whereas gaze results the main factor of interaction regulation in sighted dyads, physical contacts and verbal productions assume a prevalent role in dyads with visually impaired mothers. Moreover, visually impaired mother's children seem to be able to differentiate between their mother and sighted interaction partners, adapting differential modes of communication. The results of this study show that, in spite of the obvious differences in the modes of communication, visual impairment does not prevent a harmonious interaction with the child. © 2015 John Wiley & Sons Ltd.

Cell Adhesion-dependent Serine 85 Phosphorylation of Paxillin Modulates Focal Adhesion Formation and Haptotactic Migration via Association with the C-terminal Tail Domain of Talin*

PubMed Central

Kwak, Tae Kyoung; Lee, Mi-Sook; Ryu, Jihye; Choi, Yoon-Ju; Kang, Minkyung; Jeong, Doyoung; Lee, Jung Weon

2012-01-01

Integrin-mediated adhesion to extracellular matrix proteins is dynamically regulated during morphological changes and cell migration. Upon cell adhesion, protein-protein interactions among molecules at focal adhesions (FAs) play major roles in the regulation of cell morphogenesis and migration. Although tyrosine phosphorylation of paxillin is critically involved in adhesion-mediated signaling, the significance of paxillin phosphorylation at Ser-85 and the mechanism by which it regulates cell migration remain unclear. In this study, we examined how Ser-85 phosphorylation of paxillin affects FA formation and cell migration. We found that paxillin phosphorylation at Ser-85 occurred during HeLa cell adhesion to collagen I and was concomitant with tyrosine phosphorylation of both focal adhesion kinase and talin. However, the non-phosphorylatable S85A mutant of paxillin impaired cell spreading, FA turnover, and migration toward collagen I but not toward serum. Furthermore, whereas the (presumably indirect) interaction between paxillin and the C-terminal tail of talin led to dynamic FAs at the cell boundary, S85A paxillin did not bind talin and caused stabilized FAs in the central region of cells. Together, these observations suggest that cell adhesion-dependent Ser-85 phosphorylation of paxillin is important for its interaction with talin and regulation of dynamic FAs and cell migration. PMID:22761432

Creating Accessible Science Museums with User-Activated Environmental Audio Beacons (Ping!)

ERIC Educational Resources Information Center

Landau, Steven; Wiener, William; Naghshineh, Koorosh; Giusti, Ellen

2005-01-01

In 2003, Touch Graphics Company carried out research on a new invention that promises to improve accessibility to science museums for visitors who are visually impaired. The system, nicknamed Ping!, allows users to navigate an exhibit area, listen to audio descriptions, and interact with exhibits using a cell phone-based interface. The system…

Bacterial Adherence and Dwelling Probability: Two Drivers of Early Alveolar Infection by Streptococcus pneumoniae Identified in Multi-Level Mathematical Modeling

PubMed Central

Santos, Guido; Lai, Xin; Eberhardt, Martin; Vera, Julio

2018-01-01

Pneumococcal infection is the most frequent cause of pneumonia, and one of the most prevalent diseases worldwide. The population groups at high risk of death from bacterial pneumonia are infants, elderly and immunosuppressed people. These groups are more vulnerable because they have immature or impaired immune systems, the efficacy of their response to vaccines is lower, and antibiotic treatment often does not take place until the inflammatory response triggered is already overwhelming. The immune response to bacterial lung infections involves dynamic interactions between several types of cells whose activation is driven by intracellular molecular networks. A feasible approach to the integration of knowledge and data linking tissue, cellular and intracellular events and the construction of hypotheses in this area is the use of mathematical modeling. For this paper, we used a multi-level computational model to analyse the role of cellular and molecular interactions during the first 10 h after alveolar invasion of Streptococcus pneumoniae bacteria. By “multi-level” we mean that we simulated the interplay between different temporal and spatial scales in a single computational model. In this instance, we included the intracellular scale of processes driving lung epithelial cell activation together with the scale of cell-to-cell interactions at the alveolar tissue. In our analysis, we combined systematic model simulations with logistic regression analysis and decision trees to find genotypic-phenotypic signatures that explain differences in bacteria strain infectivity. According to our simulations, pneumococci benefit from a high dwelling probability and a high proliferation rate during the first stages of infection. In addition to this, the model predicts that during the very early phases of infection the bacterial capsule could be an impediment to the establishment of the alveolar infection because it impairs bacterial colonization. PMID:29868515

Bacterial Adherence and Dwelling Probability: Two Drivers of Early Alveolar Infection by Streptococcus pneumoniae Identified in Multi-Level Mathematical Modeling.

PubMed

Santos, Guido; Lai, Xin; Eberhardt, Martin; Vera, Julio

2018-01-01

Pneumococcal infection is the most frequent cause of pneumonia, and one of the most prevalent diseases worldwide. The population groups at high risk of death from bacterial pneumonia are infants, elderly and immunosuppressed people. These groups are more vulnerable because they have immature or impaired immune systems, the efficacy of their response to vaccines is lower, and antibiotic treatment often does not take place until the inflammatory response triggered is already overwhelming. The immune response to bacterial lung infections involves dynamic interactions between several types of cells whose activation is driven by intracellular molecular networks. A feasible approach to the integration of knowledge and data linking tissue, cellular and intracellular events and the construction of hypotheses in this area is the use of mathematical modeling. For this paper, we used a multi-level computational model to analyse the role of cellular and molecular interactions during the first 10 h after alveolar invasion of Streptococcus pneumoniae bacteria. By "multi-level" we mean that we simulated the interplay between different temporal and spatial scales in a single computational model. In this instance, we included the intracellular scale of processes driving lung epithelial cell activation together with the scale of cell-to-cell interactions at the alveolar tissue. In our analysis, we combined systematic model simulations with logistic regression analysis and decision trees to find genotypic-phenotypic signatures that explain differences in bacteria strain infectivity. According to our simulations, pneumococci benefit from a high dwelling probability and a high proliferation rate during the first stages of infection. In addition to this, the model predicts that during the very early phases of infection the bacterial capsule could be an impediment to the establishment of the alveolar infection because it impairs bacterial colonization.

SPARC expression induces cell cycle arrest via STAT3 signaling pathway in medulloblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chetty, Chandramu; Dontula, Ranadheer; Ganji, Purnachandra Nagaraju

2012-01-13

Highlights: Black-Right-Pointing-Pointer Ectopic expression of SPARC impaired cell proliferation in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression induces STAT3 mediated cell cycle arrest in medulloblastoma cells. Black-Right-Pointing-Pointer SPARC expression significantly inhibited pre-established tumor growth in nude-mice. -- Abstract: Dynamic cell interaction with ECM components has profound influence in cancer progression. SPARC is a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. We previously reported that SPARC expression significantly impairs medulloblastoma tumor growth in vivo. In this study, we demonstrate that expression of SPARC inhibits medulloblastoma cell proliferation. MTT assay indicated a dose-dependent reductionmore » in tumor cell proliferation in adenoviral mediated expression of SPARC full length cDNA (Ad-DsRed-SP) in D425 and UW228 cells. Flow cytometric analysis showed that Ad-DsRed-SP-infected cells accumulate in the G2/M phase of cell cycle. Further, immunoblot and immunoprecipitation analyses revealed that SPARC induced G2/M cell cycle arrest was mediated through inhibition of the Cyclin-B-regulated signaling pathway involving p21 and Cdc2 expression. Additionally, expression of SPARC decreased STAT3 phosphorylation at Tyr-705; constitutively active STAT3 expression reversed SPARC induced G2/M arrest. Ad-DsRed-SP significantly inhibited the pre-established orthotopic tumor growth and tumor volume in nude-mice. Immunohistochemical analysis of tumor sections from mice treated with Ad-DsRed-SP showed decreased immunoreactivity for pSTAT3 and increased immunoreactivity for p21 compared to tumor section from mice treated with mock and Ad-DsRed. Taken together our studies further reveal that STAT3 plays a key role in SPARC induced G2/M arrest in medulloblastoma cells. These new findings provide a molecular basis for the mechanistic understanding of the effects of SPARC on medulloblastoma tumor cell proliferation.« less

R-Ras Regulates Migration through an Interaction with Filamin A in Melanoma Cells

PubMed Central

Gawecka, Joanna E.; Griffiths, Genevieve S.; Ek-Rylander, Barbro; Ramos, Joe W.; Matter, Michelle L.

2010-01-01

Background Changes in cell adhesion and migration in the tumor microenvironment are key in the initiation and progression of metastasis. R-Ras is one of several small GTPases that regulate cell adhesion and migration on the extracellular matrix, however the mechanism has not been completely elucidated. Using a yeast two-hybrid approach we sought to identify novel R-Ras binding proteins that might mediate its effects on integrins. Methods and Findings We identified Filamin A (FLNa) as a candidate interacting protein. FLNa is an actin-binding scaffold protein that also binds to integrin β1, β2 and β7 tails and is associated with diverse cell processes including cell migration. Indeed, M2 melanoma cells require FLNa for motility. We further show that R-Ras and FLNa interact in co-immunoprecipitations and pull-down assays. Deletion of FLNa repeat 3 (FLNaΔ3) abrogated this interaction. In M2 melanoma cells active R-Ras co-localized with FLNa but did not co-localize with FLNa lacking repeat 3. Thus, activated R-Ras binds repeat 3 of FLNa. The functional consequence of this interaction was that active R-Ras and FLNa coordinately increased cell migration. In contrast, co-expression of R-Ras and FLNaΔ3 had a significantly reduced effect on migration. While there was enhancement of integrin activation and fibronectin matrix assembly, cell adhesion was not altered. Finally, siRNA knockdown of endogenous R-Ras impaired FLNa-dependent fibronectin matrix assembly. Conclusions These data support a model in which R-Ras functionally associates with FLNa and thereby regulates integrin-dependent migration. Thus in melanoma cells R-Ras and FLNa may cooperatively promote metastasis by enhancing cell migration. PMID:20585650

Effect of HIV-1 Tat on the formation of the mitotic spindle by interaction with ribosomal protein S3.

PubMed

Kim, Jiyoung; Kim, Yeon-Soo

2018-06-06

Human immunodeficiency virus type 1 (HIV-1) Tat, an important regulator of viral transcription, interacts with diverse cellular proteins and promotes or inhibits cell proliferation. Here, we show that ribosomal protein S3 (RPS3) plays an important role in mitosis through an interaction with α-tubulin and that Tat binds to and inhibits the localization of RPS3 in the mitotic spindle during mitosis. RPS3 colocalized with α-tubulin around chromosomes in the mitotic spindle. Depletion of RPS3 promoted α-tubulin assembly, while overexpression of RPS3 impaired α-tubulin assembly. Depletion of RPS3 resulted in aberrant mitotic spindle formation, segregation failure, and defective abscission. Moreover, ectopic expression of RPS3 rescued the cell proliferation defect in RPS3-knockdown cells. HIV-1 Tat interacted with RPS3 through its basic domain and increased the level of RPS3 in the nucleus. Expression of Tat caused defects in mitotic spindle formation and chromosome assembly in mitosis. Moreover, the localization of RPS3 in the mitotic spindle was disrupted when HIV-1 Tat was expressed in HeLa and Jurkat cells. These results suggest that Tat inhibits cell proliferation via an interaction with RPS3 and thereby disrupts mitotic spindle formation during HIV-1 infection. These results might provide insight into the mechanism underlying lymphocyte pathogenesis during HIV-1 infection.

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

PubMed

Kerkhoff, Claus; Nacken, Wolfgang; Benedyk, Malgorzata; Dagher, Marie Claire; Sopalla, Claudia; Doussiere, Jacques

2005-03-01

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

Endothelial progenitor cells inhibit platelet function in a P-selectin-dependent manner.

PubMed

Abou-Saleh, Haissam; Hachem, Ahmed; Yacoub, Daniel; Gillis, Marc-Antoine; Merhi, Yahye

2015-05-07

The role of endothelial progenitor cells (EPCs) in vascular repair is related to their recruitment at the sites of injury and their interaction with different components of the circulatory system. We have previously shown that EPCs bind and inhibit platelet function and impair thrombus formation via prostacyclin secretion, but the role of EPC binding to platelet P-selectin in this process has not been fully characterized. In the present study, we assessed the impact of EPCs on thrombus formation and we addressed the implication of P-selectin in this process. EPCs were generated from human peripheral blood mononuclear cells cultured on fibronectin in conditioned media. The impact of EPCs on platelet aggregation and thrombus formation was investigated in P-selectin deficient (P-sel(-/-)) mice and their wild-type (WT) counterparts. EPCs significantly and dose-dependently impaired collagen-induced whole blood platelet aggregation in WT mice, whereas no effects were observed in P-sel(-/-) mice. Moreover, in a ferric chloride-induced arterial thrombosis model, infusion of EPCs significantly reduced thrombus formation in WT, but not in P-sel(-/-) mice. Furthermore, the relative mass of thrombi generated in EPC-treated P-sel(-/-) mice were significantly larger than those in EPC-treated WT mice, and the number of EPCs recruited within the thrombi and along the arterial wall was reduced in P-sel(-/-) mice as compared to WT mice. This study shows that EPCs impair platelet aggregation and reduce thrombus formation via a cellular mechanism involving binding to platelet P-selectin. These findings add new insights into the role of EPC-platelet interactions in the regulation of thrombotic events during vascular repair.

p110α Inhibition Overcomes Stromal Cell-Mediated Ibrutinib Resistance in Mantle Cell Lymphoma.

PubMed

Guan, Jiyu; Huang, Dan; Yakimchuk, Konstantin; Okret, Sam

2018-05-01

Acquired resistance to cancer drugs is common, also for modern targeted drugs like the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, a new drug approved for the treatment of the highly aggressive and relapsing mantle cell lymphoma (MCL). The tumor microenvironment often impacts negatively on drug response. Here, we demonstrate that stromal cells protect MCL cells from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated downregulation of PI3K/AKT signaling. Importantly, the stromal cell-mediated ibrutinib resistance was overcome in vitro by inhibiting AKT activity using the PI3K catalytic p110α subunit-specific inhibitor BYL719. This was seen both for MCL cell lines and primary MCL cells. Furthermore, inhibition of p110α activity by BYL719 potentiated the ability of ibrutinib to inhibit MCL tumor growth in vivo in a mouse xenograft model. The stromal cell-mediated ibrutinib resistance was found to be due to a direct interaction with MCL cells and involves the integrin VLA-4, as disrupting stromal cell-MCL cell interaction using a VLA-4 blocking antibody abrogated the ibrutinib resistance. This suggests that combined treatment with ibrutinib and a p110α inhibitor, alternatively by disrupting stromal cell-MCL cell interaction, may be a promising therapeutic strategy to overcome stromal cell-mediated ibrutinib resistance in MCL. Mol Cancer Ther; 17(5); 1090-100. ©2018 AACR . ©2018 American Association for Cancer Research.

Disease-Associated Mutant Ubiquitin Causes Proteasomal Impairment and Enhances the Toxicity of Protein Aggregates

PubMed Central

Tank, Elizabeth M. H.; True, Heather L.

2009-01-01

Protein homeostasis is critical for cellular survival and its dysregulation has been implicated in Alzheimer's disease (AD) and other neurodegenerative disorders. Despite the growing appreciation of the pathogenic mechanisms involved in familial forms of AD, much less is known about the sporadic cases. Aggregates found in both familial and sporadic AD often include proteins other than those typically associated with the disease. One such protein is a mutant form of ubiquitin, UBB+1, a frameshift product generated by molecular misreading of a wild-type ubiquitin gene. UBB+1 has been associated with multiple disorders. UBB+1 cannot function as a ubiquitin molecule, and it is itself a substrate for degradation by the ubiquitin/proteasome system (UPS). Accumulation of UBB+1 impairs the proteasome system and enhances toxic protein aggregation, ultimately resulting in cell death. Here, we describe a novel model system to investigate how UBB+1 impairs UPS function and whether it plays a causal role in protein aggregation. We expressed a protein analogous to UBB+1 in yeast (Ubext) and demonstrated that it caused UPS impairment. Blocking ubiquitination of Ubext or weakening its interactions with other ubiquitin-processing proteins reduced the UPS impairment. Expression of Ubext altered the conjugation of wild-type ubiquitin to a UPS substrate. The expression of Ubext markedly enhanced cellular susceptibility to toxic protein aggregates but, surprisingly, did not induce or alter nontoxic protein aggregates in yeast. Taken together, these results suggest that Ubext interacts with more than one protein to elicit impairment of the UPS and affect protein aggregate toxicity. Furthermore, we suggest a model whereby chronic UPS impairment could inflict deleterious consequences on proper protein aggregate sequestration. PMID:19214209

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells

PubMed Central

Liu, Hebin; Schneider, Helga; Recino, Asha; Richardson, Christine; Goldberg, Martin W.; Rudd, Christopher E.

2015-01-01

Summary While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells. PMID:26321253

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells.

PubMed

Liu, Hebin; Schneider, Helga; Recino, Asha; Richardson, Christine; Goldberg, Martin W; Rudd, Christopher E

2015-09-03

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Neuroendocrine immunomodulation network dysfunction in SAMP8 mice and PrP-hAβPPswe/PS1ΔE9 mice: potential mechanism underlying cognitive impairment

PubMed Central

Wang, Jian-hui; Cheng, Xiao-rui; Zhang, Xiao-rui; Wang, Tong-xing; Xu, Wen-jian; Li, Fei; Liu, Feng; Cheng, Jun-ping; Bo, Xiao-chen; Wang, Sheng-qi; Zhou, Wen-xia; Zhang, Yong-xiang

2016-01-01

Senescence-accelerated mouse prone 8 strain (SAMP8) and PrP-hAβPPswe/PS1ΔE9 (APP/PS1) mice are classic animal models of sporadic Alzheimer's disease and familial AD respectively. Our study showed that object recognition memory, spatial learning and memory, active and passive avoidance were deteriorated and neuroendocrine immunomodulation (NIM) network was imbalance in SAMP8 and APP/PS1 mice. SAMP8 and APP/PS1 mice had their own specific phenotype of cognition, neuroendocrine, immune and NIM molecular network. The endocrine hormone corticosterone, luteinizing hormone and follicle-stimulating hormone, chemotactic factor monocyte chemotactic protein-1, macrophage inflammatory protein-1β, regulated upon activation normal T cell expressed and secreted factor and eotaxin, pro-inflammatory factor interleukin-23, and the Th1 cell acting as cell immunity accounted for cognitive deficiencies in SAMP8 mice, while adrenocorticotropic hormone and gonadotropin-releasing hormone, colony stimulating factor granulocyte colony stimulating factor, and Th2 cell acting as humoral immunity in APP/PS1 mice. On the pathway level, chemokine signaling and T cell receptor signaling pathway played the key role in cognition impairments of two models, while cytokine-cytokine receptor interaction and natural killer cell mediated cytotoxicity were more important in cognitive deterioration of SAMP8 mice than APP/PS1 mice. This mechanisms of NIM network underlying cognitive impairment is significant for further understanding the pathogenesis of AD and can provide useful information for development of AD therapeutic drug. PMID:27049828

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

PubMed

Capra, Valérie; Mauri, Mario; Guzzi, Francesca; Busnelli, Marta; Accomazzo, Maria Rosa; Gaussem, Pascale; Nisar, Shaista P; Mundell, Stuart J; Parenti, Marco; Rovati, G Enrico

2017-01-15

Thromboxane A 2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Betulin derivatives impair Leishmania braziliensis viability and host-parasite interaction.

PubMed

Alcazar, Wilmer; López, Adrian Silva; Alakurtti, Sami; Tuononen, Maija-Liisa; Yli-Kauhaluoma, Jari; Ponte-Sucre, Alicia

2014-11-01

Leishmaniasis is a public health problem in tropical and subtropical areas of the world, including Venezuela. The incidence of treatment failure and the number of cases with Leishmania-HIV co-infection underscore the importance of developing alternative, economical and effective therapies against this disease. The work presented here analyzed whether terpenoids derived from betulin are active against New World Leishmania parasites. Initially we determined the concentration that inhibits the growth of these parasites by 50% or IC50, and subsequently evaluated the chemotactic effect of four compounds with leishmanicidal activity in the sub-micromolar and micromolar range. That is, we measured the migratory capacity of Leishmania (V.) braziliensis in the presence of increasing concentrations of compounds. Finally, we evaluated their cytotoxicity against the host cell and their effect on the infectivity of L. (V.) braziliensis. The results suggest that (1) compounds 14, 17, 18, 25 and 27 are active at concentrations lower than 10 μM; (2) compound 26 inhibits parasite growth with an IC50 lower than 1 μM; (3) compounds 18, 26 and 27 inhibit parasite migration at pico- to nanomolar concentrations, suggesting that they impair host-parasite interaction. None of the tested compounds was cytotoxic against J774.A1 macrophages thus indicating their potential as starting points to develop compounds that might affect parasite-host cell interaction, as well as being leishmanicidal. Copyright © 2014 Elsevier Ltd. All rights reserved.

The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear

PubMed Central

Jacob, Daria; Hunegnaw, Ruth; Sabyrzyanova, Tatyana A.; Pushkarsky, Tatiana; Chekhov, Vladimir O.; Adzhubei, Alexei A.; Kalebina, Tatyana S.; Bukrinsky, Michael

2014-01-01

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using coimmunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The 2226DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope. PMID:24406162

skNAC, a Smyd1-interacting transcription factor, is involved in cardiac development and skeletal muscle growth and regeneration.

PubMed

Park, Chong Yon; Pierce, Stephanie A; von Drehle, Morgan; Ivey, Kathryn N; Morgan, Jayson A; Blau, Helen M; Srivastava, Deepak

2010-11-30

Cardiac and skeletal muscle development and maintenance require complex interactions between DNA-binding proteins and chromatin remodeling factors. We previously reported that Smyd1, a muscle-restricted histone methyltransferase, is essential for cardiogenesis and functions with a network of cardiac regulatory proteins. Here we show that the muscle-specific transcription factor skNAC is the major binding partner for Smyd1 in the developing heart. Targeted deletion of skNAC in mice resulted in partial embryonic lethality by embryonic day 12.5, with ventricular hypoplasia and decreased cardiomyocyte proliferation that were similar but less severe than in Smyd1 mutants. Expression of Irx4, a ventricle-specific transcription factor down-regulated in hearts lacking Smyd1, also depended on the presence of skNAC. Viable skNAC(-/-) adult mice had reduced postnatal skeletal muscle growth and impaired regenerative capacity after cardiotoxin-induced injury. Satellite cells isolated from skNAC(-/-) mice had impaired survival compared with wild-type littermate satellite cells. Our results indicate that skNAC plays a critical role in ventricular cardiomyocyte expansion and regulates postnatal skeletal muscle growth and regeneration in mice.

Functional metabolic interactions of human neuron-astrocyte 3D in vitro networks

PubMed Central

Simão, Daniel; Terrasso, Ana P.; Teixeira, Ana P.; Brito, Catarina; Sonnewald, Ursula; Alves, Paula M.

2016-01-01

The generation of human neural tissue-like 3D structures holds great promise for disease modeling, drug discovery and regenerative medicine strategies. Promoting the establishment of complex cell-cell interactions, 3D culture systems enable the development of human cell-based models with increased physiological relevance, over monolayer cultures. Here, we demonstrate the establishment of neuronal and astrocytic metabolic signatures and shuttles in a human 3D neural cell model, namely the glutamine-glutamate-GABA shuttle. This was indicated by labeling of neuronal GABA following incubation with the glia-specific substrate [2-13C]acetate, which decreased by methionine sulfoximine-induced inhibition of the glial enzyme glutamine synthetase. Cell metabolic specialization was further demonstrated by higher pyruvate carboxylase-derived labeling in glutamine than in glutamate, indicating its activity in astrocytes and not in neurons. Exposure to the neurotoxin acrylamide resulted in intracellular accumulation of glutamate and decreased GABA synthesis. These results suggest an acrylamide-induced impairment of neuronal synaptic vesicle trafficking and imbalanced glutamine-glutamate-GABA cycle, due to loss of cell-cell contacts at synaptic sites. This work demonstrates, for the first time to our knowledge, that neural differentiation of human cells in a 3D setting recapitulates neuronal-astrocytic metabolic interactions, highlighting the relevance of these models for toxicology and better understanding the crosstalk between human neural cells. PMID:27619889

Functional metabolic interactions of human neuron-astrocyte 3D in vitro networks.

PubMed

Simão, Daniel; Terrasso, Ana P; Teixeira, Ana P; Brito, Catarina; Sonnewald, Ursula; Alves, Paula M

2016-09-13

The generation of human neural tissue-like 3D structures holds great promise for disease modeling, drug discovery and regenerative medicine strategies. Promoting the establishment of complex cell-cell interactions, 3D culture systems enable the development of human cell-based models with increased physiological relevance, over monolayer cultures. Here, we demonstrate the establishment of neuronal and astrocytic metabolic signatures and shuttles in a human 3D neural cell model, namely the glutamine-glutamate-GABA shuttle. This was indicated by labeling of neuronal GABA following incubation with the glia-specific substrate [2-(13)C]acetate, which decreased by methionine sulfoximine-induced inhibition of the glial enzyme glutamine synthetase. Cell metabolic specialization was further demonstrated by higher pyruvate carboxylase-derived labeling in glutamine than in glutamate, indicating its activity in astrocytes and not in neurons. Exposure to the neurotoxin acrylamide resulted in intracellular accumulation of glutamate and decreased GABA synthesis. These results suggest an acrylamide-induced impairment of neuronal synaptic vesicle trafficking and imbalanced glutamine-glutamate-GABA cycle, due to loss of cell-cell contacts at synaptic sites. This work demonstrates, for the first time to our knowledge, that neural differentiation of human cells in a 3D setting recapitulates neuronal-astrocytic metabolic interactions, highlighting the relevance of these models for toxicology and better understanding the crosstalk between human neural cells.

Taspine derivative 12k suppressed A549 cell migration through the Wnt/β-catenin and EphrinB2 signaling pathway.

PubMed

Dai, Bingling; Ma, Yujiao; Yang, Tianfeng; Wang, Wenjie; Zhang, Yanmin

2017-03-01

12k, a taspine derivative, has been demonstrated to have the potent anti-tumor activity in lung cancer and colorectal cancer. The study aims to further explore the underlying mechanisms of 12k on A549 cell migration in vitro. Our data demonstrated that 12k negatively regulated Wnt signaling pathway by suppressing the phosphorylation of LRP5/6, and inhibiting the expression and nuclear translocation of β-catenin. 12k was shown to downregulate MMP3 and MMP7 expression which regulated by β-catenin interacts with TCF/LEF in the nucleus, and effectively impaired the related migration protein expression of MMP2 and MMP9 in A549 cells. In addition, 12k repressed the EphrinB2 and its PDZ protein, impairing the VEGFR2 and VEGFR3 expression in A549 cells, as well as inhibited the downstream of VEGFR2 included PI3K/AKT/mTOR and ERK/MAPK signaling pathways. Taken together, our findings revealed that 12k suppressed migration of A549 cells through the Wnt/β-catenin signaling pathway and EphrinB2 related signaling pathway. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

HIV infection deregulates innate immunity to malaria despite combination antiretroviral therapy.

PubMed

Finney, Constance A M; Ayi, Kodjo; Wasmuth, James D; Sheth, Prameet M; Kaul, Rupert; Loutfy, Mona R; Kain, Kevin C; Serghides, Lena

2013-01-28

Malaria and HIV-1 adversely interact, with HIV-positive individuals suffering higher parasite burdens and worse clinical outcomes. However, the mechanisms underlying these disease interactions are unclear. We hypothesized that HIV coinfection impairs the innate immune response to malaria, and that combination antiretroviral therapy (cART) may restore this response. Our aim was to examine the innate inflammatory response of natural killer (NK), natural killer T (NKT), and γδ T-cells isolated from the peripheral blood of HIV-infected therapy-naive donors to malaria parasites, and determine the effect of cART on these responses. Freshly isolated peripheral blood mononuclear cells from 25 HIV-infected individuals pre-cART (month 0) and post-cART (months 3 and 6), and HIV-negative individuals at matched time-points, were cultured in the presence of Plasmodium falciparum parasitized erythrocytes. Supernatants and cells were collected to assess cytokine production and phenotypic changes. Compared to HIV-negative participants, NKT, NK, and γδ T-cell subsets from participants with chronic HIV infection showed marked differences, including decreased production of interferon γ (IFNγ) and tumor necrosis factor (TNF) in response to malaria parasites. IFNγ production was linked to interleukin-18 receptor (IL-18R) expression in all three cell types studied. Six months of cART provided partial cellular reconstitution but had no effect on IL-18R expression, or IFNγ and TNF production. These data suggest that HIV infection impairs the inflammatory response of innate effector cells to malaria, and that the response is not fully restored within 6 months of cART. This may contribute to higher parasite burdens and ineffective immune responses, and have implications for vaccination initiatives in coinfected individuals.

Dl-3-n-Butylphthalide Inhibits NLRP3 Inflammasome and Mitigates Alzheimer's-Like Pathology via Nrf2-TXNIP-TrX Axis.

PubMed

Wang, Chun-Yan; Xu, Ye; Wang, Xu; Guo, Chuang; Wang, Tao; Wang, Zhan-You

2018-04-25

Oxidative stress and neuroinflammation play important roles in the pathology of Alzheimer's disease (AD). Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of antioxidant thioredoxin, is suspected to be an important modulator of oxidative stress and inflammation. However, the underlying mechanism involved in the abnormal homeostasis of TXNIP-thioredoxin (TrX) in AD pathogenesis remains unclear. Using the Swedish mutant form of APP (APPswe)/PSEN1dE9 transgenic mouse (APP/PS1) and human-derived neuronal cells as model systems, we disclosed the impairment of the nuclear factor erythroid 2-related factor 2 (Nrf2)-TXNIP-TrX signaling in Alzheimer's-like pathology. We observed that the immune staining of TXNIP was increased in postmortem AD brain. The chronic accumulation of inflammatory mediator in neuronal cells facilitates interactions of TXNIP-nucleotide binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) and NLRP3-ASC, which increases β-amyloid (Aβ) secretion. The antioxidant Dl-3-n-butylphthalide (Dl-NBP) is commonly used for cerebral ischemia treatment. In our study, we elucidated for new mechanisms by which Dl-NBP enhanced TrX activity, suppressed TXNIP, and ameliorated neuronal apoptosis in the APP/PS1 mouse brains. In human glioblastoma A172 cells and neuroblastoma SH-SY5Y cells, we delineated the Dl-NBP-mediated signaling pathways by which Dl-NBP-dependent upregulation of Nrf2 mediated the reciprocal regulation of reducing proinflammatory cytokine and inhibiting Aβ production in the glial and neuronal cells overexpressing APPswe. Our data provide a novel insight into the molecular mechanism that impairments of Nrf2-TXNIP-TrX system may be involved in the imbalance of cellular redox homeostasis and inflammatory damage in the AD brain. Dl-NBP treatment could suppress TXNIP-NLRP3 interaction and inhibit NLRP3 inflammasome activation via upregulating Nrf2. These findings may provide an instrumental therapeutic approach for AD. Antioxid. Redox Signal. 00, 000-000.

Cell Phones, Tablets, and Other Mobile Technology for Users with Visual Impairments

MedlinePlus

... Visual Impairments Cell Phones, Tablets, and Other Mobile Technology for Users with Visual Impairments The Mobile Revolution ... 223 Likes) Cell Phones, Tablets, and Other Mobile Technology Touchscreen Smartphone Accessibility for People with Visual Impairments ...

Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation

NASA Astrophysics Data System (ADS)

Cheng, Jingdong; Yang, Huirong; Fang, Jian; Ma, Lixiang; Gong, Rui; Wang, Ping; Li, Ze; Xu, Yanhui

2015-05-01

DNMT1 is an important epigenetic regulator that plays a key role in the maintenance of DNA methylation. Here we determined the crystal structure of DNMT1 in complex with USP7 at 2.9 Å resolution. The interaction between the two proteins is primarily mediated by an acidic pocket in USP7 and Lysine residues within DNMT1's KG linker. This intermolecular interaction is required for USP7-mediated stabilization of DNMT1. Acetylation of the KG linker Lysine residues impair DNMT1-USP7 interaction and promote the degradation of DNMT1. Treatment with HDAC inhibitors results in an increase in acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation is observed in differentiated neuronal cells and pancreatic cancer cells. Our studies reveal that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provide a structural basis for the design of inhibitors, targeting the DNMT1-USP7 interaction surface for therapeutic applications.

Radiation and stress-induced apoptosis: A role for Fas/Fas ligand interactions

PubMed Central

Reap, Elizabeth A.; Roof, Kevin; Maynor, Kenrick; Borrero, Michelle; Booker, Jessica; Cohen, Philip L.

1997-01-01

The lpr gene encodes a defective form of Fas, a cell surface protein that mediates apoptosis. This defect blocks apoptotic deletion of autoreactive T and B cells, leading to lymphoproliferation and lupus-like autoantibody production. The effects of the lpr Fas mutation on other kinds of physiologically relevant apoptosis are largely undocumented. To assess whether some of the apoptosis known to occur after ionizing radiation might be mediated by Fas/Fas ligand (FasL) interactions, we quantitated in vitro apoptosis by flow cytometry measurement of DNA content in splenic T and B cells from irradiated 5- to 8-month-old B6/lpr mice. Total apoptosis of both lpr and control cells was substantial after treatment; however there was a significant difference between B6 (73%) and lpr (25%) lymphocyte apoptosis. Thy1, CD4, CD8, and IgM cells from lpr showed much lower levels of apoptosis than control cells after irradiation. Apoptosis induced by heat shock was also impaired in lpr. The finding that γ-irradiation increased Fas expression on B6 cells and that irradiation-induced apoptosis could be blocked with a Fas–Fc fusion protein further supported the possible involvement of Fas in this form of apoptosis. Fas/FasL interactions may thus play an important role in identifying and eliminating damaged cells after γ-irradiation and other forms of injury. PMID:9159145

LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

PubMed

Zhang, Hui; Sweezey, Neil B; Kaplan, Feige

2015-02-15

Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development. Copyright © 2015 the American Physiological Society.

The evolutionarily conserved interaction between LC3 and p62 selectively mediates autophagy-dependent degradation of mutant huntingtin.

PubMed

Tung, Ying-Tsen; Hsu, Wen-Ming; Lee, Hsinyu; Huang, Wei-Pang; Liao, Yung-Feng

2010-07-01

Mammalian p62/sequestosome-1 protein binds to both LC3, the mammalian homologue of yeast Atg8, and polyubiquitinated cargo proteins destined to undergo autophagy-mediated degradation. We previously identified a cargo receptor-binding domain in Atg8 that is essential for its interaction with the cargo receptor Atg19 in selective autophagic processes in yeast. We, thus, sought to determine whether this interaction is evolutionally conserved from yeast to mammals. Using an amino acid replacement approach, we demonstrate that cells expressing mutant LC3 (LC3-K30D, LC3-K51A, or LC3-L53A) all exhibit defective lipidation of LC3, a disrupted LC3-p62 interaction, and impaired autophagic degradation of p62, suggesting that the p62-binding site of LC3 is localized within an evolutionarily conserved domain. Importantly, whereas cells expressing these LC3 mutants exhibited similar overall autophagic activity comparable to that of cells expressing wild-type LC3, autophagy-mediated clearance of the aggregation-prone mutant Huntingtin was defective in the mutant-expressing cells. Together, these results suggest that p62 directly binds to the evolutionarily conserved cargo receptor-binding domain of Atg8/LC3 and selectively mediates the clearance of mutant Huntingtin.

The Molecular Chaperone HSPA2 Plays a Key Role in Regulating the Expression of Sperm Surface Receptors That Mediate Sperm-Egg Recognition

PubMed Central

Redgrove, Kate A.; Nixon, Brett; Baker, Mark A.; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R. John

2012-01-01

A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm–egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm–zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility. PMID:23209833

Spatial and Temporal Coordination of Bone Marrow-Derived Cell Activity During Arteriogenesis: Regulation of the Endogenous Response and Therapeutic Implications

PubMed Central

Meisner, Joshua K.; Price, Richard J.

2010-01-01

Arterial occlusive disease (AOD) is the leading cause of morbidity and mortality through the developed world, which creates a significant need for effective therapies to halt disease progression. Despite success of animal and small-scale human therapeutic arteriogenesis studies, this promising concept for treating AOD has yielded largely disappointing results in large-scale clinical trials. One reason for this lack of successful translation is that endogenous arteriogenesis is highly dependent on a poorly understood sequence of events and interactions between bone marrow derived cells (BMCs) and vascular cells, which makes designing effective therapies difficult. We contend that the process follows a complex, ordered sequence of events with multiple, specific BMC populations recruited at specific times and locations. Here we present the evidence suggesting roles for multiple BMC populations from neutrophils and mast cells to progenitor cells and propose how and where these cell populations fit within the sequence of events during arteriogenesis. Disruptions in these various BMC populations can impair the arteriogenesis process in patterns that characterize specific patient populations. We propose that an improved understanding of how arteriogenesis functions as a system can reveal individual BMC populations and functions that can be targeted for overcoming particular impairments in collateral vessel development. PMID:21044213

Expression of checkpoint molecules on myeloid-derived suppressor cells.

PubMed

Ballbach, Marlene; Dannert, Angelika; Singh, Anurag; Siegmund, Darina M; Handgretinger, Rupert; Piali, Luca; Rieber, Nikolaus; Hartl, Dominik

2017-12-01

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous cell population expanded in cancer, infection and autoimmunity capable of suppressing T-cell functions. Checkpoint inhibitors have emerged as a key therapeutic strategy in immune-oncology. While checkpoint molecules were initially associated with T cell functions, recent evidence suggests a broader expression and function in innate myeloid cells. Previous studies provided first evidence for a potential role for checkpoints on MDSCs, yet the human relevance remained poorly understood. Therefore, we investigated the expression and functional relevance of checkpoint molecules in human MDSC-T-cell interactions. Our studies demonstrate that programmed death-ligand 1 (PD-L1) is expressed on granulocytic MDSCs upon co-culture with T cells. Transwell experiments showed that cell-to-cell contact was required for MDSC-T-cell interactions and antibody blocking studies showed that targeting PD-L1 partially impaired MDSC-mediated T-cell suppression. Collectively, these studies suggest a role for PD-L1 in human MDSC function and thereby expand the functionality of this checkpoint beyond T cells, which could pave the way for further understanding and therapeutic targeting of PD-1/PD-L1 in innate immune-mediated diseases. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Cytokines, hepatic cell profiling and cell interactions during bone marrow cell therapy for liver fibrosis in cholestatic mice

PubMed Central

Pinheiro, Daphne; Leirós, Luana; Dáu, Juliana Barbosa Torreão; Stumbo, Ana Carolina; Thole, Alessandra Alves; Cortez, Erika Afonso Costa; Mandarim-de-Lacerda, Carlos Alberto; de Carvalho, Lais

2017-01-01

Bone marrow cells (BMC) migrate to the injured liver after transplantation, contributing to regeneration through multiple pathways, but mechanisms involved are unclear. This work aimed to study BMC migration, characterize cytokine profile, cell populations and proliferation in mice with liver fibrosis transplanted with GFP+ BMC. Confocal microscopy analysis showed GFP+ BMC near regions expressing HGF and SDF-1 in the fibrotic liver. Impaired liver cell proliferation in fibrotic groups was restored after BMC transplantation. Regarding total cell populations, there was a significant reduction in CD68+ cells and increased Ly6G+ cells in transplanted fibrotic group. BMC contributed to the total populations of CD144, CD11b and Ly6G cells in the fibrotic liver, related to an increment of anti-fibrotic cytokines (IL-10, IL-13, IFN-γ and HGF) and reduction of pro-inflammatory cytokines (IL-17A and IL-6). Therefore, HGF and SDF-1 may represent important chemoattractants for transplanted BMC in the injured liver, where these cells can give rise to populations of extrahepatic macrophages, neutrophils and endothelial progenitor cells that can interact synergistically with other liver cells towards the modulation of an anti-fibrotic cytokine profile promoting the onset of liver regeneration. PMID:29176797

Music application alleviates short-term memory impairments through increasing cell proliferation in the hippocampus of valproic acid-induced autistic rat pups.

PubMed

Lee, Sung-Min; Kim, Bo-Kyun; Kim, Tae-Woon; Ji, Eun-Sang; Choi, Hyun-Hee

2016-06-01

Autism is a neurodevelopmental disorder and this disorder shows impairment in reciprocal social interactions, deficits in communication, and restrictive and repetitive patterns of behaviors and interests. The effect of music on short-term memory in the view of cell proliferation in the hippocampus was evaluated using valproic acid-induced autistic rat pups. Animal model of autism was made by subcutaneous injection of 400-mg/kg valproic acid into the rat pups on the postnatal day 14. The rat pups in the music-applied groups were exposed to the 65-dB comfortable classic music for 1 hr once a day, starting postnatal day 15 and continued until postnatal day 28. In the present results, short-term memory was deteriorated by autism induction. The numbers of 5-bromo-2'-deoxyridine (BrdU)-positive, Ki-67-positive, and doublecortin (DCX)-positive cells in the hippocampal dentate gyrus were decreased by autism induction. Brain-derived neurotrophic factor (BDNF) and tyrosine kinase B (TrkB) expressions in the hippocampus were also suppressed in the autistic rat pups. Music application alleviated short-term memory deficits with enhancing the numbers of BrdU-positive, Ki-67-positive, and DCX-positive cells in the autistic rat pups. Music application also enhanced BDNF and TrkB expressions in the autistic rat pups. The present study show that application of music enhanced hippocampal cell proliferation and alleviated short-term memory impairment through stimulating BDNF-TrkB signaling in the autistic rat pups. Music can be suggested as the therapeutic strategy to overcome the autism-induced memory deficits.

Role of ACTH in the Interactive/Paracrine Regulation of Adrenal Steroid Secretion in Physiological and Pathophysiological Conditions

PubMed Central

Lefebvre, Hervé; Thomas, Michaël; Duparc, Céline; Bertherat, Jérôme; Louiset, Estelle

2016-01-01

In the normal human adrenal gland, steroid secretion is regulated by a complex network of autocrine/paracrine interactions involving bioactive signals released by endothelial cells, nerve terminals, chromaffin cells, immunocompetent cells, and adrenocortical cells themselves. ACTH can be locally produced by medullary chromaffin cells and is, therefore, a major mediator of the corticomedullary functional interplay. Plasma ACTH also triggers the release of angiogenic and vasoactive agents from adrenocortical cells and adrenal mast cells and, thus, indirectly regulates steroid production through modulation of the adrenal blood flow. Adrenocortical neoplasms associated with steroid hypersecretion exhibit molecular and cellular defects that tend to reinforce the influence of paracrine regulatory loops on corticosteroidogenesis. Especially, ACTH has been found to be abnormally synthesized in bilateral macronodular adrenal hyperplasia responsible for hypercortisolism. In these tissues, ACTH is detected in a subpopulation of adrenocortical cells that express gonadal markers. This observation suggests that ectopic production of ACTH may result from impaired embryogenesis leading to abnormal maturation of the adrenogonadal primordium. Globally, the current literature indicates that ACTH is a major player in the autocrine/paracrine processes occurring in the adrenal gland in both physiological and pathological conditions. PMID:27489549

Chronic ethanol consumption inhibits glucokinase transcriptional activity by Atf3 and triggers metabolic syndrome in vivo.

PubMed

Kim, Ji Yeon; Hwang, Joo-Yeon; Lee, Dae Yeon; Song, Eun Hyun; Park, Keon Jae; Kim, Gyu Hee; Jeong, Eun Ae; Lee, Yoo Jeong; Go, Min Jin; Kim, Dae Jin; Lee, Seong Su; Kim, Bong-Jo; Song, Jihyun; Roh, Gu Seob; Gao, Bin; Kim, Won-Ho

2014-09-26

Chronic ethanol consumption induces pancreatic β-cell dysfunction through glucokinase (Gck) nitration and down-regulation, leading to impaired glucose tolerance and insulin resistance, but the underlying mechanism remains largely unknown. Here, we demonstrate that Gck gene expression and promoter activity in pancreatic β-cells were suppressed by chronic ethanol exposure in vivo and in vitro, whereas expression of activating transcription factor 3 (Atf3) and its binding to the putative Atf/Creb site (from -287 to -158 bp) on the Gck promoter were up-regulated. Furthermore, in vitro ethanol-induced Atf3 inhibited the positive effect of Pdx-1 on Gck transcriptional regulation, enhanced recruitment of Hdac1/2 and histone H3 deacetylation, and subsequently augmented the interaction of Hdac1/Pdx-1 on the Gck promoter, which were diminished by Atf3 siRNA. In vivo Atf3-silencing reversed ethanol-mediated Gck down-regulation and β-cell dysfunction, followed by the amelioration of impaired glucose tolerance and insulin resistance. Together, we identified that ethanol-induced Atf3 fosters β-cell dysfunction via Gck down-regulation and that its loss ameliorates metabolic syndrome and could be a potential therapeutic target in treating type 2 diabetes. The Atf3 gene is associated with the induction of type 2 diabetes and alcohol consumption-induced metabolic impairment and thus may be the major negative regulator for glucose homeostasis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

PHD3-mediated prolyl hydroxylation of nonmuscle actin impairs polymerization and cell motility

PubMed Central

Luo, Weibo; Lin, Benjamin; Wang, Yingfei; Zhong, Jun; O'Meally, Robert; Cole, Robert N.; Pandey, Akhilesh; Levchenko, Andre; Semenza, Gregg L.

2014-01-01

Actin filaments play an essential role in cell movement, and many posttranslational modifications regulate actin filament assembly. Here we report that prolyl hydroxylase 3 (PHD3) interacts with nonmuscle actin in human cells and catalyzes hydroxylation of actin at proline residues 307 and 322. Blocking PHD3 expression or catalytic activity by short hairpin RNA knockdown or pharmacological inhibition, respectively, decreased actin prolyl hydroxylation. PHD3 knockdown increased filamentous F-actin assembly, which was reversed by PHD3 overexpression. PHD3 knockdown increased cell velocity and migration distance. Inhibition of PHD3 prolyl hydroxylase activity by dimethyloxalylglycine also increased actin polymerization and cell migration. These data reveal a novel role for PHD3 as a negative regulator of cell motility through posttranslational modification of nonmuscle actins. PMID:25079693

Arsenic impairs insulin signaling in differentiated neuroblastoma SH-SY5Y cells.

PubMed

Niyomchan, Apichaya; Visitnonthachai, Daranee; Suntararuks, Sumitra; Ngamsiri, Pronrumpa; Watcharasit, Piyajit; Satayavivad, Jutamaad

2018-05-01

A strong correlation between chronic arsenic exposure and neuropsychological disorders leads to a growing concern about a potential risk of arsenic related neurodegeneration. Evidently, brain insulin signaling contributes to physiological effects, including energy homeostasis, and learning and memory. Arsenic has been shown to impair insulin signaling in adipocytes and myocytes, however, this impairment has not yet been explored in neurons. Here we showed that NaAsO 2 caused significant reduction in basal levels of glucose, plasma membrane glucose transporter, GLUT 3 and Akt phosphorylation in differentiated human neuroblastoma SH-SY5Y cells. NaAsO 2 significantly decreased insulin-mediated glucose uptake, as well as GLUT1 and 3 membrane translocation. Furthermore, the ability of insulin to increase Akt phosphorylation, a well-recognized insulin signaling response, was significantly lessened by NaAsO 2 treatment. In addition, the classical tyrosine phosphorylation response of insulin was reduced by NaAsO 2 , as evidenced by reduction of insulin-induced tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1(IRS-1). Moreover, NaAsO 2 lowered the ratio of p110, a catalytic subunit to p85, a regulatory subunit of PI3K causing an imbalance between p110 and p85, the conditions reported to contribute to insulin sensitivity. Additionally, increment of IRS-1 interaction with GSK3β, and p85-PI3K were observed in NaAsO 2 treated cells. These molecular modulations may be mechanistically attributed to neuronal insulin signaling impairment by arsenic. Copyright © 2018 Elsevier B.V. All rights reserved.

Divisome-dependent subcellular localization of cell-cell joining protein SepJ in the filamentous cyanobacterium Anabaena.

PubMed

Ramos-León, Félix; Mariscal, Vicente; Frías, José E; Flores, Enrique; Herrero, Antonia

2015-05-01

Heterocyst-forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA-dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two-hybrid system. We found SepJ self-interaction and a specific interaction with FtsQ, confirmed by co-purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity. © 2015 John Wiley & Sons Ltd.

Septin dynamics are essential for exocytosis.

PubMed

Tokhtaeva, Elmira; Capri, Joe; Marcus, Elizabeth A; Whitelegge, Julian P; Khuzakhmetova, Venera; Bukharaeva, Ellya; Deiss-Yehiely, Nimrod; Dada, Laura A; Sachs, George; Fernandez-Salas, Ester; Vagin, Olga

2015-02-27

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cancer exosomes and NKG2D receptor-ligand interactions: impairing NKG2D-mediated cytotoxicity and anti-tumour immune surveillance.

PubMed

Mincheva-Nilsson, Lucia; Baranov, Vladimir

2014-10-01

Human cancers constitutively produce and release endosome-derived nanometer-sized vesicles called exosomes that carry biologically active proteins, messenger and micro RNAs and serve as vehicles of intercellular communication. The tumour exosomes are present in the blood, urine and various malignant effusions such as peritoneal and pleural fluid of cancer patients and can modulate immune cells and responses thus deranging the immune system of cancer patients and giving advantage to the cancer to establish and spread itself. Here, the role of exosomes in the NKG2D receptor-ligand system's interactions is discussed. The activating NK cell receptor NKG2D and its multiple ligands, the MHC class I-related chain (MIC) A/B and the retinoic acid transcript-1/UL-16 binding proteins (RAET1/ULBP) 1-6 comprise a powerful stress-inducible danger detector system that targets infected, inflamed and malignantly transformed cells and plays a decisive role in anti-tumour immune surveillance. Mounting evidence reveals that the MIC- and RAET1/ULBP ligand family members are enriched in the endosomal compartment of various tumour cells and expressed and released into the intercellular space and bodily fluids on exosomes thus preserving their entire molecule, three-dimensional protein structure and biologic activity. The NKG2D ligand-expressing exosomes serve as decoys with a powerful ability to down regulate the cognate receptor and impair the cytotoxic function of NK-, NKT-, gamma/delta- and cytotoxic T cells. This review summarizes recent findings concerning the role of NKG2D receptor-ligand system in cancer with emphasis on regulation of NKG2D ligand expression and the immunosuppressive role of exosomally expressed NKG2D ligands. Copyright © 2014 Elsevier Ltd. All rights reserved.

β‐Taxilin participates in differentiation of C2C12 myoblasts into myotubes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakane, Hiroshi; Makiyama, Tomohiko; Nogami, Satoru

Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that β-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of β-taxilin in myogenesis. In C2C12 cells, knockdown of β-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also foundmore » that β-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of β-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that β-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes. - Highlights: • β‐Taxilin is progressively expressed during differentiation of C2C12 cell. • Knockdown of β-taxilin impaired C2C12 myotube formation. • β‐Taxilin interacted with dysbindin. • Knockdown of dysbindin promoted C2C12 myotube formation. • The function of β-taxilin in C2C12 myotube formation depends on dysbindin.« less

Discordance in the effects of Yersinia pestis on the dendritic cell functions manifested by induction of maturation and paralysis of migration.

PubMed

Velan, Baruch; Bar-Haim, Erez; Zauberman, Ayelet; Mamroud, Emanuelle; Shafferman, Avigdor; Cohen, Sara

2006-11-01

The encounter between invading microorganisms and dendritic cells (DC) triggers a series of events which include uptake and degradation of the microorganism, induction of a maturation process, and enhancement of DC migration to the draining lymph nodes. Various pathogens have developed strategies to counteract these events as a measure to evade the host defense. In the present study we found that interaction of the Yersinia pestis EV76 strain with DC has no effect on cell viability and is characterized by compliance with effective maturation, which is manifested by surface display of major histocompatibility complex class II, of costimulatory markers, and of the chemokine receptor CCR7. This is in contrast to maturation inhibition and cell death induction exerted by the related species Yersinia enterocolitica WA O:8. Y. pestis interactions with DC were found, however, to impair functions related to cytoskeleton rearrangement. DC pulsed with Y. pestis failed to adhere to solid surfaces and to migrate toward the chemokine CCL19 in an in vitro transmembrane assay. Both effects were dependent on the presence of the pCD1 virulence plasmid and on a bacterial growth shift to 37 degrees C prior to infection. Moreover, while instillation of a pCD1-cured Y. pestis strain into mouse airways triggered effective transport of alveolar DC to the mediastinal lymph node, instillation of Y. pestis harboring the plasmid failed to do so. Taken together, these results suggest that virulence plasmid-dependent impairment of DC migration is the major mechanism utilized by Y. pestis to subvert DC function.

Role of Acinetobactin-Mediated Iron Acquisition Functions in the Interaction of Acinetobacter baumannii Strain ATCC 19606T with Human Lung Epithelial Cells, Galleria mellonella Caterpillars, and Mice

PubMed Central

Gaddy, Jennifer A.; Arivett, Brock A.; McConnell, Michael J.; López-Rojas, Rafael; Pachón, Jerónimo

2012-01-01

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acquisition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial interaction of the ATCC 19606T type strain with A549 human alveolar epithelial cells is independent of the production of BasD and BauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for this strain to persist within epithelial cells and cause their apoptotic death. Infection assays using Galleria mellonella larvae showed that impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606T cells to persist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with these ex vivo and in vivo approaches were validated using a mouse sepsis model, which showed that expression of the acinetobactin-mediated iron acquisition system is critical for ATCC 19606T to establish an infection and kill this vertebrate host. These observations demonstrate that the virulence of the ATCC 19606T strain depends on the expression of a fully active acinetobactin-mediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediate virulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactin intermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of mature acinetobactin. PMID:22232188

Cellular interactions with tissue-engineered microenvironments and nanoparticles

NASA Astrophysics Data System (ADS)

Pan, Zhi

Tissue-engineered hydrogels composed of intermolecularlly crosslinked hyaluronan (HA-DTPH) and fibronectin functional domains (FNfds) were applied as a physiological relevant ECM mimic with controlled mechanical and biochemical properties. Cellular interactions with this tissue-engineered environment, especially physical interactions (cellular traction forces), were quantitatively measured by using the digital image speckle correlation (DISC) technique and finite element method (FEM). By correlating with other cell functions such as cell morphology and migration, a comprehensive structure-function relationship between cells and their environments was identified. Furthermore, spatiotemporal redistribution of cellular traction stresses was time-lapse measured during cell migration to better understand the dynamics of cell mobility. The results suggest that the reinforcement of the traction stresses around the nucleus, as well as the relaxation of nuclear deformation, are critical steps during cell migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. Besides single cell migration, en masse cell migration was studied by using agarose droplet migration assay. Cell density was demonstrated to be another important parameter to influence cell behaviors besides substrate properties. Findings from these studies will provide fundamental design criteria to develop novel and effective tissue-engineered constructs. Cellular interactions with rutile and anatase TiO2 nanoparticles were also studied. These particles can penetrate easily through the cell membrane and impair cell function, with the latter being more damaging. The exposure to nanoparticles was found to decrease cell area, cell proliferation, motility, and contractility. To prevent this, a dense grafted polymer brush coating was applied onto the nanoparticle surface. These modified nanoparticles failed to adhere to and penetrate through the cell membrane. As a consequence, the coating effectively decreased reactive oxygen species (ROS) formation and protected the cells. Considering the broad applications of these nanoparticles in personal health care products, the functionalized polymer coating will likely play an important role in protecting cells and tissue from damage.

Mediation of glucolipotoxicity in INS-1 rat insulinoma cells by small heterodimer partner interacting leucine zipper protein (SMILE).

PubMed

Lee, Kyeong-Min; Seo, Ye Jin; Kim, Mi-Kyung; Seo, Hyun-Ae; Jeong, Ji-Yun; Choi, Hueng-Sik; Lee, In-Kyu; Park, Keun-Gyu

2012-03-23

Sustained elevations of glucose and free fatty acid concentration have deleterious effects on pancreatic beta cell function. One of the hallmarks of such glucolipotoxicity is a reduction in insulin gene expression, resulting from decreased insulin promoter activity. Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, is related to the development of beta cell dysfunction caused by elevated concentrations of glucose and free fatty acid. Small heterodimer partner (SHP) interacting leucine zipper protein (SMILE), also known as Zhangfei, is a novel protein which interacts with SHP that mediates glucotoxicity in INS-1 rat insulinoma cells. Treatment of INS-1 cells with high concentrations of glucose and palmitate increased SREBP-1c and SMILE expression, and decreased insulin gene expression. Adenovirus-mediated overexpression of SREBP-1c in INS-1 cells induced SMILE expression. Moreover, adenovirus-mediated overexpression of SMILE (Ad-SMILE) in INS-1 cells impaired glucose-stimulated insulin secretion as well as insulin gene expression. Ad-SMILE overexpression also inhibited the expression of beta-cell enriched transcription factors including pancreatic duodenal homeobox factor-1, beta cell E box transactivator 2 and RIPE3b1/MafA, in INS-1 cells. Finally, in COS-1 cells, expression of SMILE inhibited the insulin promoter activity induced by these same beta-cell enriched transcription factors. These results collectively suggest that SMILE plays an important role in the development of beta cell dysfunction induced by glucolipotoxicity. Copyright © 2012 Elsevier Inc. All rights reserved.

Matrix metalloproteinase 3 promotes cellular anti-dengue virus response via interaction with transcription factor NFκB in cell nucleus.

PubMed

Zuo, Xiangyang; Pan, Wen; Feng, Tingting; Shi, Xiaohong; Dai, Jianfeng

2014-01-01

Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation.

Hepatitis B Virus X Protein Impairs Hepatic Insulin Signaling Through Degradation of IRS1 and Induction of SOCS3

PubMed Central

Kim, KyeongJin; Kim, Kook Hwan; Cheong, JaeHun

2010-01-01

Background Hepatitis B virus (HBV) is a major cause of chronic liver diseases, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. The role of HCV in associations with insulin signaling has been elucidated. However, the pathogenesis of HBV-associated insulin signaling remains to be clearly characterized. Therefore, we have attempted to determine the mechanisms underlying the HBV-associated impairment of insulin signaling. Methodology The expressions of insulin signaling components were investigated in HBx-transgenic mice, HBx-constitutive expressing cells, and transiently HBx-transfected cells. Protein and gene expression was examined by Western blot, immunohistochemistry, RT-PCR, and promoter assay. Protein-protein interaction was detected by coimmunoprecipitation. Principal Findings HBx induced a reduction in the expression of IRS1, and a potent proteasomal inhibitor blocked the downregulation of IRS1. Additionally, HBx enhanced the expression of SOCS3 and induced IRS1 ubiquitination. Also, C/EBPα and STAT3 were involved in the HBx-induced expression of SOCS3. HBx interfered with insulin signaling activation and recovered the insulin-mediated downregulation of gluconeogenic genes. Conclusions/Significance These results provide direct experimental evidences for the contribution of HBx in the impairment of insulin signaling. PMID:20351777

Acidosis-Induced Dysfunction of Cortical GABAergic Neurons through Astrocyte-Related Excitotoxicity

PubMed Central

Guan, Sudong; Zhu, Yan; Wang, Jin-Hui

2015-01-01

Background Acidosis impairs cognitions and behaviors presumably by acidification-induced changes in neuronal metabolism. Cortical GABAergic neurons are vulnerable to pathological factors and their injury leads to brain dysfunction. How acidosis induces GABAergic neuron injury remains elusive. As the glia cells and neurons interact each other, we intend to examine the role of the astrocytes in acidosis-induced GABAergic neuron injury. Results Experiments were done at GABAergic cells and astrocytes in mouse cortical slices. To identify astrocytic involvement in acidosis-induced impairment, we induced the acidification in single GABAergic neuron by infusing proton intracellularly or in both neurons and astrocytes by using proton extracellularly. Compared the effects of intracellular acidification and extracellular acidification on GABAergic neurons, we found that their active intrinsic properties and synaptic outputs appeared more severely impaired in extracellular acidosis than intracellular acidosis. Meanwhile, extracellular acidosis deteriorated glutamate transporter currents on the astrocytes and upregulated excitatory synaptic transmission on the GABAergic neurons. Moreover, the antagonists of glutamate NMDA-/AMPA-receptors partially reverse extracellular acidosis-induced injury in the GABAergic neurons. Conclusion Our studies suggest that acidosis leads to the dysfunction of cortical GABAergic neurons by astrocyte-mediated excitotoxicity, in addition to their metabolic changes as indicated previously. PMID:26474076

Defects in chromosome congression and mitotic progression in KIF18A-deficient cells are partly mediated through impaired functions of CENP-E.

PubMed

Huang, Ying; Yao, Yixin; Xu, Han-Zhang; Wang, Zhu-Gang; Lu, Luo; Dai, Wei

2009-08-15

KIF18A, a molecular motor, is an essential component in the regulation of orderly chromosome congression by attenuation of the kinetochore oscillation amplitude at the midzone during mitosis in vertebrate cells. Here we report that KIF18A depletion resulted in mitotic arrest which was accompanied by the presence of unaligned chromosomes in HeLa cells. This resembles the phenotype induced by an impaired function of CENP-E, also a mitotic kinesin essential for the formation of the mitotic spindles. Our further analysis showed that KIF18A depletion caused specific downregulation of CENP-E. Downregulation of CENP-E as the result of KIF18A silencing was not due to reduced transcription but primarily due to the enhanced protein degradation. Co-immunoprecipitation revealed that KIF18A physically interacted with CENP-E and BubR1 during mitosis. Ectopic expression of the wild-type tail domain of CENP-E, but not a corresponding mutant, significantly suppressed chromosome congression defects in mitotic cells. Together, our studies strongly suggest that chromosome congression defects as the result of KIF18A depletion is at least in part mediated through destabilizing kinetochore CENP-E.

Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy.

PubMed

Picaud, Sarah; Fedorov, Oleg; Thanasopoulou, Angeliki; Leonards, Katharina; Jones, Katherine; Meier, Julia; Olzscha, Heidi; Monteiro, Octovia; Martin, Sarah; Philpott, Martin; Tumber, Anthony; Filippakopoulos, Panagis; Yapp, Clarence; Wells, Christopher; Che, Ka Hing; Bannister, Andrew; Robson, Samuel; Kumar, Umesh; Parr, Nigel; Lee, Kevin; Lugo, Dave; Jeffrey, Philip; Taylor, Simon; Vecellio, Matteo L; Bountra, Chas; Brennan, Paul E; O'Mahony, Alison; Velichko, Sharlene; Müller, Susanne; Hay, Duncan; Daniels, Danette L; Urh, Marjeta; La Thangue, Nicholas B; Kouzarides, Tony; Prinjha, Rab; Schwaller, Jürg; Knapp, Stefan

2015-12-01

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers. ©2015 American Association for Cancer Research.

Generation of a selective small molecule inhibitor of the CBP/p300 bromodomain for leukemia therapy

PubMed Central

Jones, Katherine; Meier, Julia; Olzscha, Heidi; Monteiro, Octovia; Martin, Sarah; Philpott, Martin; Tumber, Anthony; Filippakopoulos, Panagis; Yapp, Clarence; Wells, Christopher; Che, Ka Hing; Bannister, Andrew; Robson, Samuel; Kumar, Umesh; Parr, Nigel; Lee, Kevin; Lugo, Dave; Jeffrey, Philip; Taylor, Simon; Vecellio, Matteo L.; Bountra, Chas; Brennan, Paul E.; O’Mahony, Alison; Velichko, Sharlene; Müller, Susanne; Hay, Duncan; Daniels, Danette L.; Urh, Marjeta; La Thangue, Nicholas B.; Kouzarides, Tony; Prinjha, Rab; Schwaller, Jürg; Knapp, Stefan

2016-01-01

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9+ AML cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers. PMID:26552700

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome.

PubMed

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Pan, Zhao-Yi; Wang, Su-Yun; Wang, Yan-Yi

2017-04-01

Upon viral infection, retinoic acid-inducible gene I-like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses.

GPATCH3 negatively regulates RLR-mediated innate antiviral responses by disrupting the assembly of VISA signalosome

PubMed Central

Nie, Ying; Ran, Yong; Zhang, Hong-Yan; Huang, Zhe-Fu; Wang, Su-Yun

2017-01-01

Upon viral infection, retinoic acid–inducible gene I–like receptors (RLRs) recognize viral RNA and trigger a series of signaling events, leading to the induction of type I interferons (IFNs). These processes are delicately regulated to prevent excessive and harmful immune responses. In this study, we identified G patch domain-containing protein 3 (GPATCH3) as a negative regulator of RLR-mediated antiviral signaling pathways. Overexpression of GPATCH3 impaired RNA virus- triggered induction of downstream antiviral genes, whereas its knockdown had opposite effects and attenuated viral replication. In addition, GPATCH3-deficient cells had higher IFNB1 mRNA level compared with control cells after RNA virus infection. Mechanistically, GPATCH3 was recruited to VISA in a viral infection dependent manner and the assembly of VISA/TRAF6/TBK1 signalosome was impaired in GPATCH3-overexpressing cells. In contrast, upon viral infection, the recruitment of TRAF6 and TBK1 to VISA was enhanced in GPATCH3 deficient cells. Taking together, our findings demonstrate that GPATCH3 interacts with VISA and disrupts the assembly of virus-induced VISA signalosome therefore acts as a negative regulator of RLR-mediated innate antiviral immune responses. PMID:28414768

G9a coordinates with the RPA complex to promote DNA damage repair and cell survival.

PubMed

Yang, Qiaoyan; Zhu, Qian; Lu, Xiaopeng; Du, Yipeng; Cao, Linlin; Shen, Changchun; Hou, Tianyun; Li, Meiting; Li, Zhiming; Liu, Chaohua; Wu, Di; Xu, Xingzhi; Wang, Lina; Wang, Haiying; Zhao, Ying; Yang, Yang; Zhu, Wei-Guo

2017-07-25

Histone methyltransferase G9a has critical roles in promoting cancer-cell growth and gene suppression, but whether it is also associated with the DNA damage response is rarely studied. Here, we report that loss of G9a impairs DNA damage repair and enhances the sensitivity of cancer cells to radiation and chemotherapeutics. In response to DNA double-strand breaks (DSBs), G9a is phosphorylated at serine 211 by casein kinase 2 (CK2) and recruited to chromatin. The chromatin-enriched G9a can then directly interact with replication protein A (RPA) and promote loading of the RPA and Rad51 recombinase to DSBs. This mechanism facilitates homologous recombination (HR) and cell survival. We confirmed the interaction between RPA and G9a to be critical for RPA foci formation and HR upon DNA damage. Collectively, our findings demonstrate a regulatory pathway based on CK2-G9a-RPA that permits HR in cancer cells and provide further rationale for the use of G9a inhibitors as a cancer therapeutic.

The Sla1 adaptor-clathrin interaction regulates coat formation and progression of endocytosis.

PubMed

Tolsma, Thomas O; Cuevas, Lena M; Di Pietro, Santiago M

2018-06-01

Clathrin-mediated endocytosis is a fundamental transport pathway that depends on numerous protein-protein interactions. Testing the importance of the adaptor protein-clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin-binding motif (sla1 AAA ) that disrupt clathrin binding. Live-cell imaging showed an impaired Sla1-clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1 AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3-dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1 AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1-clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

How to Hit Mesenchymal Stromal Cells and Make the Tumor Microenvironment Immunostimulant Rather Than Immunosuppressive

PubMed Central

Poggi, Alessandro; Varesano, Serena; Zocchi, Maria Raffaella

2018-01-01

Experimental evidence indicates that mesenchymal stromal cells (MSCs) may regulate tumor microenvironment (TME). It is conceivable that the interaction with MSC can influence neoplastic cell functional behavior, remodeling TME and generating a tumor cell niche that supports tissue neovascularization, tumor invasion and metastasization. In addition, MSC can release transforming growth factor-beta that is involved in the epithelial–mesenchymal transition of carcinoma cells; this transition is essential to give rise to aggressive tumor cells and favor cancer progression. Also, MSC can both affect the anti-tumor immune response and limit drug availability surrounding tumor cells, thus creating a sort of barrier. This mechanism, in principle, should limit tumor expansion but, on the contrary, often leads to the impairment of the immune system-mediated recognition of tumor cells. Furthermore, the cross-talk between MSC and anti-tumor lymphocytes of the innate and adaptive arms of the immune system strongly drives TME to become immunosuppressive. Indeed, MSC can trigger the generation of several types of regulatory cells which block immune response and eventually impair the elimination of tumor cells. Based on these considerations, it should be possible to favor the anti-tumor immune response acting on TME. First, we will review the molecular mechanisms involved in MSC-mediated regulation of immune response. Second, we will focus on the experimental data supporting that it is possible to convert TME from immunosuppressive to immunostimulant, specifically targeting MSC. PMID:29515580

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis.

PubMed

Chen, W; Wang, Q; Bai, L; Chen, W; Wang, X; Tellez, C S; Leng, S; Padilla, M T; Nyunoya, T; Belinsky, S A; Lin, Y

2014-07-01

Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.

Involvement of cyclophilin B in the replication of Japanese encephalitis virus.

PubMed

Kambara, Hiroto; Tani, Hideki; Mori, Yoshio; Abe, Takayuki; Katoh, Hiroshi; Fukuhara, Takasuke; Taguwa, Shuhei; Moriishi, Kohji; Matsuura, Yoshiharu

2011-03-30

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus that belongs to the Flaviviridae family. In this study, we have examined the effect of cyclosporin A (CsA) on the propagation of JEV. CsA exhibited potent anti-JEV activity in various mammalian cell lines through the inhibition of CypB. The propagation of JEV was impaired in the CypB-knockdown cells and this reduction was cancelled by the expression of wild-type but not of peptidylprolyl cis-trans isomerase (PPIase)-deficient CypB, indicating that PPIase activity of CypB is critical for JEV propagation. Infection of pseudotype viruses bearing JEV envelope proteins was not impaired by the knockdown of CypB, suggesting that CypB participates in the replication but not in the entry of JEV. CypB was colocalized and immunoprecipitated with JEV NS4A in infected cells. These results suggest that CypB plays a crucial role in the replication of JEV through an interaction with NS4A. Copyright © 2011 Elsevier Inc. All rights reserved.

Mind bomb-1 is an essential modulator of long-term memory and synaptic plasticity via the Notch signaling pathway

PubMed Central

2012-01-01

Background Notch signaling is well recognized as a key regulator of the neuronal fate during embryonic development, but its function in the adult brain is still largely unknown. Mind bomb-1 (Mib1) is an essential positive regulator in the Notch pathway, acting non-autonomously in the signal-sending cells. Therefore, genetic ablation of Mib1 in mature neuron would give valuable insight to understand the cell-to-cell interaction between neurons via Notch signaling for their proper function. Results Here we show that the inactivation of Mib1 in mature neurons in forebrain results in impaired hippocampal dependent spatial memory and contextual fear memory. Consistently, hippocampal slices from Mib1-deficient mice show impaired late-phase, but not early-phase, long-term potentiation and long-term depression without change in basal synaptic transmission at SC-CA1 synapses. Conclusions These data suggest that Mib1-mediated Notch signaling is essential for long-lasting synaptic plasticity and memory formation in the rodent hippocampus. PMID:23111145

Endothelin-1–Rho kinase interactions impair lung structure and cause pulmonary hypertension after bleomycin exposure in neonatal rat pups

PubMed Central

Tseng, Nancy; Seedorf, Gregory; Kuhn, Katherine; Abman, Steven H.

2016-01-01

Bronchopulmonary dysplasia (BPD) is the chronic lung disease associated with premature birth, characterized by impaired vascular and alveolar growth. In neonatal rats bleomycin decreases lung growth and causes pulmonary hypertension (PH), which is poorly responsive to nitric oxide. In the developing lung, through Rho kinase (ROCK) activation, ET-1 impairs endothelial cell function; however, whether ET-1–ROCK interactions contribute to impaired vascular and alveolar growth in experimental BPD is unknown. Neonatal rats were treated daily with intraperitoneal bleomycin with and without selective ETA (BQ123/BQ610) and ETB (BQ788) receptor blockers, nonselective ET receptor blocker (ETRB) (bosentan), or fasudil (ROCK inhibitor). At day 14, lungs were harvested for morphometrics, and measurements of Fulton's index (RV/LV+S), medial wall thickness (MWT), and vessel density. Lung ET-1 protein and ROCK activity (phospho-MYPT-1:total MYPT-1 ratio) were also measured by Western blot analysis. Bleomycin increased lung ET-1 protein expression by 65%, RV/LV+S by 60%, mean linear intercept (MLI) by 212%, and MWT by 140% and decreased radial alveolar count (RAC) and vessel density by 40 and 44%, respectively (P < 0.01 for each comparison). After bleomycin treatment, fasudil and bosentan partially restored RAC and vessel density and decreased MLI, RV/LV+S, and MWT to normal values. Bleomycin increased ROCK activity by 120%, which was restored to normal values by bosentan but not selective ETRB. We conclude that ET-1–ROCK interactions contribute to decreased alveolar and vascular growth and PH in experimental BPD. We speculate that nonselective ETRB and ROCK inhibitors may be effective in the treatment of infants with BPD and PH. PMID:27760762

Endothelin-1-Rho kinase interactions impair lung structure and cause pulmonary hypertension after bleomycin exposure in neonatal rat pups.

PubMed

Gien, Jason; Tseng, Nancy; Seedorf, Gregory; Kuhn, Katherine; Abman, Steven H

2016-12-01

Bronchopulmonary dysplasia (BPD) is the chronic lung disease associated with premature birth, characterized by impaired vascular and alveolar growth. In neonatal rats bleomycin decreases lung growth and causes pulmonary hypertension (PH), which is poorly responsive to nitric oxide. In the developing lung, through Rho kinase (ROCK) activation, ET-1 impairs endothelial cell function; however, whether ET-1-ROCK interactions contribute to impaired vascular and alveolar growth in experimental BPD is unknown. Neonatal rats were treated daily with intraperitoneal bleomycin with and without selective ET A (BQ123/BQ610) and ET B (BQ788) receptor blockers, nonselective ET receptor blocker (ETRB) (bosentan), or fasudil (ROCK inhibitor). At day 14, lungs were harvested for morphometrics, and measurements of Fulton's index (RV/LV+S), medial wall thickness (MWT), and vessel density. Lung ET-1 protein and ROCK activity (phospho-MYPT-1:total MYPT-1 ratio) were also measured by Western blot analysis. Bleomycin increased lung ET-1 protein expression by 65%, RV/LV+S by 60%, mean linear intercept (MLI) by 212%, and MWT by 140% and decreased radial alveolar count (RAC) and vessel density by 40 and 44%, respectively (P < 0.01 for each comparison). After bleomycin treatment, fasudil and bosentan partially restored RAC and vessel density and decreased MLI, RV/LV+S, and MWT to normal values. Bleomycin increased ROCK activity by 120%, which was restored to normal values by bosentan but not selective ETRB. We conclude that ET-1-ROCK interactions contribute to decreased alveolar and vascular growth and PH in experimental BPD. We speculate that nonselective ETRB and ROCK inhibitors may be effective in the treatment of infants with BPD and PH. Copyright © 2016 the American Physiological Society.

Significant Deregulated Pathways in Diabetes Type II Complications Identified through Expression Based Network Biology

NASA Astrophysics Data System (ADS)

Ukil, Sanchaita; Sinha, Meenakshee; Varshney, Lavneesh; Agrawal, Shipra

Type 2 Diabetes is a complex multifactorial disease, which alters several signaling cascades giving rise to serious complications. It is one of the major risk factors for cardiovascular diseases. The present research work describes an integrated functional network biology approach to identify pathways that get transcriptionally altered and lead to complex complications thereby amplifying the phenotypic effect of the impaired disease state. We have identified two sub-network modules, which could be activated under abnormal circumstances in diabetes. Present work describes key proteins such as P85A and SRC serving as important nodes to mediate alternate signaling routes during diseased condition. P85A has been shown to be an important link between stress responsive MAPK and CVD markers involved in fibrosis. MAPK8 has been shown to interact with P85A and further activate CTGF through VEGF signaling. We have traced a novel and unique route correlating inflammation and fibrosis by considering P85A as a key mediator of signals. The next sub-network module shows SRC as a junction for various signaling processes, which results in interaction between NF-kB and beta catenin to cause cell death. The powerful interaction between these important genes in response to transcriptionally altered lipid metabolism and impaired inflammatory response via SRC causes apoptosis of cells. The crosstalk between inflammation, lipid homeostasis and stress, and their serious effects downstream have been explained in the present analyses.

Imbalanced lignin biosynthesis promotes the sexual reproduction of homothallic oomycete pathogens.

PubMed

Quentin, Michaël; Allasia, Valérie; Pegard, Anthony; Allais, Florent; Ducrot, Paul-Henri; Favery, Bruno; Levis, Caroline; Martinet, Sophie; Masur, Clarissa; Ponchet, Michel; Roby, Dominique; Schlaich, Nikolaus L; Jouanin, Lise; Keller, Harald

2009-01-01

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-L-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-L-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment.

Imbalanced Lignin Biosynthesis Promotes the Sexual Reproduction of Homothallic Oomycete Pathogens

PubMed Central

Quentin, Michaël; Allasia, Valérie; Pegard, Anthony; Allais, Florent; Ducrot, Paul-Henri; Favery, Bruno; Levis, Caroline; Martinet, Sophie; Masur, Clarissa; Ponchet, Michel; Roby, Dominique; Schlaich, Nikolaus L.; Jouanin, Lise; Keller, Harald

2009-01-01

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-l-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-l-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment. PMID:19148278

The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.

PubMed

Athanasopoulos, Athanasios N; Economopoulou, Matina; Orlova, Valeria V; Sobke, Astrid; Schneider, Darius; Weber, Holger; Augustin, Hellmut G; Eming, Sabine A; Schubert, Uwe; Linn, Thomas; Nawroth, Peter P; Hussain, Muzaffar; Hammes, Hans-Peter; Herrmann, Mathias; Preissner, Klaus T; Chavakis, Triantafyllos

2006-04-01

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

Analyses of a Mutant Foxp3 Allele Reveal BATF as a Critical Transcription Factor in the Differentiation and Accumulation of Tissue Regulatory T Cells.

PubMed

Hayatsu, Norihito; Miyao, Takahisa; Tachibana, Masashi; Murakami, Ryuichi; Kimura, Akihiko; Kato, Takako; Kawakami, Eiryo; Endo, Takaho A; Setoguchi, Ruka; Watarai, Hiroshi; Nishikawa, Takeshi; Yasuda, Takuwa; Yoshida, Hisahiro; Hori, Shohei

2017-08-15

Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate. Copyright © 2017 Elsevier Inc. All rights reserved.

Human microRNA-1245 down-regulates the NKG2D receptor in natural killer cells and impairs NKG2D-mediated functions

PubMed Central

Espinoza, J. Luis; Takami, Akiyoshi; Yoshioka, Katsuji; Nakata, Katsuya; Sato, Tokiharu; Kasahara, Yoshihito; Nakao, Shinji

2012-01-01

Background NKG2D is an activating receptor expressed by natural killer and T cells, which have crucial functions in tumor and microbial immunosurveillance. Several cytokines have been identified as modulators of NKG2D receptor expression. However, little is known about NKG2D gene regulation. In this study, we found that microRNA 1245 attenuated the expression of NKG2D in natural killer cells. Design and Methods We investigated the potential interactions between the 3′-untranslated region of the NKG2D gene and microRNA as well as their functional roles in the regulation of NKG2D expression and cytotoxicity in natural killer cells. Results Transforming growth factor-β1, a major negative regulator of NKG2D expression, post-transcriptionally up-regulated mature microRNA-1245 expression, thus down-regulating NKG2D expression and impairing NKG2D-mediated immune responses in natural killer cells. Conversely, microRNA-1245 down-regulation significantly increased the expression of NKG2D expression in natural killer cells, resulting in more efficient NKG2D-mediated cytotoxicity. Conclusions These results reveal a novel NKG2D regulatory pathway mediated by microRNA-1245, which may represent one of the mechanisms used by transforming growth factor-β1 to attenuate NKG2D expression in natural killer cells. PMID:22491735

Interaction of Munc18c and syntaxin4 facilitates invadopodium formation and extracellular matrix invasion of tumor cells.

PubMed

Brasher, Megan I; Martynowicz, David M; Grafinger, Olivia R; Hucik, Andrea; Shanks-Skinner, Emma; Uniacke, James; Coppolino, Marc G

2017-09-29

Tumor cell invasion involves targeted localization of proteins required for interactions with the extracellular matrix and for proteolysis. The localization of many proteins during these cell-extracellular matrix interactions relies on membrane trafficking mediated in part by SNAREs. The SNARE protein syntaxin4 (Stx4) is involved in the formation of invasive structures called invadopodia; however, it is unclear how Stx4 function is regulated during tumor cell invasion. Munc18c is known to regulate Stx4 activity, and here we show that Munc18c is required for Stx4-mediated invadopodium formation and cell invasion. Biochemical and microscopic analyses revealed a physical association between Munc18c and Stx4, which was enhanced during invadopodium formation, and that a reduction in Munc18c expression decreases invadopodium formation. We also found that an N-terminal Stx4-derived peptide associates with Munc18c and inhibits endogenous interactions of Stx4 with synaptosome-associated protein 23 (SNAP23) and vesicle-associated membrane protein 2 (VAMP2). Furthermore, expression of the Stx4 N-terminal peptide decreased invadopodium formation and cell invasion in vitro Of note, cells expressing the Stx4 N-terminal peptide exhibited impaired trafficking of membrane type 1 matrix metalloproteinase (MT1-MMP) and EGF receptor (EGFR) to the cell surface during invadopodium formation. Our findings implicate Munc18c as a regulator of Stx4-mediated trafficking of MT1-MMP and EGFR, advancing our understanding of the role of SNARE function in the localization of proteins that drive tumor cell invasion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Inflammatory Th17 cells promote depression-like behavior in mice

PubMed Central

Beurel, Eléonore; Harrington, Laurie E.; Jope, Richard S.

2012-01-01

Background Recognition of substantial immune-neural interactions is revising dogmas about their insular actions and revealing that immune-neural interactions can substantially impact CNS functions. The inflammatory cytokine interleukin-6 promotes susceptibility to depression and drives production of inflammatory T helper 17 (Th17) T cells, raising the hypothesis that in mouse models Th17 cells promote susceptibility to depression-like behaviors. Methods Behavioral characteristics were measured in male mice administered Th17 cells, CD4+ cells, or vehicle, and in RORγT+/GFP mice or male mice treated with RORγT inhibitor or anti-IL-17A antibodies. Results Mouse brain Th17 cells were elevated by learned helplessness and chronic restraint stress, two common depression-like models. Th17 cell administration promoted learned helplessness in 89% of mice in a paradigm where no vehicle-treated mice developed learned helplessness, and impaired novelty suppressed feeding and social interaction behaviors. Mice deficient in the RORγT transcription factor necessary for Th17 cell production exhibited resistance to learned helplessness, identifying modulation of RORγT as a potential intervention. Treatment with the RORγT inhibitor SR1001, or anti-IL-17A antibodies to abrogate Th17 cell function, reduced Th17-dependent learned helplessness. Conclusions These findings indicate that Th17 cells are increased in the brain during depression-like states, promote depression-like behaviors in mice, and specifically inhibiting the production or function of Th17 cells reduces vulnerability to depression-like behavior, suggesting antidepressant effects may be attained by targeting Th17 cells. PMID:23174342

PI3Kδ promotes CD4(+) T-cell interactions with antigen-presenting cells by increasing LFA-1 binding to ICAM-1.

PubMed

Garçon, Fabien; Okkenhaug, Klaus

2016-05-01

Activation of T lymphocytes by peptide/major histocompatibility complex on antigen-presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo marked morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin lymphocyte function-associated antigen 1 (LFA-1) expressed by T cells to intercellular adhesion molecule (ICAM)-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with APCs. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional phosphatidylinositol(3,4,5)P3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T-cell-dependent immune responses in PI3Kδ-deficient mice.

Cross-talk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis (LAP) of apoptotic germ cells.

PubMed

Panneerdoss, Subbarayalu; Viswanadhapalli, Suryavathi; Abdelfattah, Nourhan; Onyeagucha, Benjamin C; Timilsina, Santosh; Mohammad, Tabrez A; Chen, Yidong; Drake, Michael; Vuori, Kristiina; Kumar, T Rajendra; Rao, Manjeet K

2017-09-19

Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

iPads Enhance Social Interaction Skills among Hearing-Impaired Children of Low Income Families in Saudi Arabia

ERIC Educational Resources Information Center

Bahatheg, Raja Omar

2015-01-01

This research tries to investigate the technical contribution on improving the social interaction of hearing-impaired children from low income families in Saudi Arabia. It compares the social interaction skills of hearing-impaired children who do and do not have access to iPads. To achieve the goals of the study; seventeen children aged five years…

Impaired spine formation and learning in GPCR kinase 2 interacting protein-1 (GIT1) knockout mice.

PubMed

Menon, Prashanthi; Deane, Rashid; Sagare, Abhay; Lane, Steven M; Zarcone, Troy J; O'Dell, Michael R; Yan, Chen; Zlokovic, Berislav V; Berk, Bradford C

2010-03-04

The G-protein coupled receptor (GPCR)-kinase interacting proteins 1 and 2 (GIT1 and GIT2) are scaffold proteins with ADP-ribosylating factor GTPase activity. GIT1 and GIT2 control numerous cellular functions and are highly expressed in neurons, endothelial cells and vascular smooth muscle cells. GIT1 promotes dendritic spine formation, growth and motility in cultured neurons, but its role in brain in vivo is unknown. By using global GIT1 knockout mice (GIT1 KO), we show that compared to WT controls, deletion of GIT1 results in markedly reduced dendritic length and spine density in the hippocampus by 36.7% (p<0.0106) and 35.1% (p<0.0028), respectively. This correlated with their poor adaptation to new environments as shown by impaired performance on tasks dependent on learning. We also studied the effect of GIT1 gene deletion on brain microcirculation. In contrast to findings in systemic circulation, GIT1 KO mice had an intact blood-brain barrier and normal regional cerebral blood flow as determined with radiotracers. Thus, our data suggest that GIT1 plays an important role in brain in vivo by regulating spine density involved in synaptic plasticity that is required for processes involved in learning. 2009 Elsevier B.V. All rights reserved.

Interactive relations of type 2 diabetes and abdominal obesity to cognitive impairment: A cross-sectional study in rural area of Xi'an in China.

PubMed

Li, Yanbo; Shang, Suhang; Fei, Yulang; Chen, Chen; Jiang, Yu; Dang, Liangjun; Liu, Jie; Ma, Louyan; Wei, Meng; Qu, Qiumin

2018-01-01

Type 2 diabetes and obesity, which are frequently comorbid, have been associated with cognitive impairment. We aim to examine the potential modulating effect between obesity and diabetes on cognitive impairment. We recruited 865 adults (aged ≥55years) lived in a village of Xi'an in China from October 2014 to March 2015. All participants underwent biomedical and neuropsychological assessment. Relations of diabetes and abdominal obesity to cognitive impairment were examined in multiple regression models. A total of 155 participants (17.9%) presented with the diagnosis of cognitive impairment. Diabetes or obesity alone wasn't significantly associated with cognitive impairment. Interaction analysis showed a significant interaction between abdominal obesity and diabetes on cognitive impairment. Stratified multivariate analysis revealed that the association between diabetes and cognitive impairment was positive in participants with abdominal obesity (OR 2.436, 95% CI 1.345-4.411, p=0.003, in diabetics with high WC, and OR 2.348, 95% CI 1.373-4.014, p=0.002, in diabetics with high WHR), but negative in those without abdominal obesity. Type 2 diabetes interacts with abdominal obesity to be associated with an increased risk of cognitive impairment by more than two times. Copyright © 2017 Elsevier Inc. All rights reserved.

Different Patterns of Theory of Mind Impairment in Mild Cognitive Impairment.

PubMed

Moreau, Noémie; Rauzy, Stéphane; Bonnefoi, Bernadette; Renié, Laurent; Martinez-Almoyna, Laurent; Viallet, François; Champagne-Lavau, Maud

2015-01-01

Theory of Mind refers to the ability to infer other’s mental states, their beliefs, intentions, or knowledge. To date, only two studies have reported the presence of Theory of Mind impairment in mild cognitive impairment (MCI). In the present study,we evaluated 20 MCI patients and compared them with 25 healthy control participants using two Theory of Mind tasks. The first task was a false belief paradigm as frequently used in the literature, and the second one was a referential communication task,assessing Theory of Mind in a real situation of interaction and which had never been used before in this population. The results showed that MCI patients presented difficulties inferring another person’s beliefs about reality and attributing knowledge to them in a situation of real-life interaction. Two different patterns of Theory of Mind emerged among the patients. In comparison with the control group, some MCI patients demonstrated impairment only in the interaction task and presented isolated episodicmemory impairment, while others were impaired in both Theory of Mind tasks and presented cognitive impairment impacting both episodic memory and executive functioning. Theory of Mind is thus altered in the very early stages of cognitive impairment even in real social interaction, which could impact precociously relationships in daily life.

Engaging the CD40-CD40L pathway augments T-helper cell responses and improves control of Mycobacterium tuberculosis infection

PubMed Central

Bizzell, Erica; Madan-Lala, Ranjna

2017-01-01

Mycobacterium tuberculosis (Mtb) impairs dendritic cell (DC) functions and induces suboptimal antigen-specific CD4 T cell immune responses that are poorly protective. Mucosal T-helper cells producing IFN-γ (Th1) and IL-17 (Th17) are important for protecting against tuberculosis (TB), but the mechanisms by which DCs generate antigen-specific T-helper responses during Mtb infection are not well defined. We previously reported that Mtb impairs CD40 expression on DCs and restricts Th1 and Th17 responses. We now demonstrate that CD40-dependent costimulation is required to generate IL-17 responses to Mtb. CD40-deficient DCs were unable to induce antigen-specific IL-17 responses after Mtb infection despite the production of Th17-polarizing innate cytokines. Disrupting the interaction between CD40 on DCs and its ligand CD40L on antigen-specific CD4 T cells, genetically or via antibody blockade, significantly reduced antigen-specific IL-17 responses. Importantly, engaging CD40 on DCs with a multimeric CD40 agonist (CD40LT) enhanced antigen-specific IL-17 generation in ex vivo DC-T cell co-culture assays. Further, intratracheal instillation of Mtb-infected DCs treated with CD40LT significantly augmented antigen-specific Th17 responses in vivo in the lungs and lung-draining lymph nodes of mice. Finally, we show that boosting CD40-CD40L interactions promoted balanced Th1/Th17 responses in a setting of mucosal DC transfer, and conferred enhanced control of lung bacterial burdens following aerosol challenge with Mtb. Our results demonstrate that CD40 costimulation by DCs plays an important role in generating antigen-specific Th17 cells and targeting the CD40-CD40L pathway represents a novel strategy to improve adaptive immunity to TB. PMID:28767735

Metabolic gene products have evolved to interact with the cell wall integrity pathway in Saccharomyces cerevisiae.

PubMed

Ugbogu, Eziuche A; Wang, Ke; Schweizer, Lilian M; Schweizer, Michael

2016-12-01

Two of the five unlinked genes theoretically capable of encoding 5-phosphoribosyl-1(α)-pyrophosphate (PRPP) synthetase (Prs) in Saccharomyces cerevisiae, PRS1 and PRS5, contain in-frame insertions which separate the cation- and PRPP-binding sites, diagnostic of Prs polypeptides. The impairment of cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) cascade in strains lacking PRS1 and the synthetic lethality associated with loss of PRS1 and PRS5 imply that these insertions are not gratuitous. Coimmunoprecipitation revealed that Prs1 interacts with the CWI MAPK pathway, only when Slt2 has been phosphorylated by Mkk1/2. Three serine residues identified by phosphoproteome analysis (Ficarro et al 2002) are located in one of the insertions of PRS5 thereby defining Prs5 as one of the 11 triply phosphorylated proteins in yeast. Mutation of these phosphosites compromised the transcriptional readout of one endpoint of the CWI pathway, Rlm1, as well as the expression of the gene encoding the stress-activated 1,3 β-glucan synthase, Fks2, regulated by a second endpoint of the CWI pathway, Swi4/Swi6 (SBF transcription factor). Therefore, the unexpected impairment of the CWI phenotype encountered in yeast strains either mutated or deleted for PRS1 or PRS5 can be explained by disruption of the communication between primary cell metabolism and CWI signalling. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Functional silencing is initiated and maintained in immature anti-insulin B cells.

PubMed

Henry, Rachel A; Acevedo-Suárez, Carlos A; Thomas, James W

2009-03-15

Mechanisms of B cell tolerance act during development in the bone marrow and periphery to eliminate or restrict autoreactive clones to prevent autoimmune disease. B cells in the spleens of mice that harbor anti-insulin BCR transgenes (125Tg) are maintained in a functionally silenced or anergic state by endogenous hormone, but it is not clear when and where anergy is induced. An in vitro bone marrow culture system was therefore used to probe whether small protein hormones, a critical class of autoantigens, could interact with the BCR to induce anergy early during B cell development. Upon exposure to insulin, anti-insulin (125Tg) immature B cells show similar hallmarks of anergy as those observed in mature splenic B cells. These include BCR down-regulation, impaired proliferative responses to anti-CD40, and diminished calcium mobilization upon stimulation with BCR-dependent and independent stimuli. Inhibition of calcineurin also results in reduced immature B cell proliferation in a similar manner, suggesting a potential mechanism through which reduced intracellular calcium mobilization may be altering cellular proliferation. Signs of impairment appear after short-term exposure to insulin, which are reversible upon Ag withdrawal. This suggests that a high degree of functional plasticity is maintained at this stage and that constant Ag engagement is required to maintain functional inactivation. These findings indicate that tolerance observed in mature, splenic 125Tg B cells is initiated by insulin in the developing B cell compartment and thus highlight an important therapeutic window for the prevention of insulin autoimmunity.

Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance

PubMed Central

Tischfield, Max A.; Baris, Hagit N.; Wu, Chen; Rudolph, Guenther; Van Maldergem, Lionel; He, Wei; Chan, Wai-Man; Andrews, Caroline; Demer, Joseph L.; Robertson, Richard L.; Mackey, David A.; Ruddle, Jonathan B.; Bird, Thomas D.; Gottlob, Irene; Pieh, Christina; Traboulsi, Elias I.; Pomeroy, Scott L.; Hunter, David G.; Soul, Janet S.; Newlin, Anna; Sabol, Louise J.; Doherty, Edward J.; de Uzcátegui, Clara E.; de Uzcátegui, Nicolas; Collins, Mary Louise Z.; Sener, Emin C.; Wabbels, Bettina; Hellebrand, Heide; Meitinger, Thomas; de Berardinis, Teresa; Magli, Adriano; Schiavi, Costantino; Pastore-Trossello, Marco; Koc, Feray; Wong, Agnes M.; Levin, Alex V.; Geraghty, Michael T.; Descartes, Maria; Flaherty, Maree; Jamieson, Robyn V.; Møller, H. U.; Meuthen, Ingo; Callen, David F.; Kerwin, Janet; Lindsay, Susan; Meindl, Alfons; Gupta, Mohan L.; Pellman, David; Engle, Elizabeth C.

2011-01-01

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific β-tubulin isotype III, result in a spectrum of human nervous system disorders we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves, and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate normal TUBB3 is required for axon guidance and maintenance in mammals. PMID:20074521

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4

PubMed Central

Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M.; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L.; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A.; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L.; Burgdorf, Sven

2016-01-01

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8+ T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte–associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality. PMID:27601670

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

PubMed

Schuette, Verena; Embgenbroich, Maria; Ulas, Thomas; Welz, Meike; Schulte-Schrepping, Jonas; Draffehn, Astrid M; Quast, Thomas; Koch, Katharina; Nehring, Melanie; König, Jessica; Zweynert, Annegret; Harms, Frederike L; Steiner, Nancy; Limmer, Andreas; Förster, Irmgard; Berberich-Siebelt, Friederike; Knolle, Percy A; Wohlleber, Dirk; Kolanus, Waldemar; Beyer, Marc; Schultze, Joachim L; Burgdorf, Sven

2016-09-20

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.

Plasma membrane calcium ATPase isoform 4 inhibits vascular endothelial growth factor-mediated angiogenesis through interaction with calcineurin.

PubMed

Baggott, Rhiannon R; Alfranca, Arantzazu; López-Maderuelo, Dolores; Mohamed, Tamer M A; Escolano, Amelia; Oller, Jorge; Ornes, Beatriz C; Kurusamy, Sathishkumar; Rowther, Farjana B; Brown, James E; Oceandy, Delvac; Cartwright, Elizabeth J; Wang, Weiguang; Gómez-del Arco, Pablo; Martínez-Martínez, Sara; Neyses, Ludwig; Redondo, Juan Miguel; Armesilla, Angel Luis

2014-10-01

Vascular endothelial growth factor (VEGF) has been identified as a crucial regulator of physiological and pathological angiogenesis. Among the intracellular signaling pathways triggered by VEGF, activation of the calcineurin/nuclear factor of activated T cells (NFAT) signaling axis has emerged as a critical mediator of angiogenic processes. We and others previously reported a novel role for the plasma membrane calcium ATPase (PMCA) as an endogenous inhibitor of the calcineurin/NFAT pathway, via interaction with calcineurin, in cardiomyocytes and breast cancer cells. However, the functional significance of the PMCA/calcineurin interaction in endothelial pathophysiology has not been addressed thus far. Using in vitro and in vivo assays, we here demonstrate that the interaction between PMCA4 and calcineurin in VEGF-stimulated endothelial cells leads to downregulation of the calcineurin/NFAT pathway and to a significant reduction in the subsequent expression of the NFAT-dependent, VEGF-activated, proangiogenic genes RCAN1.4 and Cox-2. PMCA4-dependent inhibition of calcineurin signaling translates into a reduction in endothelial cell motility and blood vessel formation that ultimately impairs in vivo angiogenesis by VEGF. Given the importance of the calcineurin/NFAT pathway in the regulation of pathological angiogenesis, targeted modulation of PMCA4 functionality might open novel therapeutic avenues to promote or attenuate new vessel formation in diseases that occur with angiogenesis. © 2014 American Heart Association, Inc.

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death.

PubMed

Jiang, Ke; Liu, Min; Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-05-03

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner.

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death

PubMed Central

Lin, Guibin; Mao, Beibei; Cheng, Wei; Liu, Han; Gal, Jozsef; Zhu, Haining; Yuan, Zengqiang; Deng, Wuguo; Liu, Quentin; Gong, Peng; Bi, Xiaolin; Meng, Songshu

2016-01-01

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner. PMID:27028858

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD.

PubMed

Goode, Alice; Butler, Kevin; Long, Jed; Cavey, James; Scott, Daniel; Shaw, Barry; Sollenberger, Jill; Gell, Christopher; Johansen, Terje; Oldham, Neil J; Searle, Mark S; Layfield, Robert

2016-07-02

Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.

9-Cis-Retinoic Acid Induces Growth Inhibition in Retinoid-Sensitive Breast Cancer and Sea Urchin Embryonic Cells via Retinoid X Receptor α and Replication Factor C3

PubMed Central

Maeng, Sejung; Kim, Gil Jung; Choi, Eun Ju; Yang, Hyun Ok; Lee, Dong-Sup

2012-01-01

There is widespread interest in defining factors and mechanisms that suppress the proliferation of cancer cells. Retinoic acid (RA) is a potent suppressor of mammary cancer and developmental embryonic cell proliferation. However, the molecular mechanisms by which 9-cis-RA signaling induces growth inhibition in RA-sensitive breast cancer and embryonic cells are not apparent. Here, we provide evidence that the inhibitory effect of 9-cis-RA on cell proliferation depends on 9-cis-RA-dependent interaction of retinoid X receptor α (RXRα) with replication factor C3 (RFC3), which is a subunit of the RFC heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp on DNA. An RFC3 ortholog in a sea urchin cDNA library was isolated by using the ligand-binding domain of RXRα as bait in a yeast two-hybrid screening. The interaction of RFC3 with RXRα depends on 9-cis-RA and bexarotene, but not on all-trans-RA or an RA receptor (RAR)-selective ligand. Truncation and mutagenesis experiments demonstrated that the C-terminal LXXLL motifs in both human and sea urchin RFC3 are critical for the interaction with RXRα. The transient interaction between 9-cis-RA-activated RXRα and RFC3 resulted in reconfiguration of the PCNA-RFC complex. Furthermore, we found that knockdown of RXRα or overexpression of RFC3 impairs the ability of 9-cis-RA to inhibit proliferation of MCF-7 breast cancer cells and sea urchin embryogenesis. Our results indicate that 9-cis-RA-activated RXRα suppresses the growth of RA-sensitive breast cancer and embryonic cells through RFC3. PMID:22949521

Physiologic TLR9-CpG-DNA interaction is essential for the homeostasis of the intestinal immune system.

PubMed

Hofmann, Claudia; Dunger, Nadja; Doser, Kristina; Lippert, Elisabeth; Siller, Sebastian; Edinger, Matthias; Falk, Werner; Obermeier, Florian

2014-01-01

Cytosine-guanosine dinucleotide (CpG) motifs are immunostimulatory components of bacterial DNA and activators of innate immunity through Toll-like receptor 9 (TLR9). Administration of CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. It was investigated whether physiologic CpG/TLR9 interactions are critical for the homeostasis of the intestinal immune system. Mesenteric lymph node cell and lamina propria mononuclear cell (LPMC) populations from BALB/c wild-type (wt) or TLR9 mice were assessed by flow cytometry and proteome profiling. Cytokine secretion was determined and nuclear extracts were analyzed for nuclear factor kappa B (NF-κB) and cAMP response-element binding protein activity. To assess the colitogenic potential of intestinal T cells, CD4-enriched cells from LPMC of wt or TLR9 donor mice were injected intraperitoneally in recipient CB-17 SCID mice. TLR9 deficiency was accompanied by slight changes in cellular composition and phosphorylation of signaling proteins of mesenteric lymph node cell and LPMC. LPMC from TLR9 mice displayed an increased proinflammatory phenotype compared with wt LPMC. NF-κB activity in cells from TLR9 mice was enhanced, whereas cAMP response-element binding activity was reduced compared with wt. Transfer of lamina propria CD4-enriched T cells from TLR9 mice induced severe colitis, whereas wt lamina propria CD4-enriched T cells displayed an attenuated phenotype. Lack of physiologic CpG/TLR9 interaction impairs the function of the intestinal immune system indicated by enhanced proinflammatory properties. Thus, physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counterregulating anti-inflammatory mechanisms.

A novel network analysis approach reveals DNA damage, oxidative stress and calcium/cAMP homeostasis-associated biomarkers in frontotemporal dementia

PubMed Central

Ferrari, Raffaele; Graziano, Francesca; Novelli, Valeria; Rossi, Giacomina; Galimberti, Daniela; Rainero, Innocenzo; Benussi, Luisa; Nacmias, Benedetta; Bruni, Amalia C.; Cusi, Daniele; Salvi, Erika; Borroni, Barbara; Grassi, Mario

2017-01-01

Frontotemporal Dementia (FTD) is the form of neurodegenerative dementia with the highest prevalence after Alzheimer’s disease, equally distributed in men and women. It includes several variants, generally characterized by behavioural instability and language impairments. Although few mendelian genes (MAPT, GRN, and C9orf72) have been associated to the FTD phenotype, in most cases there is only evidence of multiple risk loci with relatively small effect size. To date, there are no comprehensive studies describing FTD at molecular level, highlighting possible genetic interactions and signalling pathways at the origin FTD-associated neurodegeneration. In this study, we designed a broad FTD genetic interaction map of the Italian population, through a novel network-based approach modelled on the concepts of disease-relevance and interaction perturbation, combining Steiner tree search and Structural Equation Model (SEM) analysis. Our results show a strong connection between Calcium/cAMP metabolism, oxidative stress-induced Serine/Threonine kinases activation, and postsynaptic membrane potentiation, suggesting a possible combination of neuronal damage and loss of neuroprotection, leading to cell death. In our model, Calcium/cAMP homeostasis and energetic metabolism impairments are primary causes of loss of neuroprotection and neural cell damage, respectively. Secondly, the altered postsynaptic membrane potentiation, due to the activation of stress-induced Serine/Threonine kinases, leads to neurodegeneration. Our study investigates the molecular underpinnings of these processes, evidencing key genes and gene interactions that may account for a significant fraction of unexplained FTD aetiology. We emphasized the key molecular actors in these processes, proposing them as novel FTD biomarkers that could be crucial for further epidemiological and molecular studies. PMID:29020091

The ER Contact Proteins VAPA/B Interact with Multiple Autophagy Proteins to Modulate Autophagosome Biogenesis.

PubMed

Zhao, Yan G; Liu, Nan; Miao, Guangyan; Chen, Yong; Zhao, Hongyu; Zhang, Hong

2018-04-23

The endoplasmic reticulum (ER) is the site of biogenesis of the isolation membrane (IM, autophagosome precursor) and forms extensive contacts with IMs during their expansion into double-membrane autophagosomes. Little is known about the molecular mechanism underlying the formation and/or maintenance of the ER/IM contact. The integral ER proteins VAPA and VAPB (VAPs) participate in establishing ER contacts with multiple membranes by interacting with different tethers. Here, we demonstrate that VAPs also modulate ER/IM contact formation. Depletion of VAPs impairs progression of IMs into autophagosomes. Upon autophagy induction, VAPs are recruited to autophagosome formation sites on the ER, a process mediated by their interactions with FIP200 and PI(3)P. VAPs directly interact with FIP200 and ULK1 through their conserved FFAT motifs and stabilize the ULK1/FIP200 complex at the autophagosome formation sites on the ER. The formation of ULK1 puncta is significantly reduced by VAPA/B depletion. VAPs also interact with WIPI2 and enhance the formation of the WIPI2/FIP200 ER/IM tethering complex. Depletion of VMP1, which increases the ER/IM contact, greatly elevates the interaction of VAPs with these autophagy proteins. The VAPB P56S mutation, which is associated with amyotrophic lateral sclerosis, reduces the ULK1/FIP200 interaction and impairs autophagy at an early step, similar to the effect seen in VAPA/B-depleted cells. Our study reveals that VAPs directly interact with multiple ATG proteins, thereby contributing to ER/IM contact formation for autophagosome biogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

PubMed

Sun, Zhao; Yu, Wenjie; Sanz Navarro, Maria; Sweat, Mason; Eliason, Steven; Sharp, Thad; Liu, Huan; Seidel, Kerstin; Zhang, Li; Moreno, Myriam; Lynch, Thomas; Holton, Nathan E; Rogers, Laura; Neff, Traci; Goodheart, Michael J; Michon, Frederic; Klein, Ophir D; Chai, Yang; Dupuy, Adam; Engelhardt, John F; Chen, Zhi; Amendt, Brad A

2016-11-15

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development. © 2016. Published by The Company of Biologists Ltd.

The N-terminal region of the Plantago asiatica mosaic virus coat protein is required for cell-to-cell movement but is dispensable for virion assembly.

PubMed

Ozeki, Johji; Hashimoto, Masayoshi; Komatsu, Ken; Maejima, Kensaku; Himeno, Misako; Senshu, Hiroko; Kawanishi, Takeshi; Kagiwada, Satoshi; Yamaji, Yasuyuki; Namba, Shigetou

2009-06-01

Potexvirus cell-to-cell movement requires coat protein (CP) and movement proteins. In this study, mutations in two conserved in-frame AUG codons in the 5' region of the CP open reading frame of Plantago asiatica mosaic virus (PlAMV) were introduced, and virus accumulation of these mutants was analyzed in inoculated and upper noninoculated leaves. When CP was translated only from the second AUG codon, virus accumulation in inoculated leaves was lower than that of wild-type PlAMV, and the viral spread was impaired. Trans-complementation analysis showed that the leucine residue at the third position (Leu-3) of CP is important for cell-to-cell movement of PlAMV. The 14-amino-acid N-terminal region of CP was dispensable for virion formation. Immunoprecipitation assays conducted with an anti-TGBp1 antibody indicated that PlAMV CP interacts with TGBp1 in vivo and that this interaction is not affected by alanine substitution at Leu-3. These results support the concept that the N-terminal region of potexvirus CP can be separated into two distinct functional domains.

The Interaction Between IGF-1, Atherosclerosis and Vascular Aging

PubMed Central

Higashi, Yusuke; Quevedo, Henry C.; Tiwari, Summit; Sukhanov, Sergiy; Shai, Shaw-Yung; Anwar, Asif; Delafontaine, Patrice

2014-01-01

The process of vascular aging encompasses alterations in the function of endothelial (EC) and vascular smooth muscle cells (VSMCs) via oxidation, inflammation, cell senescence and epigenetic modifications, increasing the probability of atherosclerosis. Aged vessels exhibit decreased endothelial antithrombogenic properties, increased reactive oxygen species (ROS) generation and inflammatory signaling, increased migration of VSMCs to the subintimal space, impaired angiogenesis and increased elastin degradation. The key initiating step in atherogenesis is subendothelial accumulation of apolipoprotein-B containing low density lipoproteins resulting in activation of endothelial cells and recruitment of monocytes. Activated endothelial cells secrete “chemokines” that interact with cognate chemokine receptors on monocytes and promote directional migration. Recruitment of immune cells establishes a pro-inflammatory status, further causing elevated oxidative stress, which in turn triggers a series of events including apoptotic or necrotic death of vascular and non-vascular cells. Increased oxidative stress is also considered to be a key factor in mechanisms of aging-associated changes in tissue integrity and function. Experimental evidence indicates that insulin-like growth factor-1 (IGF-1) exerts anti-oxidant, anti-inflammatory and pro-survival effects on the vasculature, reducing atherosclerotic plaque burden and promoting features of atherosclerotic plaque stability. PMID:24943302

Interactions between MSCs and Immune Cells: Implications for Bone Healing

PubMed Central

Kovach, Tracy K.; Dighe, Abhijit S.; Lobo, Peter I.; Cui, Quanjun

2015-01-01

It is estimated that, of the 7.9 million fractures sustained in the United States each year, 5% to 20% result in delayed or impaired healing requiring therapeutic intervention. Following fracture injury, there is an initial inflammatory response that plays a crucial role in bone healing; however, prolonged inflammation is inhibitory for fracture repair. The precise spatial and temporal impact of immune cells and their cytokines on fracture healing remains obscure. Some cytokines are reported to be proosteogenic while others inhibit bone healing. Cell-based therapy utilizing mesenchymal stromal cells (MSCs) is an attractive option for augmenting the fracture repair process. Osteoprogenitor MSCs not only differentiate into bone, but they also exert modulatory effects on immune cells via a variety of mechanisms. In this paper, we review the current literature on both in vitro and in vivo studies on the role of the immune system in fracture repair, the use of MSCs in the enhancement of fracture healing, and interactions between MSCs and immune cells. Insight into this paradigm can provide valuable clues in identifying cellular and noncellular targets that can potentially be modulated to enhance both natural bone healing and bone repair augmented by the exogenous addition of MSCs. PMID:26000315

Disruption of a -35kb enhancer impairs CTCF binding and MLH1 expression in colorectal cells.

PubMed

Liu, Qing; Thoms, Julie A; Nunez, Andrea C; Huang, Yizhou; Knezevic, Kathy; Packham, Deborah; Poulos, Rebecca C; Williams, Rachel; Beck, Dominik; Hawkins, Nicholas J; Ward, Robyn L; Wong, Jason W H; Hesson, Luke B; Sloane, Mathew A; Pimanda, John

2018-06-13

MLH1 is a major tumour suppressor gene involved in the pathogenesis of Lynch syndrome and various sporadic cancers. Despite their potential pathogenic importance, genomic regions capable of regulating MLH1 expression over long distances have yet to be identified. Here we use chromosome conformation capture (3C) to screen a 650-kb region flanking the MLH1 locus to identify interactions between the MLH1 promoter and distal regions in MLH1 expressing and non-expressing cells. Putative enhancers were functionally validated using luciferase reporter assays, chromatin immunoprecipitation and CRISPR-Cas9 mediated deletion of endogenous regions. To evaluate whether germline variants in the enhancer might contribute to impaired MLH1 expression in patients with suspected Lynch syndrome, we also screened germline DNA from a cohort of 74 patients with no known coding mutations or epimutations at the MLH1 promoter. A 1.8kb DNA fragment, 35kb upstream of the MLH1 transcription start site enhances MLH1 gene expression in colorectal cells. The enhancer was bound by CTCF and CRISPR-Cas9 mediated deletion of a core binding region impairs endogenous MLH1 expression. 5.4% of suspected Lynch syndrome patients have a rare single nucleotide variant (G>A; rs143969848; 2.5% in gnomAD European, non-Finnish) within a highly conserved CTCF binding motif, which disrupts enhancer activity in SW620 colorectal carcinoma cells. A CTCF bound region within the MLH1 -35 enhancer regulates MLH1 expression in colorectal cells and is worthy of scrutiny in future genetic screening strategies for suspected Lynch syndrome associated with loss of MLH1 expression. Copyright ©2018, American Association for Cancer Research.

Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies*

PubMed Central

Kammerer, Robert; Rüttiger, Lukas; Riesenberg, Rainer; Schäuble, Constanze; Krupar, Rosemarie; Kamp, Annegret; Sunami, Kishiko; Eisenried, Andreas; Hennenberg, Martin; Grunert, Fritz; Bress, Andreas; Battaglia, Sebastiano; Schrewe, Heinrich; Knipper, Marlies; Schneider, Marlon R.; Zimmermann, Wolfgang

2012-01-01

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16−/− mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16−/− mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range. PMID:22544735

Interaction of colicin E7 with the major coat protein (g8p) may confer limited protection on colicinogenic Escherichia coli against M13 bacteriophage infection.

PubMed

Chen, Yuh-Ren; Yang, Tsung-Yeh; Lei, Guang-Sheng; Liao, Chen-Chung; Chak, Kin-Fu

2010-11-01

Colicin release provides producer strains with a competitive advantage under certain circumstances. We found that propagation of M13 bacteriophage in cells producing colicin E7 is impaired, without alteration in the efficiency of bacteriophage adsorption, as compared with non-producing cells. In contrast to the protective effect of the colicin against M13 bacteriophage infection, the endogenously expressed colicin does not confer limited protection against transfection with M13 bacteriophage DNA. Furthermore, it was found that the translocation-receptor-binding domain and toxicity domain of the colicin are able to interact with the M13 major coat protein, g8p, during bacteriophage infection. Based on these observations, we propose that interaction between colicin E7 and g8p during infection interferes with g8p depolymerizing into the cytoplasmic membrane during bacteriophage DNA penetration, thus resulting in the limited protection against M13 bacteriophage infection.

STAM Adaptor Proteins Interact with COPII Complexes and Function in ER-to-Golgi Trafficking

PubMed Central

Rismanchi, Neggy; Puertollano, Rosa; Blackstone, Craig

2009-01-01

Signal transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. Here we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein (VSVG)-GFP trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with COPII proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex. PMID:19054391

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1.

PubMed

Hinrichsen, Inga; Ernst, Benjamin Philipp; Nuber, Franziska; Passmann, Sandra; Schäfer, Dieter; Steinke, Verena; Friedrichs, Nicolaus; Plotz, Guido; Zeuzem, Stefan; Brieger, Angela

2014-01-24

Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated. Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively. MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1

PubMed Central

2014-01-01

Introduction Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated. Methods Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively. Results MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. Conclusions These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1. PMID:24456667

Store-operated Ca2+ entry controls ameloblast cell function and enamel development

PubMed Central

Eckstein, Miriam; Vaeth, Martin; Fornai, Cinzia; Vinu, Manikandan; Bromage, Timothy G.; Nurbaeva, Meerim K.; Sorge, Jessica L.; Coelho, Paulo G.; Idaghdour, Youssef; Feske, Stefan; Lacruz, Rodrigo S.

2017-01-01

Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release–activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel. PMID:28352661

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production.

PubMed

Chatel-Chaix, Laurent; Melançon, Pierre; Racine, Marie-Ève; Baril, Martin; Lamarre, Daniel

2011-11-01

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.

An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex

PubMed Central

Kracker, Sven; Di Virgilio, Michela; Schwartzentruber, Jeremy; Cuenin, Cyrille; Forveille, Monique; Deau, Marie-Céline; McBride, Kevin M.; Majewski, Jacek; Gazumyan, Anna; Seneviratne, Suranjith; Grimbacher, Bodo; Kutukculer, Necil; Herceg, Zdenko; Cavazzana, Marina; Jabado, Nada; Nussenzweig, Michel C.; Fischer, Alain; Durandy, Anne

2015-01-01

Background Immunoglobulin class-switch recombination defects (CSR-D) are rare primary immunodeficiencies characterized by impaired production of switched immunoglobulin isotypes and normal or elevated IgM levels. They are caused by impaired T:B cooperation or intrinsic B cell defects. However, many immunoglobulin CSR-Ds are still undefined at the molecular level. Objective This study's objective was to delineate new causes of immunoglobulin CSR-Ds and thus gain further insights into the process of immunoglobulin class-switch recombination (CSR). Methods Exome sequencing in 2 immunoglobulin CSR-D patients identified variations in the INO80 gene. Functional experiments were performed to assess the function of INO80 on immunoglobulin CSR. Results We identified recessive, nonsynonymous coding variations in the INO80 gene in 2 patients affected by defective immunoglobulin CSR. Expression of wild-type INO80 in patients' fibroblastic cells corrected their hypersensitivity to high doses of γ-irradiation. In murine CH12-F3 cells, the INO80 complex accumulates at Sα and Eμ regions of the IgH locus, and downregulation of INO80 as well as its partners Reptin and Pontin impaired CSR. In addition, Reptin and Pontin were shown to interact with activation-induced cytidine deaminase. Finally, an abnormal separation of sister chromatids was observed upon INO80 downregulation in CH12-F3 cells, pinpointing its role in cohesin activity. Conclusion INO80 deficiency appears to be associated with defective immunoglobulin CSR. We propose that the INO80 complex modulates cohesin function that may be required during immunoglobulin switch region synapsis. PMID:25312759

Social Interactions of Speech- and Language-Impaired Children.

ERIC Educational Resources Information Center

Rice, Mabel L.; And Others

1991-01-01

This study found that 17 preschool children with limited communication skills [specific language impairment, speech impairment, and English as a Second Language (ESL)] were more likely than normal peers to initiate with adults, shorten their responses, and use nonverbal responses. ESL children were least likely to initiate interactions.…

R-Ras2 is required for germinal center formation to aid B cells during energetically demanding processes.

PubMed

Mendoza, Pilar; Martínez-Martín, Nuria; Bovolenta, Elena R; Reyes-Garau, Diana; Hernansanz-Agustín, Pablo; Delgado, Pilar; Diaz-Muñoz, Manuel D; Oeste, Clara L; Fernández-Pisonero, Isabel; Castellano, Ester; Martínez-Ruiz, Antonio; Alonso-Lopez, Diego; Santos, Eugenio; Bustelo, Xosé R; Kurosaki, Tomohiro; Alarcón, Balbino

2018-05-29

Upon antigen recognition within peripheral lymphoid organs, B cells interact with T cells and other immune cells to transiently form morphological structures called germinal centers (GCs), which are required for B cell clonal expansion, immunoglobulin class switching, and affinity maturation. This process, known as the GC response, is an energetically demanding process that requires the metabolic reprogramming of B cells. We showed that the Ras-related guanosine triphosphate hydrolase (GTPase) R-Ras2 (also known as TC21) plays an essential, nonredundant, and B cell-intrinsic role in the GC response. Both the conversion of B cells into GC B cells and their expansion were impaired in mice lacking R-Ras2, but not in those lacking a highly related R-Ras subfamily member or both the classic H-Ras and N-Ras GTPases. In the absence of R-Ras2, activated B cells did not exhibit increased oxidative phosphorylation or aerobic glycolysis. We showed that R-Ras2 was an effector of both the B cell receptor (BCR) and CD40 and that, in its absence, B cells exhibited impaired activation of the PI3K-Akt-mTORC1 pathway, reduced mitochondrial DNA replication, and decreased expression of genes involved in glucose metabolism. Because most human B cell lymphomas originate from GC B cells or B cells that have undergone the GC response, our data suggest that R-Ras2 may also regulate metabolism in B cell malignancies. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Effect of zolpidem on human cytochrome P450 activity, and on transport mediated by P-glycoprotein.

PubMed

von Moltke, Lisa L; Weemhoff, James L; Perloff, Michael D; Hesse, Leah M; Harmatz, Jerold S; Roth-Schechter, Barbara F; Greenblatt, David J

2002-12-01

The influence of high concentrations of zolpidem (100 microM, corresponding to approximately 200 times maximum therapeutic concentrations) on the activity of six human Cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Zolpidem produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. Transport of rhodamine 123, presumed to be mediated mainly by the energy-dependent efflux transport protein P-glycoprotein, was studied in a cell culture system using a human intestinal cell line. High concentrations of zolpidem (100 microM), exceeding the usual therapeutic range by more than 100-fold, produced only modest impairment of rhodamine 123 transport. The findings indicate that zolpidem is very unlikely to cause clinical drug interactions attributable to impairment of CYP activity or P-gp mediated transport. Copyright 2002 John Wiley & Sons, Ltd.

Fas/FasL interaction mediates imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells.

PubMed

Dou, Rui; Hong, Zhenya; Tan, Xiaosheng; Hu, Fenfen; Ding, Yajie; Wang, Wei; Liang, Zhihui; Zhong, Rongrong; Wu, Xiongwen; Weng, Xiufang

2018-07-01

The rapid antitumor cytokine production and direct cytotoxicity confer invariant NKT (iNKT) cells ideal candidates for cancer therapy. However, the therapeutic potential of iNKT cells in T-cell malignant diseases remains elusive, as antigen presentation by T cells (T-T presentation) has been suggested to induce hyporesponsiveness of iNKT cells. In this study, we found discrepancies in iNKT cell responses against two T cell-origin cell lines (Jurkat and Molt-4). Human iNKT cells exhibited more intensive cytotoxicity and less efficient cytokine production in response to Fas-bearing Jurkat cells than those to the Fas-negative tumor cells (Molt-4 and myeloid-derived K562). The imbalanced cytokine/cytotoxicity responses of iNKT cells against Jurkat cells were CD1d-dependent and relied mostly on Fas/FasL interaction. The impairment in cytokine production could be overcome by Fas/FasL blocking antibodies and exogenous IL-2. Elevated CD1d levels as well as CD1d and Fas co-localization were found in T-cell lymphomas. However, defects in frequency and function of circulating iNKT cells were observed in the patients, which could be partly rescued by exogenous IL-2. Collectively, the Fas/FasL-dependent aberrant iNKT cell responses and the reversibility of the defects suggest the distinct iNKT cell manipulation in CD1d- and Fas-bearing T cell malignancies. Copyright © 2018. Published by Elsevier Ltd.

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

PubMed Central

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

Interaction of fibrinogen and muramidase-released protein promotes the development of Streptococcus suis meningitis

PubMed Central

Wang, Junping; Kong, Decong; Zhang, Shengwei; Jiang, Hua; Zheng, Yuling; Zang, Yating; Hao, Huaijie; Jiang, Yongqiang

2015-01-01

Muramidase-released protein (MRP) is as an important virulence marker of Streptococcus suis (S. suis) serotype 2. Our previous works have shown that MRP can bind human fibrinogen (hFg); however, the function of this interaction in S. suis meningitis is not known. In this study, we found that the deletion of mrp significantly impairs the hFg-mediated adherence and traversal ability of S. suis across human cerebral microvascular endothelial cells (hCMEC/D3). Measurement of the permeability to Lucifer yellow in vitro and Evans blue extravasation in vivo show that the MRP-hFg interaction significantly increases the permeability of the blood–brain barrier (BBB). In the mouse meningitis model, wild type S. suis caused higher bacterial loads in the brain and more severe histopathological signs of meningitis than the mrp mutant at day 3 post-infection. Western blot analysis and immunofluorescence observations reveal that the MRP-hFg interaction can destroy the cell adherens junction protein p120-catenin of hCMEC/D3. These results indicate that the MRP-hFg interaction is important in the development of S. suis meningitis. PMID:26441928

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

PubMed

Saso, Wakana; Tsukuda, Senko; Ohashi, Hirofumi; Fukano, Kento; Morishita, Ryo; Matsunaga, Satoko; Ohki, Mio; Ryo, Akihide; Park, Sam-Yong; Suzuki, Ryosuke; Aizaki, Hideki; Muramatsu, Masamichi; Sureau, Camille; Wakita, Takaji; Matano, Tetsuro; Watashi, Koichi

2018-06-22

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

Treg Cells Protect Dopaminergic Neurons against MPP+ Neurotoxicity via CD47-SIRPA Interaction.

PubMed

Huang, Yan; Liu, Zhan; Cao, Bei-Bei; Qiu, Yi-Hua; Peng, Yu-Ping

2017-01-01

Regulatory T (Treg) cells have been associated with neuroprotection by inhibiting microglial activation in animal models of Parkinson's disease (PD), a progressive neurodegenerative disease characterized by dopaminergic neuronal loss in the nigrostriatal system. Herein, we show that Treg cells directly protect dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity via an interaction between the two transmembrane proteins CD47 and signal regulatory protein α (SIRPA). Primary ventral mesencephalic (VM) cells or VM neurons were pretreated with Treg cells before MPP+ treatment. Transwell co-culture of Treg cells and VM neurons was used to assess the effects of the Treg cytokines transforming growth factor (TGF)-β1 and interleukin (IL)-10 on dopaminergic neurons. Live cell imaging system detected a dynamic contact of Treg cells with VM neurons that were stained with CD47 and SIRPA, respectively. Dopaminergic neuronal loss, which was assessed by the number of tyrosine hydroxylase (TH)-immunoreactive cells, was examined after silencing CD47 in Treg cells or silencing SIRPA in VM neurons. Treg cells prevented MPP+-induced dopaminergic neuronal loss and glial inflammatory responses. TGF-β1 and IL-10 secreted from Treg cells did not significantly prevent MPP+-induced dopaminergic neuronal loss in transwell co-culture of Treg cells and VM neurons. CD47 and SIRPA were expressed by Treg cells and VM neurons, respectively. CD47-labeled Treg cells dynamically contacted with SIRPA-labeled VM neurons. Silencing CD47 gene in Treg cells impaired the ability of Treg cells to protect dopaminergic neurons against MPP+ toxicity. Similarly, SIRPA knockdown in VM neurons reduced the ability of Treg cell neuroprotection. Rac1/Akt signaling pathway in VM neurons was activated by CD47-SIRPA interaction between Treg cells and the neurons. Inhibiting Rac1/Akt signaling in VM neurons compromised Treg cell neuroprotection. Treg cells protect dopaminergic neurons against MPP+ neurotoxicity by a cell-to-cell contact mechanism underlying CD47-SIRPA interaction and Rac1/Akt activation. © 2017 The Author(s)Published by S. Karger AG, Basel.

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2

PubMed Central

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-01-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of possible 18 α-chains and one of possible 8 β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalised by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalisation by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with AP2 C-µ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions. PMID:26779610

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

PubMed

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-02-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.

Zyxin-Siah2–Lats2 axis mediates cooperation between Hippo and TGF-β signalling pathways

PubMed Central

Ma, Biao; Cheng, Hongcheng; Gao, Ruize; Mu, Chenglong; Chen, Ling; Wu, Shian; Chen, Quan; Zhu, Yushan

2016-01-01

The evolutionarily conserved Hippo pathway is a regulator that controls organ size, cell growth and tissue homeostasis. Upstream signals of the Hippo pathway have been widely studied, but how microenvironmental factors coordinately regulate this pathway remains unclear. In this study, we identify LIM domain protein Zyxin, as a scaffold protein, that in response to hypoxia and TGF-β stimuli, forms a ternary complex with Lats2 and Siah2 and stabilizes their interaction. This interaction facilitates Lats2 ubiquitination and degradation, Yap dephosphorylation and subsequently activation. We show that Zyxin is required for TGF-β and hypoxia-induced Lats2 downregulation and deactivation of Hippo signalling in MDA-MB-231 cells. Depletion of Zyxin impairs the capability of cell migration, proliferation and tumourigenesis in a xenograft model. Zyxin is upregulated in human breast cancer and positively correlates with histological stages and metastasis. Our study demonstrates that Zyxin-Lats2–Siah2 axis may serve as a potential therapeutic target in cancer treatment. PMID:27030211

Testing Brain Overgrowth and Synaptic Models of Autism Using NPCs and Neurons from Patient-Derived IPS Cells

DTIC Science & Technology

2015-12-01

Award Number: W81XWH-13-1-0415 TITLE: Testing Brain Overgrowth and Synaptic Models of Autism Using NPC’s and Neurons from Patient-Derived IPS...Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Autism and autism spectrum disorders (ASD) are complex...impaired social interaction, and limited and repetitive interests and behavior. Recent studies have led to two major hypotheses for autism

The role of pleiotrophin and β-catenin in fetal lung development

PubMed Central

2010-01-01

Mammalian lung development is a complex biological process, which is temporally and spatially regulated by growth factors, hormones, and extracellular matrix proteins. Abnormal changes of these molecules often lead to impaired lung development, and thus pulmonary diseases. Epithelial-mesenchymal interactions are crucial for fetal lung development. This paper reviews two interconnected pathways, pleiotrophin and Wnt/β-catenin, which are involved in fibroblast and epithelial cell communication during fetal lung development. PMID:20565841

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Kyoung Ho; Yeo, Sang Won, E-mail: swyeo@catholic.ac.kr; Troy, Frederic A., E-mail: fatroy@ucdavis.edu

Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC withmore » epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.« less

Interactions between muscle and the immune system during modified musculoskeletal loading

NASA Technical Reports Server (NTRS)

Tidball, James G.

2002-01-01

Interactions between the immune system and skeletal muscle may play a significant role in modulating the course of muscle injury and repair after modified musculoskeletal loading. Current evidence indicates that activation of the complement system is an early event during modified loading, which then leads to inflammatory cell invasion. However, the functions of those inflammatory cells are complex and they seem to be capable of promoting additional injury and repair. Recent findings implicate an early invading neutrophil population in increasing muscle damage that is detected by the presence of muscle membrane lesions. Macrophages that invade subsequently serve to remove cellular debris, and seem to promote repair. However, macrophages also have the ability to increase damage in muscle in which there is an impaired capacity to generate nitric oxide. In vivo and in vitro evidence indicates that muscle-derived nitric oxide can serve an important role in protecting muscle from membrane damage by invading inflammatory cells. Collectively, these findings indicate that the dynamic balance between inflammatory cells, the complement system, and muscle-derived free radicals can play important roles in the secondary damage of muscle during modified musculoskeletal loading.

Studying the dynamics of SLP-76, Nck, and Vav1 multimolecular complex formation in live human cells with triple-color FRET.

PubMed

Pauker, Maor H; Hassan, Nirit; Noy, Elad; Reicher, Barak; Barda-Saad, Mira

2012-04-24

Protein-protein interactions regulate and control many cellular functions. A multimolecular complex consisting of the adaptor proteins SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kD), Nck, and the guanine nucleotide exchange factor Vav1 is recruited to the T cell side of the interface with an antigen-presenting cell during initial T cell activation. This complex is crucial for regulation of the actin machinery, antigen recognition, and signaling in T cells. We studied the interactions between these proteins as well as the dynamics of their recruitment into a complex that governs cytoskeletal reorganization. We developed a triple-color Förster resonance energy transfer (3FRET) system to observe the dynamics of the formation of this trimolecular signaling complex in live human T cells and to follow the three molecular interactions in parallel. Using the 3FRET system, we demonstrated that dimers of Nck and Vav1 were constitutively formed independently of both T cell activation and the association between SLP-76 and Nck. After T cell receptor stimulation, SLP-76 was phosphorylated, which enabled the binding of Nck. A point mutation in the proline-rich site of Vav1, which abolishes its binding to Nck, impaired actin rearrangement, suggesting that Nck-Vav1 dimers play a critical role in regulation of the actin machinery. We suggest that these findings revise the accepted model of the formation of a complex of SLP-76, Nck, and Vav1 and demonstrate the use of 3FRET as a tool to study signal transduction in live cells.

Differential monocyte responses to TLR ligands in children with autism spectrum disorders

PubMed Central

Enstrom, Amanda M; Onore, Charity E; Van de Water, Judy A; Ashwood, Paul

2010-01-01

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR2 (lipoteichoic acid; LTA), TLR3 (poly I:C), TLR4 (lipopolysaccharide; LPS), TLR5 (flagellin) and TLR9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1β, IL-6, IL-8, TNFα, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1β, IL-6 and TNFα responses following TLR2, and IL-1β response following TLR4 stimulation in monocyte cultures from children with ASD (p<0.04). Conversely, following TLR9 stimulation there was a decrease in IL-1β, IL-6, GM-CSF and TNFα responses in monocyte cell cultures from children with ASD compared with controls (p<0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD. PMID:19666104

Differential monocyte responses to TLR ligands in children with autism spectrum disorders.

PubMed

Enstrom, Amanda M; Onore, Charity E; Van de Water, Judy A; Ashwood, Paul

2010-01-01

Autism spectrum disorders (ASD) are characterized by impairment in social interactions, communication deficits, and restricted repetitive interests and behaviors. Recent evidence has suggested that impairments of innate immunity may play an important role in ASD. To test this hypothesis, we isolated peripheral blood monocytes from 17 children with ASD and 16 age-matched typically developing (TD) controls and stimulated these cell cultures in vitro with distinct toll-like receptors (TLR) ligands: TLR 2 (lipoteichoic acid; LTA), TLR 3 (poly I:C), TLR 4 (lipopolysaccharide; LPS), TLR 5 (flagellin), and TLR 9 (CpG-B). Supernatants were harvested from the cell cultures and pro-inflammatory cytokine responses for IL-1beta, IL-6, IL-8, TNFalpha, MCP-1, and GM-CSF were determined by multiplex Luminex analysis. After in vitro challenge with TLR ligands, differential cytokine responses were observed in monocyte cultures from children with ASD compared with TD control children. In particular, there was a marked increase in pro-inflammatory IL-1beta, IL-6, and TNFalpha responses following TLR 2, and IL-1beta response following TLR 4 stimulation in monocyte cultures from children with ASD (p<0.04). Conversely, following TLR 9 stimulation there was a decrease in IL-1beta, IL-6, GM-CSF, and TNFalpha responses in monocyte cell cultures from children with ASD compared with controls (p<0.05). These data indicate that, monocyte cultures from children with ASD are more responsive to signaling via select TLRs. As monocytes are key regulators of the immune response, dysfunction in the response of these cells could result in long-term immune alterations in children with ASD that may lead to the development of adverse neuroimmune interactions and could play a role in the pathophysiology observed in ASD.

The Fanconi anemia protein interaction network: casting a wide net.

PubMed

Rego, Meghan A; Kolling, Frederick W; Howlett, Niall G

2009-07-31

It has long been hypothesized that a defect in the repair of damaged DNA is central to the etiology of Fanconi anemia (FA). Indeed, an increased sensitivity of FA patient-derived cells to the lethal effects of various forms of DNA damaging agents was described over three decades ago [A.J. Fornace, Jr., J.B. Little, R.R. Weichselbaum, DNA repair in a Fanconi's anemia fibroblast cell strain, Biochim. Biophys. Acta 561 (1979) 99-109; Y. Fujiwara, M. Tatsumi, Repair of mitomycin C damage to DNA in mammalian cells and its impairment in Fanconi's anemia cells, Biochem. Biophys. Res. Commun. 66 (1975) 592-598; A.J. Rainbow, M. Howes, Defective repair of ultraviolet- and gamma-ray-damaged DNA in Fanconi's anaemia, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 31 (1977) 191-195]. Furthermore, the cytological hallmark of FA, the DNA crosslink-induced radial chromosome formation, exemplifies an innate impairment in the repair of these particularly cytotoxic DNA lesions [A.D. Auerbach, Fanconi anemia diagnosis and the diepoxybutane (DEB) test, Exp. Hematol. 21 (1993) 731-733]. Precisely defining the collective role of the FA proteins in DNA repair, however, continues to be one of the most enigmatic and challenging questions in the FA field. The first six identified FA proteins (A, C, E, F, G, and D2) harbored no recognizable enzymatic features, precluding association with a specific metabolic process. Consequently, our knowledge of the role of the FA proteins in the DNA damage response has been gleaned primarily through biochemical association studies with non-FA proteins. Here, we provide a chronological discourse of the major FA protein interaction network discoveries, with particular emphasis on the DNA damage response, that have defined our current understanding of the molecular basis of FA.

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets

PubMed Central

Pan, Yanfang; Yago, Tadayuki; Fu, Jianxin; Herzog, Brett; McDaniel, J. Michael; Mehta-D’Souza, Padmaja; Cai, Xiaofeng; Ruan, Changgeng; McEver, Rodger P.; West, Christopher; Dai, Kesheng; Chen, Hong

2014-01-01

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan–deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan–deficient endothelial cells, but not on wild-type ECs. The lymph from mice with sepsis induced by cecal ligation and puncture, which contained bacteria-derived sialidase, reduced PDPN levels on wild-type ECs. The MMP inhibitor, GM6001, rescued these reductions. Additionally, GM6001 treatment rescued the reduction of PDPN level on lymphatic endothelial cells in mice lacking endothelial core 1 O-glycan or cecal ligation and puncture-treated mice. Furthermore, core 1 O-glycan–deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by MMPs in the lymph. PMID:25336627

Small synthetic peptides homologous to segments of the first external loop of occludin impair tight junction resealing.

PubMed

Lacaz-Vieira, F; Jaeger, M M; Farshori, P; Kachar, B

1999-04-01

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca2+ removal and was characterized by a marked drop of TER. The reintroduction of Ca2+ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca2+ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions.

Inflammatory T helper 17 cells promote depression-like behavior in mice.

PubMed

Beurel, Eléonore; Harrington, Laurie E; Jope, Richard S

2013-04-01

Recognition of substantial immune-neural interactions is revising dogmas about their insular actions and revealing that immune-neural interactions can substantially impact central nervous system functions. The inflammatory cytokine interleukin-6 promotes susceptibility to depression and drives production of inflammatory T helper 17 (Th17) T cells, raising the hypothesis that in mouse models, Th17 cells promote susceptibility to depression-like behaviors. Behavioral characteristics were measured in male mice administered Th17 cells, CD4(+) cells, or vehicle and in retinoid-related orphan receptor-γT (RORγT)(+/GFP) mice or male mice treated with RORγT inhibitor or anti-interleukin-17A antibodies. Mouse brain Th17 cells were elevated by learned helplessness and chronic restraint stress, two common depression-like models. Th17 cell administration promoted learned helplessness in 89% of mice in a paradigm where no vehicle-treated mice developed learned helplessness, and impaired novelty suppressed feeding and social interaction behaviors. Mice deficient in the RORγT transcription factor necessary for Th17 cell production exhibited resistance to learned helplessness, identifying modulation of RORγT as a potential intervention. Treatment with the RORγT inhibitor SR1001, or anti-interleukin-17A antibodies to abrogate Th17 cell function, reduced Th17-dependent learned helplessness. These findings indicate that Th17 cells are increased in the brain during depression-like states, promote depression-like behaviors in mice, and specifically inhibiting the production or function of Th17 cells reduces vulnerability to depression-like behavior, suggesting antidepressant effects may be attained by targeting Th17 cells. Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Bcl-2-interacting mediator of cell death influences autoantigen-driven deletion and TCR revision.

PubMed

Hale, J Scott; Nelson, Lisa T; Simmons, Kalynn B; Fink, Pamela J

2011-01-15

Peripheral CD4(+)Vβ5(+) T cells are tolerized to an endogenous mouse mammary tumor virus superantigen either by deletion or TCR revision. Through TCR revision, RAG reexpression mediates extrathymic TCRβ rearrangement and results in a population of postrevision CD4(+)Vβ5(-) T cells expressing revised TCRβ chains. We have hypothesized that cell death pathways regulate the selection of cells undergoing TCR revision to ensure the safety and utility of the postrevision population. In this study, we investigate the role of Bcl-2-interacting mediator of cell death (Bim)-mediated cell death in autoantigen-driven deletion and TCR revision. Bim deficiency and Bcl-2 overexpression in Vβ5 transgenic (Tg) mice both impair peripheral deletion. Vβ5 Tg Bim-deficient and Bcl-2 Tg mice exhibit an elevated frequency of CD4(+) T cells expressing both the transgene-encoded Vβ5 chain and a revised TCRβ chain. We now show that these dual-TCR-expressing cells are TCR revision intermediates and that the population of RAG-expressing, revising CD4(+) T cells is increased in Bim-deficient Vβ5 Tg mice. These findings support a role for Bim and Bcl-2 in regulating the balance of survival versus apoptosis in peripheral T cells undergoing RAG-dependent TCR rearrangements during TCR revision, thereby ensuring the utility of the postrevision repertoire.

AMPK activators suppress breast cancer cell growth by inhibiting DVL3-facilitated Wnt/β-catenin signaling pathway activity.

PubMed

Zou, Yu-Feng; Xie, Chun-Wei; Yang, Shi-Xin; Xiong, Jian-Ping

2017-02-01

Adenosine monophosphate-activated protein kinase (AMPK) is a principal regulator of metabolism and the conservation of energy in cells, and protects them from exposure to various stressors. AMPK activators may exhibit therapeutic potential as suppressors of cell growth; however, the molecular mechanism underlying this phenomenon in various cancer cells remains to be fully elucidated. The present study investigated the effects of AMPK activators on breast cancer cell growth and specified the underlying molecular mechanism. In the present study, the AMPK activator metformin impaired breast cancer cell growth by reducing dishevelled segment polarity protein 3 (DVL3) and β‑catenin levels. Western blotting and immunohistochemistry demonstrated that DVL3 was recurrently upregulated in breast cancer cells that were not treated with metformin, and was significantly associated with enhanced levels of β‑catenin, c‑Myc and cyclin D1. Overexpression of DVL3 resulted in upregulation of β‑catenin and amplification of breast cancer cell growth, which confirmed that Wnt/β‑catenin activation via DVL3 is associated with breast cancer oncogenesis. To elucidate the underlying mechanism of these effects, the present study verified that metformin resulted in a downregulation of DVL3 and β‑catenin in a dose‑dependent manner, and induced phosphorylation of AMPK. Compound C is an AMPK inhibitor, which when administered alongside metformin, significantly abolished the effects of metformin on the reduction of DVL3 and activation of the phosphorylation of AMPK. Notably, the effects of metformin on the mRNA expression levels of DVL3 remain to be fully elucidated; however, a possible interaction with DVL3 at the post‑transcriptional level was observed. It has previously been suggested that the molecular mechanism underlying AMPK activator‑induced suppression of breast cancer cell growth involves an interaction with, and impairment of, DVL3 proteins. The results of the present study are of future clinical importance and advocate the use of metformin as a potential therapeutic agent against breast cancer.

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Myc requires RhoA/SRF to reprogram glutamine metabolism.

PubMed

Haikala, Heidi M; Marques, Elsa; Turunen, Mikko; Klefström, Juha

2018-05-04

RhoA regulates actin cytoskeleton but recent evidence suggest a role for this conserved Rho GTPase also in other cellular processes, including transcriptional control of cell proliferation and survival. Interestingy, loss of RhoA is synthetic lethal with oncogenic Myc, a master transcription factor that turns on anabolic metabolism to promote cell growth in many cancers. We show evidence indicating that the synthetic lethal interaction between RhoA loss and Myc arises from deficiency in glutamine utilization, resulting from impaired co-regulation of glutaminase expression and anaplerosis by Myc and RhoA - serum response factor (SRF) pathway. The results suggest metabolic coordination between Myc and RhoA/SRF in sustaining cancer cell viability and indicate RhoA/SRF as a potential vulnerability in cancer cells for therapeutic targeting.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smirnov, Sergey V.; Harbacheuski, Ryhor; Lewis-Antes, Anita

Mesenchymal stem cells (MSCs) in bone marrow (BM) regulate the differentiation and proliferation of adjacent hematopoietic precursor cells and contribute to the regeneration of mesenchymal tissues, including bone, cartilage, fat and connective tissue. BM is an important site for the pathogenesis of human cytomegalovirus (HCMV) where the virus establishes latency in hematopoietic progenitors and can transmit after reactivation to neighboring cells. Here we demonstrate that BM-MSCs are permissive to productive HCMV infection, and that HCMV alters the function of MSCs: (i) by changing the repertoire of cell surface molecules in BM-MSCs, HCMV modifies the pattern of interaction between BM-MSCs andmore » hematopoietic cells; (ii) HCMV infection of BM-MSCs undergoing adipogenic or osteogenic differentiation impaired the process of differentiation. Our results suggest that by altering BM-MSC biology, HCMV may contribute to the development of various diseases.« less

Association of neck circumference and cognitive impairment among Chinese elderly.

PubMed

Chen, Jin-Mei; Li, Qing-Wei; Jiang, Guo-Xin; Zeng, Shu-Jun; Shen, Jun; Sun, Ji; Wu, Dan-Hong; Cheng, Qi

2018-03-01

To investigate the association between neck circumference (NC) and cognitive impairment and interactions between relevant variables to the risk of cognitive impairment. A population-based survey was conducted among elderly inhabitants aged 60 years and over from a community in Shanghai suburb. Multivariate logistic regression analyses were performed to evaluate associations and log likelihood ratio tests to examine interactions. Cognitive impairment was identified in 269 (10.8%) subjects from 2,500 participants. Higher BMI (OR = 1.55; 95% CI = 1.11-2.16), higher WHR (OR = 1.44; 95% CI = 1.07-1.95), and higher total cholesterol (TC) (OR = 1.52; 95% CI = 1.09-2.13) were significantly associated with the increased risk of cognitive impairment. Significant interactions were observed between TC and a few other relevant variables, respectively. NC was associated with the high risk of cognitive impairment. Additive effects of NC with TC on cognitive impairment were observed.

Matrix Metalloproteinase 3 Promotes Cellular Anti-Dengue Virus Response via Interaction with Transcription Factor NFκB in Cell Nucleus

PubMed Central

Zuo, Xiangyang; Pan, Wen; Feng, Tingting; Shi, Xiaohong; Dai, Jianfeng

2014-01-01

Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation. PMID:24416274

Repression of HNF1α-mediated transcription by amino-terminal enhancer of split (AES)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Eun Hee; Gorman, Amanda A.; Singh, Puja

2015-12-04

HNF1α (Hepatocyte Nuclear Factor 1α) is one of the master regulators in pancreatic beta-cell development and function, and the mutations in Hnf1α are the most common monogenic causes of diabetes mellitus. As a member of the POU transcription factor family, HNF1α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge in their functional complex formation. In this study, we identified the Groucho protein AES (Amino-terminal Enhancer of Split) as a HNF1α-specific physical binding partner and functional repressor of HNF1α-mediated transcription, which has a direct link to glucose-stimulated insulin secretion in beta-cells that ismore » impaired in the HNF1α mutation-driven diabetes. - Highlights: • We identified AES as a transcriptional repressor for HNF1α in pancreatic beta-cell. • AES's repressive activity was HNF1α-specific and was not observed with HNF1β. • AES interacts with the transactivation domain of HNF1α. • Small molecules can be designed or discovered to disrupt this interaction and improve insulin secretion and glucose homeostasis.« less

Polyphenol Compound as a Transcription Factor Inhibitor.

PubMed

Park, Seyeon

2015-10-30

A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor-DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein-protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)).

Relationship of ethnicity and CD4 Count with glucose metabolism among HIV patients on Highly-Active Antiretroviral Therapy (HAART)

PubMed Central

2013-01-01

Background HIV patients on HAART are prone to metabolic abnormalities, including insulin resistance, lipodystrophy and diabetes. This study purports to investigate the relationship of ethnicity and CD4+ T cell count attained after stable highly-active antiretroviral treatment (HAART) with glucose metabolism in hyperrtriglyceridemic HIV patients without a history of diabetes. Methods Demographic, anthropometric, clinical, endocrinologic, energy expenditure and metabolic measures were obtained in 199 multiethnic, healthy but hypertriglyceridemic HIV-infected patients [46% Hispanic, 17% African-American, 37% Non-Hispanic White (NHW)] on stable HAART without a history of diabetes. The relationship of glucose and insulin responses to ethnicity, CD4 strata (low (<300/cc) or moderate-to-high (≥ 300/cc)), and their interaction was determined. Results African-Americans had significantly greater impairment of glucose tolerance (P < 0.05) and HbA1c levels (P < .001) than either Hispanics or NHWs. In multivariate models, after adjusting for confounders (age, sex, HIV/HAART duration, smoking, obesity, glucose, insulin and lipids), African-Americans and Hispanics had significantly higher HbA1c and 2-hour glucose levels than NHW’s. Demonstrating a significant interaction between ethnicity and CD4 count (P = 0.023), African Americans with CD4 <300/cc and Hispanics with CD4 ≥300/cc had the most impaired glucose response following oral glucose challenge. Conclusions Among hypertriglyceridemic HIV patients on HAART, African-Americans and Hispanics are at increased risk of developing diabetes. Ethnicity also interacts with CD4+ T cell count attained on stable HAART to affect post-challenge glycemic response. PMID:23607267

p38 Mitogen-Activated Protein Kinase/Signal Transducer and Activator of Transcription-3 Pathway Signaling Regulates Expression of Inhibitory Molecules in T Cells Activated by HIV-1–Exposed Dendritic Cells

PubMed Central

Che, Karlhans Fru; Shankar, Esaki Muthu; Muthu, Sundaram; Zandi, Sasan; Sigvardsson, Mikael; Hinkula, Jorma; Messmer, Davorka; Larsson, Marie

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1–primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules. PMID:22777388

Modeling Parkinson's disease falls associated with brainstem cholinergic systems decline.

PubMed

Kucinski, Aaron; Sarter, Martin

2015-04-01

In addition to the primary disease-defining symptoms, approximately half of patients with Parkinson's disease (PD) suffer from postural instability, impairments in gait control and a propensity for falls. Consistent with evidence from patients, we previously demonstrated that combined striatal dopamine (DA) and basal forebrain (BF) cholinergic cell loss causes falls in rats traversing dynamic surfaces. Because evidence suggests that degeneration of brainstem cholinergic neurons arising from the pedunculopontine nucleus (PPN) also contributes to impaired gait and falls, here we assessed the effects of selective cholinergic PPN lesions in combination with striatal DA loss or BF cholinergic cells loss as well as losses in all 3 regions. Results indicate that all combination losses that included the BF cholinergic system slowed traversal and increased slips and falls. However, the performance of rats with losses in all 3 regions (PPN, BF, and DA) was not more severely impaired than following combined BF cholinergic and striatal DA lesions. These results confirm the hypothesis that BF cholinergic-striatal disruption of attentional-motor interactions is a primary source of falls. Additional losses of PPN cholinergic neurons may worsen posture and gait control in situations not captured by the current testing conditions. (PsycINFO Database Record (c) 2015 APA, all rights reserved).

A peptide representing the carboxyl-terminal tail of the met receptor inhibits kinase activity and invasive growth.

PubMed

Bardelli, A; Longati, P; Williams, T A; Benvenuti, S; Comoglio, P M

1999-10-08

Interaction of the hepatocyte growth factor (HGF) with its receptor, the Met tyrosine kinase, results in invasive growth, a genetic program essential to embryonic development and implicated in tumor metastasis. Met-mediated invasive growth requires autophosphorylation of the receptor on tyrosines located in the kinase activation loop (Tyr(1234)-Tyr(1235)) and in the carboxyl-terminal tail (Tyr(1349)-Tyr(1356)). We report that peptides derived from the Met receptor tail, but not from the activation loop, bind the receptor and inhibit the kinase activity in vitro. Cell delivery of the tail receptor peptide impairs HGF-dependent Met phosphorylation and downstream signaling. In normal and transformed epithelial cells, the tail receptor peptide inhibits HGF-mediated invasive growth, as measured by cell migration, invasiveness, and branched morphogenesis. The Met tail peptide inhibits the closely related Ron receptor but does not significantly affect the epidermal growth factor, platelet-derived growth factor, or vascular endothelial growth factor receptor activities. These experiments show that carboxyl-terminal sequences impair the catalytic properties of the Met receptor, thus suggesting that in the resting state the nonphosphorylated tail acts as an intramolecular modulator. Furthermore, they provide a strategy to selectively target the MET proto-oncogene by using small, cell-permeable, peptide derivatives.

Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

PubMed

Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

2014-07-15

Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

Interaction of Macrophage Antigen 1 and CD40 Ligand Leads to IL-12 Production and Resistance in CD40-Deficient Mice Infected with Leishmania major.

PubMed

Okwor, Ifeoma; Jia, Ping; Uzonna, Jude E

2015-10-01

Although some studies indicate that the interaction of CD40 and CD40L is critical for IL-12 production and resistance to cutaneous leishmaniasis, others suggest that this pathway may be dispensable. In this article, we compared the outcome of Leishmania major infection in both CD40- and CD40L-deficient mice after treatment with rIL-12. We show that although CD40 and CD40L knockout (KO) mice are highly susceptible to L. major, treatment with rIL-12 during the first 2 wk of infection causes resolution of cutaneous lesions and control of parasite replication. Interestingly, although treated CD40 KO mice remained healed, developed long-term immunity, and were resistant to secondary L. major challenge, treated CD40L KO reactivated their lesion after cessation of rIL-12 treatment. Disease reactivation in CD40L KO mice was associated with impaired IL-12 and IFN-γ production and a concomitant increase in IL-4 production by cells from lymph nodes draining the infection site. We show that IL-12 production by dendritic cells and macrophages via CD40L-macrophage Ag 1 (Mac-1) interaction is responsible for the sustained resistance in CD40 KO mice after cessation of rIL-12 treatment. Blockade of CD40L-Mac-1 interaction with anti-Mac-1 mAb led to spontaneous disease reactivation in healed CD40 KO mice, which was associated with impaired IFN-γ response and loss of infection-induced immunity after secondary L. major challenge. Collectively, our data reveal a novel role of CD40L-Mac-1 interaction in IL-12 production, development, and maintenance of optimal Th1 immunity in mice infected with L. major. Copyright © 2015 by The American Association of Immunologists, Inc.

Biocompatibility of tungsten disulfide inorganic nanotubes and fullerene-like nanoparticles with salivary gland cells.

PubMed

Goldman, Elisheva B; Zak, Alla; Tenne, Reshef; Kartvelishvily, Elena; Levin-Zaidman, Smadar; Neumann, Yoav; Stiubea-Cohen, Raluca; Palmon, Aaron; Hovav, Avi-Hai; Aframian, Doron J

2015-03-01

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.

Biocompatibility of Tungsten Disulfide Inorganic Nanotubes and Fullerene-Like Nanoparticles with Salivary Gland Cells

PubMed Central

Goldman, Elisheva B.; Zak, Alla; Tenne, Reshef; Kartvelishvily, Elena; Levin-Zaidman, Smadar; Neumann, Yoav; Stiubea-Cohen, Raluca; Palmon, Aaron; Hovav, Avi-Hai

2015-01-01

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles. PMID:25366879

Interactive effects of diabetes and impaired kidney function on cognitive performance in old age: a population-based study.

PubMed

Yin, Zhaoxue; Yan, Zhongrui; Liang, Yajun; Jiang, Hui; Cai, Chuanzhu; Song, Aiqin; Feng, Lei; Qiu, Chengxuan

2016-01-12

The interactive effect between diabetes and impaired kidney function on cognitive impairment in older adults has not yet been reported. The aim of this study was to investigate the association of diabetes and impaired kidney function with cognitive impairment among Chinese older people living in a rural area. This cross-sectional study included 1,358 participants (age ≥60 years; 60.5% women) in the population-based Confucius Hometown Aging Project in Shandong, China. Data on demographics, lifestyle factors, health history, use of medications, global cognitive function, and kidney function were collected through structured interviews, clinical examinations, and blood tests. We defined diabetes as a fasting plasma glucose level ≥7.0 mmol/l or use of hypoglycemic agents, impaired kidney function as glomerular filtration rate estimated from cystatin C (eGFRcys) <60 ml/min/1.73 m(2). Cognitive impairment was defined using the education-based cut-off scores of Mini-Mental State Examination (MMSE). Data were analyzed using multiple general linear and logistic regression models. Cognitive impairment was defined in 197 (14.5%) persons. The multi-adjusted β coefficient of MMSE score associated with diabetes was -0.06 (95% confidence interval [CI], -0.16, 0.03); the corresponding figures associated with eGFRcys <60, 60-89.9, and ≥90 ml/min/1.73 m(2) were -0.15 (-0.28, -0.02), -0.01 (-0.10, 0.08), and 0 (reference) (Ptrend = 0.046), respectively. Diabetes and impaired kidney function showed an interactive effect on cognitive impairment ( interaction = 0.02). Compared with individuals having neither diabetes nor impaired kidney function, those with both conditions had a multi-adjusted odds ratio of 4.23 (95% CI, 2.10-8.49) for cognitive impairment. The relative excess risk due to interaction was 2.74. This study suggests that concurrent presence of diabetes and impaired kidney function is associated with a substantial likelihood for cognitive impairment in older adults.

Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation

PubMed Central

Balashova, Olga A.; Visina, Olesya

2017-01-01

Folate supplementation prevents up to 70% of neural tube defects (NTDs), which result from a failure of neural tube closure during embryogenesis. The elucidation of the mechanisms underlying folate action has been challenging. This study introduces Xenopus laevis as a model to determine the cellular and molecular mechanisms involved in folate action during neural tube formation. We show that knockdown of folate receptor 1 (Folr1; also known as FRα) impairs neural tube formation and leads to NTDs. Folr1 knockdown in neural plate cells only is necessary and sufficient to induce NTDs. Folr1-deficient neural plate cells fail to constrict, resulting in widening of the neural plate midline and defective neural tube closure. Pharmacological inhibition of folate action by methotrexate during neurulation induces NTDs by inhibiting folate interaction with its uptake systems. Our findings support a model in which the folate receptor interacts with cell adhesion molecules, thus regulating the apical cell membrane remodeling and cytoskeletal dynamics necessary for neural plate folding. Further studies in this organism could unveil novel cellular and molecular events mediated by folate and lead to new ways of preventing NTDs. PMID:28255006

CRISPR-Cas9 Mediated Telomere Removal Leads to Mitochondrial Stress and Protein Aggregation.

PubMed

Kim, Hyojung; Ham, Sangwoo; Jo, Minkyung; Lee, Gum Hwa; Lee, Yun-Song; Shin, Joo-Ho; Lee, Yunjong

2017-10-03

Aging is considered the major risk factor for neurodegenerative diseases including Parkinson's disease (PD). Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD.

Characterization of resistance to rhabdovirus and retrovirus infection in a human myeloid cell line.

PubMed

Boso, Guney; Somia, Nikunj V

2015-01-01

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.

The effects of antibodies on cells

PubMed Central

Dumonde, D. C.; Bitensky, Lucille; Cunningham, G. J.; Chayen, J.

1965-01-01

Biochemical and histochemical methods were used to study the interaction of antibodies and complement with mouse Ehrlich ascites tumour cells. In the presence of complement, both iso- and hetero-antibodies caused cell lysis with penetration of antibodies into the damaged cells, as detected by immunofluorescence; the cells were then unable to support aerobic glycolysis though they retained their ability to consume oxygen in the presence of succinate. Under these conditions there was unmasking of phospholipid particularly at the cell surface, together with lysosomal changes resulting in diffuse staining for lysosomal acid—phosphatase. In the absence of complement, antibodies did not appear to penetrate the cells which respired normally and were not lysed. However, in these cells there was intense lysosomal activation accompanied by unmasking of cytoplasmic phospholipid; it appeared that an immune reaction confined to the cell surface was able to induce changes in the cytoplasm without acutely impairing the viability of the cell. ImagesFIGS. 1-8FIGS. 9-14 PMID:14245309

Cross-over endocytosis of claudins is mediated by interactions via their extracellular loops.

PubMed

Gehne, Nora; Lamik, Agathe; Lehmann, Martin; Haseloff, Reiner F; Andjelkovic, Anuska V; Blasig, Ingolf E

2017-01-01

Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.

Social Skills of Children with Specific Language Impairment: Peer Relationships.

ERIC Educational Resources Information Center

Craig, Holly K.

1993-01-01

This paper reviews literature on peer relationships of children with specific language impairment and provides a clinical case to illustrate the social-interactional difficulties encountered. The paper analyzes the quantity and quality of peer interactions, child's knowledge of interactive access behaviors, disputes, responsiveness, assertiveness,…

Multi-chaperone function modulation and association with cytoskeletal proteins are key features of the function of AIP in the pituitary gland

PubMed Central

Hernández-Ramírez, Laura C.; Morgan, Rhodri M.L.; Barry, Sayka; D’Acquisto, Fulvio; Prodromou, Chrisostomos; Korbonits, Márta

2018-01-01

Despite the well-recognized role of loss-of-function mutations of the aryl hydrocarbon receptor interacting protein gene (AIP) predisposing to pituitary adenomas, the pituitary-specific function of this tumor suppressor remains an enigma. To determine the repertoire of interacting partners for the AIP protein in somatotroph cells, wild-type and variant AIP proteins were used for pull-down/quantitative mass spectrometry experiments against lysates of rat somatotropinoma-derived cells; relevant findings were validated by co-immunoprecipitation and co-localization. Global gene expression was studied in AIP mutation positive and negative pituitary adenomas via RNA microarrays. Direct interaction with AIP was confirmed for three known and six novel partner proteins. Novel interactions with HSPA5 and HSPA9, together with known interactions with HSP90AA1, HSP90AB1 and HSPA8, indicate that the function/stability of multiple chaperone client proteins could be perturbed by a deficient AIP co-chaperone function. Interactions with TUBB, TUBB2A, NME1 and SOD1 were also identified. The AIP variants p.R304* and p.R304Q showed impaired interactions with HSPA8, HSP90AB1, NME1 and SOD1; p.R304* also displayed reduced binding to TUBB and TUBB2A, and AIP-mutated tumors showed reduced TUBB2A expression. Our findings suggest that cytoskeletal organization, cell motility/adhesion, as well as oxidative stress responses, are functions that are likely to be involved in the tumor suppressor activity of AIP. PMID:29507682

Commensal-innate immune miscommunication in IBD pathogenesis.

PubMed

Cario, Elke

2012-01-01

Commensal microbiota plays a key role in the health and disease of the host. The innate immune system comprises an essential functional component of the intestinal mucosal barrier, maintaining hyporesponsiveness to omnipresent harmless commensals in the lumen, but rapidly recognizing and combating invading bacteria through diverse antimicrobial mechanisms. Interactions between commensals and innate immune cells are constant, multidimensional and entirely context-dependent. Environment, genetics and host defense differentially modulate commensal-innate immune effects and functions in the intestinal mucosa. In IBD, dysbiosis, mucus layer disruption, impairment in bacterial clearance, intestinal epithelial cell barrier dysfunction and/or immune cell deregulation may lead to commensal-innate immune miscommunication, which critically drives mucosal inflammation and associated cancer. Copyright © 2012 S. Karger AG, Basel.

Neuro-immune dysfunction during brain aging: new insights in microglial cell regulation.

PubMed

Matt, Stephanie M; Johnson, Rodney W

2016-02-01

Microglia, the resident immune cells of the brain, are at the center of communication between the central nervous system and immune system. While these brain-immune interactions are balanced in healthy adulthood, the ability to maintain homeostasis during aging is impaired. Microglia develop a loss of integrated regulatory networks including aberrant signaling from other brain cells, immune sensors, and epigenetic modifiers. The low-grade chronic neuroinflammation associated with this dysfunctional activity likely contributes to cognitive deficits and susceptibility to age-related pathologies. A better understanding of the underlying mechanisms responsible for neuro-immune dysregulation with age is crucial for providing targeted therapeutic strategies to support brain repair and healthy aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

The ubiquitin ligase Mdm2 controls oligodendrocyte maturation by intertwining mTOR with G protein-coupled receptor kinase 2 in the regulation of GPR17 receptor desensitization.

PubMed

Fumagalli, Marta; Bonfanti, Elisabetta; Daniele, Simona; Zappelli, Elisa; Lecca, Davide; Martini, Claudia; Trincavelli, Maria L; Abbracchio, Maria P

2015-12-01

During oligodendrocyte precursor cell (OPC) differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impair remyelination under demyelinating conditions. After the immature oligodendrocyte stage, to enable cells to complete maturation, GPR17 is physiologically down-regulated via phosphorylation/desensitization by G protein-coupled receptor kinases (GRKs); conversely, GRKs are regulated by the "mammalian target of rapamycin" mTOR. However, how GRKs and mTOR are connected to each other in modulating GPR17 function and oligodendrogenesis has remained elusive. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of the onco-suppressor p53 protein. In maturing OPCs, both rapamycin and Nutlin-3, a small molecule inhibitor of Mdm2-p53 interactions, increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. Thus, Mdm2 intertwines mTOR with GRK2 in regulating GPR17 and oligodendrogenesis and represents a novel actor in myelination. © 2015 Wiley Periodicals, Inc.

A new Caenorhabditis elegans model of human huntingtin 513 aggregation and toxicity in body wall muscles.

PubMed

Lee, Amy L; Ung, Hailey M; Sands, L Paul; Kikis, Elise A

2017-01-01

Expanded polyglutamine repeats in different proteins are the known determinants of at least nine progressive neurodegenerative disorders whose symptoms include cognitive and motor impairment that worsen as patients age. One such disorder is Huntington's Disease (HD) that is caused by a polyglutamine expansion in the human huntingtin protein (htt). The polyglutamine expansion destabilizes htt leading to protein misfolding, which in turn triggers neurodegeneration and the disruption of energy metabolism in muscle cells. However, the molecular mechanisms that underlie htt proteotoxicity have been somewhat elusive, and the muscle phenotypes have not been well studied. To generate tools to elucidate the basis for muscle dysfunction, we engineered Caenorhabditis elegans to express a disease-associated 513 amino acid fragment of human htt in body wall muscle cells. We show that this htt fragment aggregates in C. elegans in a polyglutamine length-dependent manner and is toxic. Toxicity manifests as motor impairment and a shortened lifespan. Compared to previous models, the data suggest that the protein context in which a polyglutamine tract is embedded alters aggregation propensity and toxicity, likely by affecting interactions with the muscle cell environment.

CHLORPYRIFOS DEVELOPMENTAL NEUROTOXICITY: INTERACTION WITH GLUCOCORTICOIDS IN PC12 CELLS

PubMed Central

Slotkin, Theodore A.; Card, Jennifer; Seidler, Frederic J.

2012-01-01

Prenatal coexposures to glucocorticoids and organophosphate pesticides are widespread. Glucocorticoids are elevated by maternal stress and are commonly given in preterm labor; organophosphate exposures are virtually ubiquitous. We used PC12 cells undergoing neurodifferentiation in order to assess whether dexamethasone enhances the developmental neurotoxicity of chlorpyrifos, focusing on concentrations relevant to human exposures. By themselves, each agent reduced the number of cells and the combined exposure elicited a correspondingly greater effect than with either agent alone. There was no general cytotoxicity, as cell growth was actually enhanced, and again, the combined treatment evoked greater cellular hypertrophy than with the individual compounds. The effects on neurodifferentiation were more complex. Chlorpyrifos alone had a promotional effect on neuri to genesis whereas dexamethasone impaired it; combined treatment showed an overall impairment greater than that seen with dexamethasone alone. The effect of chlorpyrifos on differentiation into specific neurotransmitter phenotypes was shifted by dexamethasone. Either agent alone promoted differentiation into the dopaminergic phenotype at the expense of the cholinergic phenotype. However, in dexamethasone-primed cells, chlorpyrifos actually enhanced cholinergic neurodifferentiation instead of suppressing this phenotype. Our results indicate that developmental exposure to glucocorticoids, either in the context of stress or the therapy of preterm labor, could enhance the developmental neurotoxicity of organophosphates and potentially of other neurotoxicants, as well as producing neurobehavioral outcomes distinct from those seen with either individual agent. PMID:22796634

Severe Impairments of Social Interaction and Associated Abnormalities in Children: Epidemiology and Classification.

ERIC Educational Resources Information Center

Wing, Lorna; Gould, Judith

1979-01-01

The prevalence of severe impairments of social interaction, language abnormalities, and repetitive stereotyped behaviors was investigated in a group of 132 children under 15 years old, consisting of a socially impaired group (more than half of whom were severely retarded) and a comparison group of sociable severely mentally retarded. Author/DLS)

3-Bromopyruvate induces rapid human prostate cancer cell death by affecting cell energy metabolism, GSH pool and the glyoxalase system.

PubMed

Valenti, Daniela; Vacca, Rosa A; de Bari, Lidia

2015-12-01

3-bromopyruvate (3-BP) is an anti-tumour drug effective on hepatocellular carcinoma and other tumour cell types, which affects both glycolytic and mitochondrial targets, depleting cellular ATP pool. Here we tested 3-BP on human prostate cancer cells showing, differently from other tumour types, efficient ATP production and functional mitochondrial metabolism. We found that 3-BP rapidly induced cultured androgen-insensitive (PC-3) and androgen-responsive (LNCaP) prostate cancer cell death at low concentrations (IC(50) values of 50 and 70 μM, respectively) with a multimodal mechanism of action. In particular, 3-BP-treated PC-3 cells showed a selective, strong reduction of glyceraldeide 3-phosphate dehydrogenase activity, due to the direct interaction of the drug with the enzyme. Moreover, 3-BP strongly impaired both glutamate/malate- and succinate-dependent mitochondrial respiration, membrane potential generation and ATP synthesis, concomitant with the inhibition of respiratory chain complex I, II and ATP synthase activities. The drastic reduction of cellular ATP levels and depletion of GSH pool, associated with significant increase in cell oxidative stress, were found after 3-BP treatment of PC-3 cells. Interestingly, the activity of both glyoxalase I and II, devoted to the elimination of the cytotoxic methylglyoxal, was strongly inhibited by 3-BP. Both N-acetylcysteine and aminoguanidine, GSH precursor and methylglyoxal scavenger, respectively, prevented 3-BP-induced PC-3 cell death, showing that impaired cell antioxidant and detoxifying capacities are crucial events leading to cell death. The provided information on the multi-target cytotoxic action of 3-BP, finally leading to PC-3 cell necrosis, might be useful for future development of 3-BP as a therapeutic option for prostate cancer treatment.

Rac1 GTPase -deficient mouse lens exhibits defects in shape, suture formation, fiber cell migration and survival

PubMed Central

Maddala, Rupalatha; Chauhan, Bharesh K.; Walker, Christopher; Zheng, Yi; Robinson, Michael L.; Lang, Richard A.; Rao, Ponugoti V.

2011-01-01

Morphogenesis and shape of the ocular lens depend on epithelial cell elongation and differentiation into fiber cells, followed by the symmetric and compact organization of fiber cells within an enclosed extracellular matrix-enriched elastic capsule. The cellular mechanisms orchestrating these different events however, remain obscure. We investigated the role of the Rac1 GTPase in these processes by targeted deletion of expression using the conditional gene knockout (cKO) approach. Rac1 cKO mice were derived from two different Cre (Le-Cre and MLR-10) transgenic mice in which lens-specific Cre expression starts at embryonic day 8.75 and 10.5, respectively, in both the lens epithelium and fiber cells. The Le-Cre/Rac1 cKO mice exhibited an early-onset (E12.5) and severe lens phenotype compared to the MLR-10/Rac1 cKO (E15.5) mice. While the Le-Cre/Rac1 cKO lenses displayed delayed primary fiber cell elongation, lenses from both Rac1 cKO strains were characterized by abnormal shape, impaired secondary fiber cell migration, sutural defects and thinning of the posterior capsule which often led to rupture. Lens fiber cell N-cadherin/β-catenin/Rap1/Nectin-based cell-cell junction formation and WAVE-2/Abi-2/Nap1-regulated actin polymerization were impaired in the Rac1 deficient mice. Additionally, the Rac1 cKO lenses were characterized by a shortened epithelial sheet, reduced levels of extracellular matrix (ECM) proteins and increased apoptosis. Taken together, these data uncover the essential role of Rac1 GTPase activity in establishment and maintenance of lens shape, suture formation and capsule integrity, and in fiber cell migration, adhesion and survival, via regulation of actin cytoskeletal dynamics, cell adhesive interactions and ECM turnover. PMID:21945075

Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.

PubMed

Uchida, Hiroaki; Shah, Waris A; Ozuer, Ali; Frampton, Arthur R; Goins, William F; Grandi, Paola; Cohen, Justus B; Glorioso, Joseph C

2009-04-01

Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.

C-terminal domain of SMYD3 serves as a unique HSP90-regulated motif in oncogenesis

PubMed Central

Harriss, June; Das, Chhaya; Zhu, Li; Edwards, Melissa; Shaaban, Salam; Tucker, Haley

2015-01-01

The SMYD3 histone methyl transferase (HMTase) and the nuclear chaperone, HSP90, have been independently implicated as proto-oncogenes in several human malignancies. We show that a degenerate tetratricopeptide repeat (TPR)-like domain encoded in the SMYD3 C-terminal domain (CTD) mediates physical interaction with HSP90. We further demonstrate that the CTD of SMYD3 is essential for its basal HMTase activity and that the TPR-like structure is required for HSP90-enhanced enzyme activity. Loss of SMYD3-HSP90 interaction leads to SMYD3 mislocalization within the nucleus, thereby losing its chromatin association. This results in reduction of SMYD3-mediated cell proliferation and, potentially, impairment of SMYD3′s oncogenic activity. These results suggest a novel approach for blocking HSP90-driven malignancy in SMYD3-overexpressing cells with a reduced toxicity profile over current HSP90 inhibitors. PMID:25738358

Mammalian autophagy degrades nuclear constituents in response to tumorigenic stress.

PubMed

Dou, Zhixun; Ivanov, Andrejs; Adams, Peter D; Berger, Shelley L

2016-08-02

During autophagy, double-membrane autophagosomes are observed in the cytoplasm. Thus, extensive studies have focused on autophagic turnover of cytoplasmic material. Whether autophagy has a role in degrading nuclear constituents is poorly understood. We reveal that the autophagy protein LC3/Atg8 directly interacts with the nuclear lamina protein LMNB1 (lamin B1), and binds to LMN/lamin-associated chromatin domains (LADs). Through these interactions, autophagy specifically mediates destruction of nuclear lamina during tumorigenic stress, such as by activated oncogenes and DNA damage. This nuclear lamina degradation upon aberrant cellular stress impairs cell proliferation by inducing cellular senescence, a stable form of cell-cycle arrest and a tumor-suppressive mechanism. Our findings thus suggest that, in response to cancer-promoting stress, autophagy degrades nuclear material to drive cellular senescence, as a means to restrain tumorigenesis. Our work provokes a new direction in studying the role of autophagy in the nucleus and in tumor suppression.

Mechanistic Aspects of Adenovirus Serotype 2 Inactivation with Free Chlorine ▿ †

PubMed Central

Page, Martin A.; Shisler, Joanna L.; Mariñas, Benito J.

2010-01-01

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery. PMID:20305026

Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity.

PubMed

Teper, Doron; Salomon, Dor; Sunitha, Sukumaran; Kim, Jung-Gun; Mudgett, Mary Beth; Sessa, Guido

2014-01-01

Effector-triggered immunity (ETI) to host-adapted pathogens is associated with rapid cell death at the infection site. The plant-pathogenic bacterium Xanthomonas euvesicatoria (Xcv) interferes with plant cellular processes by injecting effector proteins into host cells through the type III secretion system. Here, we show that the Xcv effector XopQ suppresses cell death induced by components of the ETI-associated MAP kinase cascade MAPKKKα MEK2/SIPK and by several R/avr gene pairs. Inactivation of xopQ by insertional mutagenesis revealed that this effector inhibits ETI-associated cell death induced by avirulent Xcv in resistant pepper (Capsicum annuum), and enhances bacterial growth in resistant pepper and tomato (Solanum lycopersicum). Using protein-protein interaction studies in yeast (Saccharomyces cerevisiae) and in planta, we identified the tomato 14-3-3 isoform SlTFT4 and homologs from other plant species as XopQ interactors. A mutation in the putative 14-3-3 binding site of XopQ impaired interaction of the effector with CaTFT4 in yeast and its virulence function in planta. Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv. Silencing of NbTFT4 in Nicotiana benthamiana significantly reduced cell death induced by MAPKKKα. In addition, silencing of CaTFT4 in pepper delayed the appearance of ETI-associated cell death and enhanced growth of virulent and avirulent Xcv, demonstrating the requirement of TFT4 for plant immunity to Xcv. Our results suggest that the XopQ virulence function is to suppress ETI and immunity-associated cell death by interacting with TFT4, which is an important component of ETI and a bona fide target of XopQ. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

O Antigen Modulates Insect Vector Acquisition of the Bacterial Plant Pathogen Xylella fastidiosa

PubMed Central

Rapicavoli, Jeannette N.; Kinsinger, Nichola; Perring, Thomas M.; Backus, Elaine A.; Shugart, Holly J.; Walker, Sharon

2015-01-01

Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. PMID:26386068

Choline dehydrogenase interacts with SQSTM1/p62 to recruit LC3 and stimulate mitophagy.

PubMed

Park, Sungwoo; Choi, Seon-Guk; Yoo, Seung-Min; Son, Jin H; Jung, Yong-Keun

2014-01-01

CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, we report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. We found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, our results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.

O antigen modulates insect vector acquisition of the bacterial plant pathogen Xylella fastidiosa.

PubMed

Rapicavoli, Jeannette N; Kinsinger, Nichola; Perring, Thomas M; Backus, Elaine A; Shugart, Holly J; Walker, Sharon; Roper, M Caroline

2015-12-01

Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Polycystin-1 interacts with TAZ to stimulate osteoblastogenesis and inhibit adipogenesis

DOE PAGES

Xiao, Zhousheng; Baudry, Jerome; Cao, Li; ...

2017-11-27

The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. In this paper, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanicalmore » stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2–mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Therefore, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.« less

Models and mechanisms for hippocampal dysfunction in obesity and diabetes

PubMed Central

Stranahan, Alexis M.

2015-01-01

Clinical studies suggest that obesity and type 2 (insulin resistant) diabetes impair the structural integrity of medial temporal lobe regions involved in memory and confer greater vulnerability to neurological insults. While eliminating obesity and its endocrine comorbidities would be the most straightforward way to minimize cognitive risk, structural barriers to physical activity and the widespread availability of calorically dense, highly palatable foods will likely necessitate additional strategies to maintain brain health over the lifespan. Research in rodents has identified numerous correlates of hippocampal functional impairment in obesity and diabetes, with several studies demonstrating causality in subsequent mechanistic studies. This review highlights recent work on pathways and cell-cell interactions underlying the synaptic consequences of obesity, diabetes, or in models with both pathological conditions. Although the mechanisms vary across different animal models, immune activation has emerged as a shared feature of obesity and diabetes, with synergistic exacerbation of neuroinflammation in model systems with both conditions. This Review discusses these findings with reference to the benefits of incorporating existing models from the fields of obesity and metabolic disease. Many transgenic lines with basal metabolic alterations or differential susceptibility to diet-induced obesity have yet to be characterized with respect to their cognitive and synaptic phenotype. Adopting these models, and building on the extensive knowledge base used to generate them, is a promising avenue for understanding interactions between peripheral disease states and neurodegenerative disorders. PMID:25934036

Polycystin-1 interacts with TAZ to stimulate osteoblastogenesis and inhibit adipogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Zhousheng; Baudry, Jerome; Cao, Li

The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. In this paper, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanicalmore » stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2–mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Therefore, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.« less

Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

PubMed

Silva, Wellington Luiz Ferreira da; Pagliari, Carla; Duarte, Maria Irma Seixas; Sotto, Mirian N

2016-05-01

Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis.

PubMed

Soni, Shivani; Bala, Shashi; Hanspal, Manjit

2008-01-01

Emp, erythroblast-macrophage protein was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis in the fetal liver, and fetal demise. To further address the activity of Emp in the hematopoietic lineage in adult bone marrow, we conducted fetal liver HSC reconstitution assay. Emp null fetal liver cells were transplanted into lethally irradiated wild-type sibling mice, and assessed the erythropoietic activity. We found that Emp null cells rescued lethally irradiated mice with efficiency comparable to that of wild-type cells. However, the recipients of Emp null cells showed abnormal erythropoiesis as indicated by the presence of persistent anemia, extensive extramedullary erythropoiesis, and increased apoptosis of erythroid precursors. Extramedullary erythropoiesis suggests perturbed interactions between the Emp-deficient hematopoietic cells and the wild-type niche. Furthermore, in spleen colony-forming unit assays, proliferation rates of the Emp null cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Emp null livers. Morphologic examination showed that Emp null CFU-E-derived erythroblasts were immature compared to those derived from wild-type CFU-Es, suggesting that loss of Emp function in erythroid cells results in impaired proliferation and terminal differentiation. These results demonstrate that Emp plays a cell intrinsic role in the erythroid lineage.

A Cyclin T1 point mutation that abolishes positive transcription elongation factor (P-TEFb) binding to Hexim1 and HIV tat

PubMed Central

2014-01-01

Background The positive transcription elongation factor b (P-TEFb) plays an essential role in activating HIV genome transcription. It is recruited to the HIV LTR promoter through an interaction between the Tat viral protein and its Cyclin T1 subunit. P-TEFb activity is inhibited by direct binding of its subunit Cyclin T (1 or 2) with Hexim (1 or 2), a cellular protein, bound to the 7SK small nuclear RNA. Hexim1 competes with Tat for P-TEFb binding. Results Mutations that impair human Cyclin T1/Hexim1 interaction were searched using systematic mutagenesis of these proteins coupled with a yeast two-hybrid screen for loss of protein interaction. Evolutionary conserved Hexim1 residues belonging to an unstructured peptide located N-terminal of the dimerization domain, were found to be critical for P-TEFb binding. Random mutagenesis of the N-terminal region of Cyclin T1 provided identification of single amino-acid mutations that impair Hexim1 binding in human cells. Furthermore, conservation of critical residues supported the existence of a functional Hexim1 homologue in nematodes. Conclusions Single Cyclin T1 amino-acid mutations that impair Hexim1 binding are located on a groove between the two cyclin folds and define a surface overlapping the HIV-1 Tat protein binding surface. One residue, Y175, in the centre of this groove was identified as essential for both Hexim1 and Tat binding to P-TEFb as well as for HIV transcription. PMID:24985203

Patterns of Communicative Interaction between a Child with Severe Speech and Physical Impairments and Her Caregiver during a Mealtime Activity

ERIC Educational Resources Information Center

Ferm, Ulrika; Ahlsen, Elisabeth; Bjorck-Akesson, Eva

2012-01-01

Background: Interaction between caregivers and children with severe impairments is closely related to the demands of daily activities. This study examines the relationship between interaction and the routine mealtime activity at home. Method: Patterns of interaction between a child (aged 6 years and 6 months) with severe speech and physical…

Muscle stem cell dysfunction impairs muscle regeneration in a mouse model of Down syndrome.

PubMed

Pawlikowski, Bradley; Betta, Nicole Dalla; Elston, Tiffany; Williams, Darian A; Olwin, Bradley B

2018-03-09

Down syndrome, caused by trisomy 21, is characterized by a variety of medical conditions including intellectual impairments, cardiovascular defects, blood cell disorders and pre-mature aging phenotypes. Several somatic stem cell populations are dysfunctional in Down syndrome and their deficiencies may contribute to multiple Down syndrome phenotypes. Down syndrome is associated with muscle weakness but skeletal muscle stem cells or satellite cells in Down syndrome have not been investigated. We find that a failure in satellite cell expansion impairs muscle regeneration in the Ts65Dn mouse model of Down syndrome. Ts65Dn satellite cells accumulate DNA damage and over express Usp16, a histone de-ubiquitinating enzyme that regulates the DNA damage response. Impairment of satellite cell function, which further declines as Ts65Dn mice age, underscores stem cell deficiencies as an important contributor to Down syndrome pathologies.

Transposase-Derived Proteins FHY3/FAR1 Interact with PHYTOCHROME-INTERACTING FACTOR1 to Regulate Chlorophyll Biosynthesis by Modulating HEMB1 during Deetiolation in Arabidopsis[W

PubMed Central

Tang, Weijiang; Wang, Wanqing; Chen, Dongqin; Ji, Qiang; Jing, Yanjun; Wang, Haiyang; Lin, Rongcheng

2012-01-01

Successful chlorophyll biosynthesis during initial light exposure is critical for plant survival and growth, as excess accumulation of chlorophyll precursors in darkness can cause photooxidative damage to cells. Therefore, efficient mechanisms have evolved to precisely regulate chlorophyll biosynthesis in plants. Here, we identify FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and FAR-RED IMPAIRED RESPONSE1 (FAR1), two transposase-derived transcription factors, as positive regulators of chlorophyll biosynthesis in Arabidopsis thaliana. We show that null mutations in FHY3 and FAR1 cause reduced protochlorophyllide (a precursor of chlorophyll) levels in darkness and less photobleaching in the light. We find that FHY3 directly binds to the promoter and activates expression of HEMB1, which encodes 5-aminolevulinic acid dehydratase in the chlorophyll biosynthetic pathway. We reveal that PHYTOCHROME-INTERACTING FACTOR1 physically interacts with the DNA binding domain of FHY3, thereby partly repressing FHY3/FAR1-activated HEMB1 expression. Strikingly, FHY3 expression is upregulated by white light. In addition, our genetic data indicate that overexpression, severe reduction, or lack of HEMB1 impairs plant growth and development. Together, our findings reveal a crucial role of FHY3/FAR1 in regulating chlorophyll biosynthesis, thus uncovering a new layer of regulation by which light promotes plant dark–light transition in early seedling development. PMID:22634759

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bowley, S.; Okumura, N; Lord, S

'A:a' knob-hole interactions and D:D interfacial interactions are important for fibrin polymerization. Previous studies with recombinant ?N308K fibrinogen, a substitution at the D:D interface, showed impaired polymerization. We examined the molecular basis for this loss of function by solving the crystal structure of ?N308K fragment D. In contrast to previous fragment D crystals, the ?N308K crystals belonged to a tetragonal space group with an unusually long unit cell (a = b = 95 Angstroms, c = 448.3 Angstroms). Alignment of the normal and ?N308K structures showed the global structure of the variant was not changed and the knob 'A' peptidemore » GPRP was bound as usual to hole 'a'. The substitution introduced an elongated positively charged patch in the D:D region. The structure showed novel, symmetric D:D crystal contacts between ?N308K molecules, indicating the normal asymmetric D:D interface in fibrin would be unstable in this variant. We examined GPRP binding to ?N308K in solution by plasmin protection assay. The results showed weaker peptide binding, suggesting that 'A:a' interactions were altered. We examined fibrin network structures by scanning electron microscopy and found the variant fibers were thicker and more heterogeneous than normal fibers. Considered together, our structural and biochemical studies indicate both 'A:a' and D:D interactions are weaker. We conclude that stable protofibrils cannot assemble from ?N308K monomers, leading to impaired polymerization.« less

Proinsulin atypical maturation and disposal induces extensive defects in mouse Ins2+/Akita β-cells.

PubMed

Yuan, Qingxin; Tang, Wei; Zhang, Xiaoping; Hinson, Jack A; Liu, Chao; Osei, Kwame; Wang, Jie

2012-01-01

Because of its low relative folding rate and plentiful manufacture in β-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in β-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR), metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2(+/Akita) β-cells. We used T antigen-transformed Ins2(+/Akita) and control Ins2(+/+) β-cells established from Akita and wild-type littermate mice. In Ins2(+/Akita) β-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2(+/Akita) β-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and β-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2(+/Akita) β-cells further support a role of the genetic and stress-susceptible PIHO disorder in β-cell failure and diabetes.

Proinsulin Atypical Maturation and Disposal Induces Extensive Defects in Mouse Ins2+/Akita β-Cells

PubMed Central

Zhang, Xiaoping; Hinson, Jack A.; Liu, Chao; Osei, Kwame; Wang, Jie

2012-01-01

Because of its low relative folding rate and plentiful manufacture in β-cells, proinsulin maintains a homeostatic balance of natively and plentiful non-natively folded states (i.e., proinsulin homeostasis, PIHO) through the integration of maturation and disposal processes. PIHO is susceptible to genetic and environmental influences, and its disorder has been critically linked to defects in β-cells in diabetes. To explore this hypothesis, we performed polymerase chain reaction (PCR), metabolic-labeling, immunoblotting, and histological studies to clarify what defects result from primary disorder of PIHO in model Ins2+/Akita β-cells. We used T antigen-transformed Ins2+/Akita and control Ins2+/+ β-cells established from Akita and wild-type littermate mice. In Ins2+/Akita β-cells, we found no apparent defect at the transcriptional and translational levels to contribute to reduced cellular content of insulin and its precursor and secreted insulin. Glucose response remained normal in proinsulin biosynthesis but was impaired for insulin secretion. The size and number of mature insulin granules were reduced, but the size/number of endoplasmic reticulum, Golgi, mitochondrion, and lysosome organelles and vacuoles were expanded/increased. Moreover, cell death increased, and severe oxidative stress, which manifested as increased reactive oxygen species, thioredoxin-interacting protein, and protein tyrosine nitration, occurred in Ins2+/Akita β-cells and/or islets. These data show the first clear evidence that primary PIHO imbalance induces severe oxidative stress and impairs glucose-stimulated insulin release and β-cell survival as well as producing other toxic consequences. The defects disclosed/clarified in model Ins2+/Akita β-cells further support a role of the genetic and stress-susceptible PIHO disorder in β-cell failure and diabetes. PMID:22509386

CD70-deficiency impairs effector CD8 T cell generation and viral clearance but is dispensable for the recall response to LCMV

PubMed Central

Munitic, Ivana; Kuka, Mirela; Allam, Atef; Scoville, Jonathan P.; Ashwell, Jonathan D.

2012-01-01

CD27 interactions with its ligand, CD70, are thought to be necessary for optimal primary and memory adaptive immune responses to a variety of pathogens. Thus far all studies addressing the function of the CD27-CD70 axis have been performed either in mice lacking CD27, overexpressing CD70, or in which these receptors were blocked or mimicked by antibodies or recombinant soluble CD70. Because these methods have in some cases led to divergent results, we generated CD70-deficient mice to directly assess its role in vivo. We find that lack of CD70-mediated stimulation during primary responses to LCMV lowered the magnitude of CD8 antigen-specific T cell response, resulting in impaired viral clearance, without affecting CD4 T cell responses. Unexpectedly, CD70-CD27 costimulation was not needed for memory CD8 T cell generation or the ability to mount a recall response to LCMV. Adoptive transfers of wild type (WT) memory T cells into CD70−/− or WT hosts also showed no need for CD70-mediated stimulation during the course of the recall response. Moreover, CD70-expression by CD8 T cells could not rescue endogenous CD70−/− cells from defective expansion, arguing against a role for CD70-mediated T:T help in this model. Therefore, CD70 appears to be an important factor in the initiation of a robust and effective primary response but dispensable for CD8 T cell memory responses. PMID:23269247

An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex.

PubMed

Kracker, Sven; Di Virgilio, Michela; Schwartzentruber, Jeremy; Cuenin, Cyrille; Forveille, Monique; Deau, Marie-Céline; McBride, Kevin M; Majewski, Jacek; Gazumyan, Anna; Seneviratne, Suranjith; Grimbacher, Bodo; Kutukculer, Necil; Herceg, Zdenko; Cavazzana, Marina; Jabado, Nada; Nussenzweig, Michel C; Fischer, Alain; Durandy, Anne

2015-04-01

Immunoglobulin class-switch recombination defects (CSR-D) are rare primary immunodeficiencies characterized by impaired production of switched immunoglobulin isotypes and normal or elevated IgM levels. They are caused by impaired T:B cooperation or intrinsic B cell defects. However, many immunoglobulin CSR-Ds are still undefined at the molecular level. This study's objective was to delineate new causes of immunoglobulin CSR-Ds and thus gain further insights into the process of immunoglobulin class-switch recombination (CSR). Exome sequencing in 2 immunoglobulin CSR-D patients identified variations in the INO80 gene. Functional experiments were performed to assess the function of INO80 on immunoglobulin CSR. We identified recessive, nonsynonymous coding variations in the INO80 gene in 2 patients affected by defective immunoglobulin CSR. Expression of wild-type INO80 in patients' fibroblastic cells corrected their hypersensitivity to high doses of γ-irradiation. In murine CH12-F3 cells, the INO80 complex accumulates at Sα and Eμ regions of the IgH locus, and downregulation of INO80 as well as its partners Reptin and Pontin impaired CSR. In addition, Reptin and Pontin were shown to interact with activation-induced cytidine deaminase. Finally, an abnormal separation of sister chromatids was observed upon INO80 downregulation in CH12-F3 cells, pinpointing its role in cohesin activity. INO80 deficiency appears to be associated with defective immunoglobulin CSR. We propose that the INO80 complex modulates cohesin function that may be required during immunoglobulin switch region synapsis. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The acylphloroglucinols hyperforin and myrtucommulone A cause mitochondrial dysfunctions in leukemic cells by direct interference with mitochondria.

PubMed

Wiechmann, Katja; Müller, Hans; Fischer, Dagmar; Jauch, Johann; Werz, Oliver

2015-11-01

The acylphloroglucinols hyperforin (Hypf) and myrtucommulone A (MC A) induce death of cancer cells by triggering the intrinsic/mitochondrial pathway of apoptosis, accompanied by a loss of the mitochondrial membrane potential and release of cytochrome c. However, the upstream targets and mechanisms leading to these mitochondrial events in cancer cells remain elusive. Here we show that Hypf and MC A directly act on mitochondria derived from human leukemic HL-60 cells and thus, disrupt mitochondrial functions. In isolated mitochondria, Hypf and MC A efficiently impaired mitochondrial viability (EC50 = 0.2 and 0.9 µM, respectively), caused loss of the mitochondrial membrane potential (at 0.03 and 0.1 µM, respectively), and suppressed mitochondrial ATP synthesis (IC50 = 0.2 and 0.5 µM, respectively). Consequently, the compounds activated the adenosine monophosphate-activated protein kinase (AMPK) in HL-60 cells, a cellular energy sensor involved in apoptosis of cancer cells. Side by side comparison with the protonophore CCCP and the ATP synthase inhibitor oligomycin suggest that Hypf and MC A act as protonophores that primarily dissipate the mitochondrial membrane potential by direct interaction with the mitochondrial membrane. Together, Hypf and MC A abolish the mitochondrial proton motive force that on one hand impairs mitochondrial viability and on the other cause activation of AMPK due to lowered ATP levels which may further facilitate the intrinsic mitochondrial pathway of apoptosis.

Lipid rafts are essential for peroxisome biogenesis in HepG2 cells.

PubMed

Woudenberg, Jannes; Rembacz, Krzysztof P; Hoekstra, Mark; Pellicoro, Antonella; van den Heuvel, Fiona A J; Heegsma, Janette; van Ijzendoorn, Sven C D; Holzinger, Andreas; Imanaka, Tsuneo; Moshage, Han; Faber, Klaas Nico

2010-08-01

Peroxisomes are particularly abundant in the liver and are involved in bile salt synthesis and fatty acid metabolism. Peroxisomal membrane proteins (PMPs) are required for peroxisome biogenesis [e.g., the interacting peroxisomal biogenesis factors Pex13p and Pex14p] and its metabolic function [e.g., the adenosine triphosphate-binding cassette transporters adrenoleukodystrophy protein (ALDP) and PMP70]. Impaired function of PMPs is the underlying cause of Zellweger syndrome and X-linked adrenoleukodystrophy. Here we studied for the first time the putative association of PMPs with cholesterol-enriched lipid rafts and their function in peroxisome biogenesis. Lipid rafts were isolated from Triton X-100-lysed or Lubrol WX-lysed HepG2 cells and analyzed for the presence of various PMPs by western blotting. Lovastatin and methyl-beta-cyclodextrin were used to deplete cholesterol and disrupt lipid rafts in HepG2 cells, and this was followed by immunofluorescence microscopy to determine the subcellular location of catalase and PMPs. Cycloheximide was used to inhibit protein synthesis. Green fluorescent protein-tagged fragments of PMP70 and ALDP were analyzed for their lipid raft association. PMP70 and Pex14p were associated with Triton X-100-resistant rafts, ALDP was associated with Lubrol WX-resistant rafts, and Pex13p was not lipid raft-associated in HepG2 cells. The minimal peroxisomal targeting signals in ALDP and PMP70 were not sufficient for lipid raft association. Cholesterol depletion led to dissociation of PMPs from lipid rafts and impaired sorting of newly synthesized catalase and ALDP but not Pex14p and PMP70. Repletion of cholesterol to these cells efficiently reestablished the peroxisomal sorting of catalase but not ALDP. Human PMPs are differentially associated with lipid rafts independently of the protein homology and/or their functional interaction. Cholesterol is required for peroxisomal lipid raft assembly and peroxisome biogenesis.

Environmental contaminant mixtures modulate in vitro influenza infection.

PubMed

Desforges, Jean-Pierre; Bandoro, Christopher; Shehata, Laila; Sonne, Christian; Dietz, Rune; Puryear, Wendy B; Runstadler, Jonathan A

2018-09-01

Environmental chemicals, particularly organochlorinated contaminants (OCs), are associated with a ranged of adverse health effects, including impairment of the immune system and antiviral immunity. Influenza A virus (IAV) is an infectious disease of major global public health concern and exposure to OCs can increase the susceptibility, morbidity, and mortality to disease. It is however unclear how pollutants are interacting and affecting the outcome of viral infections at the cellular level. In this study, we investigated the effects of a mixture of environmentally relevant OCs on IAV infectivity upon in vitro exposure in Madin Darby Canine Kidney (MDCK) cells and human lung epithelial cells (A549). Exposure to OCs reduced IAV infectivity in MDCK and A549 cells during both short (18-24h) and long-term (72h) infections at 0.05 and 0.5ppm, and effects were more pronounced in cells co-treated with OCs and IAV than pre-treated with OCs prior to IAV (p<0.001). Pre-treatment of host cells with OCs did not affect IAV cell surface attachment or entry. Visualization of IAV by transmission electron microscopy revealed increased envelope deformations and fewer intact virions during OC exposure. Taken together, our results suggest that disruption of IAV infection upon in vitro exposure to OCs was not due to host-cell effects influencing viral attachment and entry, but perhaps mediated by direct effects on viral particles or cellular processes involved in host-virus interactions. In vitro infectivity studies such as ours can shed light on the complex processes underlying host-pathogen-pollutant interactions. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

PubMed Central

Zhang, Min; Maddala, Rupalatha; Rao, Ponugoti Vasantha

2008-01-01

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells. PMID:18799648

The leukocyte receptor CD84 inhibits Fc epsilon RI-mediated signaling through homophilic interaction in transfected RBL-2H3 cells.

PubMed

Oliver-Vila, Irene; Saborit-Villarroya, Ifigènia; Engel, Pablo; Martin, Margarita

2008-04-01

Signaling through the high-affinity receptor for immunoglobulin E (Fc epsilon RI) results in the coordinated activation of tyrosine kinases, thus leading to calcium mobilization, degranulation, and leukotriene and cytokine synthesis. Here, we show that CD84, a member of the CD150 family of leukocyte receptors, inhibits Fc epsilon RI-mediated mast cell degranulation in CD84-transfected rat basophilic leukaemia-2H3 mast cell line cells (RBL-2H3) through homophilic interaction. There was no reduction in overall protein phosphorylation following IgE triggering in CD84 RBL-2H3 cells. Indeed, phosphorylation of Dok-1 and c-Cbl increased in CD84 RBL-2H3, suggesting that inhibition is mediated by these molecules. MAP kinase phosphorylation (ERK1/2, JNK and p38) and cytokine synthesis were impaired in CD84 RBL-2H3. This inhibitory mechanism was independent of SAP and SHP-2 recruitment. Interestingly, CD84 mutants in tyrosines (Y279F and DeltaY324) reversed this inhibitory profile. These data suggest that CD84 may play a role in modulating Fc epsilon RI-mediated signaling in mast cells. Thus, CD84 could play a protective role against undesired allergic and inflammatory responses.

Physiological and physiopathological aspects of connexins and communicating gap junctions in spermatogenesis

PubMed Central

Pointis, Georges; Gilleron, Jérome; Carette, Diane; Segretain, Dominique

2010-01-01

Spermatogenesis is a highly regulated process of germ cell proliferation and differentiation, starting from spermatogonia to spermatocytes and giving rise to spermatids, the future spermatozoa. In addition to endocrine regulation, testicular cell–cell interactions are essential for spermatogenesis. This precise control is mediated through paracrine/autocrine pathways, direct intercellular contacts and through intercellular communication channels, consisting of gap junctions and their constitutive proteins, the connexins. Gap junctions are localized between adjacent Leydig cells, between Sertoli cells and between Sertoli cells and specific germ cells. This review focuses on the distribution of connexins within the seminiferous epithelium, their participation in gap junction channel formation, the control of their expression and the physiological relevance of these junctions in both the Sertoli–Sertoli cell functional synchronization and the Sertoli–germ cell dialogue. In this review, we also discuss the potential implication of disrupted connexin in testis cancer, since impaired expression of connexin has been described as a typical feature of tumoral proliferation. PMID:20403873

ABCB5 maintains melanoma-initiating cells through a pro-inflammatory cytokine signaling circuit

PubMed Central

Wilson, Brian J.; Saab, Karim R.; Ma, Jie; Schatton, Tobias; Pütz, Pablo; Zhan, Qian; Murphy, George F.; Gasser, Martin; Waaga-Gasser, Ana Maria; Frank, Natasha Y.; Frank, Markus H.

2014-01-01

The drug efflux transporter ABCB5 identifies cancer stem-like cells (CSC) in diverse human malignancies, where its expression is associated with clinical disease progression and tumor recurrence. ABCB5 confers therapeutic resistance but other functions in tumorigenesis independent of drug efflux have not been described that might help explain why it is so broadly overexpressed in human cancer. Here we show that in melanoma-initiating cells ABCB5 controls IL-1β secretion which serves to maintain slow-cycling, chemoresistant cells through an IL-1β/IL8/CXCR1 cytokine signaling circuit. This CSC maintenance circuit involved reciprocal paracrine interactions with ABCB5-negative cancer cell populations. ABCB5 blockade induced cellular differentiation, reversed resistance to multiple chemotherapeutic agents, and impaired tumor growth in vivo. Together, our results defined a novel function for ABCB5 in CSC maintenance and tumor growth. PMID:24934811

Blood dendritic cells interact with splenic marginal zone B cells to initiate T-independent immune responses.

PubMed

Balázs, Mercedesz; Martin, Flavius; Zhou, Tong; Kearney, John

2002-09-01

Marginal zone (MZ) and B1 B lymphocytes participate jointly in the early immune response against T-independent (TI) particulate antigens. Here we show that blood-derived neutrophil granulocytes and CD11c(lo) immature dendritic cells (DC) are the primary cells that efficiently capture and transport particulate bacteria to the spleen. In a systemic infection, CD11c(lo) DC, but not neutrophils, provide critical survival signals, which can be inhibited by TACI-Fc, to antigen-specific MZ B cells and promote their differentiation into IgM-secreting plasmablasts. In a local TI response, peritoneal cavity macrophages provide similar support to B1 B-derived Ag-specific blasts. In the absence of soluble TACI ligands, Ag-activated MZ- and B1-derived blasts lack survival signals and undergo apoptosis, resulting in severely impaired antibody responses.

Immunoglobulins drive terminal maturation of splenic dendritic cells

PubMed Central

Ziętara, Natalia; Łyszkiewicz, Marcin; Puchałka, Jacek; Pei, Gang; Gutierrez, Maximiliano Gabriel; Lienenklaus, Stefan; Hobeika, Elias; Reth, Michael; Martins dos Santos, Vitor A. P.; Krueger, Andreas; Weiss, Siegfried

2013-01-01

Nature and physiological status of antigen-presenting cells, such as dendritic cells DCs, are decisive for the immune reactions elicited. Multiple factors and cell interactions have been described that affect maturation of DCs. Here, we show that DCs arising in the absence of immunoglobulins (Ig) in vivo are impaired in cross-presentation of soluble antigen. This deficiency was due to aberrant cellular targeting of antigen to lysosomes and its rapid degradation. Function of DCs could be restored by transfer of Ig irrespective of antigen specificity and isotype. Modulation of cross-presentation by Ig was inhibited by coapplication of mannan and, thus, likely to be mediated by C-type lectin receptors. This unexpected dependency of splenic DCs on Ig to cross-present antigen provides insights into the interplay between cellular and humoral immunity and the immunomodulatory capacity of Ig. PMID:23345431

Critical role of dendritic cells in T cell retention in the interfollicular region of Peyer's patches.

PubMed

Obata, Takashi; Shibata, Naoko; Goto, Yoshiyuki; Ishikawa, Izumi; Sato, Shintaro; Kunisawa, Jun; Kiyono, Hiroshi

2013-07-15

Peyer's patches (PPs) simultaneously initiate active and quiescent immune responses in the gut. The immunological function is achieved by the rigid regulation of cell distribution and trafficking, but how the cell distribution is maintained remains to be elucidated. In this study, we show that binding of stromal cell-derived lymphoid chemokines to conventional dendritic cells (cDCs) is essential for the retention of naive CD4(+) T cells in the interfollicular region (IFR) of PPs. Transitory depletion of CD11c(high) cDCs in mice rapidly impaired the IFR structure in the PPs without affecting B cell follicles or germinal centers, lymphoid chemokine production from stromal cells, or the immigration of naive T cells into the IFRs of PPs. The cDC-orchestrated retention of naive T cells was mediated by heparinase-sensitive molecules that were expressed on cDCs and bound the lymphoid chemokine CCL21 produced from stromal cells. These data collectively reveal that interactions among cDCs, stromal cells, and naive T cells are necessary for the formation of IFRs in the PPs.

Characterization of interactions between Nedd4 and beta and gammaENaC using surface plasmon resonance.

PubMed

Asher, C; Chigaev, A; Garty, H

2001-09-07

Cell surface expression of the epithelial Na(+) channel ENaC is regulated by the ubiquitin ligase Nedd4. Binding of the WW domains of Nedd4 to the PY region in the carboxy tails of beta and gammaENaC, results in channel ubiquitination and degradation. Kinetic analysis of these interactions has been done using surface plasmon resonance. Synthetic peptides corresponding to the PY regions of beta and gammaENaC were immobilized on a sensor chip and "real-time" kinetics of their binding to recombinant WW proteins was determined. Specificity of the interactions was established by competition experiment, as well as by monitoring effects of a point mutation known to impair Nedd4/ENaC binding. These data provides the first determination of association, dissociation and equilibrium constants for the interactions between WW2 and beta or gammaENaC. Copyright 2001 Academic Press.

Interactive Games with an Assistive Robotic System for Hearing-Impaired Children.

PubMed

Uluer, Pinar; Akalin, Neziha; Gurpinar, Cemal; Kose, Hatice

2017-01-01

This paper presents an assistive robotic system, which can recognize and express sign language words from a predefined set, within interactive games to communicate with and teach hearing-impaired children sign language. The robotic system uses audio, visual and tactile feedback for interaction with the children and the teacher/researcher.

Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells

PubMed Central

Jung, Oisun; Choi, Suyong; Jang, Sun-Bok; Lee, Sin-Ae; Lim, Ssang-Taek; Choi, Yoon-Ju; Kim, Hye-Jin; Kim, Do-Hee; Kwak, Tae Kyoung; Kim, Hyeonjung; Kang, Minkyung; Lee, Mi-Sook; Park, Sook Young; Ryu, Jihye; Jeong, Doyoung; Cheong, Hae-Kap; Kim, Hyun Jeong; Park, Ki Hun; Lee, Bong-Jin; Schlaepfer, David D.; Lee, Jung Weon

2012-01-01

Summary Transmembrane 4 L six family member 5 (TM4SF5) plays an important role in cell migration, and focal adhesion kinase (FAK) activity is essential for homeostatic and pathological migration of adherent cells. However, it is unclear how TM4SF5 signaling mediates the activation of cellular migration machinery, and how FAK is activated during cell adhesion. Here, we showed that direct and adhesion-dependent binding of TM4SF5 to FAK causes a structural alteration that may release the inhibitory intramolecular interaction in FAK. In turn, this may activate FAK at the cell's leading edge, to promote migration/invasion and in vivo metastasis. TM4SF5-mediated FAK activation occurred during integrin-mediated cell adhesion. TM4SF5 was localized at the leading edge of the cells, together with FAK and actin-organizing molecules, indicating a signaling link between TM4SF5/FAK and actin reorganization machinery. Impaired interactions between TM4SF5 and FAK resulted in an attenuated FAK phosphorylation (the signaling link to actin organization machinery) and the metastatic potential. Our findings demonstrate that TM4SF5 directly binds to and activates FAK in an adhesion-dependent manner, to regulate cell migration and invasion, suggesting that TM4SF5 is a promising target in the treatment of metastatic cancer. PMID:23077174

Visualizing Without Vision at the Microscale: Students With Visual Impairments Explore Cells With Touch

NASA Astrophysics Data System (ADS)

Jones, M. Gail; Minogue, James; Oppewal, Tom; Cook, Michelle P.; Broadwell, Bethany

2006-12-01

Science instruction is typically highly dependent on visual representations of scientific concepts that are communicated through textbooks, teacher presentations, and computer-based multimedia materials. Little is known about how students with visual impairments access and interpret these types of visually-dependent instructional materials. This study explored the efficacy of new haptic (simulated tactile feedback and kinesthetics) instructional technology for teaching cell morphology and function to middle and high school students with visual impairments. The study examined students' prior experiences learning about the cell and cell functions in classroom instruction, as well as how haptic feedback technology impacted students' awareness of the 3-D nature of an animal cell, the morphology and function of cell organelles, and students' interest in the haptic technology as an instructional tool. Twenty-one students with visual impairment participated in the study. Students explored a tactile model of the cell with a haptic point probe that allowed them to feel the cell and its organelles. Results showed that students made significant gains in their ability to identify cell organelles and found the technology to be highly interesting as an instructional tool. The need for additional adaptive technology for students with visual impairments is discussed.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade.

PubMed

Caldeira, Joana; Figueiredo, Joana; Brás-Pereira, Catarina; Carneiro, Patrícia; Moreira, Ana M; Pinto, Marta T; Relvas, João B; Carneiro, Fátima; Barbosa, Mário; Casares, Fernando; Janody, Florence; Seruca, Raquel

2015-10-15

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The M-phase specific hyperphosphorylation of Staufen2 involved the cyclin-dependent kinase CDK1.

PubMed

Beaujois, Rémy; Ottoni, Elizabeth; Zhang, Xin; Gagnon, Christina; HSine, Sami; Mollet, Stéphanie; Viranaicken, Wildriss; DesGroseillers, Luc

2017-07-14

Staufen2 (STAU2) is an RNA-binding protein involved in the post-transcriptional regulation of gene expression. This protein was shown to be required for organ formation and cell differentiation. Although STAU2 functions have been reported in neuronal cells, its role in dividing cells remains deeply uncharacterized. Especially, its regulation during the cell cycle is completely unknown. In this study, we showed that STAU2 isoforms display a mitosis-specific slow migration pattern on SDS-gels in all tested transformed and untransformed cell lines. Deeper analyses in hTert-RPE1 and HeLa cells further indicated that the slow migration pattern of STAU2 isoforms is due to phosphorylation. Time course studies showed that STAU2 phosphorylation occurs before prometaphase and terminates as cells exit mitosis. Interestingly, STAU2 isoforms were phosphorylated on several amino acid residues in the C-terminal half via the cyclin-dependent kinase 1 (Cdk1), an enzyme known to play crucial roles during mitosis. Introduction of phospho-mimetic or phospho-null mutations in STAU2 did not impair its RNA-binding capacity, its stability, its interaction with protein co-factors or its sub-cellular localization, suggesting that STAU2 phosphorylation in mitosis does not regulate these functions. Similarly, STAU2 phosphorylation is not likely to be crucial for cell cycle progression since expression of phosphorylation mutants in hTert-RPE1 cells did not impair cell proliferation. Altogether, these results indicate that STAU2 isoforms are phosphorylated during mitosis and that the phosphorylation process involves Cdk1. The meaning of this post-translational modification is still elusive.

Sensitivity of tumor cells towards CIGB-300 anticancer peptide relies on its nucleolar localization.

PubMed

Perera, Yasser; Costales, Heydi C; Diaz, Yakelin; Reyes, Osvaldo; Farina, Hernan G; Mendez, Lissandra; Gómez, Roberto E; Acevedo, Boris E; Gomez, Daniel E; Alonso, Daniel F; Perea, Silvio E

2012-04-01

CIGB-300 is a novel anticancer peptide that impairs the casein kinase 2-mediated phosphorylation by direct binding to the conserved phosphoacceptor site on their substrates. Previous findings indicated that CIGB-300 inhibits tumor cell proliferation in vitro and induces tumor growth delay in vivo in cancer animal models. Interestingly, we had previously demonstrated that the putative oncogene B23/nucleophosmin (NPM) is the major intracellular target for CIGB-300 in a sensitive human lung cancer cell line. However, the ability of this peptide to target B23/NPM in cancer cells with differential CIGB-300 response phenotype remained to be determined. Interestingly, in this work, we evidenced that CIGB-300's antiproliferative activity on tumor cells strongly correlates with its nucleolar localization, the main subcellular localization of the previously identified B23/NPM target. Likewise, using CIGB-300 equipotent doses (concentration that inhibits 50% of proliferation), we demonstrated that this peptide interacts and inhibits B23/NPM phosphorylation in different cancer cell lines as evidenced by in vivo pull-down and metabolic labeling experiments. Moreover, such inhibition was followed by a fast apoptosis on CIGB-300-treated cells and also an impairment of cell cycle progression mainly after 5 h of treatment. Altogether, our data not only validates B23/NPM as a main target for CIGB-300 in cancer cells but also provides the first experimental clues to explain their differential antiproliferative response. Importantly, our findings suggest that further improvements to this cell penetrating peptide-based drug should entail its more efficient intracellular delivery at such subcellular localization. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

Cellular uptake of poly(allylamine hydrochloride) microcapsules with different deformability and its influence on cell functions.

PubMed

Yu, Wei; Zhang, Wenbo; Chen, Ying; Song, Xiaoxue; Tong, Weijun; Mao, Zhengwei; Gao, Changyou

2016-03-01

It is important to understand the safety issue and cell interaction pattern of polyelectrolyte microcapsules with different deformability before their use in biomedical applications. In this study, SiO2, poly(sodium-p-styrenesulfonate) (PSS) doped CaCO3 and porous CaCO3 spheres, all about 4μm in diameter, were used as templates to prepare microcapsules with different inner structure and subsequent deformability. As a result, three kinds of covalently assembled poly(allylaminehydrochloride)/glutaraldehyde (PAH/GA) microcapsules with similar size but different deformability under external osmotic pressure were prepared. The impact of different microcapsules on cell viability and functions are studied using smooth muscle cells (SMCs), endothelial cells (ECs) and HepG2 cells. The results demonstrated that viabilities of SMCs, ECs and HepG2 cells were not significantly influenced by either of the three kinds of microcapsules. However, the adhesion ability of SMCs and ECs as well as the mobility of SMCs, ECs and HepG2 cells were significantly impaired after treatment with microcapsules in a deformability dependent manner, especially the microcapsules with lower deformability caused higher impairment on cell functions. The cellular uptake kinetics, uptake pathways, intracellular distribution of microcapsules are further investigated in SMCs to reveal the potential mechanism. The SMCs showed faster uptake rate and exocytosis rate of microcapsules with lower deformability (Cap@CaCO3/PSS and Cap@CaCO3), leading to higher intracellular accumulation of microcapsules with lower deformability and possibly larger retardation of cell functions. The results pointed out that the deformability of microcapsules is an important factor governing the biological performance of microcapsules, which requires careful adjustment for further biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

B and T lymphocyte attenuator mediates inhibition of tumor-reactive CD8+ T cells in patients after allogeneic stem cell transplantation.

PubMed

Hobo, Willemijn; Norde, Wieger J; Schaap, Nicolaas; Fredrix, Hanny; Maas, Frans; Schellens, Karen; Falkenburg, J H Frederik; Korman, Alan J; Olive, Daniel; van der Voort, Robbert; Dolstra, Harry

2012-07-01

Allogeneic stem cell transplantation (allo-SCT) can cure hematological malignancies by inducing alloreactive T cell responses targeting minor histocompatibility antigens (MiHA) expressed on malignant cells. Despite induction of robust MiHA-specific T cell responses and long-term persistence of alloreactive memory T cells specific for the tumor, often these T cells fail to respond efficiently to tumor relapse. Previously, we demonstrated the involvement of the coinhibitory receptor programmed death-1 (PD-1) in suppressing MiHA-specific CD8(+) T cell immunity. In this study, we investigated whether B and T lymphocyte attenuator (BTLA) plays a similar role in functional impairment of MiHA-specific T cells after allo-SCT. In addition to PD-1, we observed higher BTLA expression on MiHA-specific CD8(+) T cells compared with that of the total population of CD8(+) effector-memory T cells. In addition, BTLA's ligand, herpes virus entry mediator (HVEM), was found constitutively expressed by myeloid leukemia, B cell lymphoma, and multiple myeloma cells. Interference with the BTLA-HVEM pathway, using a BTLA blocking Ab, augmented proliferation of BTLA(+)PD-1(+) MiHA-specific CD8(+) T cells by HVEM-expressing dendritic cells. Notably, we demonstrated that blocking of BTLA or PD-1 enhanced ex vivo proliferation of MiHA-specific CD8(+) T cells in respectively 7 and 9 of 11 allo-SCT patients. Notably, in 3 of 11 patients, the effect of BTLA blockade was more prominent than that of PD-1 blockade. Furthermore, these expanded MiHA-specific CD8(+) T cells competently produced effector cytokines and degranulated upon Ag reencounter. Together, these results demonstrate that BTLA-HVEM interactions impair MiHA-specific T cell functionality, providing a rationale for interfering with BTLA signaling in post-stem cell transplantation therapies.

Chronic inflammation in skeletal muscle impairs satellite cells function during regeneration: can physical exercise restore the satellite cell niche?

PubMed

Perandini, Luiz Augusto; Chimin, Patricia; Lutkemeyer, Diego da Silva; Câmara, Niels Olsen Saraiva

2018-06-01

Chronic inflammation impairs skeletal muscle regeneration. Although many cells are involved in chronic inflammation, macrophages seem to play an important role in impaired muscle regeneration since these cells are associated with skeletal muscle stem cell (namely, satellite cells) activation and fibro-adipogenic progenitor cell (FAP) survival. Specifically, an imbalance of M1 and M2 macrophages seems to lead to impaired satellite cell activation, and these are the main cells that function during skeletal muscle regeneration, after muscle damage. Additionally, this imbalance leads to the accumulation of FAPs in skeletal muscle, with aberrant production of pro-fibrotic factors (e.g., extracellular matrix components), impairing the niche for proper satellite cell activation and differentiation. Treatments aiming to block the inflammatory pro-fibrotic response are partially effective due to their side effects. Therefore, strategies reverting chronic inflammation into a pro-regenerative pattern are required. In this review, we first describe skeletal muscle resident macrophage ontogeny and homeostasis, and explain how macrophages are replenished after muscle injury. We next discuss the potential role of chronic physical activity and exercise in restoring the M1 and M2 macrophage balance and consequently, the satellite cell niche to improve skeletal muscle regeneration after injury. © 2018 Federation of European Biochemical Societies.

Putting theory of mind in its place: psychological explanations of the socio-emotional-communicative impairments in autistic spectrum disorder.

PubMed

Boucher, Jill

2012-05-01

In this review, the history of the theory of mind (ToM) theory of autistic spectrum disorder (ASD) is outlined (in which ToM is indexed by success on false belief tasks), and the explanatory power and psychological causes of impaired ToM in ASD are critically discussed. It is concluded that impaired ToM by itself has only limited explanatory power, but that explorations of the psychological precursors of impaired ToM have been fruitful in increasing understanding of mindreading impairments in ASD (where 'mindreading' refers those abilities that underlie triadic interaction as well as ToM). It is argued that early explanations of impaired mindreading are untenable for various reasons, but that impairments of dyadic interaction in ASD that could lead to impaired ability to represent others' mental states may be the critical psychological cause, or causes, of impaired ToM. The complexity of causal routes to impaired ToM is emphasized.

TDP-43 Promotes Neurodegeneration by Impairing Chromatin Remodeling.

PubMed

Berson, Amit; Sartoris, Ashley; Nativio, Raffaella; Van Deerlin, Vivianna; Toledo, Jon B; Porta, Sílvia; Liu, Shichong; Chung, Chia-Yu; Garcia, Benjamin A; Lee, Virginia M-Y; Trojanowski, John Q; Johnson, F Brad; Berger, Shelley L; Bonini, Nancy M

2017-12-04

Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. Here we find, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (FTD). Copyright © 2017 Elsevier Ltd. All rights reserved.

LUBAC Formation Is Impaired in the Livers of Mice with MCD-Dependent Nonalcoholic Steatohepatitis.

PubMed

Matsunaga, Yasuka; Nakatsu, Yusuke; Fukushima, Toshiaki; Okubo, Hirofumi; Iwashita, Misaki; Sakoda, Hideyuki; Fujishiro, Midori; Yamamotoya, Takeshi; Kushiyama, Akifumi; Takahashi, Shin-Ichiro; Tsuchiya, Yoshihiro; Kamata, Hideaki; Tokunaga, Fuminori; Iwai, Kazuhiro; Asano, Tomoichiro

2015-01-01

Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic lipid accumulation followed by the inflammation-induced death of hepatocytes and fibrosis. In this process, oxidative stress contributes to the induction of several inflammatory cytokines including TNF-α andIL-1β in macrophages, while, in hepatocytes, NF-κB reportedly induces the expressions of cell survival genes for protection from apoptosis. Recently, it was reported that the new ubiquitin ligase complex termed linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on NF-κB essential modulator (NEMO) and thereby induces NF-κB pathway activation. In this study, we demonstrated the formation of LUBAC to be impaired in the livers of NASH rodent models produced by methionine and choline deficient (MCD) diet feeding, first by either gel filtration or Blue Native-PAGE, with subsequent confirmation by western blotting. The reduction of LUBAC is likely to be attributable to markedly reduced expression of SHARPIN, one of its components. Thus, impaired LUBAC formation, which would result in insufficient NF-κB activation, may be one of the molecular mechanisms underlying the enhanced apoptotic response of hepatocytes in MCD diet-induced NASH livers.

Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development

PubMed Central

Sá e Cunha, Cláudia; Nyboer, Britta; Heiss, Kirsten; Sanches-Vaz, Margarida; Fontinha, Diana; Wiedtke, Ellen; Grimm, Dirk; Przyborski, Jude Marek; Mota, Maria M.; Prudêncio, Miguel; Mueller, Ann-Kristin

2017-01-01

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite’s hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host–parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies. PMID:28137845

SOX2 O-GlcNAcylation alters its protein-protein interactions and genomic occupancy to modulate gene expression in pluripotent cells

PubMed Central

Myers, Samuel A; Peddada, Sailaja; Chatterjee, Nilanjana; Friedrich, Tara; Tomoda, Kiichrio; Krings, Gregor; Thomas, Sean; Maynard, Jason; Broeker, Michael; Thomson, Matthew; Pollard, Katherine; Yamanaka, Shinya; Burlingame, Alma L; Panning, Barbara

2016-01-01

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor. DOI: http://dx.doi.org/10.7554/eLife.10647.001 PMID:26949256

Towards force spectroscopy of single tip-link bonds

NASA Astrophysics Data System (ADS)

Koussa, Mounir A.; Sotomayor, Marcos; Wong, Wesley P.; Corey, David P.

2015-12-01

Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. Molecular dynamics simulations predict that the tip-link bond is mechanically strong enough to withstand forces in hair cells, but its experimentally determined strength is unknown. We have developed molecular tools to facilitate single-molecule force spectroscopy on the tip link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip link nanoswitches are designed to provide a signature force-extension profile. This molecular signature should allow us to identify single-molecule rupture events in pulling experiments.

Podoplanin requires sialylated O-glycans for stable expression on lymphatic endothelial cells and for interaction with platelets.

PubMed

Pan, Yanfang; Yago, Tadayuki; Fu, Jianxin; Herzog, Brett; McDaniel, J Michael; Mehta-D'Souza, Padmaja; Cai, Xiaofeng; Ruan, Changgeng; McEver, Rodger P; West, Christopher; Dai, Kesheng; Chen, Hong; Xia, Lijun

2014-12-04

O-glycosylation of podoplanin (PDPN) on lymphatic endothelial cells is critical for the separation of blood and lymphatic systems by interacting with platelet C-type lectin-like receptor 2 during development. However, how O-glycosylation controls endothelial PDPN function and expression remains unclear. In this study, we report that core 1 O-glycan-deficient or desialylated PDPN was highly susceptible to proteolytic degradation by various proteases, including metalloproteinases (MMP)-2/9. We found that the lymph contained activated MMP-2/9 and incubation of the lymph reduced surface levels of PDPN on core 1 O-glycan-deficient endothelial cells, but not on wild-type ECs. The lymph from mice with sepsis induced by cecal ligation and puncture, which contained bacteria-derived sialidase, reduced PDPN levels on wild-type ECs. The MMP inhibitor, GM6001, rescued these reductions. Additionally, GM6001 treatment rescued the reduction of PDPN level on lymphatic endothelial cells in mice lacking endothelial core 1 O-glycan or cecal ligation and puncture-treated mice. Furthermore, core 1 O-glycan-deficient or desialylated PDPN impaired platelet interaction under physiological flow. These data indicate that sialylated O-glycans of PDPN are essential for platelet adhesion and prevent PDPN from proteolytic degradation primarily mediated by MMPs in the lymph. © 2014 by The American Society of Hematology.

Factor H: A Complement Regulator in Health and Disease, and a Mediator of Cellular Interactions

PubMed Central

Kopp, Anne; Hebecker, Mario; Svobodová, Eliška; Józsi, Mihály

2012-01-01

Complement is an essential part of innate immunity as it participates in host defense against infections, disposal of cellular debris and apoptotic cells, inflammatory processes and modulation of adaptive immune responses. Several soluble and membrane-bound regulators protect the host from the potentially deleterious effects of uncontrolled and misdirected complement activation. Factor H is a major soluble regulator of the alternative complement pathway, but it can also bind to host cells and tissues, protecting them from complement attack. Interactions of factor H with various endogenous ligands, such as pentraxins, extracellular matrix proteins and DNA are important in limiting local complement-mediated inflammation. Impaired regulatory as well as ligand and cell recognition functions of factor H, caused by mutations or autoantibodies, are associated with the kidney diseases: atypical hemolytic uremic syndrome and dense deposit disease and the eye disorder: age-related macular degeneration. In addition, factor H binds to receptors on host cells and is involved in adhesion, phagocytosis and modulation of cell activation. In this review we discuss current concepts on the physiological and pathophysiological roles of factor H in light of new data and recent developments in our understanding of the versatile roles of factor H as an inhibitor of complement activation and inflammation, as well as a mediator of cellular interactions. A detailed knowledge of the functions of factor H in health and disease is expected to unravel novel therapeutic intervention possibilities and to facilitate the development or improvement of therapies. PMID:24970127

Pathogenesis of Crimean-Congo hemorrhagic fever.

PubMed

Akıncı, Esragül; Bodur, Hürrem; Leblebicioglu, Hakan

2013-07-01

Although Crimean-Congo hemorrhagic fever (CCHF) is a widespread tick-borne disease, little is known about its pathogenesis. The interaction of the virus with host cells is most likely responsible for the pathogenesis of CCHF. The main contributors are endothelial cells (ECs) and immune cells. There are 2 theories underlying the CCHF pathogenesis: One is that the virus interacts with the ECs directly and the other that it interacts indirectly via immune cells with subsequent release of soluble mediators. ECs are activated upon infection by the upregulation of soluble molecules and proinflammatory cytokines. Probably, in severe cases, deregulation and excessive release of the cytokines accompanied by endothelial activation have toxic effects, leading to increased vascular permeability, vasodilatation, and subsequently hypotension, multiple organ failure, shock, and death. Studies indicate that CCHF virus (CCHFV) also can impair the innate immune system and cause a delay in adaptive immune response, which is critical for the clearance of CCHFV. The virus has many different ways to block the immune response, leading to uncontrolled viral replication followed by systemic spread of the virus throughout the body. Partial activation of dendritic cells and macrophages, delayed induction of interferons, weak antibody response, apoptosis of lymphocytes, and hemophagocytosis are some of these tactics. However, there are many points waiting for clarification about the pathogenesis of CCHF. Although the high risk of contagiousness limits research, we need more studies to understand the CCHF pathogenesis better. Here we review the main characteristics of the pathogenesis of CCHF.

Psychosocial outcomes in a cohort of perinatally HIV-infected adolescents in Western Jamaica.

PubMed

Evans-Gilbert, Tracy; Kasimbie, Kazie; Reid, Gail; Williams, Shelly Ann

2018-02-05

Background Psychosocial factors interact with adolescent development and affect the ability of HIV-infected adolescents to cope with and adhere to treatment. Aim To evaluate psychosocial outcomes in perinatally HIV-infected adolescents (PHIVAs) in Western Jamaica after psychosocial intervention. Methods The Bright Futures Paediatric Symptom Checklist (BF-PSC) was used for psychological screening of PHIVAs in Western Jamaica. Referred patients were evaluated using the Youth version of the Columbia Impairment Scale (CIS). Demographic, laboratory and clinical data obtained between July 2014 and June 2016 were evaluated retrospectively and outcomes were reviewed before and after psychosocial intervention. Results Sixty PHIVAs were enrolled and 36 (60%) had a positive BF-PSC score that necessitated referral. The BF-PSC correctly identified 89% of patients with impaired psychosocial assessment by CIS scores. Referred patients were less likely to adhere to treatment, to be virologically suppressed or to have a CD4+ count of >500 cells/μl, and were more likely to be in the late teenage group or to be of orphan status. After intervention, the prevalence of viral suppression increased and median viral load decreased. A difference in mean CD4+ cell count was detected before but not after intervention in teenage and orphan groups. Conclusions The BF-PSC identified at-risk PHIVAs with impaired psychosocial functioning. Increased vulnerability was noted in orphans and older teenagers. Psychosocial interventions (including family therapy) reduced psychosocial impairment and improved virological suppression. Mental health intervention should be instituted to facilitate improved clinical outcomes, autonomy of care and transition to adult care.

Cellular senescence and autophagy in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF).

PubMed

Kuwano, Kazuyoshi; Araya, Jun; Hara, Hiromichi; Minagawa, Shunsuke; Takasaka, Naoki; Ito, Saburo; Kobayashi, Kenji; Nakayama, Katsutoshi

2016-11-01

Aging is associated with impairments in homeostasis. Although aging and senescence are not equivalent, the number of senescent cells increases with aging. Cellular senescence plays important roles in tissue repair or remodeling, as well as embryonic development. Autophagy is a process of lysosomal self-degradation that maintains a homeostatic balance between the synthesis, degradation, and recycling of cellular proteins. Autophagy diminishes with aging; additionally, accelerated aging can be attributed to reduced autophagy. Cellular senescence has been widely implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), a disease of accelerated lung aging, presumably by impairing cell repopulation and by aberrant cytokine secretion in the senescence-associated secretory phenotype. The possible participation of autophagy in the pathogenic sequence of COPD has been extensively explored. Although it has been reported that increased autophagy may induce epithelial cell death, an insufficient reserve of autophagy can induce cellular senescence in bronchial epithelial cells of COPD. Furthermore, advanced age is one of the most important risk factors for the development of idiopathic pulmonary fibrosis (IPF). Telomere shortening is found in blood leukocytes and alveolar epithelial cells from patients with IPF. Accelerated senescence of epithelial cells plays a role in IPF pathogenesis by perpetuating abnormal epithelial-mesenchymal interactions. Insufficient autophagy may be an underlying mechanism of accelerated epithelial cell senescence and myofibroblast differentiation in IPF. Herein, we review the molecular mechanisms of cellular senescence and autophagy and summarize the role of cellular senescence and autophagy in both COPD and IPF. Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Dual inhibition of mTORC1 and mTORC2 perturbs cytoskeletal organization and impairs endothelial cell elongation.

PubMed

Tsuji-Tamura, Kiyomi; Ogawa, Minetaro

2018-02-26

Elongation of endothelial cells is an important process in vascular formation and is expected to be a therapeutic target for inhibiting tumor angiogenesis. We have previously demonstrated that inhibition of mTORC1 and mTORC2 impaired endothelial cell elongation, although the mechanism has not been well defined. In this study, we analyzed the effects of the mTORC1-specific inhibitor everolimus and the mTORC1/mTORC2 dual inhibitor KU0063794 on the cytoskeletal organization and morphology of endothelial cell lines. While both inhibitors equally inhibited cell proliferation, KU0063794 specifically caused abnormal accumulation of F-actin and disordered distribution of microtubules, thereby markedly impairing endothelial cell elongation and tube formation. The effects of KU0063794 were phenocopied by paclitaxel treatment, suggesting that KU0063794 might impair endothelial cell morphology through over-stabilization of microtubules. Although mTORC1 is a key signaling molecule in cell proliferation and has been considered a target for preventing angiogenesis, mTORC1 inhibitors have not been sufficient to suppress angiogenesis. Our results suggest that mTORC1/mTORC2 dual inhibition is more effective for anti-angiogenic therapy, as it impairs not only endothelial cell proliferation, but also endothelial cell elongation. Copyright © 2018 Elsevier Inc. All rights reserved.

Diamond, graphite, and graphene oxide nanoparticles decrease migration and invasiveness in glioblastoma cell lines by impairing extracellular adhesion

PubMed Central

Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Grodzik, Marta; Strojny, Barbara; Kurantowicz, Natalia; Zdunek, Krzysztof; Chodun, Rafał; Chwalibog, André; Sawosz, Ewa

2017-01-01

The highly invasive nature of glioblastoma is one of the most significant problems regarding the treatment of this tumor. Diamond nanoparticles (ND), graphite nanoparticles (NG), and graphene oxide nanoplatelets (nGO) have been explored for their biomedical applications, especially for drug delivery. The objective of this research was to assess changes in the adhesion, migration, and invasiveness of two glioblastoma cell lines, U87 and U118, after ND, NG, and nGO treatment. All treatments affected the cell surface structure, adhesion-dependent EGFR/AKT/mTOR, and β-catenin signaling pathways, decreasing the migration and invasiveness of both glioblastoma cell lines. The examined nanoparticles did not show strong toxicity but effectively deregulated cell migration. ND was effectively taken up by cells, whereas nGO and NG strongly interacted with the cell surface. These results indicate that nanoparticles could be used in biomedical applications as a low toxicity active compound for glioblastoma treatment. PMID:29042773

Diamond, graphite, and graphene oxide nanoparticles decrease migration and invasiveness in glioblastoma cell lines by impairing extracellular adhesion.

PubMed

Wierzbicki, Mateusz; Jaworski, Sławomir; Kutwin, Marta; Grodzik, Marta; Strojny, Barbara; Kurantowicz, Natalia; Zdunek, Krzysztof; Chodun, Rafał; Chwalibog, André; Sawosz, Ewa

2017-01-01

The highly invasive nature of glioblastoma is one of the most significant problems regarding the treatment of this tumor. Diamond nanoparticles (ND), graphite nanoparticles (NG), and graphene oxide nanoplatelets (nGO) have been explored for their biomedical applications, especially for drug delivery. The objective of this research was to assess changes in the adhesion, migration, and invasiveness of two glioblastoma cell lines, U87 and U118, after ND, NG, and nGO treatment. All treatments affected the cell surface structure, adhesion-dependent EGFR/AKT/mTOR, and β-catenin signaling pathways, decreasing the migration and invasiveness of both glioblastoma cell lines. The examined nanoparticles did not show strong toxicity but effectively deregulated cell migration. ND was effectively taken up by cells, whereas nGO and NG strongly interacted with the cell surface. These results indicate that nanoparticles could be used in biomedical applications as a low toxicity active compound for glioblastoma treatment.

Endocardial Hippo signaling regulates myocardial growth and cardiogenesis.

PubMed

Artap, Stanley; Manderfield, Lauren J; Smith, Cheryl L; Poleshko, Andrey; Aghajanian, Haig; See, Kelvin; Li, Li; Jain, Rajan; Epstein, Jonathan A

2018-08-01

The Hippo signaling pathway has been implicated in control of cell and organ size, proliferation, and endothelial-mesenchymal transformation. This pathway impacts upon two partially redundant transcription cofactors, Yap and Taz, that interact with other factors, including members of the Tead family, to affect expression of downstream genes. Yap and Taz have been shown to regulate, in a cell-autonomous manner, myocardial proliferation, myocardial hypertrophy, regenerative potential, and overall size of the heart. Here, we show that Yap and Taz also play an instructive, non-cell-autonomous role in the endocardium of the developing heart to regulate myocardial growth through release of the paracrine factor, neuregulin. Without endocardial Yap and Taz, myocardial growth is impaired causing early post-natal lethality. Thus, the Hippo signaling pathway regulates cell size via both cell-autonomous and non-cell-autonomous mechanisms. Furthermore, these data suggest that Hippo may regulate organ size via a sensing and paracrine function in endothelial cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Vaccine-elicited memory CD4+ T cell expansion is impaired in the lungs during tuberculosis.

PubMed

Carpenter, Stephen M; Yang, Jason D; Lee, Jinhee; Barreira-Silva, Palmira; Behar, Samuel M

2017-11-01

Immunological memory is the key biological process that makes vaccines possible. Although tuberculosis vaccines elicit protective immunity in animals, few provide durable protection. To understand why protection is transient, we evaluated the ability of memory CD4+ T cells to expand, differentiate, and control Mycobacterium tuberculosis. Both naïve and memory CD4+ T cells initially proliferated exponentially, and the accumulation of memory T cells in the lung correlated with early bacterial control. However, later during infection, memory CD4+ T cell proliferation was curtailed and no protection was observed. We show that memory CD4+ T cells are first activated in the LN and their recruitment to the lung attenuates bacterial growth. However, their interaction with Mtb-infected macrophages does not promote continued proliferation. We conclude that a lack of sustained expansion by memory-derived T cells in the lung limits the durability of their protection, linking their slower expansion with transient protection in vaccinated mice.

AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

PubMed

Lecoq, Anne-Lise; Viengchareun, Say; Hage, Mirella; Bouligand, Jérôme; Young, Jacques; Boutron, Audrey; Zizzari, Philippe; Lombès, Marc; Chanson, Philippe; Kamenický, Peter

2016-05-01

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner. © 2016 Society for Endocrinology.

Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells.

PubMed

Chavira-Suárez, Erika; Sandoval, Alejandro; Felix, Ricardo; Lamas, Mónica

2011-01-14

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy. Copyright © 2010 Elsevier Inc. All rights reserved.

Viscoelastic response of a model endothelial glycocalyx

NASA Astrophysics Data System (ADS)

Nijenhuis, Nadja; Mizuno, Daisuke; Spaan, Jos A. E.; Schmidt, Christoph F.

2009-06-01

Many cells cover themselves with a multifunctional polymer coat, the pericellular matrix (PCM), to mediate mechanical interactions with the environment. A particular PCM, the endothelial glycocalyx (EG), is formed by vascular endothelial cells at their luminal side, forming a mechanical interface between the flowing blood and the endothelial cell layer. The glycosaminoglycan (GAG) hyaluronan (HA) is involved in the main functions of the EG, mechanotransduction of fluid shear stress and molecular sieving. HA, due to its length, is the only GAG in the EG or any other PCM able to form an entangled network. The mechanical functions of the EG are, however, impaired when any one of its components is removed. We here used microrheology to measure the effect of the EG constituents heparan sulfate, chondroitin sulfate, whole blood plasma and albumin on the high-bandwidth mechanical properties of a HA solution. Furthermore, we probed the effect of the hyaldherin aggrecan, a constituent of the PCM of chondrocytes, and very similar to versican (present in the PCM of various cells, and possibly in the EG). We show that components directly interacting with HA (chondroitin sulfate and aggrecan) can increase the viscoelastic shear modulus of the polymer composite.

Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion.

PubMed

Shen, Bang; Sibley, L David

2014-03-04

Gliding motility and host-cell invasion by apicomplexan parasites depend on cell-surface adhesins that are translocated via an actin-myosin motor beneath the membrane. The current model posits that fructose-1,6-bisphosphate aldolase (ALD) provides a critical link between the cytoplasmic tails of transmembrane adhesins and the actin-myosin motor. Here we tested this model using the Toxoplasma gondii apical membrane protein 1 (TgAMA1), which binds to aldolase in vitro. TgAMA1 cytoplasmic tail mutations that disrupt ALD binding in vitro showed no correlation with host-cell invasion, indicating this interaction is not essential. Furthermore, ALD-depleted parasites were impaired when grown in glucose, yet they showed normal gliding and invasion in glucose-free medium. Depletion of ALD in the presence of glucose led to accumulation of fructose-1,6-bisphosphate, which has been associated with toxicity in other systems. Finally, TgALD knockout parasites and an ALD mutant that specifically disrupts adhesin binding in vitro also supported normal invasion when cultured in glucose-free medium. Taken together, these results suggest that ALD is primarily important for energy metabolism rather than interacting with microneme adhesins, challenging the current model for apicomplexan motility and invasion.

The metabolites in peripheral blood mononuclear cells showed greater differences between patients with impaired fasting glucose or type 2 diabetes and healthy controls than those in plasma.

PubMed

Kim, Minjoo; Kim, Minkyung; Han, Ji Yun; Lee, Sang-Hyun; Jee, Sun Ha; Lee, Jong Ho

2017-03-01

To determine differences between peripheral blood mononuclear cells and the plasma metabolites in patients with impaired fasting glucose or type 2 diabetes and healthy controls. In all, 65 nononobese patients (aged 30-70 years) with impaired fasting glucose or type 2 diabetes and 65 nonobese sex-matched healthy controls were included, and fasting peripheral blood mononuclear cell and plasma metabolomes were profiled. The diabetic or impaired fasting glucose patients showed higher circulating and peripheral blood mononuclear cell lipoprotein phospholipase A 2 activities, high-sensitivity C-reactive protein and tumour necrosis factor-α than controls. Compared with controls, impaired fasting glucose or diabetic subjects showed increases in 11 peripheral blood mononuclear cell metabolites: six amino acids (valine, leucine, methionine, phenylalanine, tyrosine and tryptophan), l-pyroglutamic acid, two fatty acid amides containing palmitic amide and oleamide and two lysophosphatidylcholines. In impaired fasting glucose or diabetic patients, peripheral blood mononuclear cell lipoprotein phospholipase A 2 positively associated with peripheral blood mononuclear cell lysophosphatidylcholines and circulating inflammatory markers, including tumour necrosis factor-α, high-sensitivity C-reactive protein and lipoprotein phospholipase A 2 activities. In plasma metabolites between patients and healthy controls, we observed significant increases in only three amino acids (proline, valine and leucine) and decreases in only five lysophosphatidylcholines. This study demonstrates significant differences in the peripheral blood mononuclear cell metabolome in patients with impaired fasting glucose or diabetes compared with healthy controls. These differences were greater than those observed in the plasma metabolome. These data suggest peripheral blood mononuclear cells as a useful tool to better understand the inflammatory pathophysiology of diabetes.

Decreased function of survival motor neuron protein impairs endocytic pathways.

PubMed

Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S; O'Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C Q; Cook, Steven J; Poulogiannis, George; Atwood, Walter J; Hall, David H; Hart, Anne C

2016-07-26

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.

Decreased function of survival motor neuron protein impairs endocytic pathways

PubMed Central

Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S.; O’Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C. Q.; Cook, Steven J.; Poulogiannis, George; Atwood, Walter J.; Hall, David H.; Hart, Anne C.

2016-01-01

Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death. PMID:27402754

TiO2 nanoparticle-induced ROS correlates with modulated immune cell function

NASA Astrophysics Data System (ADS)

Maurer-Jones, Melissa A.; Christenson, Jenna R.; Haynes, Christy L.

2012-12-01

Design of non-toxic nanoparticles will be greatly facilitated by understanding the nanoparticle-cell interaction mechanism on a cell function level. Mast cells are important cells for the immune system's first line of defense, and we can utilize their exocytotic behavior as a model cellular function as it is a conserved process across cell types and species. Perturbations in exocytosis can also have implications for whole organism health. One proposed mode of toxicity is nanoparticle-induced reactive oxygen species (ROS), particularly for titanium dioxide (TiO2) nanoparticles. Herein, we have correlated changes in ROS with the perturbation of the critical cell function of exocytosis, using UV light to induce greater levels of ROS in TiO2 exposed cells. The primary culture mouse peritoneal mast cells (MPMCs) were exposed to varying concentrations of TiO2 nanoparticles for 24 h. ROS content was determined using 2,7-dihydrodichlorofluorescein diacetate (DCFDA). Cellular viability was determined with the MTT and Trypan blue assays, and exocytosis was measured by the analytical electrochemistry technique of carbon-fiber microelectrode amperometry. MPMCs exposed to TiO2 nanoparticles experienced a dose-dependent increase in total ROS content. While there was minimal impact of ROS on cellular viability, there is a correlation between ROS amount and exocytosis perturbation. As nanoparticle-induced ROS increases, there is a significant decrease (45 %) in the number of serotonin molecules being released during exocytosis, increase (26 %) in the amount of time for each exocytotic granule to release, and decrease (28 %) in the efficiency of granule trafficking and docking. This is the first evidence that nanoparticle-induced ROS correlates with chemical messenger molecule secretion, possibly making a critical connection between functional impairment and mechanisms contributing to that impairment.

Interleukin 6 Influences Germinal Center Development and Antibody Production via a Contribution of C3 Complement Component

PubMed Central

Kopf, Manfred; Herren, Suzanne; Wiles, Michael V.; Pepys, Mark B.; Kosco-Vilbois, Marie H.

1998-01-01

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses. PMID:9815267

Inhibition of latent membrane protein 1 impairs the growth and tumorigenesis of latency II Epstein-Barr virus-transformed T cells.

PubMed

Ndour, Papa Alioune; Brocqueville, Guillaume; Ouk, Tan-Sothéa; Goormachtigh, Gautier; Morales, Olivier; Mougel, Alexandra; Bertout, Julie; Melnyk, Oleg; Fafeur, Véronique; Feuillard, Jean; Coll, Jean; Adriaenssens, Eric

2012-04-01

Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV.

Inhibition of Latent Membrane Protein 1 Impairs the Growth and Tumorigenesis of Latency II Epstein-Barr Virus-Transformed T Cells

PubMed Central

Ndour, Papa Alioune; Brocqueville, Guillaume; Ouk, Tan-Sothéa; Goormachtigh, Gautier; Morales, Olivier; Mougel, Alexandra; Bertout, Julie; Melnyk, Oleg; Fafeur, Véronique; Feuillard, Jean; Coll, Jean

2012-01-01

Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV. PMID:22258264

Cellular reprogramming: a novel tool for investigating autism spectrum disorders.

PubMed

Kim, Kun-Yong; Jung, Yong Wook; Sullivan, Gareth J; Chung, Leeyup; Park, In-Hyun

2012-08-01

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in reciprocal social interaction and communication, as well as the manifestation of stereotyped behaviors. Despite much effort, ASDs are not yet fully understood. Advanced genetics and genomics technologies have recently identified novel ASD genes, and approaches using genetically engineered murine models or postmortem human brain have facilitated understanding ASD. Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) provides unprecedented opportunities in generating human disease models. Here, we present an overview of applying iPSCs in developing cellular models for understanding ASD. We also discuss future perspectives in the use of iPSCs as a source of cell therapy and as a screening platform for identifying small molecules with efficacy for alleviating ASD. Copyright © 2012. Published by Elsevier Ltd.

Fetal Alcohol Spectrum Disorder (FASD) Associated Neural Defects: Complex Mechanisms and Potential Therapeutic Targets

PubMed Central

Muralidharan, Pooja; Sarmah, Swapnalee; Zhou, Feng C.; Marrs, James A.

2013-01-01

Fetal alcohol spectrum disorder (FASD), caused by prenatal alcohol exposure, can result in craniofacial dysmorphism, cognitive impairment, sensory and motor disabilities among other defects. FASD incidences are as high as 2% to 5 % children born in the US, and prevalence is higher in low socioeconomic populations. Despite various mechanisms being proposed to explain the etiology of FASD, the molecular targets of ethanol toxicity during development are unknown. Proposed mechanisms include cell death, cell signaling defects and gene expression changes. More recently, the involvement of several other molecular pathways was explored, including non-coding RNA, epigenetic changes and specific vitamin deficiencies. These various pathways may interact, producing a wide spectrum of consequences. Detailed understanding of these various pathways and their interactions will facilitate the therapeutic target identification, leading to new clinical intervention, which may reduce the incidence and severity of these highly prevalent preventable birth defects. This review discusses manifestations of alcohol exposure on the developing central nervous system, including the neural crest cells and sensory neural placodes, focusing on molecular neurodevelopmental pathways as possible therapeutic targets for prevention or protection. PMID:24961433

A protein interaction mechanism for suppressing the mechanosensitive Piezo channels.

PubMed

Zhang, Tingxin; Chi, Shaopeng; Jiang, Fan; Zhao, Qiancheng; Xiao, Bailong

2017-11-27

Piezo proteins are bona fide mammalian mechanotransduction channels for various cell types including endothelial cells. The mouse Piezo1 of 2547 residues forms a three-bladed, propeller-like homo-trimer comprising a central pore-module and three propeller-structures that might serve as mechanotransduction-modules. However, the mechanogating and regulation of Piezo channels remain unclear. Here we identify the sarcoplasmic /endoplasmic-reticulum Ca 2+ ATPase (SERCA), including the widely expressed SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses Piezo1 via acting on a 14-residue-constituted intracellular linker connecting the pore-module and mechanotransduction-module. Mutating the linker impairs mechanogating and SERCA2-mediated modulation of Piezo1. Furthermore, the synthetic linker-peptide disrupts the modulatory effects of SERCA2, demonstrating the key role of the linker in mechanogating and regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent migration of endothelial cells. Collectively, we identify SERCA-mediated regulation of Piezos and the functional significance of the linker, providing important insights into the mechanogating and regulation mechanisms of Piezo channels.

Fetal Alcohol Spectrum Disorder (FASD) Associated Neural Defects: Complex Mechanisms and Potential Therapeutic Targets.

PubMed

Muralidharan, Pooja; Sarmah, Swapnalee; Zhou, Feng C; Marrs, James A

2013-06-19

Fetal alcohol spectrum disorder (FASD), caused by prenatal alcohol exposure, can result in craniofacial dysmorphism, cognitive impairment, sensory and motor disabilities among other defects. FASD incidences are as high as 2% to 5 % children born in the US, and prevalence is higher in low socioeconomic populations. Despite various mechanisms being proposed to explain the etiology of FASD, the molecular targets of ethanol toxicity during development are unknown. Proposed mechanisms include cell death, cell signaling defects and gene expression changes. More recently, the involvement of several other molecular pathways was explored, including non-coding RNA, epigenetic changes and specific vitamin deficiencies. These various pathways may interact, producing a wide spectrum of consequences. Detailed understanding of these various pathways and their interactions will facilitate the therapeutic target identification, leading to new clinical intervention, which may reduce the incidence and severity of these highly prevalent preventable birth defects. This review discusses manifestations of alcohol exposure on the developing central nervous system, including the neural crest cells and sensory neural placodes, focusing on molecular neurodevelopmental pathways as possible therapeutic targets for prevention or protection.

Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus.

PubMed

Deschamps, Thibaut; Kalamvoki, Maria

2017-05-01

Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2'3'-cyclic GAMP (2'3'-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway. IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1. Copyright © 2017 American Society for Microbiology.

Impaired STING Pathway in Human Osteosarcoma U2OS Cells Contributes to the Growth of ICP0-Null Mutant Herpes Simplex Virus

PubMed Central

Deschamps, Thibaut

2017-01-01

ABSTRACT Human herpes simplex virus 1 (HSV-1) is a widespread pathogen, with 80% of the population being latently infected. To successfully evade the host, the virus has evolved strategies to counteract antiviral responses, including the gene-silencing and innate immunity machineries. The immediately early protein of the virus, infected cell protein 0 (ICP0), plays a central role in these processes. ICP0 blocks innate immunity, and one mechanism is by degrading hostile factors with its intrinsic E3 ligase activity. ICP0 also functions as a promiscuous transactivator, and it blocks repressor complexes to enable viral gene transcription. For these reasons, the growth of a ΔICP0 virus is impaired in most cells, except cells of the human osteosarcoma cell line U2OS, and it is only partially impaired in cells of the human osteosarcoma cell line Saos-2. We found that the two human osteosarcoma cell lines that supported the growth of the ΔICP0 virus failed to activate innate immune responses upon treatment with 2′3′-cyclic GAMP (2′3′-cGAMP), the natural agonist of STING (i.e., stimulator of interferon genes) or after infection with the ΔICP0 mutant virus. Innate immune responses were restored in these cells by transient expression of the STING protein but not after overexpression of interferon-inducible protein 16 (IFI16). Restoration of STING expression resulted in suppression of ΔICP0 virus gene expression and a decrease in viral yields. Overexpression of IFI16 also suppressed ΔICP0 virus gene expression, albeit to a lesser extent than STING. These data suggest that the susceptibility of U2OS and Saos-2 cells to the ΔICP0 HSV-1 is in part due to an impaired STING pathway. IMPORTANCE The DNA sensor STING plays pivotal role in controlling HSV-1 infection both in cell culture and in mice. The HSV-1 genome encodes numerous proteins that are dedicated to combat host antiviral responses. The immediate early protein of the virus ICP0 plays major role in this process as it targets hostile host proteins for degradation with its E3 ligase activity, and it disrupts repressor complexes via protein-protein interaction to enable viral gene transcription. Therefore, the ΔICP0 HSV-1 virus is defective for growth in most cells, except the human osteosarcoma cell lines U2OS and Saos-2. We found that both cell lines that support ΔICP0 virus infection have defects in the STING DNA-sensing pathway, which partially accounts for the rescue of the ΔICP0 virus growth. Restoration of STING expression in these cells rescued innate immunity and suppressed ΔICP0 virus infection. This study underscores the importance of STING in the control of HSV-1. PMID:28179534

Biophysical markers of the peripheral vasoconstriction response to pain in sickle cell disease

PubMed Central

Khaleel, Maha; Sunwoo, John; Shah, Payal; Detterich, Jon A.; Kato, Roberta M.; Thuptimdang, Wanwara; Meiselman, Herbert J.; Sposto, Richard; Tsao, Jennie; Wood, John C.; Zeltzer, Lonnie; Coates, Thomas D.; Khoo, Michael C. K.

2017-01-01

Painful vaso-occlusive crisis (VOC), a complication of sickle cell disease (SCD), occurs when sickled red blood cells obstruct flow in the microvasculature. We postulated that exaggerated sympathetically mediated vasoconstriction, endothelial dysfunction and the synergistic interaction between these two factors act together to reduce microvascular flow, promoting regional vaso-occlusions, setting the stage for VOC. We previously found that SCD subjects had stronger vasoconstriction response to pulses of heat-induced pain compared to controls but the relative degrees to which autonomic dysregulation, peripheral vascular dysfunction and their interaction are present in SCD remain unknown. In the present study, we employed a mathematical model to decompose the total vasoconstriction response to pain into: 1) the neurogenic component, 2) the vascular response to blood pressure, 3) respiratory coupling and 4) neurogenic-vascular interaction. The model allowed us to quantify the contribution of each component to the total vasoconstriction response. The most salient features of the components were extracted to represent biophysical markers of autonomic and vascular impairment in SCD and controls. These markers provide a means of phenotyping severity of disease in sickle-cell anemia that is based more on underlying physiology than on genotype. The marker of the vascular component (BMv) showed stronger contribution to vasoconstriction in SCD than controls (p = 0.0409), suggesting a dominant myogenic response in the SCD subjects as a consequence of endothelial dysfunction. The marker of neurogenic-vascular interaction (BMn-v) revealed that the interaction reinforced vasoconstriction in SCD but produced vasodilatory response in controls (p = 0.0167). This marked difference in BMn-v suggests that it is the most sensitive marker for quantifying combined alterations in autonomic and vascular function in SCD in response to heat-induced pain. PMID:28542469

SPAK kinase is a substrate and target of PKCθ in T-cell receptor-induced AP-1 activation pathway

PubMed Central

Li, Yingqiu; Hu, Junru; Vita, Randi; Sun, Binggang; Tabata, Hiroki; Altman, Amnon

2004-01-01

Protein kinase C-θ (PKCθ) plays an important role in T-cell activation via stimulation of AP-1 and NF-κB. Here we report the isolation of SPAK, a Ste20-related upstream mitogen-activated protein kinase (MAPK), as a PKCθ-interacting kinase. SPAK interacted with PKCθ (but not with PKCα) via its 99 COOH-terminal residues. TCR/CD28 costimulation enhanced this association and stimulated the catalytic activity of SPAK. Recombinant SPAK was phosphorylated on Ser-311 in its kinase domain by PKCθ, but not by PKCα. The magnitude and duration of TCR/CD28-induced endogenous SPAK activation were markedly impaired in PKCθ-deficient T cells. Transfected SPAK synergized with constitutively active PKCθ to activate AP-1, but not NF-κB. This synergistic activity, as well as the receptor-induced SPAK activation, required the PKCθ-interacting region of SPAK, and Ser-311 mutation greatly reduced these activities of SPAK. Conversely, a SPAK-specific RNAi or a dominant-negative SPAK mutant inhibited PKCθ- and TCR/CD28-induced AP-1, but not NF-κB, activation. These results define SPAK as a substrate and target of PKCθ in a TCR/CD28-induced signaling pathway leading selectively to AP-1 (but not NF-κB) activation. PMID:14988727

Human T-cell leukemia virus type 1 Tax interacts with Chk1 and attenuates DNA-damage induced G2 arrest mediated by Chk1.

PubMed

Park, Hyeon Ung; Jeong, Jae-Hoon; Chung, Jay H; Brady, John N

2004-06-24

Checkpoint kinase 1 (Chk1) mediates diverse cellular responses to genotoxic stress, regulating the network of genome-surveillance pathways that coordinate cell cycle progression with DNA repair. Chk1 is essential for mammalian development and viability, and has been shown to be important for both S and G(2) checkpoints. We now present evidence that the HTLV-1 Tax protein interacts directly with Chk1 and impairs its kinase activities in vitro and in vivo. The direct and physical interaction of Chk1 and Tax was observed in HTLV-1-infected T cells (C81, HuT 102 and MT-2) and transfected fibroblasts (293 T) by coimmunoprecipitation and by in vitro GST pull-down assays. Interestingly, Tax inhibited the kinase activity of Chk1 protein in in vitro and in vivo kinase assays. Consistent with these results, Tax inhibited the phosphorylation-dependent degradation of Cdc25A and G(2) arrest in response to gamma-irradiation (IR) in a dose-dependent manner in vivo. The G(2) arrest did not require Chk2 or p53. These studies provide the first example of a viral transforming protein targeting Chk1 and provide important insights into checkpoint pathway regulation.

Bio-inspired engineering of cell- and virus-like nanoparticles for drug delivery.

PubMed

Parodi, Alessandro; Molinaro, Roberto; Sushnitha, Manuela; Evangelopoulos, Michael; Martinez, Jonathan O; Arrighetti, Noemi; Corbo, Claudia; Tasciotti, Ennio

2017-12-01

The engineering of future generations of nanodelivery systems aims at the creation of multifunctional vectors endowed with improved circulation, enhanced targeting and responsiveness to the biological environment. Moving past purely bio-inert systems, researchers have begun to create nanoparticles capable of proactively interacting with the biology of the body. Nature offers a wide-range of sources of inspiration for the synthesis of more effective drug delivery platforms. Because the nano-bio-interface is the key driver of nanoparticle behavior and function, the modification of nanoparticles' surfaces allows the transfer of biological properties to synthetic carriers by imparting them with a biological identity. Modulation of these surface characteristics governs nanoparticle interactions with the biological barriers they encounter. Building off these observations, we provide here an overview of virus- and cell-derived biomimetic delivery systems that combine the intrinsic hallmarks of biological membranes with the delivery capabilities of synthetic carriers. We describe the features and properties of biomimetic delivery systems, recapitulating the distinctive traits and functions of viruses, exosomes, platelets, red and white blood cells. By mimicking these biological entities, we will learn how to more efficiently interact with the human body and refine our ability to negotiate with the biological barriers that impair the therapeutic efficacy of nanoparticles. Copyright © 2017. Published by Elsevier Ltd.

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses

PubMed Central

Vijayakrishnan, Niranjana; Woodruff, Elvin A.; Broadie, Kendal

2009-01-01

Summary Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis within minutes following a temperature shift from 25°C to 37°C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbots displays a strong synergistic interaction with the Syntaxin-1A TS allele syx3-69, recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbots showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbots;syx3-69 double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbots synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbots displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis. PMID:19066280

Rolling blackout is required for bulk endocytosis in non-neuronal cells and neuronal synapses.

PubMed

Vijayakrishnan, Niranjana; Woodruff, Elvin A; Broadie, Kendal

2009-01-01

Rolling blackout (RBO) is a Drosophila EFR3 integral membrane lipase. A conditional temperature-sensitive (TS) mutant (rbo(ts)) displays paralysis within minutes following a temperature shift from 25 degrees C to 37 degrees C, an impairment previously attributed solely to blocked synaptic-vesicle exocytosis. However, we found that rbo(ts) displays a strong synergistic interaction with the Syntaxin-1A TS allele syx(3-69), recently shown to be a dominant positive mutant that increases Syntaxin-1A function. At neuromuscular synapses, rbo(ts) showed a strong defect in styryl-FM-dye (FM) endocytosis, and rbo(ts);syx(3-69) double mutants displayed a synergistic, more severe, endocytosis impairment. Similarly, central rbo(ts) synapses in primary brain culture showed severely defective FM endocytosis. Non-neuronal nephrocyte Garland cells showed the same endocytosis defect in tracer-uptake assays. Ultrastructurally, rbo(ts) displayed a specific defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbo(ts) synapse, there was a total blockade of endosome formation via activity-dependent bulk endocytosis. Clathrin-mediated endocytosis was not affected; indeed, there was a significant increase in direct vesicle formation. Together, these results demonstrate that RBO is required for constitutive and/or bulk endocytosis and/or macropinocytosis in both neuronal and non-neuronal cells, and that, at the synapse, this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.

The mitochondrial voltage-dependent anion channel 1 in tumor cells.

PubMed

Shoshan-Barmatz, Varda; Ben-Hail, Danya; Admoni, Lee; Krelin, Yakov; Tripathi, Shambhoo Sharan

2015-10-01

VDAC1 is found at the crossroads of metabolic and survival pathways. VDAC1 controls metabolic cross-talk between mitochondria and the rest of the cell by allowing the influx and efflux of metabolites, ions, nucleotides, Ca2+ and more. The location of VDAC1 at the outer mitochondrial membrane also enables its interaction with proteins that mediate and regulate the integration of mitochondrial functions with cellular activities. As a transporter of metabolites, VDAC1 contributes to the metabolic phenotype of cancer cells. Indeed, this protein is over-expressed in many cancer types, and silencing of VDAC1 expression induces an inhibition of tumor development. At the same time, along with regulating cellular energy production and metabolism, VDAC1 is involved in the process of mitochondria-mediated apoptosis by mediating the release of apoptotic proteins and interacting with anti-apoptotic proteins. The engagement of VDAC1 in the release of apoptotic proteins located in the inter-membranal space involves VDAC1 oligomerization that mediates the release of cytochrome c and AIF to the cytosol, subsequently leading to apoptotic cell death. Apoptosis can also be regulated by VDAC1, serving as an anchor point for mitochondria-interacting proteins, such as hexokinase (HK), Bcl2 and Bcl-xL, some of which are also highly expressed in many cancers. By binding to VDAC1, HK provides both a metabolic benefit and apoptosis-suppressive capacity that offer the cell a proliferative advantage and increase its resistance to chemotherapy. Thus, these and other functions point to VDAC1 as an excellent target for impairing the re-programed metabolism of cancer cells and their ability to evade apoptosis. Here, we review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to both cancer development and therapy. In addressing the recently solved 3D structures of VDAC1, this review will point to structure-function relationships of VDAC as critical for deciphering how this channel can perform such a variety of roles, all of which are important for cell life and death. Finally, this review will also provide insight into VDAC function in Ca2+ homeostasis, protection against oxidative stress, regulation of apoptosis and involvement in several diseases, as well as its role in the action of different drugs. We will discuss the use of VDAC1-based strategies to attack the altered metabolism and apoptosis of cancer cells. These strategies include specific siRNA able to impair energy and metabolic homeostasis, leading to arrested cancer cell growth and tumor development, as well VDAC1-based peptides that interact with anti-apoptotic proteins to induce apoptosis, thereby overcoming the resistance of cancer cell to chemotherapy. Finally, small molecules targeting VDAC1 can induce apoptosis. VDAC1 can thus be considered as standing at the crossroads between mitochondrial metabolite transport and apoptosis and hence represents an emerging cancer drug target. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. Copyright © 2014 Elsevier B.V. All rights reserved.

Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria

PubMed Central

Das, Soumita; Owen, Katherine A.; Ly, Kim T.; Park, Daeho; Black, Steven G.; Wilson, Jeffrey M.; Sifri, Costi D.; Ravichandran, Kodi S.; Ernst, Peter B.; Casanova, James E.

2011-01-01

Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gram-negative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection. PMID:21245295

The impact of IUGR on pancreatic islet development and β-cell function.

PubMed

Boehmer, Brit H; Limesand, Sean W; Rozance, Paul J

2017-11-01

Placental insufficiency is a primary cause of intrauterine growth restriction (IUGR). IUGR increases the risk of developing type 2 diabetes mellitus (T2DM) throughout life, which indicates that insults from placental insufficiency impair β-cell development during the perinatal period because β-cells have a central role in the regulation of glucose tolerance. The severely IUGR fetal pancreas is characterized by smaller islets, less β-cells, and lower insulin secretion. Because of the important associations among impaired islet growth, β-cell dysfunction, impaired fetal growth, and the propensity for T2DM, significant progress has been made in understanding the pathophysiology of IUGR and programing events in the fetal endocrine pancreas. Animal models of IUGR replicate many of the observations in severe cases of human IUGR and allow us to refine our understanding of the pathophysiology of developmental and functional defects in islet from IUGR fetuses. Almost all models demonstrate a phenotype of progressive loss of β-cell mass and impaired β-cell function. This review will first provide evidence of impaired human islet development and β-cell function associated with IUGR and the impact on glucose homeostasis including the development of glucose intolerance and diabetes in adulthood. We then discuss evidence for the mechanisms regulating β-cell mass and insulin secretion in the IUGR fetus, including the role of hypoxia, catecholamines, nutrients, growth factors, and pancreatic vascularity. We focus on recent evidence from experimental interventions in established models of IUGR to understand better the pathophysiological mechanisms linking placental insufficiency with impaired islet development and β-cell function. © 2017 Society for Endocrinology.

Nonmuscle Myosin II Is Required for Internalization of the Epidermal Growth Factor Receptor and Modulation of Downstream Signaling*

PubMed Central

Kim, Jong Hyun; Wang, Aibing; Conti, Mary Anne; Adelstein, Robert S.

2012-01-01

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. Here, we demonstrate that nonmuscle myosin II (NM II) is required for the internalization of the EGFR and to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II by co-immunoprecipitation and mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR. Two paralogs of NM II, NM II-A, and NM II-B can act to internalize the EGFR, depending on the cell type and paralog content of the cell line. Loss (siRNA) or inhibition (25 μm blebbistatin) of NM II attenuates the internalization of the EGFR and impairs EGFR-dependent activation of ERK and AKT. Both internalization of the EGFR and downstream signaling to ERK and AKT can be partially restored in siRNA-treated cells by introduction of wild type (WT) GFP-NM II, but cannot be restored by motor mutant NM II. Taken together, these results suggest that NM II plays a role in the internalization of the EGFR and EGFR-mediated signaling pathways. PMID:22718763

Mouse superkiller‐2‐like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses

PubMed Central

Goubau, Delphine; van der Veen, Annemarthe G.; Chakravarty, Probir; Lin, Rongtuan; Rogers, Neil; Rehwinkel, Jan; Deddouche, Safia; Rosewell, Ian; Hiscott, John

2015-01-01

Abstract IFN‐α/β allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2‐like DExH‐box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN‐α/β by retinoic acid inducible gene 1‐like receptors (RLRs) that detect the presence of RNA viruses in a cell‐intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN‐α/β induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60‐deficient mice revealed no impairment in IFN‐α/β production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60‐deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus‐1. These results put in question the reported role of DDX60 as a broad‐acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses. PMID:26457795

APR3 modulates oxidative stress and mitochondrial function in ARPE-19 cells.

PubMed

Li, Yuan; Zou, Xuan; Gao, Jing; Cao, Ke; Feng, Zhihui; Liu, Jiankang

2018-05-24

Impairment of retinal pigment epithelial (RPE) cells is considered a key contributor to the development of age-related macular degeneration. Apoptosis-related protein 3 (APR3) was recently discovered after treatment with all- trans retinoic acid, a pivotal molecule in RPE cells. However, the function of APR3 remains poorly understood. In the present study, we found that APR3 could interact with nuclear factor (erythroid-derived 2)-like 2, which is a regulator of phase II enzymes, and that knockdown of APR3 promoted nuclear factor (erythroid-derived 2)-like 2 nuclear translocation and activated expression of phase II enzymes, which was accompanied by improved redox status and mitochondrial activity. Overexpression of APR3 revealed its mitochondrial localization and induced a robust production of reactive oxygen species that was accompanied by impaired mitochondrial oxygen consumption, complex activity, and lower ATP content, resulting in significant changes in mitochondrial structure, which may contribute to cell apoptosis. High doses of all- trans retinoic acid treatment were found to significantly induce APR3 expression, increase reactive oxygen species levels, and decrease ATP content, which were abolished by knockdown of APR3. These results indicate that APR3 plays a vital role in regulating redox status and mitochondrial activity and thus suggest APR3 might be a potential novel target for study of treatment of age-related macular degeneration.-Li, Y., Zou, X., Gao, J., Cao, K., Feng, Z., Liu, J. APR3 modulates oxidative stress and mitochondrial function in ARPE-19 cells.

Why does interactional justice promote organizational loyalty, job performance, and prevent mental impairment? The role of social support and social stressors.

PubMed

Otto, Kathleen; Mamatoglu, Nihal

2015-01-01

Using social exchange theory as a conceptual framework, we investigated the relationship between interactional justice and the outcomes organizational loyalty (affective commitment, turnover intentions), perceived job performance (self-rated performance, personal accomplishment), and mental impairment (cognitive irritation, emotional exhaustion) in an online survey of 218 employees working in the field of computer technology. Specifically, we predicted that interactional justice would heighten the quality of social exchange relationships and therefore expected perceived social support (POS) and bullying to mediate the proposed relationships. We tested our hypotheses applying a latent structural equation model. Our findings revealed that POS mediated the relationship between interactional justice and organizational loyalty, whereas bullying mediated the relationship between interactional justice and mental impairment. Practical implications are discussed concerning how to foster interactional justice and POS and how to weaken bullying behavior.

Respiratory proteins contribute differentially to Campylobacter jejuni’s survival and in vitro interaction with hosts’ intestinal cells

PubMed Central

2012-01-01

Background The genetic features that facilitate Campylobacter jejuni’s adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni’s adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni’s i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens). Results C. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA’s motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H2O2), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H2O2 was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division. Conclusions We conclude that the RPs contribute to C. jejuni’s motility, H2O2 resistance, biofilm formation, and in vitro interactions with hosts’ intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen’s remarkable potential for adaptation. PMID:23148765

Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

PubMed Central

Malina, Halina Z; Hess, Otto M

2004-01-01

Background Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid. Results Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption. Conclusion Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions. PMID:15068490

T Cell CX3CR1 Mediates Excess Atherosclerotic Inflammation in Renal Impairment

PubMed Central

Dong, Lei; Nordlohne, Johannes; Ge, Shuwang; Hertel, Barbara; Melk, Anette; Rong, Song; Haller, Hermann

2016-01-01

Reduced kidney function increases the risk for atherosclerosis and cardiovascular death. Leukocytes in the arterial wall contribute to atherosclerotic plaque formation. We investigated the role of fractalkine receptor CX3CR1 in atherosclerotic inflammation in renal impairment. Apoe−/− (apolipoprotein E) CX3CR1−/− mice with renal impairment were protected from increased aortic atherosclerotic lesion size and macrophage accumulation. Deficiency of CX3CR1 in bone marrow, only, attenuated atherosclerosis in renal impairment in an independent atherosclerosis model of LDL receptor–deficient (LDLr−/−) mice as well. Analysis of inflammatory leukocytes in atherosclerotic mixed bone-marrow chimeric mice (50% wild-type/50% CX3CR1−/− bone marrow into LDLr−/− mice) showed that CX3CR1 cell intrinsically promoted aortic T cell accumulation much more than CD11b+CD11c+ myeloid cell accumulation and increased IL-17-producing T cell counts. In vitro, fewer TH17 cells were obtained from CX3CR1−/− splenocytes than from wild-type splenocytes after polarization with IL-6, IL-23, and TGFβ. Polarization of TH17 or TREG cells, or stimulation of splenocytes with TGFβ alone, increased T cell CX3CR1 reporter gene expression. Furthermore, TGFβ induced CX3CR1 mRNA expression in wild-type cells in a dose- and time-dependent manner. In atherosclerotic LDLr−/− mice, CX3CR1+/− T cells upregulated CX3CR1 and IL-17A production in renal impairment, whereas CX3CR1−/− T cells did not. Transfer of CX3CR1+/− but not Il17a−/− T cells into LDLr−/−CX3CR1−/− mice increased aortic lesion size and aortic CD11b+CD11c+ myeloid cell accumulation in renal impairment. In summary, T cell CX3CR1 expression can be induced by TGFβ and is instrumental in enhanced atherosclerosis in renal impairment. PMID:26449606

Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

PubMed

Bedarida, Tatiana; Domingues, Alison; Baron, Stephanie; Ferreira, Chrystophe; Vibert, Francoise; Cottart, Charles-Henry; Paul, Jean-Louis; Escriou, Virginie; Bigey, Pascal; Gaussem, Pascale; Leguillier, Teddy; Nivet-Antoine, Valerie

2018-06-01

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIP fl/fl cdh5 cre ). Control (TXNIP fl/fl ) and TXNIP fl/fl cdh5 cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIP fl/fl and TXNIP fl/fl cdh5 cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIP fl/fl cdh5 cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1β. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

RP1 Is a Phosphorylation Target of CK2 and Is Involved in Cell Adhesion

PubMed Central

Göttig, Stephan; Henschler, Reinhard; Markuly, Norbert; Kleber, Sascha; Faust, Michael; Mischo, Axel; Bauer, Stefan; Zweifel, Martin; Knuth, Alexander; Renner, Christoph; Wadle, Andreas

2013-01-01

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser236 in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP236 show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser236 by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association. PMID:23844040

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion.

PubMed

Stenner, Frank; Liewen, Heike; Göttig, Stephan; Henschler, Reinhard; Markuly, Norbert; Kleber, Sascha; Faust, Michael; Mischo, Axel; Bauer, Stefan; Zweifel, Martin; Knuth, Alexander; Renner, Christoph; Wadle, Andreas

2013-01-01

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1.

PubMed

Ivie, Susan E; McClain, Mark S

2012-09-25

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1.

Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

PubMed Central

Ivie, Susan E.; McClain, Mark S.

2012-01-01

Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

Heterozygous Vangl2Looptail mice reveal novel roles for the planar cell polarity pathway in adult lung homeostasis and repair

PubMed Central

Poobalasingam, Thanushiyan; Yates, Laura L.; Walker, Simone A.; Pereira, Miguel; Gross, Nina Y.; Ali, Akmol; Kolatsi-Joannou, Maria; Jarvelin, Marjo-Riitta; Pekkanen, Juha; Papakrivopoulou, Eugenia; Long, David A.; Griffiths, Mark; Wagner, Darcy; Königshoff, Melanie; Hind, Matthew; Minelli, Cosetta; Lloyd, Clare M.

2017-01-01

ABSTRACT Lung diseases impose a huge economic and health burden worldwide. A key aspect of several adult lung diseases, such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), including emphysema, is aberrant tissue repair, which leads to an accumulation of damage and impaired respiratory function. Currently, there are few effective treatments available for these diseases and their incidence is rising. The planar cell polarity (PCP) pathway is critical for the embryonic development of many organs, including kidney and lung. We have previously shown that perturbation of the PCP pathway impairs tissue morphogenesis, which disrupts the number and shape of epithelial tubes formed within these organs during embryogenesis. However, very little is known about the role of the PCP pathway beyond birth, partly because of the perinatal lethality of many PCP mouse mutant lines. Here, we investigate heterozygous Looptail (Lp) mice, in which a single copy of the core PCP gene, Vangl2, is disrupted. We show that these mice are viable but display severe airspace enlargement and impaired adult lung function. Underlying these defects, we find that Vangl2Lp/+ lungs exhibit altered distribution of actin microfilaments and abnormal regulation of the actin-modifying protein cofilin. In addition, we show that Vangl2Lp/+ lungs exhibit many of the hallmarks of tissue damage, including an altered macrophage population, abnormal elastin deposition and elevated levels of the elastin-modifying enzyme, Mmp12, all of which are observed in emphysema. In vitro, disruption of VANGL2 impairs directed cell migration and reduces the rate of repair following scratch wounding of human alveolar epithelial cells. Moreover, using population data from a birth cohort of young adults, all aged 31, we found evidence of an interactive effect between VANGL2 and smoking on lung function. Finally, we show that PCP genes VANGL2 and SCRIB are significantly downregulated in lung tissue from patients with emphysema. Our data reveal an important novel role for the PCP pathway in adult lung homeostasis and repair and shed new light on the genetic factors which may modify destructive lung diseases such as emphysema. PMID:28237967

Acute Viral Respiratory Infection Rapidly Induces a CD8+ T Cell Exhaustion-like Phenotype.

PubMed

Erickson, John J; Lu, Pengcheng; Wen, Sherry; Hastings, Andrew K; Gilchuk, Pavlo; Joyce, Sebastian; Shyr, Yu; Williams, John V

2015-11-01

Acute viral infections typically generate functional effector CD8(+) T cells (TCD8) that aid in pathogen clearance. However, during acute viral lower respiratory infection, lung TCD8 are functionally impaired and do not optimally control viral replication. T cells also become unresponsive to Ag during chronic infections and cancer via signaling by inhibitory receptors such as programmed cell death-1 (PD-1). PD-1 also contributes to TCD8 impairment during viral lower respiratory infection, but how it regulates TCD8 impairment and the connection between this state and T cell exhaustion during chronic infections are unknown. In this study, we show that PD-1 operates in a cell-intrinsic manner to impair lung TCD8. In light of this, we compared global gene expression profiles of impaired epitope-specific lung TCD8 to functional spleen TCD8 in the same human metapneumovirus-infected mice. These two populations differentially regulate hundreds of genes, including the upregulation of numerous inhibitory receptors by lung TCD8. We then compared the gene expression of TCD8 during human metapneumovirus infection to those in acute or chronic lymphocytic choriomeningitis virus infection. We find that the immunophenotype of lung TCD8 more closely resembles T cell exhaustion late into chronic infection than do functional effector T cells arising early in acute infection. Finally, we demonstrate that trafficking to the infected lung alone is insufficient for TCD8 impairment or inhibitory receptor upregulation, but that viral Ag-induced TCR signaling is also required. Our results indicate that viral Ag in infected lungs rapidly induces an exhaustion-like state in lung TCD8 characterized by progressive functional impairment and upregulation of numerous inhibitory receptors. Copyright © 2015 by The American Association of Immunologists, Inc.

CD8+ T Cells Induce Fatal Brainstem Pathology during Cerebral Malaria via Luminal Antigen-Specific Engagement of Brain Vasculature

PubMed Central

Swanson, Phillip A.; Hart, Geoffrey T.; Russo, Matthew V.; Nayak, Debasis; Yazew, Takele; Peña, Mirna; Khan, Shahid M.; Pierce, Susan K.; McGavern, Dorian B.

2016-01-01

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection that results in thousands of deaths each year, mostly in African children. The in vivo mechanisms underlying this fatal condition are not entirely understood. Using the animal model of experimental cerebral malaria (ECM), we sought mechanistic insights into the pathogenesis of CM. Fatal disease was associated with alterations in tight junction proteins, vascular breakdown in the meninges / parenchyma, edema, and ultimately neuronal cell death in the brainstem, which is consistent with cerebral herniation as a cause of death. At the peak of ECM, we revealed using intravital two-photon microscopy that myelomonocytic cells and parasite-specific CD8+ T cells associated primarily with the luminal surface of CNS blood vessels. Myelomonocytic cells participated in the removal of parasitized red blood cells (pRBCs) from cerebral blood vessels, but were not required for the disease. Interestingly, the majority of disease-inducing parasite-specific CD8+ T cells interacted with the lumen of brain vascular endothelial cells (ECs), where they were observed surveying, dividing, and arresting in a cognate peptide-MHC I dependent manner. These activities were critically dependent on IFN-γ, which was responsible for activating cerebrovascular ECs to upregulate adhesion and antigen-presenting molecules. Importantly, parasite-specific CD8+ T cell interactions with cerebral vessels were impaired in chimeric mice rendered unable to present EC antigens on MHC I, and these mice were in turn resistant to fatal brainstem pathology. Moreover, anti-adhesion molecule (LFA-1 / VLA-4) therapy prevented fatal disease by rapidly displacing luminal CD8+ T cells from cerebrovascular ECs without affecting extravascular T cells. These in vivo data demonstrate that parasite-specific CD8+ T cell-induced fatal vascular breakdown and subsequent neuronal death during ECM is associated with luminal, antigen-dependent interactions with cerebrovasculature. PMID:27907215

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade

PubMed Central

Caldeira, Joana; Figueiredo, Joana; Brás-Pereira, Catarina; Carneiro, Patrícia; Moreira, Ana M.; Pinto, Marta T.; Relvas, João B.; Carneiro, Fátima; Barbosa, Mário; Casares, Fernando; Janody, Florence; Seruca, Raquel

2015-01-01

Epithelial-cadherin (Ecad) deregulation affects cell–cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell–matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and βPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), β1 and β4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression. PMID:26246502

Impaired cellulose synthase guidance leads to stem torsion and twists phyllotactic patterns in Arabidopsis.

PubMed

Landrein, Benoît; Lathe, Rahul; Bringmann, Martin; Vouillot, Cyril; Ivakov, Alexander; Boudaoud, Arezki; Persson, Staffan; Hamant, Olivier

2013-05-20

The parallel alignment of stiff cellulose microfibrils in plant-cell walls mediates anisotropic growth. This is largely controlled by cortical microtubules, which drive the insertion and trajectory of the cellulose synthase (CESA) complex at the plasma membrane. The CESA interactive protein 1 (CSI1) acts as a physical linker between CESA and cortical microtubules. Here we show that the inflorescence stems of csi1 mutants exhibit subtle right-handed torsion. Because cellulose deposition is largely uncoupled from cortical microtubules in csi1, we hypothesize that strictly transverse deposition of microfibrils in the wild-type is replaced by a helical orientation of uniform handedness in the mutant and that the helical microfibril alignment generates torsion. Interestingly, both elastic and viscous models for an expanding cell predict that a net helical orientation of microfibrils gives rise to a torque. We indeed observed tilted microfibrils in csi1 cells, and the torsion was almost absent in a csi1 prc1 background with impaired cellulose synthesis. In addition, the stem torsion led to a novel bimodal and robust phyllotactic pattern in the csi1 mutant, illustrating how growth perturbations can replace one robust mathematical pattern with a different, equally robust pattern. Copyright © 2013 Elsevier Ltd. All rights reserved.

Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche.

PubMed

Chakrabarti, Rumela; Celià-Terrassa, Toni; Kumar, Sushil; Hang, Xiang; Wei, Yong; Choudhury, Abrar; Hwang, Julie; Peng, Jia; Nixon, Briana; Grady, John J; DeCoste, Christina; Gao, Jie; van Es, Johan H; Li, Ming O; Aifantis, Iannis; Clevers, Hans; Kang, Yibin

2018-05-17

The stem cell niche is a specialized environment that dictates stem cell function during development and homeostasis. Here, we show that Dll1, a Notch pathway ligand, is enriched in mammary gland stem cells (MaSCs) and mediates critical interactions with stromal macrophages in the surrounding niche. Conditional deletion of Dll1 reduced the number of MaSCs and impaired ductal morphogenesis in the mammary gland. Moreover, MaSC-expressed Dll1 activates Notch signaling in stromal macrophages, increasing their expression of Wnt family ligands such as Wnt3, Wnt10A, and Wnt16, thereby initiating a feed back loop that promotes the function of Dll1 + MaSCs. Together, these findings reveal functionally important cross-talk between MaSCs and their macrophageal niche through Dll1/Notch-mediated signaling. Copyright © 2018, American Association for the Advancement of Science.

CHIP promotes thyroid cancer proliferation via activation of the MAPK and AKT pathways.

PubMed

Zhang, Li; Liu, Lianyong; He, Xiaohua; Shen, Yunling; Liu, Xuerong; Wei, Jing; Yu, Fang; Tian, Jianqing

2016-08-26

The carboxyl terminus of Hsp70-interacting protein (CHIP) is a U box-type ubiquitin ligase that plays crucial roles in various biological processes, including tumor progression. To date, the functional mechanism of CHIP in thyroid cancer remains unknown. Here, we obtained evidence of upregulation of CHIP in thyroid cancer tissues and cell lines. CHIP overexpression markedly enhanced thyroid cancer cell viability and colony formation in vitro and accelerated tumor growth in vivo. Conversely, CHIP knockdown impaired cell proliferation and tumor growth. Notably, CHIP promoted cell growth through activation of MAPK and AKT pathways, subsequently decreasing p27 and increasing cyclin D1 and p-FOXO3a expression. Our findings collectively indicate that CHIP functions as an oncogene in thyroid cancer, and is therefore a potential therapeutic target for this disease. Copyright © 2016 Elsevier Inc. All rights reserved.

Deficiency of β-arrestin1 ameliorates collagen-induced arthritis with impaired TH17 cell differentiation

PubMed Central

Li, Juan; Wei, Bin; Guo, Ao; Liu, Chang; Huang, Shichao; Du, Fang; Fan, Wei; Bao, Chunde; Pei, Gang

2013-01-01

Rheumatoid arthritis (RA) is an inflammatory disease in which interleukin 17 (IL-17)-producing T helper 17 (TH17) cells have been critically involved. We show that in patients with RA, the expression of a multifunctional regulator β-arrestin1 was significantly up-regulated in peripheral and synovial CD4+ T cells, which correlated well with active phases of RA. In collagen-induced arthritis, deficiency of β-arrestin1 ameliorated disease with decreased TH17 cell differentiation, proinflammatory cytokine production, synovitis, and cartilage and bone destruction. Further mechanistic study reveals that β-arrestin1 promoted signal transducer and activator of transcription 3 (STAT3) activation required for TH17 cell differentiation through scaffolding the interaction of Janus kinase 1 and STAT3. These findings indicate a critical role for β-arrestin1 in the pathogenesis of collagen-induced arthritis and TH17 cell differentiation and suggest β-arrestin1 as a potential diagnostic biomarker and therapeutic target for RA. PMID:23589893

Topographical Control of Ocular Cell Types for Tissue Engineering

PubMed Central

McHugh, Kevin J.; Saint-Geniez, Magali; Tao, Sarah L.

2014-01-01

Visual impairment affects over 285 million people worldwide and has a major impact on an individual’s quality of life. Tissue engineering has the potential to increase quality of life for many of these patients by preventing vision loss or restoring vision using cell-based therapies. However, these strategies will require an understanding of the microenvironmental factors that influence cell behavior. The eye is a well-organized organ whose structural complexity is essential for proper function. Interactions between ocular cells and their highly ordered extracellular matrix are necessary for maintaining key tissue properties including corneal transparency and retinal lamination. Therefore, it is not surprising that culturing these cells in vitro on traditional flat substrates result in irregular morphology. Instead, topographically patterned biomaterials better mimic native extracellular matrix and have been shown to elicit in vivo-like morphology and gene expression which is essential for tissue engineering. Herein we review multiple methods for producing well-controlled topography and discuss optimal biomaterial scaffold design for cells of the cornea, retina, and lens. PMID:23744715

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner

PubMed Central

Hughes, K. R.; Harnisch, L. C.; Alcon-Giner, C.; Mitra, S.; Wright, C. J.; Ketskemety, J.

2017-01-01

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi—a process termed ‘cell shedding’. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. PMID:28123052

Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

PubMed

Hughes, K R; Harnisch, L C; Alcon-Giner, C; Mitra, S; Wright, C J; Ketskemety, J; van Sinderen, D; Watson, A J M; Hall, L J

2017-01-01

Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. © 2017 The Authors.

Specific binding of the WASP N-terminal domain to Btk is critical for TLR2 signaling in macrophages.

PubMed

Sakuma, Chisato; Sato, Mitsuru; Takenouchi, Takato; Kitani, Hiroshi

2015-02-01

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we revealed that WASP is involved in lipopolysaccharide-TLR4 signaling in macrophages by association of Bruton's tyrosine kinase (Btk) with the WASP N-terminal domain. Btk has been shown to play important roles in the signaling of several TLRs and to modulate the inflammatory response in macrophages. In this study, we evaluated the importance of the interaction between Btk and WASP in TLR2 signaling by using bone marrow-derived macrophage cell lines from transgenic (Tg) mice expressing anti-WASP N-terminal domain single-chain variable fragment (scFv) or VL single-domain intrabodies. In this Tg bone marrow-derived macrophages, specific interaction between WASP and Btk were strongly inhibited by masking of the binding site in the WASP N-terminal domain. There was impairment of gene expression of TNF-α, IL-6, and IL-1β and phosphorylation of inhibitor of κB α/β (IKKα/β) and nuclear factor (NF)-κB upon stimulation with TLR2 ligands. Furthermore, tyrosine phosphorylation of WASP following TLR2-ligand stimulation was severely inhibited in the Tg bone marrow-derived macrophages, as shown by the impairment in WASP tyrosine phosphorylation following lipopolysaccharide stimulation. These results strongly suggest that the association between the WASP N-terminal domain and Btk plays an important role in the TLR2-signaling pathway in macrophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks

PubMed Central

Stante, Maria; Minopoli, Giuseppina; Passaro, Fabiana; Raia, Maddalena; Vecchio, Luigi Del; Russo, Tommaso

2009-01-01

Fe65 is a binding partner of the Alzheimer's β-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites. PMID:19282473

Peripheral vascular reactivity and serum BDNF responses to aerobic training are impaired by the BDNF Val66Met polymorphism.

PubMed

Lemos, José R; Alves, Cleber R; de Souza, Sílvia B C; Marsiglia, Julia D C; Silva, Michelle S M; Pereira, Alexandre C; Teixeira, Antônio L; Vieira, Erica L M; Krieger, José E; Negrão, Carlos E; Alves, Guilherme B; de Oliveira, Edilamar M; Bolani, Wladimir; Dias, Rodrigo G; Trombetta, Ivani C

2016-02-01

Besides neuronal plasticity, the neurotrophin brain-derived neurotrophic factor (BDNF) is also important in vascular function. The BDNF has been associated with angiogenesis through its specific receptor tropomyosin-related kinase B (TrkB). Additionally, Val66Met polymorphism decreases activity-induced BDNF. Since BDNF and TrkB are expressed in vascular endothelial cells and aerobic exercise training can increase serum BDNF, this study aimed to test the hypotheses: 1) Serum BDNF levels modulate peripheral blood flow; 2) The Val66Met BDNF polymorphism impairs exercise training-induced vasodilation. We genotyped 304 healthy male volunteers (Val66Val, n = 221; Val66Met, n = 83) who underwent intense aerobic exercise training on a running track three times/wk for 4 mo. We evaluated pre- and post-exercise training serum BDNF and proBDNF concentration, heart rate (HR), mean blood pressure (MBP), forearm blood flow (FBF), and forearm vascular resistance (FVR). In the pre-exercise training, BDNF, proBDNF, BDNF/proBDNF ratio, FBF, and FVR were similar between genotypes. After exercise training, functional capacity (V̇o2 peak) increased and HR decreased similarly in both groups. Val66Val, but not Val66Met, increased BDNF (interaction, P = 0.04) and BDNF/proBDNF ratio (interaction, P < 0.001). Interestingly, FBF (interaction, P = 0.04) and the FVR (interaction, P = 0.01) responses during handgrip exercise (HG) improved in Val66Val compared with Val66Met, even with similar responses of HR and MBP. There were association between BDNF/proBDNF ratio and FBF (r = 0.64, P < 0.001) and FVR (r = -0.58, P < 0.001) during HG exercise. These results show that peripheral vascular reactivity and serum BDNF responses to exercise training are impaired by the BDNF Val66Met polymorphism and such responsiveness is associated with serum BDNF concentrations in healthy subjects. Copyright © 2016 the American Physiological Society.

Mouse superkiller-2-like helicase DDX60 is dispensable for type I IFN induction and immunity to multiple viruses.

PubMed

Goubau, Delphine; van der Veen, Annemarthe G; Chakravarty, Probir; Lin, Rongtuan; Rogers, Neil; Rehwinkel, Jan; Deddouche, Safia; Rosewell, Ian; Hiscott, John; Reis E Sousa, Caetano

2015-12-01

IFN-α/β allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. One of these, the Ski2-like DExH-box helicase DDX60, was recently implicated in resistance of human cells to hepatitis C virus, as well as in induction of IFN-α/β by retinoic acid inducible gene 1-like receptors (RLRs) that detect the presence of RNA viruses in a cell-intrinsic manner. Here, we sought to investigate the role of DDX60 in IFN-α/β induction and in resistance to virus infection. Analysis of fibroblasts and myeloid cells from Ddx60-deficient mice revealed no impairment in IFN-α/β production in response to RLR agonists, RNA viruses, or other stimuli. Moreover, overexpression of DDX60 did not potentiate IFN induction and DDX60 did not interact with RLRs or capture RLR agonists from virally infected cells. We also failed to identify any impairment in Ddx60-deficient murine cells or mice in resistance to infection with influenza A virus, encephalomyocarditis virus, Sindbis virus, vaccinia virus, or herpes simplex virus-1. These results put in question the reported role of DDX60 as a broad-acting positive regulator of RLR responses and hint at the possibility that it may function as a restriction factor highly specific for a particular virus or class of viruses. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Maternal Interactions with a Hearing and Hearing-Impaired Twin: Similarities and Differences in Speech Input, Interaction Quality, and Word Production

ERIC Educational Resources Information Center

Lam, Christa; Kitamura, Christine

2010-01-01

Purpose: This study examined a mother's speech style and interactive behaviors with her twin sons: 1 with bilateral hearing impairment (HI) and the other with normal hearing (NH). Method: The mother was video-recorded interacting with her twin sons when the boys were 12.5 and 22 months of age. Mean F0, F0 range, duration, and F1/F2 vowel space of…

SOXF factors regulate murine satellite cell self-renewal and function through inhibition of β-catenin activity.

PubMed

Alonso-Martin, Sonia; Auradé, Frédéric; Mademtzoglou, Despoina; Rochat, Anne; Zammit, Peter S; Relaix, Frédéric

2018-06-08

Muscle satellite cells are the primary source of stem cells for postnatal skeletal muscle growth and regeneration. Understanding genetic control of satellite cell formation, maintenance, and acquisition of their stem cell properties is on-going, and we have identified SOXF (SOX7, SOX17, SOX18) transcriptional factors as being induced during satellite cell specification. We demonstrate that SOXF factors regulate satellite cell quiescence, self-renewal and differentiation. Moreover, ablation of Sox17 in the muscle lineage impairs postnatal muscle growth and regeneration. We further determine that activities of SOX7, SOX17 and SOX18 overlap during muscle regeneration, with SOXF transcriptional activity requisite. Finally, we show that SOXF factors also control satellite cell expansion and renewal by directly inhibiting the output of β-catenin activity, including inhibition of Ccnd1 and Axin2 . Together, our findings identify a key regulatory function of SoxF genes in muscle stem cells via direct transcriptional control and interaction with canonical Wnt/β-catenin signaling. © 2018, Alonso-Martin et al.

Inhibition of melanoma cell motility by the snake venom disintegrin eristostatin

PubMed Central

Tian, Jing; Paquette-Straub, Carrie; Sage, E. Helene; Funk, Sarah E.; Patel, Vivek; Galileo, Deni; McLane, Mary Ann

2007-01-01

Eristostatin, an RGD-containing disintegrin isolated from the venom of Eristicophis macmahoni, inhibits lung or liver colonization of melanoma cells in a mouse model. In this study, transwell migration and in vitro wound closure assays were used to determine the effect of eristostatin on the migration of melanoma cells. Eristostatin significantly impaired the migration of 5 human melanoma cell lines. Furthermore, it specifically inhibited cell migration on fibronectin in a concentration-dependent manner, but not that on collagen IV or laminin. In contrast, eristostatin was found to have no effect on cell proliferation or angiogenesis. These results indicate that the interaction between eristostatin and melanoma cells may involve fibronectin-binding integrins that mediate cell migration. Mutations to alanine of seven residues within the RGD loop of eristostatin and four residues outside the RGD loop of eristostatin resulted in significantly less potency in both platelet aggregation and wound closure assays. For six of the mutations, however, decreased activity was found only in the latter assay. We conclude that a different mechanism and/or integrin is involved in these two cell activities. PMID:17316731

Infant sex, obstetric risk factors, and 2-year neurodevelopmental outcome among preterm infants.

PubMed

Spinillo, Arsenio; Montanari, Laura; Gardella, Barbara; Roccio, Marianna; Stronati, Mauro; Fazzi, Elisa

2009-07-01

To evaluate the effect of the interaction between fetal sex and obstetric variables on the risk of neurodevelopmental impairment among preterm infants. A cohort study of 394 male and 360 female surviving infants born at 24 to 33 completed weeks of gestational age. Neurological examination and cognitive assessment of the infants (Bayley Scales of Infant Development) were performed at 2 years corrected age. Mean gestational age was 30.4 weeks (SD 2.4). Rates of mild and moderate-to-severe neurodevelopmental impairment were 14.6% (110/754) and 7% (53/754) respectively. In logistic analysis, male sex was associated with an increased risk of neurodevelopmental impairment (odds ratio 1.8, 95% confidence interval 1.21-2.68) compared with females. The excess risk of neurodevelopmental impairment associated with male sex was higher among preeclamptic than normotensive pregnancies (p for interaction=0.004), among infants who were either small for gestational age or delivered to a mother with preeclampsia (p for interaction=0.001) and in iatrogenic as opposed to spontaneous preterm birth or preterm premature rupture of membranes (p for interaction=0.035). The excess risk of neurodevelopmental impairment associated with male sex among preterm infants is modulated by obstetric risk factors.

CDK5-A Novel Role in Prostate Cancer Immunotherapy

DTIC Science & Technology

2017-10-01

of the involvement of a T cell antitumor response in impaired growth of prostate cancer in immunocompetent murine models of prostate cancer, and...of immune system activation by Cdk5 deletion in prostate cancer. We will confirm the involvement of a T cell antitumor response in impaired growth of...project? Major Task 1: Involvement of T cell anticancer immune response in impaired growth of TRAMP Cdk5-/- model. Months 1-10. Completed, month 10

Inhibition of autophagy and glycolysis by nitric oxide during hypoxia-reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons.

PubMed

Benavides, Gloria A; Liang, Qiuli; Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

2013-12-01

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated. © 2013 Elsevier Inc. All rights reserved.

Leishmania donovani Utilize Sialic Acids for Binding and Phagocytosis in the Macrophages through Selective Utilization of Siglecs and Impair the Innate Immune Arm.

PubMed

Roy, Saptarshi; Mandal, Chitra

2016-08-01

Leishmania donovani, belonging to a unicellular protozoan parasite, display the differential level of linkage-specific sialic acids on their surface. Sialic acids binding immunoglobulin-like lectins (siglecs) are a class of membrane-bound receptors present in the haematopoetic cell lineages interact with the linkage-specific sialic acids. Here we aimed to explore the utilization of sialic acids by Leishmania donovani for siglec-mediated binding, phagocytosis, modulation of innate immune response and signaling pathways for establishment of successful infection in the host. We have found enhanced binding of high sialic acids containing virulent strains (AG83+Sias) with siglec-1 and siglec-5 present on macrophages compared to sialidase treated AG83+Sias (AG83-Sias) and low sialic acids-containing avirulent strain (UR6) by flow cytometry. This specific receptor-ligand interaction between sialic acids and siglecs were further confirmed by confocal microscopy. Sialic acids-siglec-1-mediated interaction of AG83+Sias with macrophages induced enhanced phagocytosis. Additionally, sialic acids-siglec-5 interaction demonstrated reduced ROS, NO generation and Th2 dominant cytokine response upon infection with AG83+Sias in contrast to AG83-Sias and UR6. Sialic acids-siglecs binding also facilitated multiplication of intracellular amastigotes. Moreover, AG83+Sias induced sialic acids-siglec-5-mediated upregulation of host phosphatase SHP-1. Such sialic acids-siglec interaction was responsible for further downregulation of MAPKs (p38, ERK and JNK) and PI3K/Akt pathways followed by the reduced translocation of p65 subunit of NF-κβ to the nucleus from cytosol in the downstream signaling pathways. This sequence of events was reversed in AG83-Sias and UR6-infected macrophages. Besides, siglec-knockdown macrophages also showed the reversal of AG83+Sias infection-induced effector functions and downstream signaling events. Taken together, this study demonstrated that virulent parasite (AG83+Sias) establish a unique sialic acids-mediated binding and subsequent phagocytosis in the host cell through the selective exploitation of siglec-1. Additionally, sialic acids-siglec-5 interaction altered the downstream signaling pathways which contributed impairment of immune effector functions of macrophages. To the best of our knowledge, this is a comprehensive report describing sialic acids-siglec interactions and their role in facilitating uptake of the virulent parasite within the host.

Impaired NK Cell Responses to Pertussis and H1N1 Influenza Vaccine Antigens in Human Cytomegalovirus-Infected Individuals

PubMed Central

Nielsen, Carolyn M.; White, Matthew J.; Bottomley, Christian; Lusa, Chiara; Rodríguez-Galán, Ana; Turner, Scarlett E. G.; Goodier, Martin R.

2015-01-01

NK cells contribute to postvaccination immune responses after activation by IL-2 from Ag-specific memory T cells or by cross-linking of the low-affinity IgG receptor, CD16, by Ag–Ab immune complexes. Sensitivity of NK cells to these signals from the adaptive immune system is heterogeneous and influenced by their stage of differentiation. CD56dimCD57+ NK cells are less responsive to IL-2 and produce less IFN-γ in response to T cell–mediated activation than do CD56bright or CD56dimCD57− NK cells. Conversely, NK cell cytotoxicity, as measured by degranulation, is maintained across the CD56dim subsets. Human CMV (HCMV), a highly prevalent herpes virus causing lifelong, usually latent, infections, drives the expansion of the CD56dimCD57+NKG2C+ NK cell population, skewing the NK cell repertoire in favor of cytotoxic responses at the expense of cytokine-driven responses. We hypothesized, therefore, that HCMV seropositivity would be associated with altered NK cell responses to vaccine Ags. In a cross-sectional study of 152 U.K. adults, with HCMV seroprevalence rate of 36%, we find that HCMV seropositivity is associated with lower NK cell IFN-γ production and degranulation after in vitro restimulation with pertussis or H1N1 influenza vaccine Ags. Higher expression of CD57/NKG2C and lower expression of IL-18Rα on NK cells from HCMV seropositive subjects do not fully explain these impaired responses, which are likely the result of multiple receptor–ligand interactions. This study demonstrates for the first time, to our knowledge, that HCMV serostatus influences NK cell contributions to adaptive immunity and raises important questions regarding the impact of HCMV infection on vaccine efficacy. PMID:25855356

Pyruvate dehydrogenase subunit β of Lactobacillus plantarum is a collagen adhesin involved in biofilm formation.

PubMed

Salzillo, Marzia; Vastano, Valeria; Capri, Ugo; Muscariello, Lidia; Marasco, Rosangela

2017-04-01

Multi-functional surface proteins have been observed in a variety of pathogenic bacteria, where they mediate host cell adhesion and invasion, as well as in commensal bacterial species, were they mediate positive interaction with the host. Among these proteins, some glycolytic enzymes, expressed on the bacterial cell surface, can bind human extracellular matrix components (ECM). A major target for them is collagen, an abundant glycoprotein of connective tissues. We have previously shown that the enolase EnoA1 of Lactobacillus plantarum, one of the most predominant species in the gut microbiota of healthy individuals, is involved in binding with collagen type I (CnI). In this study, we found that PDHB, a component of the pyruvate dehydrogenase complex, contributes to the L. plantarum LM3 adhesion to CnI. By a cellular adhesion assay to immobilized CnI, we show that LM3-B1 cells, carrying a null mutation in the pdhB gene, bind to CnI - coated surfaces less efficiently than wild-type cells. Moreover, we show that the PDHB-CnI interaction requires a native state for PDHB. We also analyzed the ability to develop biofilm in wild-type and mutant strains and we found that the lack of the PDHB on cell surface generates cells partially impaired in biofilm development. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovering the infectome of human endothelial cells challenged with Aspergillus fumigatus applying a mass spectrometry label-free approach.

PubMed

Curty, N; Kubitschek-Barreira, P H; Neves, G W; Gomes, D; Pizzatti, L; Abdelhay, E; Souza, G H M F; Lopes-Bezerra, L M

2014-01-31

Blood vessel invasion is a key feature of invasive aspergillosis. This angioinvasion process contributes to tissue thrombosis, which can impair the access of leukocytes and antifungal drugs to the site of infection. It has been demonstrated that human umbilical vein endothelial cells (HUVECs) are activated and assume a prothrombotic phenotype following contact with Aspergillus fumigatus hyphae or germlings, a process that is independent of fungus viability. However, the molecular mechanisms by which this pathogen can activate endothelial cells, together with the endothelial pathways that are involved in this process, remain unknown. Using a label-free approach by High Definition Mass Spectrometry (HDMS(E)), differentially expressed proteins were identified during HUVEC-A. fumigatus interaction. Among these, 89 proteins were determined to be up- or down-regulated, and another 409 proteins were exclusive to one experimental condition: the HUVEC control or HUVEC:AF interaction. The in silico predictions provided a general view of which biological processes and/or pathways were regulated during HUVEC:AF interaction, and they mainly included cell signaling, immune response and hemostasis pathways. This work describes the first global proteomic analysis of HUVECs following interaction with A. fumigatus germlings, the fungus morphotype that represents the first step of invasion and dissemination within the host. A. fumigatus causes the main opportunistic invasive fungal infection related to neutropenic hematologic patients. One of the key steps during the establishment of invasive aspergillosis is angioinvasion but the mechanism associated with the interaction of A. fumigatus with the vascular endothelium remains unknown. The identification of up- and down-regulated proteins expressed by human endothelial cells in response to the fungus infection can contribute to reveal the mechanism of endothelial response and, to understand the physiopathology of this high mortality disease. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. © 2013 Elsevier B.V. All rights reserved.

Measles virus attachment proteins with impaired ability to bind CD46 interact more efficiently with the homologous fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Corey, Elizabeth A.; Iorio, Ronald M.; Program in Immunology and Virology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655

2009-01-05

Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation ofmore » fusion by these two paramyxoviruses.« less

Inhibition of polyomavirus ori-dependent DNA replication by mSin3B.

PubMed

Xie, An-Yong; Folk, William R

2002-12-01

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) ori-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.

Exacerbation of spontaneous autoimmune nephritis following regulatory T cell depletion in B cell lymphoma 2‐interacting mediator knock‐out mice

PubMed Central

Wang, Y. M.; Zhang, G. Y.; Wang, Y.; Hu, M.; Zhou, J. J.; Sawyer, A.; Cao, Q.; Wang, Y.; Zheng, G.; Lee, V. W. S.; Harris, D. C. H.

2017-01-01

Summary Regulatory T cells (Tregs) have been recognized as central mediators for maintaining peripheral tolerance and limiting autoimmune diseases. The loss of Tregs or their function has been associated with exacerbation of autoimmune disease. However, the temporary loss of Tregs in the chronic spontaneous disease model has not been investigated. In this study, we evaluated the role of Tregs in a novel chronic spontaneous glomerulonephritis model of B cell lymphoma 2‐interacting mediator (Bim) knock‐out mice by transient depleting Tregs. Bim is a pro‐apoptotic member of the B cell lymphoma 2 (Bcl‐2) family. Bim knock‐out (Bim–/–) mice fail to delete autoreactive T cells in thymus, leading to chronic spontaneous autoimmune kidney disease. We found that Treg depletion in Bim–/– mice exacerbated the kidney injury with increased proteinuria, impaired kidney function, weight loss and greater histological injury compared with wild‐type mice. There was a significant increase in interstitial infiltrate of inflammatory cells, antibody deposition and tubular damage. Furthermore, the serum levels of cytokines interleukin (IL)−2, IL‐4, IL‐6, IL‐10, IL‐17α, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α were increased significantly after Treg depletion in Bim–/– mice. This study demonstrates that transient depletion of Tregs leads to enhanced self‐reactive T effector cell function followed by exacerbation of kidney disease in the chronic spontaneous kidney disease model of Bim‐deficient mice. PMID:28152566

Predicting the impact of combined therapies on myeloma cell growth using a hybrid multi-scale agent-based model.

PubMed

Ji, Zhiwei; Su, Jing; Wu, Dan; Peng, Huiming; Zhao, Weiling; Nlong Zhao, Brian; Zhou, Xiaobo

2017-01-31

Multiple myeloma is a malignant still incurable plasma cell disorder. This is due to refractory disease relapse, immune impairment, and development of multi-drug resistance. The growth of malignant plasma cells is dependent on the bone marrow (BM) microenvironment and evasion of the host's anti-tumor immune response. Hence, we hypothesized that targeting tumor-stromal cell interaction and endogenous immune system in BM will potentially improve the response of multiple myeloma (MM). Therefore, we proposed a computational simulation of the myeloma development in the complicated microenvironment which includes immune cell components and bone marrow stromal cells and predicted the effects of combined treatment with multi-drugs on myeloma cell growth. We constructed a hybrid multi-scale agent-based model (HABM) that combines an ODE system and Agent-based model (ABM). The ODEs was used for modeling the dynamic changes of intracellular signal transductions and ABM for modeling the cell-cell interactions between stromal cells, tumor, and immune components in the BM. This model simulated myeloma growth in the bone marrow microenvironment and revealed the important role of immune system in this process. The predicted outcomes were consistent with the experimental observations from previous studies. Moreover, we applied this model to predict the treatment effects of three key therapeutic drugs used for MM, and found that the combination of these three drugs potentially suppress the growth of myeloma cells and reactivate the immune response. In summary, the proposed model may serve as a novel computational platform for simulating the formation of MM and evaluating the treatment response of MM to multiple drugs.

The HLA-A2 Restricted T Cell Epitope HCV Core35–44 Stabilizes HLA-E Expression and Inhibits Cytolysis Mediated by Natural Killer Cells

PubMed Central

Nattermann, Jacob; Nischalke, Hans Dieter; Hofmeister, Valeska; Ahlenstiel, Golo; Zimmermann, Henning; Leifeld, Ludger; Weiss, Elisabeth H.; Sauerbruch, Tilman; Spengler, Ulrich

2005-01-01

Impaired activity of natural killer cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. Natural cytotoxicity is regulated by interactions of HLA-E with inhibitory CD94/NKG2A receptors on natural killer (NK) cells. Here, we studied whether HCV core encodes peptides that bind to HLA-E and inhibit natural cytotoxicity. We analyzed 30 HCV core-derived peptides. Peptide-induced stabilization of HLA-E expression was measured flow cytometrically after incubating HLA-E-transfected cells with peptides. NK cell function was studied with a 51chromium-release-assay. Intrahepatic HLA-E expression was analyzed by an indirect immunoperoxidase technique and flow cytometry of isolated cells using a HLA-E-specific antibody. We identified peptide aa35–44, a well-characterized HLA-A2 restricted T cell epitope, as a peptide stabilizing HLA-E expression and thereby inhibiting NK cell-mediated lysis. Blocking experiments confirmed that this inhibitory effect of peptide aa35–44 on natural cytotoxicity was mediated via interactions between CD94/NKG2A receptors and enhanced HLA-E expression. In line with these in vitro data we found enhanced intrahepatic HLA-E expression on antigen-presenting cells in HCV-infected patients. Our data indicate the existence of T cell epitopes that can be recognized by HLA-A2 and HLA-E. This dual recognition may contribute to viral persistence in hepatitis C. PMID:15681828

Defective PDI release from platelets and endothelial cells impairs thrombus formation in Hermansky-Pudlak syndrome

PubMed Central

Sharda, Anish; Kim, Sarah H.; Jasuja, Reema; Gopal, Srila; Flaumenhaft, Robert; Furie, Barbara C.

2015-01-01

Protein disulfide isomerase (PDI), secreted from platelets and endothelial cells after injury, is required for thrombus formation. The effect of platelet and endothelial cell granule contents on PDI-mediated thrombus formation was studied by intravital microscopy using a mouse model of Hermansky-Pudlak syndrome in which platelet dense granules are absent. Platelet deposition and fibrin generation were nearly absent, and extracellular PDI was significantly reduced in HPS6−/− mice after vascular injury. HPS6−/− platelets displayed impaired PDI secretion and impaired exocytosis of α granules, lysosomes, and T granules due to decreased sensitivity to thrombin, but these defects could be corrected by addition of subthreshold amounts of adenosine 5′-diphosphate (ADP). Human Hermansky-Pudlak syndrome platelets demonstrated similar characteristics. Infusion of wild-type platelets rescued thrombus formation in HPS6−/− mice. Human umbilical vein endothelial cells in which the HPS6 gene was silenced displayed impaired PDI secretion and exocytosis of Weibel-Palade bodies. Defective thrombus formation in Hermansky-Pudlak syndrome, associated with impaired exocytosis of residual granules in endothelial cells and platelets, the latter due to deficiency of ADP, is characterized by a defect in T granule secretion, a deficiency in extracellular PDI secretion, and impaired fibrin generation and platelet aggregation. Hermansky-Pudlak syndrome is an example of a hereditary disease whereby impaired PDI secretion contributes to a bleeding phenotype. PMID:25593336

Impaired Calcium Entry into Cells Is Associated with Pathological Signs of Zinc Deficiency12

PubMed Central

O’Dell, Boyd L.; Browning, Jimmy D.

2013-01-01

Zinc is an essential trace element whose deficiency gives rise to specific pathological signs. These signs occur because an essential metabolic function is impaired as the result of failure to form or maintain a specific metal-ion protein complex. Although zinc is a component of many essential metalloenzymes and transcription factors, few of these have been identified with a specific sign of incipient zinc deficiency. Zinc also functions as a structural component of other essential proteins. Recent research with Swiss murine fibroblasts, 3T3 cells, has shown that zinc deficiency impairs calcium entry into cells, a process essential for many cell functions, including proliferation, maturation, contraction, and immunity. Impairment of calcium entry and the subsequent failure of cell proliferation could explain the growth failure associated with zinc deficiency. Defective calcium uptake is associated with impaired nerve transmission and pathology of the peripheral nervous system, as well as the failure of platelet aggregation and the bleeding tendency of zinc deficiency. There is a strong analogy between the pathology of genetic diseases that result in impaired calcium entry and other signs of zinc deficiency, such as decreased and cyclic food intake, taste abnormalities, abnormal water balance, skin lesions, impaired reproduction, depressed immunity, and teratogenesis. This analogy suggests that failure of calcium entry is involved in these signs of zinc deficiency as well. PMID:23674794

Neutrophil stunning by metoprolol reduces infarct size

PubMed Central

García-Prieto, Jaime; Villena-Gutiérrez, Rocío; Gómez, Mónica; Bernardo, Esther; Pun-García, Andrés; García-Lunar, Inés; Crainiciuc, Georgiana; Fernández-Jiménez, Rodrigo; Sreeramkumar, Vinatha; Bourio-Martínez, Rafael; García-Ruiz, José M; del Valle, Alfonso Serrano; Sanz-Rosa, David; Pizarro, Gonzalo; Fernández-Ortiz, Antonio; Hidalgo, Andrés; Fuster, Valentín; Ibanez, Borja

2017-01-01

The β1-adrenergic-receptor (ADRB1) antagonist metoprolol reduces infarct size in acute myocardial infarction (AMI) patients. The prevailing view has been that metoprolol acts mainly on cardiomyocytes. Here, we demonstrate that metoprolol reduces reperfusion injury by targeting the haematopoietic compartment. Metoprolol inhibits neutrophil migration in an ADRB1-dependent manner. Metoprolol acts during early phases of neutrophil recruitment by impairing structural and functional rearrangements needed for productive engagement of circulating platelets, resulting in erratic intravascular dynamics and blunted inflammation. Depletion of neutrophils, ablation of Adrb1 in haematopoietic cells, or blockade of PSGL-1, the receptor involved in neutrophil–platelet interactions, fully abrogated metoprolol's infarct-limiting effects. The association between neutrophil count and microvascular obstruction is abolished in metoprolol-treated AMI patients. Metoprolol inhibits neutrophil–platelet interactions in AMI patients by targeting neutrophils. Identification of the relevant role of ADRB1 in haematopoietic cells during acute injury and the protective role upon its modulation offers potential for developing new therapeutic strategies. PMID:28416795

Epilepsy and intellectual disability linked protein Shrm4 interaction with GABABRs shapes inhibitory neurotransmission

PubMed Central

Zapata, Jonathan; Moretto, Edoardo; Hannan, Saad; Murru, Luca; Longatti, Anna; Mazza, Davide; Benedetti, Lorena; Fossati, Matteo; Heise, Christopher; Ponzoni, Luisa; Valnegri, Pamela; Braida, Daniela; Sala, Mariaelvina; Francolini, Maura; Hildebrand, Jeffrey; Kalscheuer, Vera; Fanelli, Francesca; Sala, Carlo; Bettler, Bernhard; Bassani, Silvia; Smart, Trevor G.; Passafaro, Maria

2017-01-01

Shrm4, a protein expressed only in polarized tissues, is encoded by the KIAA1202 gene, whose mutations have been linked to epilepsy and intellectual disability. However, a physiological role for Shrm4 in the brain is yet to be established. Here, we report that Shrm4 is localized to synapses where it regulates dendritic spine morphology and interacts with the C terminus of GABAB receptors (GABABRs) to control their cell surface expression and intracellular trafficking via a dynein-dependent mechanism. Knockdown of Shrm4 in rat severely impairs GABABR activity causing increased anxiety-like behaviour and susceptibility to seizures. Moreover, Shrm4 influences hippocampal excitability by modulating tonic inhibition in dentate gyrus granule cells, in a process involving crosstalk between GABABRs and extrasynaptic δ-subunit-containing GABAARs. Our data highlights a role for Shrm4 in synaptogenesis and in maintaining GABABR-mediated inhibition, perturbation of which may be responsible for the involvement of Shrm4 in cognitive disorders and epilepsy. PMID:28262662

Real-time imaging of Huntingtin aggregates diverting target search and gene transcription

PubMed Central

Li, Li; Liu, Hui; Dong, Peng; Li, Dong; Legant, Wesley R; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert; Liu, Zhe

2016-01-01

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells – 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells. DOI: http://dx.doi.org/10.7554/eLife.17056.001 PMID:27484239

Mycobacterium leprae–host-cell interactions and genetic determinants in leprosy: an overview

PubMed Central

Pinheiro, Roberta Olmo; de Souza Salles, Jorgenilce; Sarno, Euzenir Nunes; Sampaio, Elizabeth Pereira

2011-01-01

Leprosy, also known as Hansen’s disease, is a chronic infectious disease caused by Mycobacterium leprae in which susceptibility to the mycobacteria and its clinical manifestations are attributed to the host immune response. Even though leprosy prevalence has decreased dramatically, the high number of new cases indicates active transmission. Owing to its singular features, M. leprae infection is an attractive model for investigating the regulation of human immune responses to pathogen-induced disease. Leprosy is one of the most common causes of nontraumatic peripheral neuropathy worldwide. The proportion of patients with disabilities is affected by the type of leprosy and delay in diagnosis. This article briefly reviews the clinical features as well as the immunopathological mechanisms related to the establishment of the different polar forms of leprosy, the mechanisms related to M. leprae–host cell interactions and prophylaxis and diagnosis of this complex disease. Host genetic factors are summarized and the impact of the development of interventions that prevent, reverse or limit leprosy-related nerve impairments are discussed. PMID:21366421

Computer-Assisted Learning for the Hearing Impaired: An Interactive Written Language Enviroment.

ERIC Educational Resources Information Center

Ward, R. D.; Rostron, A. B.

1983-01-01

To help hearing-impaired children develop their linguistic competence, a computer system that can process sentences and give feedback about their acceptability was developed. Suggestions are made of ways to use the system as an environment for interactive written communication. (Author/CL)

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

PubMed

Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Perturbation of auxin homeostasis by overexpression of wild-type IAA15 results in impaired stem cell differentiation and gravitropism in roots.

PubMed

Yan, Da-Wei; Wang, Jing; Yuan, Ting-Ting; Hong, Li-Wei; Gao, Xiang; Lu, Ying-Tang

2013-01-01

Aux/IAAs interact with auxin response factors (ARFs) to repress their transcriptional activity in the auxin signaling pathway. Previous studies have focused on gain-of-function mutations of domain II and little is known about whether the expression level of wild-type Aux/IAAs can modulate auxin homeostasis. Here we examined the perturbation of auxin homeostasis by ectopic expression of wild-type IAA15. Root gravitropism and stem cell differentiation were also analyzed. The transgenic lines were less sensitive to exogenous auxin and exhibited low-auxin phenotypes including failures in gravity response and defects in stem cell differentiation. Overexpression lines also showed an increase in auxin concentration and reduced polar auxin transport. These results demonstrate that an alteration in the expression of wild-type IAA15 can disrupt auxin homeostasis.

Modeling autism spectrum disorders with human neurons.

PubMed

Beltrão-Braga, Patricia C B; Muotri, Alysson R

2017-02-01

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impaired social communication and interactions and by restricted and repetitive behaviors. Although ASD is suspected to have a heritable or sporadic genetic basis, its underlying etiology and pathogenesis are not well understood. Therefore, viable human neurons and glial cells produced using induced pluripotent stem cells (iPSC) to reprogram cells from individuals affected with ASD provide an unprecedented opportunity to elucidate the pathophysiology of these disorders, providing novel insights regarding ASD and a potential platform to develop and test therapeutic compounds. Herein, we discuss the state of art with regards to ASD modeling, including limitations of this technology, as well as potential future directions. This article is part of a Special Issue entitled SI: Exploiting human neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

Monocyte:T cell interaction regulates human T cell activation through a CD28/CD46 crosstalk

PubMed Central

Charron, Lauren; Doctrinal, Axelle; Choileain, Siobhan Ni; Astier, Anne L.

2015-01-01

T cell activation requires engagement of the T cell receptor and of at least one costimulatory molecule. The key role of CD28 in inducing T cell activation has been reported several decades ago and the molecular mechanisms involved well described. The complement regulator CD46 also acts as a costimulatory molecule for T cells but, in contrast to CD28, has the ability to drive T cell differentiation from producing some IFNγ to secreting some potent anti-inflammatory IL-10, acquiring a so-called Type I regulatory phenotype (Tr1). Proteolytic cleavage of CD46 occurs upon costimulation and is important for T cell activation and IL-10 production. The observation that CD46 cleavage was reduced when PBMC were costimulated compared to purified naive T cells led us to hypothesize that interactions between different cell types within the PBMC were able to modulate the CD46 pathway. We show that CD46 downregulation is also reduced when CD4+ T cells are co-cultured with autologous monocytes. Indeed, monocyte:T cell co-cultures impaired CD46–mediated T cell differentiation and coactivation, by reducing downregulation of surface CD46, lowering induction of the early activation marker CD69, as well as reducing the levels of IL-10 secretion. Blocking of CD86 could partly restore CD69 expression and cytokine secretion, demonstrating that the CD28-CD86 pathway regulates CD46 activation. Direct concomitant ligation of CD28 and CD46 on CD4+ T cells also modulated CD46 expression and regulated cytokine production. These data identify a crosstalk between two main costimulatory pathways and provide novel insights into the regulation of human T cell activation. PMID:25787182

Heart Failure With Preserved Ejection Fraction in Diabetes: Mechanisms and Management.

PubMed

Meagher, Patrick; Adam, Mohamed; Civitarese, Robert; Bugyei-Twum, Antoinette; Connelly, Kim A

2018-05-01

Diabetes mellitus (DM) is a major cause of heart failure in the Western world, either secondary to coronary artery disease or from a distinct entity known as "diabetic cardiomyopathy." Furthermore, heart failure with preserved ejection fraction (HFpEF) is emerging as a significant clinical problem for patients with DM. Current clinical data suggest that between 30% and 40% of patients with HFpEF suffer from DM. The typical structural phenotype of the HFpEF heart consists of endothelial dysfunction, increased interstitial and perivascular fibrosis, cardiomyocyte stiffness, and hypertrophy along with advanced glycation end products deposition. There is a myriad of mechanisms that result in the phenotypical HFpEF heart including impaired cardiac metabolism and substrate utilization, altered insulin signalling leading to protein kinase C activation, advanced glycated end products deposition, prosclerotic cytokine activation (eg, transforming growth factor-β activation), along with impaired nitric oxide production from the endothelium. Moreover, recent investigations have focused on the role of endothelial-myocyte interactions. Despite intense research, current therapeutic strategies have had little effect on improving morbidity and mortality in patients with DM and HFpEF. Possible explanations for this include a limited understanding of the role that direct cell-cell communication or indirect cell-cell paracrine signalling plays in the pathogenesis of DM and HFpEF. Additionally, integrins remain another important mediator of signals from the extracellular matrix to cells within the failing heart and might play a significant role in cell-cell cross-talk. In this review we discuss the characteristics and mechanisms of DM and HFpEF to stimulate potential future research for patients with this common, and morbid condition. Copyright © 2018 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.

Role of lipid phosphate phosphatase 3 in human aortic endothelial cell function

PubMed Central

Touat-Hamici, Zahia; Weidmann, Henri; Blum, Yuna; Proust, Carole; Durand, Hervé; Iannacci, Francesca; Codoni, Veronica; Gaignard, Pauline; Thérond, Patrice; Civelek, Mete; Karabina, Sonia A.; Lusis, Aldons J.; Cambien, François; Ninio, Ewa

2016-01-01

Aims Lipid phosphate phosphatase 3; type 2 phosphatidic acid phosphatase β (LPP3; PPAP2B) is a transmembrane protein dephosphorylating and thereby terminating signalling of lipid substrates including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Human LPP3 possesses a cell adhesion motif that allows interaction with integrins. A polymorphism (rs17114036) in PPAP2B is associated with coronary artery disease, which prompted us to investigate the possible role of LPP3 in human endothelial dysfunction, a condition promoting atherosclerosis. Methods and results To study the role of LPP3 in endothelial cells we used human primary aortic endothelial cells (HAECs) in which LPP3 was silenced or overexpressed using either wild type or mutated cDNA constructs. LPP3 silencing in HAECs enhanced secretion of inflammatory cytokines, leucocyte adhesion, cell survival, and migration and impaired angiogenesis, whereas wild-type LPP3 overexpression reversed these effects and induced apoptosis. We also demonstrated that LPP3 expression was negatively correlated with vascular endothelial growth factor expression. Mutations in either the catalytic or the arginine-glycine-aspartate (RGD) domains impaired endothelial cell function and pharmacological inhibition of S1P or LPA restored it. LPA was not secreted in HAECs under silencing or overexpressing LPP3. However, the intra- and extra-cellular levels of S1P tended to be correlated with LPP3 expression, indicating that S1P is probably degraded by LPP3. Conclusions We demonstrated that LPP3 is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in HAECs, suggesting a protective role of LPP3 against endothelial dysfunction in humans. Both the catalytic and the RGD functional domains were involved and S1P, but not LPA, might be the endogenous substrate of LPP3. PMID:27694435

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

PubMed

Terzaghi, L; Tessaro, I; Raucci, F; Merico, V; Mazzini, G; Garagna, S; Zuccotti, M; Franciosi, F; Lodde, V

2016-08-02

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

Heterogeneity and proliferation of invasive cancer subclones in game theory models of the Warburg effect.

PubMed

Archetti, M

2015-04-01

The Warburg effect, a switch from aerobic energy production to anaerobic glycolysis, promotes tumour proliferation and motility by inducing acidification of the tumour microenvironment. Therapies that reduce acidity could impair tumour growth and invasiveness. I analysed the dynamics of cell proliferation and of resistance to therapies that target acidity, in a population of cells, under the Warburg effect. The dynamics of mutant cells with increased glycolysis and motility has been assessed in a multi-player game with collective interactions in the framework of evolutionary game theory. Perturbations of the level of acidity in the microenvironment have been used to simulate the effect of therapies that target glycolysis. The non-linear effects of glycolysis induce frequency-dependent clonal selection leading to coexistence of glycolytic and non-glycolytic cells within a tumour. Mutants with increased motility can invade such a polymorphic population and spread within the tumour. While reducing acidity may produce a sudden reduction in tumour cell proliferation, frequency-dependent selection enables it to adapt to the new conditions and can enable the tumour to restore its original levels of growth and invasiveness. The acidity produced by glycolysis acts as a non-linear public good that leads to coexistence of cells with high and low glycolysis within the tumour. Such a heterogeneous population can easily adapt to changes in acidity. Therapies that target acidity can only be effective in the long term if the cost of glycolysis is high, that is, under non-limiting oxygen concentrations. Their efficacy, therefore, is reduced when combined with therapies that impair angiogenesis. © 2015 The Authors Cell Proliferation Published by John Wiley & Sons Ltd.

The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

PubMed Central

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

PubMed

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.

Physical Association of Saccharomyces cerevisiae Polo-like Kinase Cdc5 with Chromosomal Cohesin Facilitates DNA Damage Response.

PubMed

Pakchuen, Sujiraporn; Ishibashi, Mai; Takakusagi, Emi; Shirahige, Katsuhiko; Sutani, Takashi

2016-08-12

At the onset of anaphase, a protease called separase breaks the link between sister chromatids by cleaving the cohesin subunit Scc1. This irreversible step in the cell cycle is promoted by degradation of the separase inhibitor, securin, and polo-like kinase (Plk) 1-dependent phosphorylation of the Scc1 subunit. Plk could recognize substrates through interaction between its phosphopeptide interaction domain, the polo-box domain, and a phosphorylated priming site in the substrate, which has been generated by a priming kinase beforehand. However, the physiological relevance of this targeting mechanism remains to be addressed for many of the Plk1 substrates. Here, we show that budding yeast Plk1, Cdc5, is pre-deposited onto cohesin engaged in cohesion on chromosome arms in G2/M phase cells. The Cdc5-cohesin association is mediated by direct interaction between the polo-box domain of Cdc5 and Scc1 phosphorylated at multiple sites in its middle region. Alanine substitutions of the possible priming phosphorylation sites (scc1-15A) impair Cdc5 association with chromosomal cohesin, but they make only a moderate impact on mitotic cell growth even in securin-deleted cells (pds1Δ), where Scc1 phosphorylation by Cdc5 is indispensable. The same scc1-15A pds1Δ double mutant, however, exhibits marked sensitivity to the DNA-damaging agent phleomycin, suggesting that the priming phosphorylation of Scc1 poses an additional layer of regulation that enables yeast cells to adapt to genotoxic environments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MXD1 localizes in the nucleolus, binds UBF and impairs rRNA synthesis.

PubMed

Lafita-Navarro, Maria Del Carmen; Blanco, Rosa; Mata-Garrido, Jorge; Liaño-Pons, Judit; Tapia, Olga; García-Gutiérrez, Lucía; García-Alegría, Eva; Berciano, María T; Lafarga, Miguel; León, Javier

2016-10-25

MXD1 is a protein that interacts with MAX, to form a repressive transcription factor. MXD1-MAX binds E-boxes. MXD1-MAX antagonizes the transcriptional activity of the MYC oncoprotein in most models. It has been reported that MYC overexpression leads to augmented RNA synthesis and ribosome biogenesis, which is a relevant activity in MYC-mediated tumorigenesis. Here we describe that MXD1, but not MYC or MNT, localizes to the nucleolus in a wide array of cell lines derived from different tissues (carcinoma, leukemia) as well as in embryonic stem cells. MXD1 also localizes in the nucleolus of primary tissue cells as neurons and Sertoli cells. The nucleolar localization of MXD1 was confirmed by co-localization with UBF. Co-immunoprecipitation experiments showed that MXD1 interacted with UBF and proximity ligase assays revealed that this interaction takes place in the nucleolus. Furthermore, chromatin immunoprecipitation assays showed that MXD1 was bound in the transcribed rDNA chromatin, where it co-localizes with UBF, but also in the ribosomal intergenic regions. The MXD1 involvement in rRNA synthesis was also suggested by the nucleolar segregation upon rRNA synthesis inhibition by actinomycin D. Silencing of MXD1 with siRNAs resulted in increased synthesis of pre-rRNA while enforced MXD1 expression reduces it. The results suggest a new role for MXD1, which is the control of ribosome biogenesis. This new MXD1 function would be important to curb MYC activity in tumor cells.

MXD1 localizes in the nucleolus, binds UBF and impairs rRNA synthesis

PubMed Central

Lafita-Navarro, Maria del Carmen; Blanco, Rosa; Mata-Garrido, Jorge; Liaño-Pons, Judit; Tapia, Olga; García-Gutiérrez, Lucía; García-Alegría, Eva; Berciano, María T.; Lafarga, Miguel; León, Javier

2016-01-01

MXD1 is a protein that interacts with MAX, to form a repressive transcription factor. MXD1-MAX binds E-boxes. MXD1-MAX antagonizes the transcriptional activity of the MYC oncoprotein in most models. It has been reported that MYC overexpression leads to augmented RNA synthesis and ribosome biogenesis, which is a relevant activity in MYC-mediated tumorigenesis. Here we describe that MXD1, but not MYC or MNT, localizes to the nucleolus in a wide array of cell lines derived from different tissues (carcinoma, leukemia) as well as in embryonic stem cells. MXD1 also localizes in the nucleolus of primary tissue cells as neurons and Sertoli cells. The nucleolar localization of MXD1 was confirmed by co-localization with UBF. Co-immunoprecipitation experiments showed that MXD1 interacted with UBF and proximity ligase assays revealed that this interaction takes place in the nucleolus. Furthermore, chromatin immunoprecipitation assays showed that MXD1 was bound in the transcribed rDNA chromatin, where it co-localizes with UBF, but also in the ribosomal intergenic regions. The MXD1 involvement in rRNA synthesis was also suggested by the nucleolar segregation upon rRNA synthesis inhibition by actinomycin D. Silencing of MXD1 with siRNAs resulted in increased synthesis of pre-rRNA while enforced MXD1 expression reduces it. The results suggest a new role for MXD1, which is the control of ribosome biogenesis. This new MXD1 function would be important to curb MYC activity in tumor cells. PMID:27588501

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajenova, Olga, E-mail: o.bazhenova@spbu.ru; Department of Genetics and Biotechnology, St. Petersburg State University, St. Petersburg 199034; Department of Surgery and Biomedical Sciences, Creighton University, Omaha, NE 68178

2014-06-10

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA andmore » beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer. - Highlights: • Elevated level of CEA increases the release of soluble E-cadherin during the progression of colorectal cancer. • CEA over-expression alters the binding preferences between E-cadherin and its partners: α-, β- and p120 catenins in adherens junction complexes. • CEA produced by colorectal cancer cells interacts with beta-catenin protein. • CEA over-expression triggers the increase in nuclear beta-catenin. • CEA over-expression alters the splicing of p120 catenin protein.« less

NADPH oxidase promotes Parkinsonian phenotypes by impairing autophagic flux in an mTORC1-independent fashion in a cellular model of Parkinson’s disease

PubMed Central

Pal, Rituraj; Bajaj, Lakshya; Sharma, Jaiprakash; Palmieri, Michela; Di Ronza, Alberto; Lotfi, Parisa; Chaudhury, Arindam; Neilson, Joel; Sardiello, Marco; Rodney, George G.

2016-01-01

Oxidative stress and aberrant accumulation of misfolded proteins in the cytosol are key pathological features associated with Parkinson’s disease (PD). NADPH oxidase (Nox2) is upregulated in the pathogenesis of PD; however, the underlying mechanism(s) of Nox2-mediated oxidative stress in PD pathogenesis are still unknown. Using a rotenone-inducible cellular model of PD, we observed that a short exposure to rotenone (0.5 μM) resulted in impaired autophagic flux through activation of a Nox2 dependent Src/PI3K/Akt axis, with a consequent disruption of a Beclin1-VPS34 interaction that was independent of mTORC1 activity. Sustained exposure to rotenone at a higher dose (10 μM) decreased mTORC1 activity; however, autophagic flux was still impaired due to dysregulation of lysosomal activity with subsequent induction of the apoptotic machinery. Cumulatively, our results highlight a complex pathogenic mechanism for PD where short- and long-term oxidative stress alters different signaling pathways, ultimately resulting in anomalous autophagic activity and disease phenotype. Inhibition of Nox2-dependent oxidative stress attenuated the impaired autophagy and cell death, highlighting the importance and therapeutic potential of these pathways for treating patients with PD. PMID:26960433

Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.

PubMed

Carlini, Leslie E; Getz, Michael J; Strauch, Arthur R; Kelm, Robert J

2002-03-08

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

The Association of Cortactin with Profilin-1 Is Critical for Smooth Muscle Contraction*

PubMed Central

Wang, Ruping; Cleary, Rachel A.; Wang, Tao; Li, Jia; Tang, Dale D.

2014-01-01

Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation. PMID:24700464

MiR-17-5p impairs trafficking of H-ERG K+ channel protein by targeting multiple er stress-related chaperones during chronic oxidative stress.

PubMed

Wang, Qi; Hu, Weina; Lei, Mingming; Wang, Yong; Yan, Bing; Liu, Jun; Zhang, Ren; Jin, Yuanzhe

2013-01-01

To investigate if microRNAs (miRNAs) play a role in regulating h-ERG trafficking in the setting of chronic oxidative stress as a common deleterious factor for many cardiac disorders. We treated neonatal rat ventricular myocytes and HEK293 cells with stable expression of h-ERG with H2O2 for 12 h and 48 h. Expression of miR-17-5p seed miRNAs was quantified by real-time RT-PCR. Protein levels of chaperones and h-ERG trafficking were measured by Western blot analysis. Luciferase reporter gene assay was used to study miRNA and target interactions. Whole-cell patch-clamp techniques were employed to record h-ERG K(+) current. H-ERG trafficking was impaired by H2O2 after 48 h treatment, accompanied by reciprocal changes of expression between miR-17-5p seed miRNAs and several chaperones (Hsp70, Hsc70, CANX, and Golga2), with the former upregulated and the latter downregulated. We established these chaperones as targets for miR-17-5p. Application miR-17-5p inhibitor rescued H2O2-induced impairment of h-ERG trafficking. Upregulation of endogenous by H2O2 or forced miR-17-5p expression either reduced h-ERG current. Sequestration of AP1 by its decoy molecule eliminated the upregulation of miR-17-5p, and ameliorated impairment of h-ERG trafficking. Collectively, deregulation of the miR-17-5p seed family miRNAs can cause severe impairment of h-ERG trafficking through targeting multiple ER stress-related chaperones, and activation of AP1 likely accounts for the deleterious upregulation of these miRNAs, in the setting of prolonged duration of oxidative stress. These findings revealed the role of miRNAs in h-ERG trafficking, which may contribute to the cardiac electrical disturbances associated with oxidative stress.

APP/Go protein Gβγ-complex signaling mediates Aβ degeneration and cognitive impairment in Alzheimer's disease models.

PubMed

Bignante, Elena Anahi; Ponce, Nicolás Eric; Heredia, Florencia; Musso, Juliana; Krawczyk, María C; Millán, Julieta; Pigino, Gustavo F; Inestrosa, Nibaldo C; Boccia, Mariano M; Lorenzo, Alfredo

2018-04-01

Deposition of amyloid-β (Aβ), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the direct involvement of APP in the mechanism of Aβ-induced degeneration in AD remains on debate. Here, we analyzed the interaction of APP with heterotrimeric Go protein in primary hippocampal cultures and found that Aβ deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aβ toxicity by a mechanism that required Go-Gβγ complex signaling and p38-mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gβγ complex, inhibited Aβ-induced dendritic and axonal dystrophy, abnormal tau phosphorylation, synaptic loss, and neuronal cell death in hippocampal neurons expressing endogenous protein levels. In the 3xTg-AD mice, intrahippocampal application of gallein reversed memory impairment associated with early Aβ pathology. Our data provide further evidence for the involvement of APP/Go protein in Aβ-induced degeneration and reveal that Gβγ complex is a signaling target potentially relevant for developing therapies for halting Aβ degeneration in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

Speech and neurology-chemical impairment correlates

NASA Astrophysics Data System (ADS)

Hayre, Harb S.

2002-05-01

Speech correlates of alcohol/drug impairment and its neurological basis is presented with suggestion for further research in impairment from poly drug/medicine/inhalent/chew use/abuse, and prediagnosis of many neuro- and endocrin-related disorders. Nerve cells all over the body detect chemical entry by smoking, injection, drinking, chewing, or skin absorption, and transmit neurosignals to their corresponding cerebral subsystems, which in turn affect speech centers-Broca's and Wernick's area, and motor cortex. For instance, gustatory cells in the mouth, cranial and spinal nerve cells in the skin, and cilia/olfactory neurons in the nose are the intake sensing nerve cells. Alcohol depression, and brain cell damage were detected from telephone speech using IMPAIRLYZER-TM, and the results of these studies were presented at 1996 ASA meeting in Indianapolis, and 2001 German Acoustical Society-DEGA conference in Hamburg, Germany respectively. Speech based chemical Impairment measure results were presented at the 2001 meeting of ASA in Chicago. New data on neurotolerance based chemical impairment for alcohol, drugs, and medicine shall be presented, and shown not to fully support NIDA-SAMSHA drug and alcohol threshold used in drug testing domain.

Social Stress in Young People with Specific Language Impairment

ERIC Educational Resources Information Center

Wadman, Ruth; Durkin, Kevin; Conti-Ramsden, Gina

2011-01-01

Social interactions can be a source of social stress for adolescents. Little is known about how adolescents with developmental difficulties, such as specific language impairment (SLI), feel when interacting socially. Participants included 28 adolescents with SLI and 28 adolescents with typical language abilities (TL). Self-report measures of…

Chemosensory interaction: acquired olfactory impairment is associated with decreased taste function.

PubMed

Landis, Basile N; Scheibe, Mandy; Weber, Cornelia; Berger, Robert; Brämerson, Annika; Bende, Mats; Nordin, Steven; Hummel, Thomas

2010-08-01

Olfaction, taste and trigeminal function are three distinct modalities. However, in daily life they are often activated concomitantly. In health and disease, it has been shown that in two of these senses, the trigeminal and olfactory senses, modification of one sense leads to changes in the other sense and vice versa. The objective of the study was to investigate whether and (if so) how, the third modality, taste, is influenced by olfactory impairment. We tested 210 subjects with normal (n = 107) or impaired (n = 103) olfactory function for their taste identification capacities. Validated tests were used for olfactory and gustatory testing (Sniffin' Sticks, Taste Strips). In an additional experiment, healthy volunteers underwent reversible olfactory cleft obstruction to investigate short-time changes of gustatory function after olfactory alteration. Mean gustatory identification (taste strip score) for the subjects with impaired olfaction was 19.4 +/- 0.6 points and 22.9 +/- 0.5 points for those with normal olfactory function (t = 4.6, p < 0.001). The frequencies of both, smell and taste impairments interacted significantly (Chi(2), F = 16.4, p < 0.001), and olfactory and gustatory function correlated (r (210) = 0.30, p < 0.001). Neither age nor olfactory impairment cause effects interfered with this olfactory-gustatory interaction. In contrast, after short-lasting induced olfactory decrease, gustatory function remained unchanged. The present study suggests that longstanding impaired olfactory function is associated with decreased gustatory function. These findings seem to extend previously described mutual chemosensory interactions also to smell and taste. It further raises the question whether chemical senses in general decrease mutually after acquired damage.

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

PubMed Central

Cai, Qing; Brissova, Marcela; Reinert, Rachel B.; Pan, Fong Cheng; Brahmachary, Priyanka; Jeansson, Marie; Shostak, Alena; Radhika, Aramandla; Poffenberger, Greg; Quaggin, Susan E.; Jerome, W. Gray; Dumont, Daniel J.; Powers, Alvin C.

2012-01-01

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a “tet-on” inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that 1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; 2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; 3) Angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization. PMID:22546694

Medication and finance management among HIV-infected adults: the impact of age and cognition.

PubMed

Thames, April D; Kim, Michelle S; Becker, Brian W; Foley, Jessica M; Hines, Lindsay J; Singer, Elyse J; Heaton, Robert K; Castellon, Steven A; Hinkin, Charles H

2011-02-01

This study examined the effects of aging and cognitive impairment on medication and finance management in an HIV sample. We observed main effects of age (older < younger) and neuropsychological impairment on functional task performance. Interactions between age and cognition demonstrated that older impaired individuals performed significantly more poorly than all other comparison groups. There were no relationships between laboratory performance and self-reported medication and finance management. The interaction of advancing age and cognitive impairment may confer significant functional limitations for HIV individuals that may be better detected by performance-based measures of functional abilities rather than patient self-report.

Medication and finance management among HIV-infected adults: The impact of age and cognition

PubMed Central

Thames, April D.; Kim, Michelle S.; Becker, Brian W.; Foley, Jessica M.; Hines, Lindsay J.; Singer, Elyse J.; Heaton, Robert K.; Castellon, Steven A.; Hinkin, Charles H.

2013-01-01

This study examined the effects of aging and cognitive impairment on medication and finance management in an HIV sample. We observed main effects of age (older < younger) and neuropsychological impairment on functional task performance. Interactions between age and cognition demonstrated that older impaired individuals performed significantly more poorly than all other comparison groups. There were no relationships between laboratory performance and self-reported medication and finance management. The interaction of advancing age and cognitive impairment may confer significant functional limitations for HIV individuals that may be better detected by performance-based measures of functional abilities rather than patient self-report. PMID:20694873

Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction.

PubMed

Javeed, Naureen; Sagar, Gunisha; Dutta, Shamit K; Smyrk, Thomas C; Lau, Julie S; Bhattacharya, Santanu; Truty, Mark; Petersen, Gloria M; Kaufman, Randal J; Chari, Suresh T; Mukhopadhyay, Debabrata

2015-04-01

Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. ©2014 American Association for Cancer Research.

Natural neural projection dynamics underlying social behavior

PubMed Central

Gunaydin, Lisa A.; Grosenick, Logan; Finkelstein, Joel C.; Kauvar, Isaac V.; Fenno, Lief E.; Adhikari, Avishek; Lammel, Stephan; Mirzabekov, Julie J.; Airan, Raag D.; Zalocusky, Kelly A.; Tye, Kay M.; Anikeeva, Polina; Malenka, Robert C.; Deisseroth, Karl

2014-01-01

Social interaction is a complex behavior essential for many species, and is impaired in major neuropsychiatric disorders. Pharmacological studies have implicated certain neurotransmitter systems in social behavior, but circuit-level understanding of endogenous neural activity during social interaction is lacking. We therefore developed and applied a new methodology, termed fiber photometry, to optically record natural neural activity in genetically- and connectivity-defined projections to elucidate the real-time role of specified pathways in mammalian behavior. Fiber photometry revealed that activity dynamics of a ventral tegmental area (VTA)-to-nucleus accumbens (NAc) projection could encode and predict key features of social but not novel-object interaction. Consistent with this observation, optogenetic control of cells specifically contributing to this projection was sufficient to modulate social behavior, which was mediated by type-1 dopamine receptor signaling downstream in the NAc. Direct observation of projection-specific activity in this way captures a fundamental and previously inaccessible dimension of circuit dynamics. PMID:24949967

Lipid-Polymer Nanoparticles Encapsulating Curcumin for Modulating the Vascular Deposition of Breast Cancer Cells

PubMed Central

Palange, Anna L.; Di Mascolo, Daniele; Carallo, Claudio; Gnasso, Agostino; Decuzzi, Paolo

2014-01-01

Vascular adhesion and endothelial transmigration are critical steps in the establishment of distant metastasis by circulating tumor cells (CTCs). Also, vascular inflammation plays a pivotal role in steering CTCs out of the blood stream. Here, long circulating lipid-polymer nanoparticles encapsulating curcumin (NANOCurc) are proposed for modulating the vascular deposition of CTCs. Upon treatment with NANOCurc, the adhesion propensity of highly metastatic breast cancer cells (MDA-MB-231) onto TNF-α stimulated endothelial cells (HUVECs) reduces by ~ 70%, in a capillary flow. Remarkably, the CTC vascular deposition already reduces up to ~ 50% by treating solely the inflamed HUVECs. The CTC arrest is mediated by the interaction between ICAM-1 on HUVECs and MUC-1 on cancer cells, and moderate doses of curcumin down-regulate the expression of both molecules. This suggests that NANOCurc could prevent metastasis and limit the progression of the disease by modulating vascular inflammation and impairing the CTC arrest. PMID:24566270

PAF-Myc-Controlled Cell Stemness Is Required for Intestinal Regeneration and Tumorigenesis.

PubMed

Kim, Moon Jong; Xia, Bo; Suh, Han Na; Lee, Sung Ho; Jun, Sohee; Lien, Esther M; Zhang, Jie; Chen, Kaifu; Park, Jae-Il

2018-03-12

The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/β-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/β-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells. Copyright © 2018 Elsevier Inc. All rights reserved.

The skeletal vascular system - Breathing life into bone tissue.

PubMed

Stegen, Steve; Carmeliet, Geert

2017-08-26

During bone development, homeostasis and repair, a dense vascular system provides oxygen and nutrients to highly anabolic skeletal cells. Characteristic for the vascular system in bone is the serial organization of two capillary systems, each typified by specific morphological and physiological features. Especially the arterial capillaries mediate the growth of the bone vascular system, serve as a niche for skeletal and hematopoietic progenitors and couple angiogenesis to osteogenesis. Endothelial cells and osteoprogenitor cells interact not only physically, but also communicate to each other by secretion of growth factors. A vital angiogenic growth factor is vascular endothelial growth factor and its expression in skeletal cells is controlled by osteogenic transcription factors and hypoxia signaling, whereas the secretion of angiocrine factors by endothelial cells is regulated by Notch signaling, blood flow and possibly hypoxia. Bone loss and impaired fracture repair are often associated with reduced and disorganized blood vessel network and therapeutic targeting of the angiogenic response may contribute to enhanced bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Endothelial progenitor cells bind and inhibit platelet function and thrombus formation.

PubMed

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2009-12-01

Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and thrombosis.

Activation, Impaired Tumor Necrosis Factor-α Production, and Deficiency of Circulating Mucosal-Associated Invariant T Cells in Patients with Scrub Typhus.

PubMed

Kang, Seung-Ji; Jin, Hye-Mi; Won, Eun Jeong; Cho, Young-Nan; Jung, Hyun-Ju; Kwon, Yong-Soo; Kee, Hae Jin; Ju, Jae Kyun; Kim, Jung-Chul; Kim, Uh Jin; Jang, Hee-Chang; Jung, Sook-In; Kee, Seung-Jung; Park, Yong-Wook

2016-07-01

Mucosal-associated invariant T (MAIT) cells contribute to protection against certain microorganism infections. However, little is known about the role of MAIT cells in Orientia tsutsugamushi infection. Hence, the aims of this study were to examine the level and function of MAIT cells in patients with scrub typhus and to evaluate the clinical relevance of MAIT cell levels. Thirty-eight patients with scrub typhus and 53 health control subjects were enrolled in the study. The patients were further divided into subgroups according to disease severity. MAIT cell level and function in the peripheral blood were measured by flow cytometry. Circulating MAIT cell levels were found to be significantly reduced in scrub typhus patients. MAIT cell deficiency reflects a variety of clinical conditions. In particular, MAT cell levels reflect disease severity. MAIT cells in scrub typhus patients displayed impaired tumor necrosis factor (TNF)-α production, which was restored during the remission phase. In addition, the impaired production of TNF-α by MAIT cells was associated with elevated CD69 expression. This study shows that circulating MAIT cells are activated, numerically deficient, and functionally impaired in TNF-α production in patients with scrub typhus. These abnormalities possibly contribute to immune system dysregulation in scrub typhus infection.

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells*

PubMed Central

Dai, Feihan F.; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B.

2015-01-01

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H+-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca2+ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. PMID:26272612

Analytical cell adhesion chromatography reveals impaired persistence of metastatic cell rolling adhesion to P-selectin

PubMed Central

Oh, Jaeho; Edwards, Erin E.; McClatchey, P. Mason; Thomas, Susan N.

2015-01-01

ABSTRACT Selectins facilitate the recruitment of circulating cells from the bloodstream by mediating rolling adhesion, which initiates the cell–cell signaling that directs extravasation into surrounding tissues. To measure the relative efficiency of cell adhesion in shear flow for in vitro drug screening, we designed and implemented a microfluidic-based analytical cell adhesion chromatography system. The juxtaposition of instantaneous rolling velocities with elution times revealed that human metastatic cancer cells, but not human leukocytes, had a reduced capacity to sustain rolling adhesion with P-selectin. We define a new parameter, termed adhesion persistence, which is conceptually similar to migration persistence in the context of chemotaxis, but instead describes the capacity of cells to resist the influence of shear flow and sustain rolling interactions with an adhesive substrate that might modulate the probability of extravasation. Among cell types assayed, adhesion persistence to P-selectin was specifically reduced in metastatic but not leukocyte-like cells in response to a low dose of heparin. In conclusion, we demonstrate this as an effective methodology to identify selectin adhesion antagonist doses that modulate homing cell adhesion and engraftment in a cell-subtype-selective manner. PMID:26349809

Vasodilator-Stimulated Phosphoprotein (VASP) depletion from breast cancer MDA-MB-231 cells inhibits tumor spheroid invasion through downregulation of Migfilin, β-catenin and urokinase-plasminogen activator (uPA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gkretsi, Vasiliki; Stylianou, Andreas; Stylianopoulos, Triantafyllos, E-mail: tstylian@ucy.ac.cy

A hallmark of cancer cells is their ability to invade surrounding tissues and form metastases. Cell-extracellular matrix (ECM)-adhesion proteins are crucial in metastasis, connecting tumor ECM with actin cytoskeleton thus enabling cells to respond to mechanical cues. Vasodilator-stimulated phosphoprotein (VASP) is an actin-polymerization regulator which interacts with cell-ECM adhesion protein Migfilin, and regulates cell migration. We compared VASP expression in MCF-7 and MDA-MB-231 breast cancer (BC) cells and found that more invasive MDA-MB-231 cells overexpress VASP. We then utilized a 3-dimensional (3D) approach to study metastasis in MDA-MB-231 cells using a system that considers mechanical forces exerted by the ECM.more » We prepared 3D collagen I gels of increasing concentration, imaged them by atomic force microscopy, and used them to either embed cells or tumor spheroids, in the presence or absence of VASP. We show, for the first time, that VASP silencing downregulated Migfilin, β-catenin and urokinase plasminogen activator both in 2D and 3D, suggesting a matrix-independent mechanism. Tumor spheroids lacking VASP demonstrated impaired invasion, indicating VASP’s involvement in metastasis, which was corroborated by Kaplan-Meier plotter showing high VASP expression to be associated with poor remission-free survival in lymph node-positive BC patients. Hence, VASP may be a novel BC metastasis biomarker. - Highlights: • More invasive MDA-MB-231 overexpress VASP compared to MCF-7 breast cancer cells. • We prepared 3D collagen I gels of increasing concentration and characterized them. • VASP silencing downregulated Migfilin, β-catenin and uPA both in 2D and 3D culture. • Tumor spheroids lacking VASP demonstrated impaired invasion. • Kaplan-Meier plotter shows association of high VASP expression with poor survival.« less

Mucolipidoses

MedlinePlus

... cells occurs, causing symptoms that range from mild learning disabilities to severe intellectual impairment and skeletal deformities. The ... cells occurs, causing symptoms that range from mild learning disabilities to severe intellectual impairment and skeletal deformities. The ...

Fat-soluble vitamin intestinal absorption: absorption sites in the intestine and interactions for absorption.

PubMed

Goncalves, Aurélie; Roi, Stéphanie; Nowicki, Marion; Dhaussy, Amélie; Huertas, Alain; Amiot, Marie-Josèphe; Reboul, Emmanuelle

2015-04-01

The interactions occurring at the intestinal level between the fat-soluble vitamins A, D, E and K (FSVs) are poorly documented. We first determined each FSV absorption profile along the duodenal-colonic axis of mouse intestine to clarify their respective absorption sites. We then investigated the interactions between FSVs during their uptake by Caco-2 cells. Our data show that vitamin A was mostly absorbed in the mouse proximal intestine, while vitamin D was absorbed in the median intestine, and vitamin E and K in the distal intestine. Significant competitive interactions for uptake were then elucidated among vitamin D, E and K, supporting the hypothesis of common absorption pathways. Vitamin A also significantly decreased the uptake of the other FSVs but, conversely, its uptake was not impaired by vitamins D and K and even promoted by vitamin E. These results should be taken into account, especially for supplement formulation, to optimise FSV absorption. Copyright © 2014 Elsevier Ltd. All rights reserved.

Insulin and wound healing.

PubMed

Hrynyk, Michael; Neufeld, Ronald J

2014-12-01

Skin is a dynamic and complex organ that relies on the interaction of different cell types, biomacromolecules and signaling molecules. Injury triggers a cascade of events designed to quickly restore skin integrity. Depending on the size and severity of the wound, extensive physiological and metabolic changes can occur, resulting in impaired wound healing and increased morbidity resulting in higher rates of death. While wound dressings provide a temporary barrier, they are inherently incapable of significantly restoring metabolic upsets, post-burn insulin resistance, and impaired wound healing in patients with extensive burns. Exogenous insulin application has therefore been investigated as a potential therapeutic intervention for nearly a century to improve wound recovery. This review will highlight the important achievements that demonstrate insulin's ability to stimulate cellular migration and burn wound recovery, as well as providing a perspective on future therapeutic applications and research directions. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Diabetes-related dysfunction of the small intestine and the colon: focus on motility.

PubMed

Horváth, Viktor József; Putz, Zsuzsanna; Izbéki, Ferenc; Körei, Anna Erzsébet; Gerő, László; Lengyel, Csaba; Kempler, Péter; Várkonyi, Tamás

2015-11-01

In contrast to gastric dysfunction, diabetes-related functional impairments of the small and large intestine have been studied less intensively. The gastrointestinal tract accomplishes several functions, such as mixing and propulsion of luminal content, absorption and secretion of ions, water, and nutrients, defense against pathogens, and elimination of waste products. Diverse functions of the gut are regulated by complex interactions among its functional elements, including gut microbiota. The network-forming tissues, the enteric nervous system) and the interstitial cells of Cajal, are definitely impaired in diabetic patients, and their loss of function is closely related to the symptoms in diabetes, but changes of other elements could also play a role in the development of diabetes mellitus-related motility disorders. The development of our understanding over the recent years of the diabetes-induced dysfunctions in the small and large intestine are reviewed in this article.

The pathobiology of vascular dementia

PubMed Central

Iadecola, Costantino

2013-01-01

Vascular cognitive impairment defines alterations in cognition, ranging from subtle deficits to full-blown dementia, attributable to cerebrovascular causes. Often coexisting with Alzheimer’s disease, mixed vascular and neurodegenerative dementia has emerged as the leading cause of age-related cognitive impairment. Central to the disease mechanism is the crucial role that cerebral blood vessels play in brain health, not only for the delivery of oxygen and nutrients, but also for the trophic signaling that links inextricably the well being of neurons and glia to that of cerebrovascular cells. This review will examine how vascular damage disrupts these vital homeostatic interactions, focusing on the hemispheric white matter, a region at heightened risk for vascular damage, and on the interplay between vascular factors and Alzheimer’s disease. Finally, preventative and therapeutic prospects will be examined, highlighting the importance of midlife vascular risk factor control in the prevention of late-life dementia. PMID:24267647

Interaction between nuclear insulin receptor substrate-2 and NF-κB in IGF-1 induces response in breast cancer cells.

PubMed

Wu, Shufang; Zhou, Bo; Xu, Lin; Sun, Hongzhi

2010-12-01

Despite significant homology between IRS-1 and IRS-2, recent studies have revealed distinct functions for these adaptor proteins in regulating breast cancer progression. Thus far, most of the studies on breast cancer have focused upon IRS-1, the biological pattern of IRS-2 is limited. We demonstrated that depletion of endogenous IRS-2 by antisense strategies impaired cell proliferation after serum withdrawal, blunted PI3K/Akt and NF-κB activation in IGF-1 induced response in MCF-7 and BT-20 breast cancer cells. In addition, IGF-1 promote nuclear translocation of IRS-2 and NF-κB in MCF-7 and BT-20 cells. Nuclear IRS-2 interaction with NF-κB-p65 and PI3K binding tyrosine residues of IRS-2 are crucial for the NF-κB activities. Moreover, nuclear IRS-2 is recruited to the cyclin D1 promoter both in MCF-7 and BT-20 cells. The selective inhibition of NF-κB-65 abolished the occupancy of IRS-2 to the cyclin D1 promoters. Our studies suggest that IRS-2 plays a significant role by activating, at least in part, NF-κB via PI3K/Akt pathway in IGF-1-induced responses in breast cancer cells and the crosstalk between nuclear IRS-2 and NF-κB might be responsible for transcriptional progression of the breast cancer cells.

The Ia.2 Epitope Defines a Subset of Lipid Raft Resident MHC Class II Molecules Crucial to Effective Antigen Presentation1

PubMed Central

Busman-Sahay, Kathleen; Sargent, Elizabeth; Harton, Jonathan A.; Drake, James R.

2016-01-01

Previous work has established that binding of the 11-5.2 anti-I-Ak mAb, which recognizes the Ia.2 epitope on I-Ak class II molecules, elicits MHC class II signaling, whereas binding of two other anti-I-Ak mAb that recognize the Ia.17 epitope fail to elicit signaling. Using a biochemical approach, we establish that the Ia.2 epitope recognized by the widely used 11-5.2 mAb defines a subset of cell surface I-Ak molecules predominantly found within membrane lipid rafts. Functional studies demonstrate that the Ia.2 bearing subset of I-Ak class II molecules is critically necessary for effective B cell–T cell interactions especially at low antigen doses, a finding consistent with published studies on the role of raft-resident class II molecules in CD4 T cell activation. Interestingly, B cells expressing recombinant I-Ak class II molecules possessing a β chain-tethered HEL peptide lack the Ia.2 epitope and fail to partition into lipid rafts. Moreover, cells expressing Ia.2 negative tethered peptide-class II molecules are severely impaired in their ability to present both tethered peptide or peptide derived from exogenous antigen to CD4 T cells. These results establish the Ia.2 epitope as defining a lipid raft-resident MHC class II confomer vital to the initiation of MHC class II restricted B cell–T cell interactions. PMID:21543648

Suppressive role of OGT-mediated O-GlcNAcylation of BAP1 in retinoic acid signaling.

PubMed

Moon, Seungtae; Lee, Yong-Kyu; Lee, Sang-Wang; Um, Soo-Jong

2017-10-07

BRCA1-associated protein 1 (BAP1) has been implicated in diverse biological functions, including tumor suppression. However, its regulation via glycosylation and its role in embryonic stem (ES) cells are poorly defined. BAP1 was recently reported to interact with O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). Here, we confirmed the physical interaction and investigated its functional significance. The O-GlcNAcylation of BAP1, which requires OGT, was examined in vivo and in vitro, and was proven using alloxan, an OGT inhibitor. OGT promoted the BAP1-induced repression of retinoic acid (RA)-induced RA receptor (RAR) activation. The repressive activity of BAP1 was relieved by alloxan but exacerbated by PUGNAc, an O-GlcNAcase (OGA) inhibitor. Finally, we addressed the role of O-GlcNAcylation in the RA-induced differentiation of murine ES cells. Alkaline phosphatase staining revealed the cooperation of RA and alloxan for impairing the pluripotency of ES cells. This cooperation was also observed by measuring the size of embryonic bodies and the expression of Sox2, a pluripotency marker. Overall, our data suggest that OGT-mediated O-GlcNAcylation of BAP1 prefers the maintenance of pluripotency, whereas its inhibition facilitates RA-induced differentiation in ES cells. Copyright © 2017 Elsevier Inc. All rights reserved.

A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction

PubMed Central

Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O.

2015-01-01

The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR•uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently-labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 µM and inhibited the tight uPAR•uPAATF interaction with an IC50 of 18 µM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR•uPA interface, also inhibited the distal VTN•uPAR interaction. In cell culture, 7 blocked the uPAR•uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells, and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTN•uPARSMB interaction through more favorable electrostatics and entropy. Disruption of the uPAR•VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB•uPAR interaction was less favorable in the VTNSMB•uPAR•7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology. PMID:25671694

A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction.

PubMed

Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O

2015-06-19

The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 μM and inhibited the tight uPAR·uPAATF interaction with an IC50 of 18 μM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB·uPAR interaction was less favorable in the VTNSMB·uPAR·7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology.

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging.

PubMed

Baar, Marjolein P; Brandt, Renata M C; Putavet, Diana A; Klein, Julian D D; Derks, Kasper W J; Bourgeois, Benjamin R M; Stryeck, Sarah; Rijksen, Yvonne; van Willigenburg, Hester; Feijtel, Danny A; van der Pluijm, Ingrid; Essers, Jeroen; van Cappellen, Wiggert A; van IJcken, Wilfred F; Houtsmuller, Adriaan B; Pothof, Joris; de Bruin, Ron W F; Madl, Tobias; Hoeijmakers, Jan H J; Campisi, Judith; de Keizer, Peter L J

2017-03-23

The accumulation of irreparable cellular damage restricts healthspan after acute stress or natural aging. Senescent cells are thought to impair tissue function, and their genetic clearance can delay features of aging. Identifying how senescent cells avoid apoptosis allows for the prospective design of anti-senescence compounds to address whether homeostasis can also be restored. Here, we identify FOXO4 as a pivot in senescent cell viability. We designed a FOXO4 peptide that perturbs the FOXO4 interaction with p53. In senescent cells, this selectively causes p53 nuclear exclusion and cell-intrinsic apoptosis. Under conditions where it was well tolerated in vivo, this FOXO4 peptide neutralized doxorubicin-induced chemotoxicity. Moreover, it restored fitness, fur density, and renal function in both fast aging Xpd TTD/TTD and naturally aged mice. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred, and in doing so tissue homeostasis can effectively be restored. Copyright © 2017 Elsevier Inc. All rights reserved.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

PubMed Central

Kuschal, Christiane; Botta, Elena; Orioli, Donata; Digiovanna, John J.; Seneca, Sara; Keymolen, Kathelijn; Tamura, Deborah; Heller, Elizabeth; Khan, Sikandar G.; Caligiuri, Giuseppina; Lanzafame, Manuela; Nardo, Tiziana; Ricotti, Roberta; Peverali, Fiorenzo A.; Stephens, Robert; Zhao, Yongmei; Lehmann, Alan R.; Baranello, Laura; Levens, David; Kraemer, Kenneth H.; Stefanini, Miria

2016-01-01

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP. PMID:26996949

The SpoIIQ‐SpoIIIAH complex of C lostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis

PubMed Central

Serrano, Mónica; Crawshaw, Adam D.; Dembek, Marcin; Monteiro, João M.; Pereira, Fátima C.; Pinho, Mariana Gomes; Fairweather, Neil F.

2016-01-01

Summary Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc‐binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQH120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQH120S, binds Zn2+, and metal absence alters the SpoIIQ‐SpoIIIAH complex in vitro. Possibly, SpoIIQH120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post‐engulfment, forespore and mother cell‐specific gene expression, suggesting a channel‐like function. Both engulfment and a channel‐like function may be ancestral functions of SpoIIQ‐SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms. PMID:26690930

Recipient Glycemic Micro-environments Govern Therapeutic Effects of Mesenchymal Stem Cell Infusion on Osteopenia

PubMed Central

Sui, Bing-Dong; Hu, Cheng-Hu; Zheng, Chen-Xi; Shuai, Yi; He, Xiao-Ning; Gao, Ping-Ping; Zhao, Pan; Li, Meng; Zhang, Xin-Yi; He, Tao; Xuan, Kun; Jin, Yan

2017-01-01

Therapeutic effects of mesenchymal stem cell (MSC) infusion have been revealed in various human disorders, but impacts of diseased micro-environments are only beginning to be noticed. Donor diabetic hyperglycemia is reported to impair therapeutic efficacy of stem cells. However, whether recipient diabetic condition also affects MSC-mediated therapy is unknown. We and others have previously shown that MSC infusion could cure osteopenia, particularly in ovariectomized (OVX) mice. Here, we discovered impaired MSC therapeutic effects on osteopenia in recipient type 1 diabetes (T1D). Through intensive glycemic control by daily insulin treatments, therapeutic effects of MSCs on osteopenia were maintained. Interestingly, by only transiently restoration of recipient euglycemia using single insulin injection, MSC infusion could also rescue T1D-induced osteopenia. Conversely, under recipient hyperglycemia induced by glucose injection in OVX mice, MSC-mediated therapeutic effects on osteopenia were diminished. Mechanistically, recipient hyperglycemic micro-environments reduce anti-inflammatory capacity of MSCs in osteoporotic therapy through suppressing MSC interaction with T cells via the Adenosine monophosphate-activated protein kinase (AMPK) pathway. We further revealed in diabetic micro-environments, double infusion of MSCs ameliorated osteopenia by anti-inflammation, attributed to the first transplanted MSCs which normalized the recipient glucose homeostasis. Collectively, our findings uncover a previously unrecognized role of recipient glycemic conditions controlling MSC-mediated therapy, and unravel that fulfillment of potent therapeutic effects of MSCs requires tight control of recipient micro-environments. PMID:28435461

Glioblastoma: A Pathogenic Crosstalk between Tumor Cells and Pericytes

PubMed Central

Redondo-Garcia, Carolina; Martinez, Salvador

2014-01-01

Cancers likely originate in progenitor zones containing stem cells and perivascular stromal cells. Much evidence suggests stromal cells play a central role in tumor initiation and progression. Brain perivascular cells (pericytes) are contractile and function normally to regulate vessel tone and morphology, have stem cell properties, are interconvertible with macrophages and are involved in new vessel formation during angiogenesis. Nevertheless, how pericytes contribute to brain tumor infiltration is not known. In this study we have investigated the underlying mechanism by which the most lethal brain cancer, Glioblastoma Multiforme (GBM) interacts with pre-existing blood vessels (co-option) to promote tumor initiation and progression. Here, using mouse xenografts and laminin-coated silicone substrates, we show that GBM malignancy proceeds via specific and previously unknown interactions of tumor cells with brain pericytes. Two-photon and confocal live imaging revealed that GBM cells employ novel, Cdc42-dependent and actin-based cytoplasmic extensions, that we call flectopodia, to modify the normal contractile activity of pericytes. This results in the co-option of modified pre-existing blood vessels that support the expansion of the tumor margin. Furthermore, our data provide evidence for GBM cell/pericyte fusion-hybrids, some of which are located on abnormally constricted vessels ahead of the tumor and linked to tumor-promoting hypoxia. Remarkably, inhibiting Cdc42 function impairs vessel co-option and converts pericytes to a phagocytic/macrophage-like phenotype, thus favoring an innate immune response against the tumor. Our work, therefore, identifies for the first time a key GBM contact-dependent interaction that switches pericyte function from tumor-suppressor to tumor-promoter, indicating that GBM may harbor the seeds of its own destruction. These data support the development of therapeutic strategies directed against co-option (preventing incorporation and modification of pre-existing blood vessels), possibly in combination with anti-angiogenesis (blocking new vessel formation), which could lead to improved vascular targeting not only in Glioblastoma but also for other cancers. PMID:25032689

Alcohol-impaired driving: average quantity consumed and frequency of drinking do matter.

PubMed

Birdsall, William C; Reed, Beth Glover; Huq, Syeda S; Wheeler, Laura; Rush, Sarah

2012-01-01

The objective of this article is to estimate and validate a logistic model of alcohol-impaired driving using previously ignored alcohol consumption behaviors, other risky behaviors, and demographic characteristics as independent variables. The determinants of impaired driving are estimated using the US Centers for Disease Control and Prevention's (CDC) Behavioral Risk Factor Surveillance System (BRFSS) surveys. Variables used in a logistic model to explain alcohol-impaired driving are not only standard sociodemographic variables and bingeing but also frequency of drinking and average quantity consumed, as well as other risky behaviors. We use interactions to understand how being female and being young affect impaired driving. Having estimated our model using the 1997 survey, we validated our model using the BRFSS data for 1999. Drinking 9 or more times in the past month doubled the odds of impaired driving. The greater average consumption of alcohol per session, the greater the odds of driving impaired, especially for persons in the highest quartile of alcohol consumed. Bingeing has the greatest effect on impaired driving. Seat belt use is the one risky behavior found to be related to such driving. Sociodemographic effects are consistent with earlier research. Being young (18-30) interacts with two of the alcohol consumption variables and being a woman interacts with always wearing a seat belt. Our model was robust in the validation analysis. All 3 dimensions of drinking behavior are important determinants of alcohol-impaired driving, including frequency and average quantity consumed. Including these factors in regressions improves the estimates of the effects of all variables.

Interaction between memory impairment and depressive symptoms can exacerbate anosognosia: a comparison of Alzheimer's disease with mild cognitive impairment.

PubMed

Oba, Hikaru; Matsuoka, Teruyuki; Imai, Ayu; Fujimoto, Hiroshi; Kato, Yuka; Shibata, Keisuke; Nakamura, Kaeko; Narumoto, Jin

2018-03-12

To investigate the effects of interactions between memory impairment, depressive symptoms, and anosognosia. Anosognosia for memory impairment was assessed in 118 patients with Alzheimer's disease (AD), 47 patients with mild cognitive impairment (MCI), and 17 non-diagnosed controls (NC) using a questionnaire and evaluation of the anosognosia score as the discrepancy between ratings of the patient and a relative. Demographic characteristics, such as the relationship of the patient with the relative and the activities of daily living (ADL) were evaluated. Memory impairment was evaluated with the Rivermead Behavioral Memory Test (RBMT), depressive symptoms were evaluated using the Geriatric Depression Scale (GDS) 15 items version. In the MCI group, a stepwise multiple regression analysis showed an interaction between RBMT and GDS scores, and simple slope analysis indicated that scores for RBMT at low GDS (-1 standard deviation) were positively correlated with self-rated memory impairment. In the AD group, the relationship of the patient with the relative, ADL, and GDS and RBMT scores were associated with the anosognosia score. Patients with MCI who have no depressive symptoms may be able to more accurately evaluate their memory impairment than those who have depressive symptoms and patients with AD. The evaluation by relatives, depressive symptoms or ADL of patients may distort evaluation of anosognosia for memory impairment in patients with AD or MCI. It seems necessary to include not only depression scale scores but also results of objective memory tests in the patients' medical information for the correct assessment of anosognosia.

MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways.

PubMed

Hu, Zhaoyong; Klein, Janet D; Mitch, William E; Zhang, Liping; Martinez, Ivan; Wang, Xiaonan H

2014-03-01

The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.

The SpoIIQ-SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis.

PubMed

Serrano, Mónica; Crawshaw, Adam D; Dembek, Marcin; Monteiro, João M; Pereira, Fátima C; Pinho, Mariana Gomes; Fairweather, Neil F; Salgado, Paula S; Henriques, Adriano O

2016-04-01

Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc-binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQ(H120S) interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQ(H120S) , binds Zn(2+) , and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQ(H120S) supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post-engulfment, forespore and mother cell-specific gene expression, suggesting a channel-like function. Both engulfment and a channel-like function may be ancestral functions of SpoIIQ-SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Cathepsin B Contributes to Autophagy-related 7 (Atg7)-induced Nod-like Receptor 3 (NLRP3)-dependent Proinflammatory Response and Aggravates Lipotoxicity in Rat Insulinoma Cell Line

PubMed Central

Li, Shali; Du, Leilei; Zhang, Lu; Hu, Yue; Xia, Wenchun; Wu, Jia; Zhu, Jing; Chen, Lingling; Zhu, Fengqi; Li, Chunxian; Yang, SiJun

2013-01-01

Impairment of glucose-stimulated insulin secretion caused by the lipotoxicity of palmitate was found in β-cells. Recent studies have indicated that defects in autophagy contribute to pathogenesis in type 2 diabetes. Here, we report that autophagy-related 7 (Atg7) induced excessive autophagic activation in INS-1(823/13) cells exposed to saturated fatty acids. Atg7-induced cathepsin B (CTSB) overexpression resulted in an unexpected significant increase in proinflammatory chemokine and cytokine production levels of IL-1β, monocyte chemotactic protein-1, IL-6, and TNF-α. Inhibition of receptor-interacting protein did not affect the inflammatory response, ruling out involvement of necrosis. CTSB siRNA suppressed the inflammatory response but did not affect apoptosis significantly, suggesting that CTSB was a molecular linker between autophagy and the proinflammatory response. Blocking caspase-3 suppressed apoptosis but did not affect the inflammatory response, suggesting that CTSB induced inflammatory effects independently of apoptosis. Silencing of Nod-like receptor 3 (NLRP3) completely abolished both IL-1β secretion and the down-regulation effects of Atg7-induced CTSB overexpression on glucose-stimulated insulin secretion impairment, thus identifying the NLRP3 inflammasome as an autophagy-responsive element in the pancreatic INS-1(823/13) cell line. Combined together, our results indicate that CTSB contributed to the Atg7-induced NLRP3-dependent proinflammatory response, resulting in aggravation of lipotoxicity, independently of apoptosis in the pancreatic INS-1(823/13) cell line. PMID:23986436

Nanosized aluminum altered immune function.

PubMed

Braydich-Stolle, Laura K; Speshock, Janice L; Castle, Alicia; Smith, Marcus; Murdock, Richard C; Hussain, Saber M

2010-07-27

On the basis of their uses in jet fuels and munitions, the most likely scenario for aluminum nanoparticle (NP) exposure is inhalation. NPs have been shown to be capable of penetrating deep into the alveolar regions of the lung, and therefore human alveolar macrophages (U937) with human type II pneumocytes (A549) were cultured together and exposed to NPs dispersed in an artificial lung surfactant to more accurately mimic the lung microenvironment. Two types of NPs were evaluated: aluminum (Al) and aluminum oxide (Al2O3). Following a 24-h incubation, cell viability was assessed using MTS, and mild toxicity was observed at higher doses with the U937 cells affected more than the A549. Since the U937 cells provided protection from NP toxicity, the cocultures were exposed to a benign concentration of NPs and infected with the respiratory pathogen community-associated methicillin-resistant Staphylococcus aureus (ca-MRSA) to determine any changes in cellular function. Phagocytosis assays demonstrated that the NPs impaired phagocytic function, and bacterial growth curves confirmed that this reduction in phagocytosis was not related to NP-bacteria interactions. Furthermore, NFkappaB PCR arrays and an IL-6 and TNF-alpha real time PCR demonstrated that both types of NPs altered immune response activation. This change was confirmed by ELISA assays that evaluated the secretion of IL-6, IL-8, IL-10, IL-1beta, and TNF-alpha and illustrated that the NPs repressed secretion of these cytokines. Therefore, although the NPs were not toxic to the cells, they did impair the cell's natural ability to respond to a respiratory pathogen regardless of NP composition.

Ecological photodynamic therapy: new trend to disrupt the intricate networks within tumor ecosystem.

PubMed

Rumie Vittar, N Belén; Lamberti, María Julia; Pansa, María Florencia; Vera, Renzo E; Rodriguez, M Exequiel; Cogno, I Sol; Milla Sanabria, Laura N; Rivarola, Viviana A

2013-01-01

As with natural ecosystems, species within the tumor microenvironment are connected by pairwise interactions (e.g. mutualism, predation) leading to a strong interdependence of different populations on each other. In this review we have identified the ecological roles played by each non-neoplastic population (macrophages, endothelial cells, fibroblasts) and other abiotic components (oxygen, extracellular matrix) directly involved with neoplastic development. A way to alter an ecosystem is to affect other species within the environment that are supporting the growth and survival of the species of interest, here the tumor cells; thus, some features of ecological systems could be exploited for cancer therapy. We propose a well-known antitumor therapy called photodynamic therapy (PDT) as a novel modulator of ecological interactions. We refer to this as "ecological photodynamic therapy." The main goal of this new strategy is the improvement of therapeutic efficiency through the disruption of ecological networks with the aim of destroying the tumor ecosystem. It is therefore necessary to identify those interactions from which tumor cells get benefit and those by which it is impaired, and then design multitargeted combined photodynamic regimes in order to orchestrate non-neoplastic populations against their neoplastic counterpart. Thus, conceiving the tumor as an ecological system opens avenues for novel approaches on treatment strategies. Copyright © 2012 Elsevier B.V. All rights reserved.

Self-organisation after embryonic kidney dissociation is driven via selective adhesion of ureteric epithelial cells.

PubMed

Lefevre, James G; Chiu, Han S; Combes, Alexander N; Vanslambrouck, Jessica M; Ju, Ali; Hamilton, Nicholas A; Little, Melissa H

2017-03-15

Human pluripotent stem cells, after directed differentiation in vitro , can spontaneously generate complex tissues via self-organisation of the component cells. Self-organisation can also reform embryonic organ structure after tissue disruption. It has previously been demonstrated that dissociated embryonic kidneys can recreate component epithelial and mesenchymal relationships sufficient to allow continued kidney morphogenesis. Here, we investigate the timing and underlying mechanisms driving self-organisation after dissociation of the embryonic kidney using time-lapse imaging, high-resolution confocal analyses and mathematical modelling. Organotypic self-organisation sufficient for nephron initiation was observed within a 24 h period. This involved cell movement, with structure emerging after the clustering of ureteric epithelial cells, a process consistent with models of random cell movement with preferential cell adhesion. Ureteric epithelialisation rapidly followed the formation of ureteric cell clusters with the reformation of nephron-forming niches representing a later event. Disruption of P-cadherin interactions was seen to impair this ureteric epithelial cell clustering without affecting epithelial maturation. This understanding could facilitate improved regulation of patterning within organoids and facilitate kidney engineering approaches guided by cell-cell self-organisation. © 2017. Published by The Company of Biologists Ltd.

A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

PubMed

Lee, S; Parent, C A; Insall, R; Firtel, R A

1999-09-01

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Androgens Exert a Cysticidal Effect upon Taenia crassiceps by Disrupting Flame Cell Morphology and Function

PubMed Central

Ambrosio, Javier R.; Valverde-Islas, Laura; Nava-Castro, Karen E.; Palacios- Arreola, M. Isabel; Ostoa-Saloma, Pedro; Reynoso-Ducoing, Olivia; Escobedo, Galileo; Ruíz-Rosado, Azucena; Dominguez-Ramírez, Lenin; Morales-Montor, Jorge

2015-01-01

The effects of testosterone (T4) and dihydrotestosterone (DHT) on the survival of the helminth cestode parasite Taenia crassiceps, as well as their effects on actin, tubulin and myosin expression and their assembly into the excretory system of flame cells are described in this paper. In vitro evaluations on parasite viability, flow cytometry, confocal microscopy, video-microscopy of live flame cells, and docking experiments of androgens interacting with actin, tubulin, and myosin were conducted. Our results show that T4 and DHT reduce T. crassiceps viability in a dose- and time-dependent fashion, reaching 90% of mortality at the highest dose used (40 ng/ml) and time exposed (10 days) in culture. Androgen treatment does not induce differences in the specific expression pattern of actin, tubulin, and myosin isoforms as compared with control parasites. Confocal microscopy demonstrated a strong disruption of the parasite tegument, with reduced assembly, shape, and motion of flame cells. Docking experiments show that androgens are capable of affecting parasite survival and flame cell morphology by directly interacting with actin, tubulin and myosin without altering their protein expression pattern. We show that both T4 and DHT are able to bind actin, tubulin, and myosin affecting their assembly and causing parasite intoxication due to impairment of flame cell function. Live flame cell video microscopy showing a reduced motion as well changes in the shape of flame cells are also shown. In summary, T4 and DHT directly act on T. crassiceps cysticerci through altering parasite survival as well as the assembly and function of flame cells. PMID:26076446

The Saccharomyces cerevisiae anaphase-promoting complex interacts with multiple histone-modifying enzymes to regulate cell cycle progression.

PubMed

Turner, Emma L; Malo, Mackenzie E; Pisclevich, Marnie G; Dash, Megan D; Davies, Gerald F; Arnason, Terra G; Harkness, Troy A A

2010-10-01

The anaphase-promoting complex (APC), a large evolutionarily conserved ubiquitin ligase complex, regulates cell cycle progression through mitosis and G(1). Here, we present data suggesting that APC-dependent cell cycle progression relies on a specific set of posttranslational histone-modifying enzymes. Multiple APC subunit mutants were impaired in total and modified histone H3 protein content. Acetylated H3K56 (H3K56(Ac)) levels were as reduced as those of total H3, indicating that loading histones with H3K56(Ac) is unaffected in APC mutants. However, under restrictive conditions, H3K9(Ac) and dimethylated H3K79 (H3K79(me2)) levels were more greatly reduced than those of total H3. In a screen for histone acetyltransferase (HAT) and histone deacetylase (HDAC) mutants that genetically interact with the apc5(CA) (chromatin assembly) mutant, we found that deletion of GCN5 or ELP3 severely hampered apc5(CA) temperature-sensitive (ts) growth. Further analyses showed that (i) the elp3Δ gcn5Δ double mutant ts defect was epistatic to that observed in apc5(CA) cells; (ii) gcn5Δ and elp3Δ mutants accumulate in mitosis; and (iii) turnover of the APC substrate Clb2 is not impaired in elp3Δ gcn5Δ cells. Increased expression of ELP3 and GCN5, as well as genes encoding the HAT Rtt109 and the chromatin assembly factors Msi1 and Asf1, suppressed apc5(CA) defects, while increased APC5 expression partially suppressed elp3Δ gcn5Δ growth defects. Finally, we demonstrate that Gcn5 is unstable during G(1) and following G(1) arrest and is stabilized in APC mutants. We present our working model in which Elp3/Gcn5 and the APC work together to facilitate passage through mitosis and G(1). To progress into S, we propose that at least Gcn5 must then be targeted for degradation in an APC-dependent fashion.

The tip link protein Cadherin-23: From Hearing Loss to Cancer.

PubMed

Vanniya S, Paridhy; Srisailapathy, C R Srikumari; Kunka Mohanram, Ramkumar

2018-04-01

Cadherin-23 is an atypical member of the cadherin superfamily, with a distinctly long extracellular domain. It has been known to be a part of the tip links of the inner ear mechanosensory hair cells. Several studies have been carried out to understand the role of Cadherin-23 in the hearing mechanism and defects in the CDH23 have been associated with hearing impairment resulting from defective or absence of tip links. Recent studies have highlighted the role of Cadherin-23 in several pathological conditions, including cancer, suggesting the presence of several unknown functions. Initially, it was proposed that Cadherin-23 represents a yet unspecified subtype of Cadherins; however, no other proteins with similar characteristics have been identified, till date. It has a unique cytoplasmic domain that does not bear a β-catenin binding region, but has been demonstrated to mediate cell-cell adhesions. Several protein interacting partners have been identified for Cadherin-23 and the roles of their interactions in various cellular mechanisms are yet to be explored. This review summarizes the characteristics of Cadherin-23 and its roles in several pathologies including cancer. Copyright © 2018 Elsevier Ltd. All rights reserved.

Internal Model Deficits Impair Joint Action in Children and Adolescents with Autism Spectrum Disorders

ERIC Educational Resources Information Center

Stoit, Astrid M. B.; van Schie, Hein T.; Riem, Madelon; Meulenbroek, Ruud G. J.; Newman-Norlund, Roger D.; Slaats-Willemse, Dorine I. E.; Bekkering, Harold; Buitelaar, Jan K.

2011-01-01

Qualitative differences in social interaction and communication are diagnostic hallmarks in autism spectrum disorders (ASD). The present study investigated the hypothesis that impaired social interaction in ASD reflects a deficit to internally model the behavior of a co-actor. Children and adolescents with ASD and matched controls performed a…

Fas cell surface death receptor controls hepatic lipid metabolism by regulating mitochondrial function.

PubMed

Item, Flurin; Wueest, Stephan; Lemos, Vera; Stein, Sokrates; Lucchini, Fabrizio C; Denzler, Rémy; Fisser, Muriel C; Challa, Tenagne D; Pirinen, Eija; Kim, Youngsoo; Hemmi, Silvio; Gulbins, Erich; Gross, Atan; O'Reilly, Lorraine A; Stoffel, Markus; Auwerx, Johan; Konrad, Daniel

2017-09-07

Nonalcoholic fatty liver disease is one of the most prevalent metabolic disorders and it tightly associates with obesity, type 2 diabetes, and cardiovascular disease. Reduced mitochondrial lipid oxidation contributes to hepatic fatty acid accumulation. Here, we show that the Fas cell surface death receptor (Fas/CD95/Apo-1) regulates hepatic mitochondrial metabolism. Hepatic Fas overexpression in chow-fed mice compromises fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes promoting hepatic lipid accumulation and insulin resistance. In line, hepatocyte-specific ablation of Fas improves mitochondrial function and ameliorates high-fat-diet-induced hepatic steatosis, glucose tolerance, and insulin resistance. Mechanistically, Fas impairs fatty acid oxidation via the BH3 interacting-domain death agonist (BID). Mice with genetic or pharmacological inhibition of BID are protected from Fas-mediated impairment of mitochondrial oxidation and hepatic steatosis. We suggest Fas as a potential novel therapeutic target to treat obesity-associated fatty liver and insulin resistance.Hepatic steatosis is a common disease closely associated with metabolic syndrome and insulin resistance. Here Item et al. show that Fas, a member of the TNF receptor superfamily, contributes to mitochondrial dysfunction, steatosis development, and insulin resistance under high fat diet.

Theory of mind reasoning in schizophrenia patients and non-psychotic relatives.

PubMed

Cassetta, Briana; Goghari, Vina

2014-08-15

Research consistently demonstrates that schizophrenia patients have theory of mind (ToM) impairments. Additionally, there is some evidence that family members of schizophrenia patients also demonstrate impairments in ToM, suggesting a genetic vulnerability for the disorder. This study assessed ToM abilities (i.e., sarcasm comprehension) in schizophrenia patients and their first-degree biological relatives during video-taped social interactions, to be representative of real-world interactions and to assess for disease-specific and/or genetic liability effects. Additionally, we assessed whether ToM abilities predicted social and global functioning in schizophrenia patients, and whether symptoms were associated with ToM deficits. Schizophrenia patients demonstrated impairments in sarcasm comprehension compared to controls and relatives, whereas relatives showed intact comprehension. Symptoms of schizophrenia significantly predicted worse ToM abilities. Furthermore, in schizophrenia patients, impaired ToM reasoning predicted worse social and global functioning. Given schizophrenia patients demonstrated impairments in ToM reasoning in a task that resembles real-life interactions, this might be a key area for remediation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing.

PubMed

Xie, Zhongcong; Dong, Yuanlin; Maeda, Uta; Xia, Weiming; Tanzi, Rudolph E

2012-03-22

Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided.

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta

2011-11-18

Highlights: Black-Right-Pointing-Pointer Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. Black-Right-Pointing-Pointer Dysferlin interacts with key signaling proteins for transcytosis in EC. Black-Right-Pointing-Pointer Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, suchmore » as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.« less

Molecular and functional interactions between AKT and SOX2 in breast carcinoma

PubMed Central

Mir, Perihan; Konantz, Martina; Pereboom, Tamara C.; Paczulla, Anna M.; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C.; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

2015-01-01

The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

RSRC1 mutation affects intellect and behaviour through aberrant splicing and transcription, downregulating IGFBP3.

PubMed

Perez, Yonatan; Menascu, Shay; Cohen, Idan; Kadir, Rotem; Basha, Omer; Shorer, Zamir; Romi, Hila; Meiri, Gal; Rabinski, Tatiana; Ofir, Rivka; Yeger-Lotem, Esti; Birk, Ohad S

2018-04-01

RSRC1, whose polymorphism is associated with altered brain function in schizophrenia, is a member of the serine and arginine rich-related protein family. Through homozygosity mapping and whole exome sequencing we show that RSRC1 mutation causes an autosomal recessive syndrome of intellectual disability, aberrant behaviour, hypotonia and mild facial dysmorphism with normal brain MRI. Further, we show that RSRC1 is ubiquitously expressed, and that the RSRC1 mutation triggers nonsense-mediated mRNA decay of the RSRC1 transcript in patients' fibroblasts. Short hairpin RNA (shRNA)-mediated lentiviral silencing and overexpression of RSRC1 in SH-SY5Y cells demonstrated that RSRC1 has a role in alternative splicing and transcription regulation. Transcriptome profiling of RSRC1-silenced cells unravelled specific differentially expressed genes previously associated with intellectual disability, hypotonia and schizophrenia, relevant to the disease phenotype. Protein-protein interaction network modelling suggested possible intermediate interactions by which RSRC1 affects gene-specific differential expression. Patient-derived induced pluripotent stem cells, differentiated into neural progenitor cells, showed expression dynamics similar to the RSRC1-silenced SH-SY5Y model. Notably, patient neural progenitor cells had 9.6-fold downregulated expression of IGFBP3, whose brain expression is affected by MECP2, aberrant in Rett syndrome. Interestingly, Igfbp3-null mice have behavioural impairment, abnormal synaptic function and monoaminergic neurotransmission, likely correlating with the disease phenotype.

Increased B7-H1 expression on dendritic cells correlates with programmed death 1 expression on T cells in simian immunodeficiency virus-infected macaques and may contribute to T cell dysfunction and disease progression.

PubMed

Xu, Huanbin; Wang, Xiaolei; Pahar, Bapi; Moroney-Rasmussen, Terri; Alvarez, Xavier; Lackner, Andrew A; Veazey, Ronald S

2010-12-15

Suppression of dendritic cell (DC) function in HIV-1 infection is thought to contribute to inhibition of immune responses and disease progression, but the mechanism of this suppression remains undetermined. Using the rhesus macaque model, we show B7-H1 (programmed death [PD]-L1) is expressed on lymphoid and mucosal DCs (both myeloid DCs and plasmacytoid DCs), and its expression significantly increases after SIV infection. Meanwhile, its receptor, PD-1, is upregulated on T cells in both peripheral and mucosal tissues and maintained at high levels on SIV-specific CD8(+) T cell clones in chronic infection. However, both B7-H1 and PD-1 expression in SIV controllers was similar to that of controls. Expression of B7-H1 on both peripheral myeloid DCs and plasmacytoid DCs positively correlated with levels of PD-1 on circulating CD4(+) and CD8(+) T cells, viremia, and declining peripheral CD4(+) T cell levels in SIV-infected macaques. Importantly, blocking DC B7-H1 interaction with PD-1(+) T cells could restore SIV-specific CD4(+) and CD8(+) T cell function as evidenced by increased cytokine secretion and proliferative capacity. Combined, the results indicate that interaction of B7-H1-PD-1 between APCs and T cells correlates with impairment of CD4(+) Th cells and CTL responses in vivo, and all are associated with disease progression in SIV infection. Blockade of this pathway may have therapeutic implications for HIV-infected patients.

Assessing the role of peripheral CD8 T cells in neurocognitive impairment in HIV-infected men who have sex with men: data from the MSM Neurocog Study.

PubMed

Rawson, T M; Dubb, S; Pozniak, A; Kelleher, W P; Mandalia, S; Gazzard, B; Barber, T J

2015-02-01

Studies have suggested CD8 lymphocytes may be a possible marker for inflammation, which is believed to be a contributing factor to neurocognitive impairment. Individuals enrolled in the MSM Neurocog Study were analysed. Those with depression, anxiety or mood disorders were excluded. Individuals with neurocognitive impairment were identified using the Brief NeuroCognitive Screen and compared to those with normal scores. CD4 and CD8 T cell values and CD4:CD8 ratios were compared between groups. In all, 144 men, aged 18-50 years, were included in the analysis. Twenty were diagnosed with neurocognitive impairment. We were unable to identify any significant difference between current, nadir or peak CD4 and CD8 counts. CD4:CD8 ratios and CD4:CD8 ratio inversion (<1) were also found to be similar between both groups. However, neurocognitive impairment subjects were 8% more likely to have inversion of CD4:CD8 ratio and higher median peak CD8 cell counts reported compared to non-impaired subjects. Analysis of data from the MSM Neurocog Study, demonstrated trends in peripheral CD8 counts and CD4:CD8 ratios. However, we are unable to demonstrate any significant benefit. Plasma biomarkers of neurocognitive impairment in HIV-infected subjects would be of great benefit over current methods of invasive CSF analysis and technical neuroimaging used in the diagnosis of neurocognitive impairment. Future, prospective, longitudinal work with large numbers of neurocognitive impairment subjects is required to further investigate the role of peripheral CD8 T cells as markers of neurocognitive impairment. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Inhibition of Wnt Signaling Pathways Impairs Chlamydia trachomatis Infection in Endometrial Epithelial Cells

PubMed Central

Kintner, Jennifer; Moore, Cheryl G.; Whittimore, Judy D.; Butler, Megan; Hall, Jennifer V.

2017-01-01

Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis. PMID:29322031

Social Cognition in Schizophrenia: From Social Stimuli Processing to Social Engagement

PubMed Central

Billeke, Pablo; Aboitiz, Francisco

2013-01-01

Social cognition consists of several skills which allow us to interact with other humans. These skills include social stimuli processing, drawing inferences about others’ mental states, and engaging in social interactions. In recent years, there has been growing evidence of social cognitive impairments in patients with schizophrenia. Apparently, these impairments are separable from general neurocognitive impairments, such as attention, memory, and executive functioning. Moreover, social cognition seems to be a main determinant of functional outcome and could be used as a guide to elaborate new pharmacological and psychological treatments. However, most of these studies focus on individual mechanisms and observational perspectives; only few of them study schizophrenic patients during interactive situations. We first review evidences of social cognitive impairments both in social stimuli processing and in mental state attribution. We focus on the relationship between these functions and both general cognitive impairments and functional outcome. We next review recent game theory approaches to the study of how social engagement occurs in schizophrenic patients. The advantage of using game theory is that game-oriented tasks can assess social decision making in an interactive everyday situation model. Finally, we review proposed theoretical models used to explain social alterations and their underlying biological mechanisms. Based on interactive studies, we propose a framework which takes into account the dynamic nature of social processes. Thus, understanding social skills as a result of dynamical systems could facilitate the development of both basic research and clinical applications oriented to psychiatric populations. PMID:23444313

ALCOHOL AND DISTRACTION INTERACT TO IMPAIR DRIVING PERFORMANCE

PubMed Central

Harrison, Emily L. R.; Fillmore, Mark T.

2011-01-01

Background Recognition of the risks associated with alcohol intoxication and driver distraction has led to a wealth of simulated driving research aimed at studying the adverse effects of each of these factors. Research on driving has moved beyond the individual, separate examination of these factors to the examination of potential interactions between alcohol intoxication and driver distraction. In many driving situations, distractions are commonplace and might have little or no disruptive influence on primary driving functions. Yet, such distractions might become disruptive to a driver who is intoxicated. Methods The present study examined the interactive impairing effects of alcohol intoxication and driver distraction on simulated driving performance in 40 young adult drivers using a divided attention task as a distracter activity. The interactive influence of alcohol and distraction was tested by having drivers perform the driving task under four different conditions: 0.65 g/kg alcohol; 0.65 g/kg alcohol + divided attention; placebo; and placebo + divided attention. Results As hypothesized, divided attention had no impairing effect on driving performance in sober drivers. However, under alcohol, divided attention exacerbated the impairing effects of alcohol on driving precision. Conclusions Alcohol and distraction continue to be appropriate targets for research into ways to reduce the rates of driving-related fatalities and injuries. Greater consideration of how alcohol and distraction interact to impair aspects of driving performance can further efforts to create prevention and intervention measures to protect drivers, particularly young adults. PMID:21277119

Alcohol and distraction interact to impair driving performance.

PubMed

Harrison, Emily L R; Fillmore, Mark T

2011-08-01

Recognition of the risks associated with alcohol intoxication and driver distraction has led to a wealth of simulated driving research aimed at studying the adverse effects of each of these factors. Research on driving has moved beyond the individual, separate examination of these factors to the examination of potential interactions between alcohol intoxication and driver distraction. In many driving situations, distractions are commonplace and might have little or no disruptive influence on primary driving functions. Yet, such distractions might become disruptive to a driver who is intoxicated. The present study examined the interactive impairing effects of alcohol intoxication and driver distraction on simulated driving performance in 40 young adult drivers using a divided attention task as a distracter activity. The interactive influence of alcohol and distraction was tested by having drivers perform the driving task under four different conditions: 0.65 g/kg alcohol; 0.65 g/kg alcohol+divided attention; placebo; and placebo+divided attention. As hypothesized, divided attention had no impairing effect on driving performance in sober drivers. However, under alcohol, divided attention exacerbated the impairing effects of alcohol on driving precision. Alcohol and distraction continue to be appropriate targets for research into ways to reduce the rates of driving-related fatalities and injuries. Greater consideration of how alcohol and distraction interact to impair aspects of driving performance can further efforts to create prevention and intervention measures to protect drivers, particularly young adults. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Renal Impairment with Sublethal Tubular Cell Injury in a Chronic Liver Disease Mouse Model

PubMed Central

Ishida, Tokiko; Kotani, Hirokazu; Miyao, Masashi; Kawai, Chihiro; Jemail, Leila; Abiru, Hitoshi; Tamaki, Keiji

2016-01-01

The pathogenesis of renal impairment in chronic liver diseases (CLDs) has been primarily studied in the advanced stages of hepatic injury. Meanwhile, the pathology of renal impairment in the early phase of CLDs is poorly understood, and animal models to elucidate its mechanisms are needed. Thus, we investigated whether an existing mouse model of CLD induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) shows renal impairment in the early phase. Renal injury markers, renal histology (including immunohistochemistry for tubular injury markers and transmission electron microscopy), autophagy, and oxidative stress were studied longitudinally in DDC- and standard diet–fed BALB/c mice. Slight but significant renal dysfunction was evident in DDC-fed mice from the early phase. Meanwhile, histological examinations of the kidneys with routine light microscopy did not show definitive morphological findings, and electron microscopic analyses were required to detect limited injuries such as loss of brush border microvilli and mitochondrial deformities. Limited injuries have been recently designated as sublethal tubular cell injury. As humans with renal impairment, either with or without CLD, often show almost normal tubules, sublethal injury has been of particular interest. In this study, the injuries were associated with mitochondrial aberrations and oxidative stress, a possible mechanism for sublethal injury. Intriguingly, two defense mechanisms were associated with this injury that prevent it from progressing to apparent cell death: autophagy and single-cell extrusion with regeneration. Furthermore, the renal impairment of this model progressed to chronic kidney disease with interstitial fibrosis after long-term DDC feeding. These findings indicated that DDC induces renal impairment with sublethal tubular cell injury from the early phase, leading to chronic kidney disease. Importantly, this CLD mouse model could be useful for studying the pathophysiological mechanisms of sublethal tubular cell injury. PMID:26752420

In Silico Analysis of Usher Encoding Genes in Klebsiella pneumoniae and Characterization of Their Role in Adhesion and Colonization

PubMed Central

Khater, Fida; Balestrino, Damien; Charbonnel, Nicolas; Dufayard, Jean François; Brisse, Sylvain; Forestier, Christiane

2015-01-01

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches. PMID:25751658

Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine.

PubMed

Pagel, René; Bär, Florian; Schröder, Torsten; Sünderhauf, Annika; Künstner, Axel; Ibrahim, Saleh M; Autenrieth, Stella E; Kalies, Kathrin; König, Peter; Tsang, Anthony H; Bettenworth, Dominik; Divanovic, Senad; Lehnert, Hendrik; Fellermann, Klaus; Oster, Henrik; Derer, Stefanie; Sina, Christian

2017-11-01

Endogenous circadian clocks regulate 24-h rhythms of physiology and behavior. Circadian rhythm disruption (CRD) is suggested as a risk factor for inflammatory bowel disease. However, the underlying molecular mechanisms remain unknown. Intestinal biopsies from Per1/2 mutant and wild-type (WT) mice were investigated by electron microscopy, immunohistochemistry, and bromodeoxyuridine pulse-chase experiments. TNF-α was injected intraperitoneally, with or without necrostatin-1, into Per1/2 mice or rhythmic and externally desynchronized WT mice to study intestinal epithelial cell death. Experimental chronic colitis was induced by oral administration of dextran sodium sulfate. In vitro , caspase activity was assayed in Per1/2-specific small interfering RNA-transfected cells. Wee1 was overexpressed to study antiapoptosis and the cell cycle. Genetic ablation of circadian clock function or environmental CRD in mice increased susceptibility to severe intestinal inflammation and epithelial dysregulation, accompanied by excessive necroptotic cell death and a reduced number of secretory epithelial cells. Receptor-interacting serine/threonine-protein kinase (RIP)-3-mediated intestinal necroptosis was linked to increased mitotic cell cycle arrest via Per1/2-controlled Wee1, resulting in increased antiapoptosis via cellular inhibitor of apoptosis-2. Together, our data suggest that circadian rhythm stability is pivotal for the maintenance of mucosal barrier function. CRD increases intestinal necroptosis, thus rendering the gut epithelium more susceptible to inflammatory processes.-Pagel, R., Bär, F., Schröder, T., Sünderhauf, A., Künstner, A., Ibrahim, S. M., Autenrieth, S. E., Kalies, K., König, P., Tsang, A. H., Bettenworth, D., Divanovic, S., Lehnert, H., Fellermann, K., Oster, H., Derer, S., Sina, C. Circadian rhythm disruption impairs tissue homeostasis and exacerbates chronic inflammation in the intestine. © FASEB.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wei; Fu, Jianfang; Zhang, Shun

Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from themore » elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli–germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli–germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. - Highlights: • Intermittent treatment with BTZ caused fertility impairment in adult mice. • BTZ treatment elicited apoptosis during early phase of testicular recovery. • Up-regulation of oxidative stress by BTZ treatment disrupted AJs dynamics. • BTZ treatment stimulated AMPK activity during late phase of testicular recovery. • AMPK-dependent mechanisms counteract FSH proliferative effects in BTZ-exposed SCs.« less

The effects of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor-gamma2 gene on glucose/insulin metabolism interact with prenatal exposure to famine.

PubMed

de Rooij, Susanne R; Painter, Rebecca C; Phillips, David I W; Osmond, Clive; Tanck, Michael W T; Defesche, Joep C; Bossuyt, Patrick M M; Michels, Robert P J; Bleker, Otto P; Roseboom, Tessa J

2006-05-01

An adverse fetal environment may permanently modify the effects of specific genes on glucose tolerance, insulin secretion, and insulin sensitivity. In the present study, we assessed a possible interaction of the peroxisome proliferator-activated receptor (PPAR)-gamma2 Pro12Ala polymorphism with prenatal exposure to famine on glucose and insulin metabolism. We measured plasma glucose and insulin concentrations after an oral glucose tolerance test and determined the PPAR-gamma2 genotype among 675 term singletons born around the time of the 1944-1945 Dutch famine. A significant interaction effect between exposure to famine during midgestation and the PPAR-gamma2 Pro12Ala polymorphism was found on the prevalence of impaired glucose tolerance and type 2 diabetes. The Ala allele of the PPAR-gamma2 gene was associated with a higher prevalence of impaired glucose tolerance and type 2 diabetes but only in participants who had been prenatally exposed to famine during midgestation. Similar interactions were found for area under the curve for insulin and insulin increment ratio, which were lower for Ala carriers exposed to famine during midgestation. The effects of the PPAR-gamma2 Pro12Ala polymorphism on glucose and insulin metabolism may be modified by prenatal exposure to famine during midgestation. This is possibly due to a combined deficit in insulin secretion, as conferred by pancreatic beta-cell maldevelopment and carrier type of the Ala allele in the PPAR-gamma2 gene.

Terreic acid, a quinone epoxide inhibitor of Bruton’s tyrosine kinase

PubMed Central

Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

1999-01-01

Bruton’s tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors. PMID:10051623

CVID-associated TACI mutations affect autoreactive B cell selection and activation

PubMed Central

Romberg, Neil; Chamberlain, Nicolas; Saadoun, David; Gentile, Maurizio; Kinnunen, Tuure; Ng, Yen Shing; Virdee, Manmeet; Menard, Laurence; Cantaert, Tineke; Morbach, Henner; Rachid, Rima; Martinez-Pomar, Natalia; Matamoros, Nuria; Geha, Raif; Grimbacher, Bodo; Cerutti, Andrea; Cunningham-Rundles, Charlotte; Meffre, Eric

2013-01-01

Common variable immune deficiency (CVID) is an assorted group of primary diseases that clinically manifest with antibody deficiency, infection susceptibility, and autoimmunity. Heterozygous mutations in the gene encoding the tumor necrosis factor receptor superfamily member TACI are associated with CVID and autoimmune manifestations, whereas two mutated alleles prevent autoimmunity. To assess how the number of TACI mutations affects B cell activation and tolerance checkpoints, we analyzed healthy individuals and CVID patients carrying one or two TACI mutations. We found that TACI interacts with the cleaved, mature forms of TLR7 and TLR9 and plays an important role during B cell activation and the central removal of autoreactive B cells in healthy donors and CVID patients. However, only subjects with a single TACI mutation displayed a breached immune tolerance and secreted antinuclear antibodies (ANAs). These antibodies were associated with the presence of circulating B cell lymphoma 6–expressing T follicular helper (Tfh) cells, likely stimulating autoreactive B cells. Thus, TACI mutations may favor CVID by altering B cell activation with coincident impairment of central B cell tolerance, whereas residual B cell responsiveness in patients with one, but not two, TACI mutations enables autoimmune complications. PMID:24051380

Increased lipolysis, diminished adipose tissue insulin sensitivity and impaired B-cell function relative to adipose tissue insulin sensitivity in obese youth with impaired glucose tolerance (IGT)

USDA-ARS?s Scientific Manuscript database

Despite evidence of insulin resistance and B-cell dysfunction in glucose metabolism in youth with prediabetes, the relationship between adipose tissue insulin sensitivity (ATIS) and B-cell function remains unknown. We investigated whole-body lipolysis, ATIS and B-cell function relative to ATIS [adip...

Heart and brain interaction in patients with heart failure: overview and proposal for a taxonomy. A position paper from the Study Group on Heart and Brain Interaction of the Heart Failure Association.

PubMed

Doehner, Wolfram; Ural, Dilek; Haeusler, Karl Georg; Čelutkienė, Jelena; Bestetti, Reinaldo; Cavusoglu, Yuksel; Peña-Duque, Marco A; Glavas, Duska; Iacoviello, Massimo; Laufs, Ulrich; Alvear, Ricardo Marmol; Mbakwem, Amam; Piepoli, Massimo F; Rosen, Stuart D; Tsivgoulis, Georgios; Vitale, Cristiana; Yilmaz, M Birhan; Anker, Stefan D; Filippatos, Gerasimos; Seferovic, Petar; Coats, Andrew J S; Ruschitzka, Frank

2018-02-01

Heart failure (HF) is a complex clinical syndrome with multiple interactions between the failing myocardium and cerebral (dys-)functions. Bi-directional feedback interactions between the heart and the brain are inherent in the pathophysiology of HF: (i) the impaired cardiac function affects cerebral structure and functional capacity, and (ii) neuronal signals impact on the cardiovascular continuum. These interactions contribute to the symptomatic presentation of HF patients and affect many co-morbidities of HF. Moreover, neuro-cardiac feedback signals significantly promote aggravation and further progression of HF and are causal in the poor prognosis of HF. The diversity and complexity of heart and brain interactions make it difficult to develop a comprehensive overview. In this paper a systematic approach is proposed to develop a comprehensive atlas of related conditions, signals and disease mechanisms of the interactions between the heart and the brain in HF. The proposed taxonomy is based on pathophysiological principles. Impaired perfusion of the brain may represent one major category, with acute (cardio-embolic) or chronic (haemodynamic failure) low perfusion being sub-categories with mostly different consequences (i.e. ischaemic stroke or cognitive impairment, respectively). Further categories include impairment of higher cortical function (mood, cognition), of brain stem function (sympathetic over-activation, neuro-cardiac reflexes). Treatment-related interactions could be categorized as medical, interventional and device-related interactions. Also interactions due to specific diseases are categorized. A methodical approach to categorize the interdependency of heart and brain may help to integrate individual research areas into an overall picture. © 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.

Socio-demographic characteristics, lifestyle factors and burden of morbidity associated with self-reported hearing and vision impairments in older British community-dwelling men: a cross-sectional study.

PubMed

Liljas, A E M; Wannamethee, S G; Whincup, P H; Papacosta, O; Walters, K; Iliffe, S; Lennon, L T; Carvalho, L A; Ramsay, S E

2016-06-01

Hearing and vision problems are common in older adults. We investigated the association of self-reported sensory impairment with lifestyle factors, chronic conditions, physical functioning, quality of life and social interaction. A population-based cross-sectional study of participants of the British Regional Heart Study aged 63-85 years. A total of 3981 men (82% response rate) provided data. Twenty-seven per cent (n = 1074) reported hearing impairment including being able to hear with aid (n = 482), being unable to hear (no aid) (n = 424) and being unable to hear despite aid (n = 168). Three per cent (n = 124) reported vision impairment. Not being able to hear, irrespective of use of hearing aid, was associated with poor quality of life, poor social interaction and poor physical functioning. Men who could not hear despite hearing aid were more likely to report coronary heart disease (CHD) [age-adjusted odds ratios (ORs) 1.89 (95% confidence interval 1.36-2.63)]. Vision impairment was associated with symptoms of CHD including breathlessness [OR 2.06 (1.38-3.06)] and chest pain [OR 1.58 (1.07-2.35)]. Vision impairment was also associated with poor quality of life, poor social interaction and poor physical functioning. Sensory impairment is associated with poor physical functioning, poor health and poor social interaction in older men. Further research is warranted on pathways underlying these associations. © The Author 2015. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Aging-induced dysregulation of dicer1-dependent microRNA expression impairs angiogenic capacity of rat cerebromicrovascular endothelial cells.

PubMed

Ungvari, Zoltan; Tucsek, Zsuzsanna; Sosnowska, Danuta; Toth, Peter; Gautam, Tripti; Podlutsky, Andrej; Csiszar, Agnes; Losonczy, Gyorgy; Valcarcel-Ares, M Noa; Sonntag, William E; Csiszar, Anna

2013-08-01

Age-related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment, but the underlying mechanisms remain elusive. To test the hypothesis that dysregulation of Dicer1 (ribonuclease III, a key enzyme of the microRNA [miRNA] machinery) impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells (CMVECs) were isolated from young (3 months old) and aged (24 months old) Fischer 344 × Brown Norway rats. We found an age-related downregulation of Dicer1 expression both in CMVECs and in small cerebral vessels isolated from aged rats. In aged CMVECs, Dicer1 expression was increased by treatment with polyethylene glycol-catalase. Compared with young cells, aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing technology), and impaired ability to form capillary-like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs, downregulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration, and tube formation, mimicking the aging phenotype. Collectively, we found that Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age-related dysregulation of Dicer1-dependent miRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.

An Epistatic Interaction between Themis1 and Vav1 Modulates Regulatory T Cell Function and Inflammatory Bowel Disease Development.

PubMed

Pedros, Christophe; Gaud, Guillaume; Bernard, Isabelle; Kassem, Sahar; Chabod, Marianne; Lagrange, Dominique; Andréoletti, Olivier; Dejean, Anne S; Lesourne, Renaud; Fournié, Gilbert J; Saoudi, Abdelhadi

2015-08-15

The development of inflammatory diseases depends on complex interactions between several genes and various environmental factors. Discovering new genetic risk factors and understanding the mechanisms whereby they influence disease development is of paramount importance. We previously reported that deficiency in Themis1, a new actor of TCR signaling, impairs regulatory T cell (Treg) function and predisposes Brown-Norway (BN) rats to spontaneous inflammatory bowel disease (IBD). In this study, we reveal that the epistasis between Themis1 and Vav1 controls the occurrence of these phenotypes. Indeed, by contrast with BN rats, Themis1 deficiency in Lewis rats neither impairs Treg suppressive functions nor induces pathological manifestations. By using congenic lines on the BN genomic background, we show that the impact of Themis1 deficiency on Treg suppressive functions depends on a 117-kb interval coding for a R63W polymorphism that impacts Vav1 expression and functions. Indeed, the introduction of a 117-kb interval containing the Lewis Vav1-R63 variant restores Treg function and protects Themis1-deficient BN rats from spontaneous IBD development. We further show that Themis1 binds more efficiently to the BN Vav1-W63 variant and is required to stabilize its recruitment to the transmembrane adaptor LAT and to fully promote the activation of Erk kinases. Together, these results highlight the importance of the signaling pathway involving epistasis between Themis1 and Vav1 in the control of Treg suppressive function and susceptibility to IBD development. Copyright © 2015 by The American Association of Immunologists, Inc.

Structural insights into the interaction between a potent anti-inflammatory protein, viral CC chemokine inhibitor (vCCI), and the human CC chemokine, Eotaxin-1.

PubMed

Kuo, Nai-Wei; Gao, Yong-Guang; Schill, Megan S; Isern, Nancy; Dupureur, Cynthia M; Liwang, Patricia J

2014-03-07

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines.

Structural Insights into the Interaction between a Potent Anti-inflammatory Protein, Viral CC Chemokine Inhibitor (vCCI), and the Human CC Chemokine, Eotaxin-1*

PubMed Central

Kuo, Nai-Wei; Gao, Yong-Guang; Schill, Megan S.; Isern, Nancy; Dupureur, Cynthia M.; LiWang, Patricia J.

2014-01-01

Chemokines play important roles in the immune system, not only recruiting leukocytes to the site of infection and inflammation but also guiding cell homing and cell development. The soluble poxvirus-encoded protein viral CC chemokine inhibitor (vCCI), a CC chemokine inhibitor, can bind to human CC chemokines tightly to impair the host immune defense. This protein has no known homologs in eukaryotes and may represent a potent method to stop inflammation. Previously, our structure of the vCCI·MIP-1β (macrophage inflammatory protein-1β) complex indicated that vCCI uses negatively charged residues in β-sheet II to interact with positively charged residues in the MIP-1β N terminus, 20s region and 40s loop. However, the interactions between vCCI and other CC chemokines have not yet been fully explored. Here, we used NMR and fluorescence anisotropy to study the interaction between vCCI and eotaxin-1 (CCL11), a CC chemokine that is an important factor in the asthma response. NMR results reveal that the binding pattern is very similar to the vCCI·MIP-1β complex and suggest that electrostatic interactions provide a major contribution to binding. Fluorescence anisotropy results on variants of eotaxin-1 further confirm the critical roles of the charged residues in eotaxin-1. In addition, the binding affinity between vCCI and other wild type CC chemokines, MCP-1 (monocyte chemoattractant protein-1), MIP-1β, and RANTES (regulated on activation normal T cell expressed and secreted), were determined as 1.1, 1.2, and 0.22 nm, respectively. To our knowledge, this is the first work quantitatively measuring the binding affinity between vCCI and multiple CC chemokines. PMID:24482230

Eya2 Is Required to Mediate the Pro-Metastatic Functions of Six1 Via the Induction of TGF-β Signaling, Epithelial-Mesenchymal Transition, and Cancer Stem Cell Properties

PubMed Central

Farabaugh, Susan M.; Micalizzi, Douglas S.; Jedlicka, Paul; Zhao, Rui; Ford, Heide L.

2011-01-01

Six1 is a critical regulator of embryonic development that requires interaction with the Eya family of proteins (Eya1-4) to activate the transcription of genes involved in neurogenesis, myogenesis, and nephrogenesis. While expression of Six1 and Eya family members is predominantly observed in development, their overexpression is observed in numerous cancers. Importantly, both Six1 and Eya have independently been shown to mediate breast cancer metastasis, but whether they functionally interact during tumor progression has not been explored. Herein we demonstrate that knockdown of Eya2 in MCF7 mammary carcinoma cells reverses the ability of Six1 to induce TGF-β signaling, as well as to induce characteristics associated with epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs), suggesting that Six1 is dependent on Eya2 to mediate numerous pro-metastatic characteristics. The importance of the Six1/Eya interaction in human breast cancer is underscored by the finding that high levels of Six1 correlate with shortened time to relapse and metastasis as well as decreased survival only when co-expressed with high levels of Eya2. Overall, these data implicate Eya2 as a necessary cofactor for many of the metastasis promoting functions of Six1, suggesting that targeting the Six1/Eya interaction may inhibit breast cancer progression. Since Six1 and Eya2 are not highly expressed in most adult tissues, the Six1-Eya interaction may be a valuable future therapeutic target whose inhibition would be expected to impair breast cancer progression while conferring limited side effects. PMID:21706047

Investigating Ebola virus pathogenicity using molecular dynamics.

PubMed

Pappalardo, Morena; Collu, Francesca; Macpherson, James; Michaelis, Martin; Fraternali, Franca; Wass, Mark N

2017-08-11

Ebolaviruses have been known to cause deadly disease in humans for 40 years and have recently been demonstrated in West Africa to be able to cause large outbreaks. Four Ebolavirus species cause severe disease associated with high mortality in humans. Reston viruses are the only Ebolaviruses that do not cause disease in humans. Conserved amino acid changes in the Reston virus protein VP24 compared to VP24 of other Ebolaviruses have been suggested to alter VP24 binding to host cell karyopherins resulting in impaired inhibition of interferon signalling, which may explain the difference in human pathogenicity. Here we used protein structural analysis and molecular dynamics to further elucidate the interaction between VP24 and KPNA5. As a control experiment, we compared the interaction of wild-type and R137A-mutant (known to affect KPNA5 binding) Ebola virus VP24 with KPNA5. Results confirmed that the R137A mutation weakens direct VP24-KPNA5 binding and enables water molecules to penetrate at the interface. Similarly, Reston virus VP24 displayed a weaker interaction with KPNA5 than Ebola virus VP24, which is likely to reduce the ability of Reston virus VP24 to prevent host cell interferon signalling. Our results provide novel molecular detail on the interaction of Reston virus VP24 and Ebola virus VP24 with human KPNA5. The results indicate a weaker interaction of Reston virus VP24 with KPNA5 than Ebola virus VP24, which is probably associated with a decreased ability to interfere with the host cell interferon response. Hence, our study provides further evidence that VP24 is a key player in determining Ebolavirus pathogenicity.

Pulmonary and generalized lysosomal storage induced by amphiphilic drugs.

PubMed Central

Hruban, Z

1984-01-01

Administration of amphiphilic drugs to experimental animals causes formation of myelinoid bodies in many cell types, accumulation of foamy macrophages in pulmonary alveoli and pulmonary alveolar proteinosis. These changes are the result of an interaction between the drugs and phospholipids which leads to an alteration in physicochemical properties of the phospholipids. Impairment of the digestion of altered pulmonary secretions in phagosomes of macrophages results in accumulation of foam cells in pulmonary alveoli. Impairment of the metabolism of altered phospholipids removed by autophagy induces an accumulation of myelinoid bodies. The administration of amphiphilic compounds thus causes pulmonary intra-alveolar histiocytosis which is a part of a drug-induced lysosomal storage or generalized lipidosis. The accumulation of drug-lipid complexes in myelinoid bodies and in pulmonary foam cells may lead to alteration of cellular functioning and to clinical disease. Currently over 50 amphiphilic drugs are known. Unique pharmacological properties necessitate clinical use of some of these drugs. The occurrence and severity of potential clinical side effects depend on the nature of each drug, dosage and duration of treatment, simultaneous administration of other drugs and foods, individual metabolic pattern of the patient and other factors. Further studies on factors preventing and potentiating adverse effects of amphiphilic drugs are indicated. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. PMID:6376111

Molecular and functional characterization of CD133+ stem/progenitor cells infused in patients with end-stage liver disease reveals their interplay with stromal liver cells.

PubMed

Catani, Lucia; Sollazzo, Daria; Bianchi, Elisa; Ciciarello, Marilena; Antoniani, Chiara; Foscoli, Licia; Caraceni, Paolo; Giannone, Ferdinando Antonino; Baldassarre, Maurizio; Giordano, Rosaria; Montemurro, Tiziana; Montelatici, Elisa; D'Errico, Antonia; Andreone, Pietro; Giudice, Valeria; Curti, Antonio; Manfredini, Rossella; Lemoli, Roberto Massimo

2017-12-01

Growing evidence supports the therapeutic potential of bone marrow (BM)-derived stem/progenitor cells for end-stage liver disease (ESLD). We recently demonstrated that CD133 + stem/progenitor cell (SPC) reinfusion in patients with ESLD is feasible and safe and improve, albeit transiently, liver function. However, the mechanism(s) through which BM-derived SPCs may improve liver function are not fully elucidated. Here, we characterized the circulating SPCs compartment of patients with ESLD undergoing CD133 + cell therapy. Next, we set up an in vitro model mimicking SPCs/liver microenvironment interaction by culturing granulocyte colony-stimulating factor (G-CSF)-mobilized CD133 + and LX-2 hepatic stellate cells. We found that patients with ESLD show normal basal levels of circulating hematopoietic and endothelial progenitors with impaired clonogenic ability. After G-CSF treatment, patients with ESLD were capable to mobilize significant numbers of functional multipotent SPCs, and interestingly, this was associated with increased levels of selected cytokines potentially facilitating SPC function. Co-culture experiments showed, at the molecular and functional levels, the bi-directional cross-talk between CD133 + SPCs and human hepatic stellate cells LX-2. Human hepatic stellate cells LX-2 showed reduced activation and fibrotic potential. In turn, hepatic stellate cells enhanced the proliferation and survival of CD133 + SPCs as well as their endothelial and hematopoietic function while promoting an anti-inflammatory profile. We demonstrated that the interaction between CD133 + SPCs from patients with ESLD and hepatic stellate cells induces significant functional changes in both cellular types that may be instrumental for the improvement of liver function in cirrhotic patients undergoing cell therapy. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Activation, Impaired Tumor Necrosis Factor-α Production, and Deficiency of Circulating Mucosal-Associated Invariant T Cells in Patients with Scrub Typhus

PubMed Central

Won, Eun Jeong; Cho, Young-Nan; Jung, Hyun-Ju; Kwon, Yong-Soo; Kee, Hae Jin; Ju, Jae Kyun; Kim, Jung-Chul; Kim, Uh Jin; Jang, Hee-Chang; Jung, Sook-In; Kee, Seung-Jung; Park, Yong-Wook

2016-01-01

Background Mucosal-associated invariant T (MAIT) cells contribute to protection against certain microorganism infections. However, little is known about the role of MAIT cells in Orientia tsutsugamushi infection. Hence, the aims of this study were to examine the level and function of MAIT cells in patients with scrub typhus and to evaluate the clinical relevance of MAIT cell levels. Methodology/Principal Findings Thirty-eight patients with scrub typhus and 53 health control subjects were enrolled in the study. The patients were further divided into subgroups according to disease severity. MAIT cell level and function in the peripheral blood were measured by flow cytometry. Circulating MAIT cell levels were found to be significantly reduced in scrub typhus patients. MAIT cell deficiency reflects a variety of clinical conditions. In particular, MAT cell levels reflect disease severity. MAIT cells in scrub typhus patients displayed impaired tumor necrosis factor (TNF)-α production, which was restored during the remission phase. In addition, the impaired production of TNF-α by MAIT cells was associated with elevated CD69 expression. Conclusions This study shows that circulating MAIT cells are activated, numerically deficient, and functionally impaired in TNF-α production in patients with scrub typhus. These abnormalities possibly contribute to immune system dysregulation in scrub typhus infection. PMID:27463223

Entry of Porphyromonas gingivalis outer membrane vesicles into epithelial cells causes cellular functional impairment.

PubMed

Furuta, Nobumichi; Takeuchi, Hiroki; Amano, Atsuo

2009-11-01

Porphyromonas gingivalis, a periodontal pathogen, secretes outer membrane vesicles (MVs) that contain major virulence factors, including proteases termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]). We recently showed that P. gingivalis MVs swiftly enter host epithelial cells via an endocytosis pathway and are finally sorted to lytic compartments. However, it remains unknown whether MV entry impairs cellular function. Herein, we analyzed cellular functional impairment following entry of P. gingivalis into epithelial cells, including HeLa and immortalized human gingival epithelial (IHGE) cells. After being taken up by endocytic vacuoles, MVs degraded the cellular transferrin receptor (TfR) and integrin-related signaling molecules, such as paxillin and focal adhesion kinase (FAK), which resulted in depletion of intracellular transferrin and inhibition of cellular migration. Few Rgp-null MVs entered the cells, and these negligibly degraded TfR, whereas paxillin and FAK degradation was significant. In contrast, Kgp-null MVs clearly entered the cells and degraded TfR, while they scarcely degraded paxillin and FAK. In addition, both wild-type and Kgp-null MVs significantly impaired cellular migration, whereas the effect of Rgp-null MVs was limited. Our findings suggest that, following entry of P. gingivalis MVs into host cells, MV-associated gingipains degrade cellular functional molecules such as TfR and paxillin/FAK, resulting in cellular impairment, indicating that P. gingivalis MVs are potent vehicles for transmission of virulence factors into host cells and are involved in the etiology of periodontitis.

Blind Spots: The Communicative Performance of Visual Impairment in Relationships and Social Interaction

ERIC Educational Resources Information Center

Frame, Melissa J.

2004-01-01

The purpose of this book is to understand the experiences of persons who are visually impaired, including those who are invisibly visually impaired. Through the use of a survey questionnaire and interviews and employing a cross-sectional survey design, the study examines the experiences of a large number of visually impaired respondents. The…

The adaptor molecule SAP plays essential roles during invariant NKT cell cytotoxicity and lytic synapse formation.

PubMed

Das, Rupali; Bassiri, Hamid; Guan, Peng; Wiener, Susan; Banerjee, Pinaki P; Zhong, Ming-Chao; Veillette, André; Orange, Jordan S; Nichols, Kim E

2013-04-25

The adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) plays critical roles during invariant natural killer T (iNKT) cell ontogeny. As a result, SAP-deficient humans and mice lack iNKT cells. The strict developmental requirement for SAP has made it difficult to discern its possible involvement in mature iNKT cell functions. By using temporal Cre recombinase-mediated gene deletion to ablate SAP expression after completion of iNKT cell development, we demonstrate that SAP is essential for T-cell receptor (TCR)-induced iNKT cell cytotoxicity against T-cell and B-cell leukemia targets in vitro and iNKT-cell-mediated control of T-cell leukemia growth in vivo. These findings are not restricted to the murine system: silencing RNA-mediated suppression of SAP expression in human iNKT cells also significantly impairs TCR-induced cytolysis. Mechanistic studies reveal that iNKT cell killing requires the tyrosine kinase Fyn, a known SAP-binding protein. Furthermore, SAP expression is required within iNKT cells to facilitate their interaction with T-cell targets and induce reorientation of the microtubule-organizing center to the immunologic synapse (IS). Collectively, these studies highlight a novel and essential role for SAP during iNKT cell cytotoxicity and formation of a functional IS.

CD22 Promotes B-1b Cell Responses to T Cell-Independent Type 2 Antigens.

PubMed

Haas, Karen M; Johnson, Kristen L; Phipps, James P; Do, Cardinal

2018-03-01

CD22 (Siglec-2) is a critical regulator of B cell activation and survival. CD22 -/- mice generate significantly impaired Ab responses to T cell-independent type 2 (TI-2) Ags, including haptenated Ficoll and pneumococcal polysaccharides, Ags that elicit poor T cell help and activate BCR signaling via multivalent epitope crosslinking. This has been proposed to be due to impaired marginal zone (MZ) B cell development/maintenance in CD22 -/- mice. However, mice expressing a mutant form of CD22 unable to bind sialic acid ligands generated normal TI-2 Ab responses, despite significantly reduced MZ B cells. Moreover, mice treated with CD22 ligand-binding blocking mAbs, which deplete MZ B cells, had little effect on TI-2 Ab responses. We therefore investigated the effects of CD22 deficiency on B-1b cells, an innate-like B cell population that plays a key role in TI-2 Ab responses. B-1b cells from CD22 -/- mice had impaired BCR-induced proliferation and significantly increased intracellular Ca 2+ concentration responses following BCR crosslinking. Ag-specific B-1b cell expansion and plasmablast differentiation following TI-2 Ag immunization was significantly impaired in CD22 -/- mice, consistent with reduced TI-2 Ab responses. We generated CD22 -/- mice with reduced CD19 levels (CD22 -/- CD19 +/- ) to test the hypothesis that augmented B-1b cell BCR signaling in CD22 -/- mice contributes to impaired TI-2 Ab responses. BCR-induced proliferation and intracellular Ca 2+ concentration responses were normalized in CD22 -/- CD19 +/- B-1b cells. Consistent with this, TI-2 Ag-specific B-1b cell expansion, plasmablast differentiation, survival, and Ab responses were rescued in CD22 -/- CD19 +/- mice. Thus, CD22 plays a critical role in regulating TI-2 Ab responses through regulating B-1b cell signaling thresholds. Copyright © 2018 by The American Association of Immunologists, Inc.

Map Learning with a 3D Printed Interactive Small-Scale Model: Improvement of Space and Text Memorization in Visually Impaired Students.

PubMed

Giraud, Stéphanie; Brock, Anke M; Macé, Marc J-M; Jouffrais, Christophe

2017-01-01

Special education teachers for visually impaired students rely on tools such as raised-line maps (RLMs) to teach spatial knowledge. These tools do not fully and adequately meet the needs of the teachers because they are long to produce, expensive, and not versatile enough to provide rapid updating of the content. For instance, the same RLM can barely be used during different lessons. In addition, those maps do not provide any interactivity, which reduces students' autonomy. With the emergence of 3D printing and low-cost microcontrollers, it is now easy to design affordable interactive small-scale models (SSMs) which are adapted to the needs of special education teachers. However, no study has previously been conducted to evaluate non-visual learning using interactive SSMs. In collaboration with a specialized teacher, we designed a SSM and a RLM representing the evolution of the geography and history of a fictitious kingdom. The two conditions were compared in a study with 24 visually impaired students regarding the memorization of the spatial layout and historical contents. The study showed that the interactive SSM improved both space and text memorization as compared to the RLM with braille legend. In conclusion, we argue that affordable home-made interactive small scale models can improve learning for visually impaired students. Interestingly, they are adaptable to any teaching situation including students with specific needs.

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis.

PubMed

Campagne, Cécile; Ripoll, Léa; Gilles-Marsens, Floriane; Raposo, Graça; Delevoye, Cédric

2018-02-14

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease.

AP-1/KIF13A Blocking Peptides Impair Melanosome Maturation and Melanin Synthesis

PubMed Central

Campagne, Cécile; Ripoll, Léa; Gilles-Marsens, Floriane; Raposo, Graça

2018-01-01

Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells. Incubating these peptides with human MNT-1 cells or 3D-reconstructed pigmented epidermis decreases pigmentation by impacting the maturation of melanosomes in fully pigmented organelles. This study highlights that peptides targeting the intracellular trafficking of melanocytes are candidate molecules to tune pigmentation in health and disease. PMID:29443872